Calcium Signalling in Cancer-Sh Erbet

Calcium Signalling in Cancer

Gajanan V. Sherbet
Cancer Research Unit The Medical School University of Newcastle upon Tyne,UK and The Institute for Molecular Medicine Huntington Beach, CA USA
CRC Press Boca Raton London New York Washington, D.C.
Library of Congress Cataloging-in-Publication Data

Sherbet, G. V. (Gajanan V.) Calcium signalling in cancer : the role of calcium binding proteins in signal transduction, cell proliferation, invasion, and metastasis / Gajanan V. Sherbet. p. cm. Includes bibliographical references and index. ISBN 0-8493-0982-4 (alk. paper) 1. Carcinogenesis. 2. Calcium-binding proteins. 3. Metastasis. 4. Cancer invasiveness. I. Title. [DNLM: 1. Neoplasm Proteinsphysiology. 2. Calcium Signalingphysiology. 3. Calcium-Binding Proteinsphysiology. 4. Cell Differentiationphysiology. 5. Neoplasm Invasiveness. 6. Neoplasm Metastasis. QZ 200 S551c 2000] RC268.5 .S53 2000 616.99407dc21 00-062115
This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microlming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specic permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identication and explanation, without intent to infringe.
2001 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-0982-4 Library of Congress Card Number 00-062115 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper
Dedication
to Shri Sai Baba
Preface
The perfect messenger commands respect, delivers the message uncorrupted, at the right time, at the right place, with integrity. Thiru Valluvar (Tamil poet, second century, India) Thirukkural, Chapter 69, verse 688
Calcium signalling occupies a preeminent position in the signal transduction system of the cell by virtue of its participation in a wide range of physiological functions and in the biological events associated with genetic expression, cell proliferation and apoptosis, and cell differentiation and morphogenesis. Calcium signalling is an important feature of cell adhesion and motility, and of cancer invasion and metastasis. The calcium ion virtually unies several pathways of signal transduction. It has rightly been described as a second messenger. Calcium-binding proteins are an important link in the calcium-signalling pathway. These proteins not only maintain the integrity of the calcium signal, but they are also responsible for transmitting the message in a temporally and spatially coordinated manner. It follows therefore that the integrity of the calcium binding proteins themselves is a basic requirement of normal biological function. This concept is encapsulated most eloquently in the Tamil quotation given above. If their integrity is compromised or lost from abnormal or inappropriate expression, or by genetic changes, that could lead to a profound deregulation of signal transduction with dramatic and wide-ranging effects on the life of the cell and its biological behaviour. That is the simple and singular justication for focusing on this protein species in this book. This volume is a natural sequel to my previous works. They are: The Biochemical and Biophysical Characterisation of the Cell Surface (Academic Press, 1978), The Biology of Tumour Malignancy (Academic Press, 1982), The Metastatic Spread of Cancer (Macmillan, 1987), and The Genetics of Cancer (Academic Press, 1997). In these one can discern the evolution of thought relating to the biological behaviour of cells and the pathogenesis of cancer, from inception of the tumour to its progression to the overt metastatic state. This book has not been written with the specialist only in mind. The discussion of topics is self-contained, as far as it was practicable, and I believe, therefore, it would be useful to students and research scientists alike.
Also, the presentation and discussions cater to the needs of scientists in related but peripheral elds of discipline. I had considerable help from my colleagues in the preparation of this book. Professor P.A. Riley of the University College School of Medicine and Dr. M.S. Lakshmi very kindly read the entire manuscript. Their comments and criticisms have been taken and consequently the manuscript has greatly improved in presentation and coverage. Dr. Lakshmi also kindly provided the Tamil quotation and its translation. I wish to thank them most sincerely for their help. It is needless to say, however, that the responsibility for errors and omissions rests solely with me. I thank Paula Rutter of the Audio-visual Centre for preparing the gures and for the patience and skill that she has displayed. CRC Press LLC received this book most enthusiastically and with great fervour. It has been a considerable pleasure working with them, especially with Fequiere Vilsaint, on this project. Finally, the North of England Cancer Research Campaign largely supported the research work in my laboratory and I wish to record my gratitude to them. Gajanan V. Sherbet Cancer Research Unit The Medical School University of Newcastle upon Tyne, UK and The Institute for Molecular Medicine Huntington Beach, CA, USA
Abbreviations
APP ABP ACTH ACV aFGF ALS ANN APC AR BA-1p BAE BDNF bFGF BMD bp BPH BrDU CAI CaM cAMP CAMPK Capn CAR CBD CBD9K CBP cdk cGMP CH CHO CK CNS CP CRE CREB CS DAG DAGK db-cAMP -amyloid precursor protein Amyloid- protein Adrenocorticotropic hormone Acyclovir Acidic broblast growth factor Amyotrophic lateral sclerosis Articial neural network Adenomatous polyposis coli (protein) Androgen receptor Bone-specic alkaline phosphatase Bovine aortic endothelial (cell) Brain-derived neurotropic factor Basic broblast growth factor Becker muscular dystrophy Base pair(s) Benign prostatic hyperplasia Bromo-deoxyuridine Carboxyamido-triazole Calmodulin Cyclic 3,4-adenosine monophosphate Calcium/calmodulin-dependent protein kinase Calpain Cancer-associated retinopathy Calbindin D-28K Calbindin D-9K Calcium-binding protein Cyclin-dependent protein kinase Cyclic guanosine monophosphate Calponin homology (domain) Chinese hamster ovary (cell line) Casein kinase Central nervous system (Actin) capping protein Cyclic AMP response element Cyclic AMP response element binding protein Cowdens syndrome 1,2-Diacylglycerol Diacylglycerol kinase Dibutyryl cyclic AMP
DD DMD DMSO EAE EC ECM EDC EFABP EGF EGFr eNOS Ep-ICAM ER ER ERK ES FC FN FGF FLG FSP1 GABA GAP GC GCAP GDNF GDP GFAP GRP GRPr GTP GTPase 4-HNE HO-1 HSP HUVEC ICAM ICE ICTP IF IFN IGF IL IP3 IP3R IP3R-P
Dariers disease Duchenne muscular dystrophy Dimethyl sulphoxide Experimental allergic encephalomyelitis Endothelial cell Extracellular matrix Epidermal differentiation complex Epidermal-type fatty acid-binding protein Epidermal growth factor Epidermal growth factor receptor Endothelial nitric oxide synthase Epithelial intercellular adhesion molecule Endoplasmic reticulum Oestrogen receptor Extracellular signal-regulated receptor kinase (or MAPK) Embryonic stem (cells) Follistatin-like (domain) Fibronectin Fibroblast growth factor Prolaggrin Fibroblast-specic protein 1 (S100A4) Gamma-amino butyric acid GTPase-activating protein Guanylate cyclase Guanylate cyclase-activating protein Glial cell-derived growth factor Guanosine diphosphate Glial brillary acidic protein Gastrin-releasing peptide Gastrin-releasing peptide receptor Guanosine triphosphate Guanosine triphosphatase 4-Hydroxynonenal Heme oxygenase-1 Heat shock protein Human umbilical vein endothelial cell Intercellular adhesion molecule Interleukin-1-converting enzyme Pyridinoline cross-linked telopeptide of type I procollagen Intermediate laments Interferon Insulin-like growth factor Interleukin Inositol 1,4,5-trisphosphate Inositol 1,4,5-trisphosphate receptor Phosphorylated form of inositol 1,4,5-trisphosphate receptor
JAK LAK LGMD L-NAME LOH LPS MAP MAPK MB-40 MBP MBPK 5-MC MDBK MHC MHC MLC MLCK MMP MMTV MS MSH MTase NAD NCAM NCBP NDP NF2 NF-AT NFT NGF NMDA NOS NSCLC NT3 NMU OA OM ORF OSE OSE-bp OT PA PAI PARP PCNA PCR
Janus tyrosine kinase Lymphokine-activated killer (cells) Limb girdle muscular dystrophy NG-Nitro-l-arginine methyl ester Loss of heterozygosity Lipopolysaccharide Microtubule associated protein Mitogen-activated protein kinase (or ERK) Basement membrane-40 protein (SPARC, osteonectin) Myelin basic protein Myelin basic protein kinase 5-Methylcytosine Madin-Darby bovine kidney (cell line) Major histocompatibility complex Myosin heavy chain Myosin light chain Myosin light chain kinase Matrix metalloproteinases Murine mammary tumour virus Multiple sclerosis Melanocyte-stimulating hormone Methyltransferase Nicotinamide adenine dinucleotide Neural cell adhesion molecule Neural calcium binding protein Nucleoside diphosphate Neurobromatosis type 2 Nuclear factor of activated T cells Neurobrillary tangles Nerve growth factor N-Methyl-d-aspartate Nitric oxide synthase Non-small cell lung carcinoma Neurotropin-3 N-methyl-N-nitrosourea Osteoarthritis Oncomodulin Open reading frame Osteonectin silencer element Osteonectin silencer element-binding protein Oxytocin Plasminogen activator Plasminogen activator inhibitor Poly (ADP-ribose) polymerase Proliferating cell nuclear antigen Polymerase chain reaction
PDE PDGF PDI PEP1 PEST PGE PgR PH PHF PI3K PICP PIIINP PIP PIP2 PIP3 PIP4 PKA PKC PLA2 PLC PMA PMCA PMN PP-1A/PP-1B PS PSA PSA-NCAM PT PTTH PV RA RA rb RBP RCN RGS RP rptn RSV RT RTK RT-PCR RV RXR RyR SCCA
Phosphodiesterase Platelet-derived growth factor Protein disulphide isomerase Prolaggrin endopeptidase 1 Pro, Asp/Glu, Ser, Thr sequence (in proteins) Prostaglandin E Progesterone receptor Pleckstrin homology [domain] Paired helical laments Phosphoinositide-3 kinase C-Terminal peptide of type I procollagen N-Terminal peptide of type III procollagen Phosphatidyl inositol-3 phosphate Phosphatidyl inositol 4,5-bisphosphate Phosphatidyl inositol 3,4,5-trisphosphate Phosphatidyl inositol 1,3,4,5-tetrakisphosphate Protein kinase A Protein kinase C Phospholipase A2 Phospholipase C Phorbol 12-myristate 13-acetate Plasma membrane Ca2+-ATPase Polymorphonuclear leukocyte Protein phosphatases 1A and 1B Presenilin (genes 1 and 2 in Alzheimers disease) Prostate-specic antigen Polysialylated NCAM Demyelinating paralytic tremor rabbit mutant Prothoracicotropic hormone Parvalbumin Retinoic acid Rheumatoid arthritis Retinoblastoma susceptibility (gene product) Retinol-binding protein Recoverin Regulator of G-protein signalling (proteins) Retinitis pigmentosa Repetin Rous sarcoma virus Reverse transcriptase Receptor tyrosine kinase Reverse transcriptase polymerase chain reaction Rubella virus Retinoid X receptor Ryanodine receptor Squamous cell lung carcinoma antigen
SCLC SDK SERCA SLE SMS SPARC SR STAT SVZ T TCR TGF THH TIMP TK Tn TNF TPA tPA uPA VASP VCAM VD3 VDR VDRE VEGF VGCC VILIP VP WAS WASP
Small cell lung carcinoma Sphingosine-dependent kinase Sarcoplasmicendoplasmic reticulum Ca2+-ATPase Systemic lupus erythematosus SmithMagenis syndrome Secreted protein, acidic, rich in cysteine (osteonectin; BM-40) Sarcoplasmic reticulum Signal transducer and activator of transcription factors Subventricular zone Beta thymosins T-cell antigen receptor Transforming growth factor Trichohyalin Tissue inhibitor of metalloproteinase Thymidine kinase Troponin Tumour necrosis factor 12-O-Tetradecanoyl phorbol 13-acetate Tissue plasminogen activator Urokinase-type plasminogen activator Vasodilator-stimulated phosphoprotein Vascular cell adhesion molecule Vitamin D3 Vitamin D3 receptor Vitamin D response element Vascular endothelial growth factor Voltage-gated calcium channel Visinin-like protein Vasopressin WiskottAldrich syndrome WiskottAldrich syndrome protein
Author
Dr. Gajanan Sherbet is a professor at the Institute for Molecular Medicine, Huntington Beach, CA. He received his D.Sc. and M.Sc. degrees from the University of London, and Ph.D., M.Sc., and B.Sc. degrees from the University of Poona. Dr. Sherbet was reader in Experimental Oncology and deputy director of the Cancer Research Unit in the Medical School of the University of Newcastle upon Tyne, England. Previous to this Dr. Sherbet was a staff member of the Chester Beatty Research Institute, Institute of Cancer Research, and University College Hospital Medical School in London. He has held prestigious fellowships such as the Beit Memorial and Williams Fellowship of the University of London. He held a career fellowship awarded by the North of England Cancer Research Campaign. For a brief period, he was a fellow of Harvard University, Cambridge, MA. Dr. Sherbet is a fellow of the Royal College of Pathologists (FRCPath), Royal Society of Chemistry (FRSC), and The Institute of Biology (FIBiol) of U.K. He served as editor of Oncology and Experimental Cell Biology and was senior editor of Pathobiology. Currently, he is a member of the editorial boards of Pathobiology and Anticancer Research. Dr. Sherbets major interest is in cancer metastasis. In recent years he has been investigating the role of the calcium binding protein S100A4 in cell proliferation, cancer invasion, and metastasis, focusing mainly on melanomas, neuroectodermal tumors, and breast cancer. He recently demonstrated the potential value of S100A4 as a marker for assessing the progression of breast cancer. He is also studying the potential of articial neural networks in the management of breast cancer, especially the analysis of expression of cancer markers and image cytometric data of breast cancer by using articial neural networks. Dr. Sherbet has published numerous scientic papers in international journals. He has written and edited several books on cancer. Notable among them are The Metastatic Spread of Cancer (Macmillan, 1987), The Biology of Tumour Malignancy (Academic Press, 1982), and The Biochemical and Biophysical Characterisation of the Cell Surface (Academic Press, 1978). His latest book, The Genetics of Cancer (Academic Press, 1997), was co-authored with Dr. M.S. Lakshmi. He was guest editor of Retinoids: Their Physiological Function and Therapeutic Potential (1997). Dr. Sherbet is co-editor of Articial Neural Networks in Cancer Diagnosis, Prognosis, and Patient Management to be published soon by CRC Press LLC.
Contents
Preface Abbreviations Chapter 1 Introduction................................................................................................................1 Chapter 2 The Calcium Signalling Pathway..............................................................................5 Homeostasis of Cell Calcium...........................................................................5 The Plasma Membrane Ca2+-ATPase Pump ........................................5 The Sarcoplasmic-Endoplasmic Reticulum Ca2+-ATPase Pump .....................................................................................................7 Voltage-Gated Calcium Channels.........................................................8 The Deregulation of Calcium Homeostasis as a Primary Event in Carcinogenesis .......................................................................9 Phospholipid Signalling..................................................................................10 PTEN Phosphatase in the Regulation of Lipid Signalling ............................11 The Protein Kinase C Pathway ......................................................................13 Protein Kinase C and Its Isoforms in Signal Transduction ...........................14 Inositol Phosphates in Calcium Signal Transduction ....................................16 Deregulation of Inositol 1,4,5-Trisphosphate Pathway and Its Consequences ............................................................................................18 Ryanodine and Related Receptors in Calcium Mobilisation.........................20 Cyclic AMP in Calcium Signalling ...............................................................21 Architectural Aspects of the Signal Transduction Machinery.......................25 The Role of Caveolae in Signal Transduction ...................................25 Caveolin Expression in Cancer ..........................................................28 Chapter 3 Calcium Binding Proteins and Their Natural Classication ..................................29 Chapter 4 Non-EF-Hand Calcium Binding Proteins ...............................................................35 Annexins .........................................................................................................35 Structure..............................................................................................35 Biologic Functions..............................................................................36 Annexins in Cancer Growth and Progression....................................38 Annexins in Morphogenesis and Differentiation ...............................39
The Gelsolin Family of Calcium Binding Proteins .......................................40 Gelsolin in Severing and Capping of Actin Filaments ......................40 Gelsolin in Embryonic Development and Morphogenesis ................41 Gelsolin Expression in Amyloidosis ..................................................42 Gelsolin in Cancer ..............................................................................42 Severin and Cytoskeletal Reorganisation...........................................44 Villin in Differentiation and Neoplasia..............................................44 Calreticulin and Its Functional Diversity .......................................................46 Structure and Molecular Features of Calreticulin..............................46 Regulation of Calreticulin Expression ...............................................46 Phosphorylation of Calreticulin..........................................................47 Intracellular Distribution of Calreticulin............................................48 Calreticulin in Intracellular Calcium Storage ....................................48 Calreticulin and Calnexin as Molecular Chaperones.........................49 Calreticulin in Cell Proliferation and Differentiation ........................50 Calreticulin in Cell Adhesion .............................................................50 Calreticulin in Neoplasia ....................................................................51 Immunological Implications of Calreticulin Function.......................52 Calsequestrin and Intracellular Calcium Storage...........................................53 Osteocalcin in Bone Metabolism and Osteotropism of Cancer ....................54 The Biology of Osteocalcin ...............................................................54 Calcium-Binding Properties of Osteocalcin.......................................55 Osteocalcin Gene Structure and Function..........................................55 Regulation of Osteocalcin by Vitamin D3 .........................................56 Osteocalcin in Cell Proliferation and Differentiation ........................57 Osteotropism of Metastatic Dissemination ........................................60 Chapter 5 The EF-Hand Calcium-Binding Proteins ................................................................63 Molecular Organisation of Calcium Binding EF-Hand Proteins ..................63 Calcium Binding and the Molecular Conguration of Calcium-Binding Proteins ...........................................................................................................65 The Structure and Organisation of S100 Family Genes................................68 Alternatively Spliced Variants of S100A4 .....................................................68 Functional Signicance of Alternatively Spliced Isoforms ...........................69 Regulation of Expression of S100 Family Genes..........................................71 Transcriptional Regulation of S100 Genes ........................................71 Regulation of Gene Expression by DNA Methylation......................72 DNA Methylation in Cancer ..............................................................72 Regulation of S100 Gene Transcription by Methylation...................74 Chapter 6 The Calmodulin Family of Calcium Binding Proteins...........................................75 Calmodulin and Its Physiological Function...................................................75 Structure and Mode of Action of Calmodulin ...................................75
Calmodulin-Mediated Signal Transduction........................................76 Calmodulin and Cell Proliferation .....................................................77 Calmodulin in Neoplasia ....................................................................78 Recoverin Subfamily of Neural Calcium-Binding Proteins and Their Function ..........................................................................................................79 The G-Protein Signalling Pathway.....................................................79 Recoverin and Its Function ................................................................80 Mode of Action of Recoverin.............................................................82 Post-translational Modication of Recoverin ....................................82 Recoverin and Cancer-Associated Retinopathy .................................83 Recoverin and Cancer-Associated Retinopathy in Small Cell Lung Cancer ......................................................83 Retinopathy Associated with Other Forms of Human Cancer .....................................................................84 Is Recoverin Involved in Retinitis Pigmentosa? .................85 Guanylate Cyclase-Activating Proteins..........................................................85 Chapter 7 The Structure of Contractile Proteins .....................................................................87 The Actin Component of Contractile Machinery of the Cell........................87 Actin Isoforms ....................................................................................87 Regulation of Actin Dynamics ...........................................................88 Colin in the Regulation of Actin Dynamics ....................................88 Prolin in the Regulation of Actin Dynamics ...................................90 Rho GTPases in Actin Dynamics and Signal Transduction ..............90 Interaction of Formin with Prolin and Rho GTPases......................92 The Role of Thymosin Family Actin-Binding Proteins in Actin Dynamics ..............................................................................................93 Sequestration of Actin by Thymosins ................................................93 Effects of Thymosins on Cell Proliferation .......................................93 Thymosins and Cell Motility and Differentiation .............................94 Expression of Thymosins in Embryonic Development .....................94 Potential Role of Thymosins in Cancer Progression.........................95 The Fimbrin Family of Actin-Binding Proteins............................................. 96 Molecular Features of Fimbrin...........................................................96 Function of Fimbrin in Cytoskeletal Organisation ............................97 Regulation of Fimbrin Expression .....................................................99 Is Fimbrin Involved in Cancer?........................................................100 Modulation of Actin Dynamics and Cancer Cell Dissemination ...................................................................................101 -Actinin.......................................................................................................102 Molecular Structure of -Actinin ....................................................102 -Actinin Isoforms ...........................................................................103 Function of -Actinin.......................................................................104
Actinins in Cell Adhesion, Motility, and Signal Transduction........104 The CadherinCatenin Complex in Signal Transduction and Cell Adhesion ...................................................................................104 Myosin Filaments .........................................................................................110 Myosin Heavy Chain (MHC) Isoforms ...........................................111 Actomyosin Assembly......................................................................112 Myosin Light Chain (MLC) Phosphorylation and Function ...........115 Troponins and Tropomyosins in the Regulation of Muscle Cell Contraction....................................................................................................118 The Regulatory Role of Troponins and Tropomyosins in Muscle Contraction........................................................................................118 Tropomyosin Isoforms in Benign and Malignant Cells ..................119 The Regulatory Role of Caldesmon.................................................121 Calponin: Its Function and Regulation ............................................122 Caltropin-Mediated Reversal of Myosin-ATPase Inhibition by Caldesmon and Calponin..................................................................124 Chapter 8 Structure and Biology of Calbindin ......................................................................125 Calbindin in Neuronal Populations ..............................................................125 Neural Cell Lineage and the Regulation of Calbindin Expression .............125 Calbindin Expression in Embryonic Development and Ageing..................126 Physiological Function of Calbindin............................................................127 Neuroprotective Function of Calbindin........................................................129 Calbindin Expression and the Metastatic Phenotype ..................................129 Chapter 9 Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker......................................................................................................131 Calretinin and Its Alternatively Spliced Isoforms........................................131 Regulation of Calretinin Expression ............................................................132 Calretinin Expression in Cell Proliferation and Differentiation..................133 Calretinin and Its Possible Neuroprotective Property..................................133 Calretinin as a Potential Tumour Marker.....................................................133 Chapter 10 Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading ................135 Molecular Features of Calcineurin...............................................................135 Calcineurin in Cell Proliferation and Adhesion-Related Phenomena .........136 Putative Role of Calcineurin in Cell Cycle Progression .................136 The Effects of Calcineurin on Cell Adhesion and Motility ............138 Calcineurin in Alzheimers Disease .............................................................140 Calcineurin in Immunosuppression..............................................................141
Chapter 11 Centrins (Caltractins) and Their Biological Functions .........................................145 Chapter 12 Reticulocalbin Family of EF-Hand Proteins.........................................................149 Molecular Features of Reticulocalbin Homologues ....................................150 Putative Functions of Reticulocalbin and Its Homologues .........................150 Chapter 13 Calpains in Normal and Aberrant Cell Physiology ..............................................153 The Calpain Family of Calcium-Binding Proteins ......................................153 Molecular Organisation of Calpains ............................................................154 Regulation of Physiological Events by Proteolytic Function......................155 Involvement of Calpains in Development and Differentiation....................157 Calpains in Cell Proliferation and Apoptosis ..............................................158 Calpains in Cell Spreading and Migration ..................................................160 Calpains in Integrin-Mediated Cell Adhesion and Signal Transduction..................................................................................................161 Calpains in Cancer Growth and Progression ...............................................162 Calpains in Myelodegenerative Diseases .....................................................163 Calpains in Muscular Dystrophy..................................................................165 Association of Calpains with Duchenne Muscular Dystrophy .......165 Calpains and Limb Girdle Muscular Dystrophy..............................166 Chapter 14 Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia .................169 Caspases in Apoptotic Cell Death................................................................169 Poly (ADP-Ribose) Polymerase as a Marker of Apoptosis ........................172 Caspase-Mediated Apoptosis and Cell Growth Inhibition in Tumour Expansion........................................................................................173 Caspase-Mediated Proteolysis of Fodrin: Implications for Apoptosis, Cell Adhesion, Cell Migration, and Neoplastic Transformation .................176 Caspases and Neuronal Loss in Alzheimers Disease .................................177 Chapter 15 Parvalbumins in Neuronal Development, Differentiation, and Proliferation .......181 Chapter 16 Osteonectin in Cell Function and Behaviour........................................................183 Molecular Structure of Osteonectin .............................................................183 Functions and Functional Domains of Osteonectin.....................................184 Regulation of Osteonectin Expression .........................................................184 Osteonectin in the Remodelling of the Extracellular Matrix ......................186
Osteonectin in Embryonic Development and Differentiation .....................187 Modulation of Cellular Adhesion, Cell Shape, and Motility by Osteonectin ..............................................................................................188 Modulation of Cell Proliferation by Osteonectin ........................................190 Effects of Osteonectin on Angiogenesis ......................................................191 Osteonectin Expression in Cancer Development and Progression..............193 Osteonectin Involvement in Other Disease States .......................................196 Osteonectin Homologues and Their Putative Tumour Suppressor Properties ......................................................................................................197 Chapter 17 S100 Proteins: Their Biological Function and Role in Pathogenesis ..................199 S100 Proteins in Cell Differentiation, Motility, and Cancer Invasion ........202 Prolaggrin (FLG) in Keratinocyte Differentiation.........................202 The Molecular Characteristics of Prolaggrin..................202 Trichohyalin (THH)..........................................................................205 Effects of S100 Proteins on Cell Deformability and Cellular Morphology ..................................................................................................205 Cell Adhesion and Invasive Potential of Cancer Cells ....................210 S100 Proteins in Remodelling of the Extracellular Matrix.............213 S100 Proteins in Cell Proliferation ..................................................214 Cell Cycle-Related Expression of S100 Proteins ............................217 Postulated Mechanism of Cell Cycle Control by S100A4..............219 S100A Isoforms............................................................................................222 S100A2 as a Putative Tumour Suppressor...................................................223 S100A3 Expression in Cell Differentiation and Neoplasia.........................224 Molecular Features of S100A3 ........................................................224 S100A3 Expression in Cell Differentiation and Human Gliomas .............................................................................................225 S100A4 in Cancer Development and Progression.......................................225 S100A4 Expression and Metastatic Potential of Cancers ...............225 Clinical Potential of S100A4 as a Marker for Cancer Prognosis...........................................................................................230 S100A4 in Human Breast Cancer ....................................................230 S100A4 in Other Forms of Human Cancer .....................................234 S100A6 (Calcyclin) in Cancer .....................................................................235 The Biological Properties of S100A7 (Psoriasin) .......................................236 Structure and Molecular Properties of S100A7...........................................236 S100A7 in Skin Pathology ...............................................................237 S100A7 in Neoplastic Disease .........................................................238 S100A8 and S100A9 Proteins in Inammatory Diseases ...........................239 S100A11 (S100C) and Possible Modes of its Function..............................239 S100P in Cancer Progression .......................................................................241 S100P and Its Putative Functions.....................................................241
Potential Value of S100 Proteins as Markers of Cancer Progression and Prognosis.......................................................................................................243 Epilogue ................................................................................................................245 References .............................................................................................................249 Index......................................................................................................................349
Introduction
Calcium-binding proteins (CBPs) are a family of unique importance in normal and aberrant cell biology, by virtue of their participation in a wide spectrum of physiological processes. These proteins appear to function from the inception of life, participating in sperm maturation and motility and implantation of the fertilised ovum, to cell differentiation and morphogenesis. Among other important physiological functions of CBPs are their involvement in Ca2+ transport and the maintenance of intracellular Ca2+ levels, as well as metabolic processes such as nucleotide metabolism. Signal transduction is another area of cellular physiology in which CBPs are heavily involved. Defective signal transduction is often associated with aberrant cell cycle regulation, which in turn can lead to the development and progression of cancer. Besides, several CBPs, especially those belonging to the S100 protein family, have recently been demonstrated to be actively engaged in the regulation of the cell cycle in normal as well as in aberrant cell proliferation and in apoptotic cell death. It follows, therefore, that CBPs may be associated with the pathogenesis of several diseases, including neoplastic disease. Directly relevant in the context of carcinogenesis is the accumulation of a substantial body of evidence demonstrating that CBPs participate in the formation and emergence of neoplastic foci of cells and in the invasive and metastatic spread of these cells constituting the progressive phase of the disease. A major pathway by which CBPs affect neoplastic progression is by altering cell proliferation via the modulation of the process of transduction of proliferative signals imparted by extracellular growth factors and hormones, and thus the regulation of the cell cycle traverse and apoptotic cell death. Inuencing cell adhesion and motility by modulating the function of enzymes implicated in the remodelling of the extracellular matrix, leading to metastatic dissemination of cancer cells, are also important features of CBP function in cancer. CBPs are associated with other pathological conditions of the central nervous system (CNS) and skin, such as amyloidosis, Alzheimers disease, and SmithMagenis syndrome (SMS), among others. The purview of the present volume is to review, analyse, and assimilate these apparently diverse functions of CBPs into a coherent picture. The conceptual basis of this book, summarised in Figure 1, is to recognise, redene, and establish CBPs as second messengers themselves. Intracellular calcium levels modulate in response to extracellular signals and calcium transport across the cell membrane. CBPs play a crucial role in calcium homeostasis, by calcium buffering, calcium transport, and release of calcium from intracellular stores. They carry the information downstream to activate the phosphorylation of target proteins, which leads to enzyme function and metabolism, muscle contraction, and a host of other physiological functions. They also inuence cytoskeletal dynamics, actively participate in the remodelling of the extracellular matrix, and consequentially affect cell morphology, motility, and intercellular interactions. A recent development is the
1
FIGURE 1 Summary of the conceptual basis of this book, providing an outline of the postulate that forms the core of this work: calcium binding proteins (CBPs) are second messengers participating in the calcium signalling cascade. CBPs not only regulate intracellular calcium levels, but they also are involved in the regulation of normal cell physiology, as well as cell behaviour, proliferation, apoptosis, tumorigenesis, and the secondary spread of cancer.
demonstration that certain CBPs may actively control the progression of the cell cycle in consort with other proteins such as p53 and stathmin. Apoptosis or programmed cell death is a calcium-dependent phenomenon, and CBPs can activate several pathways of molecular degradation leading to apoptosis. In light of these multifarious functions, the thesis developed here is that CBPs are an integral component of the mechanisms of cell population homeostasis and tumour growth. Genetic activation is the coup de grace of signal transduction. The inappropriate expression, temporally and spatially, of some CBPs induces invasive behaviour and
Introduction
secondary spread of immortalised cells. These genetic and phenotypic changes induced by CBPs are integrated here to provide a postulate for the growth and progression of cancers. The formulation of this postulate underpins the evolution and development of new strategies for controlling the growth of the tumour and its metastatic spread.
The Calcium Signalling Pathway
HOMEOSTASIS OF CELL CALCIUM

Ion channels are involved in a large number of cell functions and in the induction of cellular responses to extracellular stimuli. There are three types of voltagesensitive ion channels: the sodium channel, the potassium channel, and the calcium channel. These function in a coordinated fashion in determining cellular responses to extraneous signals. The Na+ channel is involved in the generation of action potentials and the K+ channel is involved in the regulation of membrane potential and is signicantly involved in synaptic plasticity. The regulation of intracellular levels of calcium is an important element in the signalling process mediated by calcium as a second messenger. There are several mechanisms by which calcium levels in the cell are exquisitely controlled and calcium homeostasis is achieved. These are listed together with their characteristic features and properties in Table 1. The Ca2+-ATPase pumps in the plasma membrane and the sarcoplasmicendoplasmic reticulum play a highly signicant part in the regulation of calcium homeostasis. Calcium homeostasis also involves the ligandgated and voltage-gated channels. Calcium-binding proteins are of special interest in the present context because of their ability to regulate intracellular calcium levels.
THE PLASMA MEMBRANE CA2+-ATPASE PUMP

The plasma membrane Ca2+-ATPase extrusion pump (PMCA) and the sarcoplasmicendoplasmic reticulum Ca2+-ATPase pump (SERCA) are specically targeted to the two membrane systems of the cell. The N-terminal region of the SERCA molecule contains a domain that has an endoplasmic reticulum (ER)-retention signal sequence. Certain mutations of the PMCA can cause it to become localised to the ER (Guerini et al. 1998). There are four genes which code for four isoforms. These isoforms show differential localisation in different organs and the subcellular compartment. PMCA-1 and PMCA-2 are expressed ubiquitously, but PMCA-3 and PMCA-4 occur specically in neural cells (Carafoli et al. 1996). The expression of PMCA genes seems to be developmentally regulated. Thus, in mouse development, PMCA genes show an overlapping, but, nonetheless, a distinctive pattern of expression (Zacharias and Kapper, 1999). Furthermore, PMCA and SERCA genes are co-ordinately regulated. The differentiative effects of nerve growth factor (NGF) on PC12 cells are an example of this. NGF not only up-regulates PMCA but also down-regulates SERCA expression (Keller and Grover, 2000).
TABLE 1 Mechanisms of Homeostasis of Cell Calcium

Mode of Homeostasis Calcium Pumps Na+/Ca2+ exchanger PMCA Ca2+-ATPase pump SERCA Ca2+-ATPase pump Ligand-gated calcium channels Voltage-gated calcium channels (VGCCs) Subtypes: T L N P/Q R Calcium binding proteins Characteristics
Plasma membraneassociated low afnity mechanism; pumps out large quantities of Ca2+ Plasma membrane calcium-ATPase pump involved in calcium extrusion High afnity ER-located mechanism; leads to luminal accumulation of Ca2+ and reduces cytosolic levels Initiation of calcium inux upon binding of ligands to respective receptors; direct inux of Ca2+ or mediated by IP3 Ca2+ inux/release from intracellular stores
Transient currents in response to membrane depolarisation Prolonged currents in response to depolarisation Inactivate in response to moderate, not severe depolarisation; not sensitive to intracellular cations Nerve terminals; initiation of neurotransmitter release Regulation of VGCC, PMCA and SERCA (e.g., by CaM); NMDA receptor channel by CaM and references cited in the text
Note: CaM, calmodulin; ER, endoplasmic reticulum; IP3, inositol 1,4,5-triphosphate; NMDA, N-methyl-D-aspartate; PMCA, plasma membrane Ca2+-ATPase; SERCA, sarcoplasmicendoplasmic reticulum Ca2+-ATPase. Source: Collated from Tsien et al. (1988), Tsunoda (1993), Campbell et al. (1988), Marin et al. (1998); Pascale and Etcheberrigaray (1999); Tsien et al. (1991); Dunlap et al. (1995); Ehlers et al. (1996), and references cited in the text.
Membrane depolarisation can also differentially regulate PMCA and SERCA expression (Guerini et al. 1999). Kuo et al. (1997) had previously reported that their expression might indeed be interdependent. The N-terminal region of PMCA molecule is highly variable and is essential for enzymatic activity. There seems to be an absolute requirement of a specic amino acid sequence for its function (Talgham and Adamo, 1999). The transcription of the PMCA isoforms is controlled by calcium itself. Calciumbinding proteins are intricately connected with this process. In developing neurones calcineurin seems to regulate PMCA expression (Carafoli et al. 1999). Besides, PMCA isoforms show a marked differential sensitivity to calcium, calmodulin (CaM), ATP, and kinases. The calcium pump activity appears to be regulated by phosphorylation, and phosphorylation might be the means by which different
isoforms become functional in a temporal dimension (Monteith et al. 1998). PMCA is activated by Ca2+/CaM, and the isoforms also show marked differences in their sensitivity to Ca2+/CaM (Elwess et al. 1997, 1998). Human PMCA-4a is phosphorylated at a serine residue that occurs within the CAM-binding domain (Verma et al. 1999; Carafoli, 1997; Carafoli et al. 1996). Phosphorylation appears to prevent CaM binding and lead to the inactivation of the pump (Enyedi et al. 1997; Penniston and Enyedi, 1998).
THE SARCOPLASMICENDOPLASMIC RETICULUM CA2+-ATPASE PUMP

SERCA is one of the modes that controls the homeostasis of intracellular calcium. SERCA2 transports Ca2+ into the lumen of the reticulum by an ATP-dependent mechanism. By virtue of this ability, SERCA2 plays an important part in normal physiological function such as the contraction and relaxation cycle of cardiac muscle, as well as in the pathogenesis of certain diseases. In addition, the correct localisation of the SERCA protein in the ER is of the utmost importance. As stated above, the localisation signal occurs in a stretch of 28 amino acids at the N-terminus of the molecule (Guerini et al. 1998). Ankyrin, a cytoskeletal protein, also seems to be involved in the targeting of SERCA. This is indicated by its abnormal localisation in ankyrin / mice. In these mutant mice, the absence of ankyrin also affects the localisation of ryanodine and inositol, 1, 4, 5-trisphophate (IP3) receptors (IP3R), both involved in calcium mobilisation (Tuvia et al. 1999). The ATP2A2 gene encodes the sarcoplasmicendoplasm reticulum ATPase isoform type 2. Periasamy et al. (1999) have demonstrated the loss of calcium sequestering activity and consequent impairment of cardiac function in heterozygous ATP2A2 gene mutants. Mutations of the ATP2A2 gene have been associated with the pathogenesis of Dariers disease (DD) (keratosis follicularis) (Sakuntabhai et al. 1999a, 1999b). DD is an autosomal dominantly inherited skin disorder. It is characterised by the presence of keratotic papules, and in histology is distinguished by acantholytic dyskeratosis. The keratotic papules occur mainly in the upper trunk, scalp, and palmar pits. Nail dystrophy is also a prominent feature of DD (Soroush and Gurevitch, 1997). Because ATP2A2 controls calcium homeostasis in the cell, it is conceivable that its deregulation by mutation of the ATP2A2 gene might represent an early event in carcinogenesis. It was demonstrated many years ago that thapsigargin, an inhibitor of SERCA2, not only alters intracellular calcium levels, but also functions as a tumour promoter (Hakii, 1986; Thastrup et al. 1990). With the demonstration that ATP2A2 mutations are associated with the genesis of DD, there have also been several attempts to investigate the incidence of neoplasia in association with DD. Soroush and Gurevitch (1997) have pointed out that basal cell carcinomas and other skin neoplasms occur in patients with DD. Downs et al. (1997) described the occurrence of a subungual squamous cell carcinoma in a DD patient. However, in this case the human papilloma virus might have been the aetiological agent. In general, neoplasia may be associated only infrequently with DD. There is an obvious common feature between DD and neoplasia that deserves a mention. This concerns the abnormal expression of the calcium-binding transmembrane glycoprotein cadherin. Some isoforms of cadherin have been regarded as
invasion suppressors, because there is a signicant body of evidence that associates the acquisition of invasive ability by tumour cells with the loss of cadherins. E-type cadherin is found in the cell membrane of epidermal cells. E-cadherin is distributed in the plasma membrane of keratinocytes, in the intercellular space between desmosomes. Interestingly, this cadherin is expressed at greatly reduced levels in the acatholytic cells of DD and HaileyHailey disease (benign pemphigus) (Furukawa et al. 1997). Desmosomes provide the adhesion machinery in most epithelia, and abnormalities associated with them may be a key feature of inherited diseases such as DD and HaileyHailey disease. Indeed, the loss of cadherin may be the determining factor that causes the characteristic loss of adhesion between epidermal cells encountered in DD. Furukawa et al. (1997) also point out that both E- and Pcadherins are absent in squamous carcinomas, malignant melanomas and in Pagets disease. But basal cell carcinomas, which are noninvasive, do not show a loss of cadherin. The above discussion underscores the importance of the SERCA2 calcium pump and intracellular calcium homeostasis in the calcium-signalling events associated with cell differentiation and dedifferentiation, and in the pathogenesis of DD and neoplasia.
VOLTAGEGATED CALCIUM CHANNELS

The Voltage-gated calcium channels (VGCCs) are located in the plasma membrane. The high voltage-activated channel subtypes L,N, P, Q, and R occur as heterodimers of four subunits. The largest of the subunits is the 1 subunit, which spans the plasma membrane and the auxiliary subunits, , , and 2 (Isom et al. 1994). The properties of the calcium channel appear to be determined by the differential expression of 1, which is the pore-forming subunit. The subunit seems to regulate channel properties and the targeting of 1. The subunit, of which four isoforms (14) have been identied, is said to interact with 1. This interaction is mediated by certain highly conserved domains (Pragnall et al. 1994; De Waard et al. 1994). The interaction between these subunits appears to regulate channel activity and the diversity of calcium currents (Varadi et al. 1991; Isom et al. 1994; Olcese et al. 1994). The subunits are believed to modulate the kinetics of channel activation and inactivation by means of phosphorylation. Calcium-binding proteins feature prominently in calcium homeostasis as important regulators of calcium channel activity. The elevation of intracellular levels of calcium can provide a negative feedback and close the channel. On the other hand, the feedback can have a positive element or facilitation, which opens the calcium channel. Zuhlke et al. (1999) have shown that CaM functions as a sensor for both negative and positive regulation of L-type channel activity. CaM appears to do this by binding to the consensus sequence called the IQ motif that occurs in the Cterminal 1C subunit of the channel protein. They substituted the isoleucine residue with alanine and reduced Ca2+-dependent inactivation and enhanced facilitation of the channel. Both inactivation and facilitation were abrogated when the isoleucine residue was converted to glutamate. CaM binds to the IQ motifs of other channel subtypes N, P/Q, and R as well (Peterson et al. 1999). Indeed, calcium-mediated
modulation of P/Q channel subtypes also seems to involve CaM function. Again the channel activity is regulated CaM binding to the IQ motif of the 1A channel subunit (A. Lee et al. 1999).
THE DEREGULATION OF CALCIUM HOMEOSTASIS AS A PRIMARY EVENT IN CARCINOGENESIS

The operation of ligand-gated calcium channels and the role that calcium binding proteins play in calcium homeostasis and as messengers of the calcium signal provide the major focus for this work. There is much justication for the view held by many investigators that the deregulation of calcium homeostasis in the cell might be a primary event in cell transformation and carcinogenesis. Indeed, several agents that block or retard the inux of calcium into cells appear to be able to inhibit the invasive ability of cancer cells, alter their adhesion properties, and inhibit tumour growth and stabilise the disease. Carboxyamido-triazole (CAI) is one such compound. CAI has been reported to be able to inhibit the invasive behaviour of breast carcinoma cell lines in vitro. The matrix metalloproteinases associated with these cells also appear to be inhibited in parallel (Lambert et al. 1997). Not surprising, therefore, is the nding that CAI inhibits the adhesion of glioma cells to collagen type IV coated substrata and also inhibits their invasion in vitro. CAI also appears to be capable of inhibiting cell proliferation (Jacobs et al. 1997). The antiproliferation and anti-invasion properties of CAI have also been described by Wasilenko et al. (1996) using human prostate cancer cell lines. These are clear indications of the variety of downstream pathways activated by calcium inux that are inhibited by CAI. Finally, CAI has been reported to stabilise the disease in patients with a variety of refractory solid tumours (Kohn et al. 1996). Verapamil, a selective L-type calcium channel blocker, also has demonstrable anti-proliferative and anti-invasive properties. These were recognised some years ago. Thus verapamil inhibits tumour growth in vitro (Brocchieri et al. 1996) and seems to arrest cells in the G0G1 phase of the cell cycle (Zeitler et al. 1997). The antiproliferative activity has been conrmed by Hoffman et al. (1998) on retinal pigment cells. Verapamil is able to inhibit the migration of CD4+ and CD8+ T lymphocytes across the endothelium (Blaheta et al. 2000). Other calcium antagonists such as mibefradil can also inhibit the adhesion and diapedesis of lymphocytes across the endothelium (Blaheta et al. 1998). Earlier Hailer et al. (1994) had noticed that the expression of endothelial adhesion molecules was unaltered by verapamil. It may be too simplistic to interpret these ndings merely in terms of blocking of calcium channels. Nonetheless, some of these publications also indicate that the Factin levels of the lymphocytes are reduced by the treatment. This could be due to changes in cytoskeletal dynamics that occur after verapamil-mediated modulation of intracellular calcium levels. This in turn could alter cellular motility. Other factors will have serious effects on the permeability of the endothelium. For instance, CD4+ lymphocytes form two subsets known as Th1 and Th2 cells. Upon activation both subsets produce a wide range of cytokines. Th1 cells secrete interleukin (IL)-2 and (IFN)-, and Th2 cells secrete IL-4, 5, 10, and 13. Some of these lymphokines are
10
bound to affect endothelial permeability. An additional factor that must be reckoned with is the generation of nitric oxide, which is known to inhibit the adhesion and spreading of endothelial cells. Nitric oxide also affects the formation of stress bres in the cells, which is bound to inuence cellular motility. Nitric oxide also actively inuences angiogenesis. IFN- is a powerful inducer of nitric oxide synthase (NOS), whereas IL-4 from Th2 cells inhibits NOS. It would be well to remember here that endothelial NOS is a Ca2+/CaM-inducible enzyme. It is activated when it is binds the Ca2+/CaM complex. More recently, the effects of verapamil have been tested on the local invasion and metastasis of a murine carcinoma cell line in BALB/C mice. Farias et al. (1998) found that it inhibited local invasion and also produced a 50% reduction in both spontaneous and experimental metastasis. In common with CAI, the cytostatic and anti-invasive effects may be due to the inhibition of membrane-associated proteinases such as matrix metalloproteinases and urokinase-type plasminogen activator (Farias et al. 1998). These events are virtually terminal events in the process of inhibition of adhesion and invasiveness by verapamil. More upstream in the pathway, verapamil has been shown to down-regulate the expression of S100A4 (Parker and Sherbet, 1992), which, as discussed elsewhere in this book, is closely allied with p53 and stathmin genes in the control of cell cycle progression. Furthermore, S100A4 expression is closely linked with the remodelling of the extracellular matrix.
PHOSPHOLIPID SIGNALLING
A host of regulatory molecules alter cell behaviour and physiological events. Thus extracellular signals imparted to the cells regulate their growth, proliferation, and apoptosis. They also regulate intercellular adhesion and cell adhesion to substrata, and consequently cell migration and invasive behaviour. A family of second messengers, the inositol phospholipids, transduces the regulatory signals across the membrane. Phospholipids (phosphatides) are a major structural component of the cell membrane and also subserve important functions of transducing the extracellular signals. The second messengers phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and IP3 are generated from these phospholipids by the action of phospholipases A and C (PLC), which are activated by the extracellular signals, and kinases. An intermediary in this process is the phosphatidylinositol (PI) transfer protein, which presents the PI metabolites to lipid kinases. The phosphorylation of the inositol ring at the 3-position gives rise to phosphoinositides PIP (phosphatidylinositol), PIP2, and PIP3. From here the transduction of the signal occurs by the interaction of these second messengers with target proteins that possess specic phosphoinositide-binding domains, such as an FYVE domain or pleckstrin homology (PH) domain. PIP2 and PIP3 show specic binding to PH domains (see Leevers et al. 1999). The second pathway is the generation of IP3 from PIP2 by PLC. As discussed below, IP3 then mediates the mobilisation of calcium from intracellular stores. The depletion of the intracellular stores triggers the so-called capacitative entry of calcium into the cell. Naturally, a deregulation of these pathways will have serious consequences in terms of differentiation, intercellular relationships, adhesion, and
11
cellular morphology. Such profound changes might indeed lead to the development of cancers, their invasion, and the formation of metastases.
PTEN PHOSPHATASE IN THE REGULATION OF LIPID SIGNALLING

Phosphoinositide-3 kinase (PI3K) is responsible for the phosphorylation and generation of PIP2 and PIP3. This phosphorylation is antagonised by a phosphatase that is encoded by the gene called PTEN/MMAC1/TEP1 located on chromosome 10q23. The phosphatase, referred to here as PTEN, shows sequence homology to tensin (Steck et al. 1997). The phosphatase activity is essential for the normal functioning of PTEN (Tamura et al. 1999b). PTEN regulates the levels of the phosphoinositol phosphates by dephosphorylation of the phosphate group at the 3-position of the inositol ring (Maehama and Dixon, 1998). This seems to inhibit cell proliferation and migration (Furnari et al. 1997; J. Li et al. 1998; Ramaswamy et al. 1999; Tamura et al. 1999c; Podsypanina et al. 1999). Ramaswamy et al. (1999) transfected cells that lack PTEN with wild-type PTEN cDNA and found that cells were arrested in the G1 phase of the cell cycle. Similar transfection studies have been carried out on the glioma cell line U373, which lacks the PTEN gene. In these cells, wild-type PTEN inhibits growth and reduces saturation density of U373 cultures. However, PTEN mutants lacking phosphatase activity have the opposite effect and they indeed enhance cell growth in soft agar (Morimoto et al. 1999), which merely conrms the potential suppressor function of PTEN. PTEN also seems to be able to regulate the apoptotic pathway. PIP3 is known to activate the akt serine/threonine kinase, which has anti-apoptotic properties. It has been shown to inhibit akt kinase activity (Maehama and Dixon, 1999; Ramaswamy et al. 1999), which could push cells into the apoptotic pathway. For instance, extraneous PTEN induces apoptosis of breast cancer cells, which correlates with the down-regulation of akt. Furthermore, akt can rescue cells from PTENinduced apoptosis (J. Li et al. 1998). PTEN also increases the expression of p27kip1 cyclin-dependent kinase (cdk) inhibitor (Y.L. Lu et al. 1999). The cdk inhibitors, kip1 and kip2, inhibit the phosphorylation of rb protein and this prevents the entry of the cells into S-phase. This is consistent with the recent nding of Paramio et al. (1999) that PTEN cannot produce growth arrest in rb-decient cells, but it induces growth arrest when functional rb is reintroduced. This could provide an alternative mechanism by which PTEN induces cell arrest in the G1 phase. The overall effects of growth arrest could be a combination of G1 arrest and apoptosis (Weng et al. 1999), but Paramio et al. (1999) believe that apoptosis might not contribute to this. It appears that PTEN might regulate cell adhesion and alterations in cell shape and motility by inuencing the downstream of the integrin-mediated signalling pathway (Tamura et al. 1999c). Tamura et al. (1999a) have reported that PTEN also may interact with focal adhesion kinase and regulate its phosphorylation and focal adhesions. A further suggestion is that PTEN function might take the nitrogenactivated protein kinase (MAPK) pathway in the regulation of motility (Gu et al. 1999). It ought to be recognised, nonetheless, that these pathways may not be
12
mutually exclusive in facilitating the ow of information, and one or more pathways may be involved in the process. Consistent with this, Morimoto et al. (2000) have shown that PTEN not only affects PLC-mediated signalling, but also inhibits the kinases that regulate integrin function as well as the anti-apoptosis kinase akt. PTEN is required at certain stages of embryonic development. Its loss appears to be lethal in early embryogenesis and to lead to abnormalities of the skin, the gastrointestinal tract, and the pancreas. PTEN / cells have shown marked changes in their ability to differentiate into cell types characteristic of the three germ layers (i.e., the ectoderm, the endoderm, and the mesoderm) (Di Cristofano et al. 1998). Presumably, inactivation of the gene affects the state of competence of the cells to respond to differentiation-inducing stimuli (see Sherbet, 1982). Thus the abnormalities in the various organs seem to have an early developmental history. The PTEN gene is mutated, or there is a consistent loss of heterozygosity (LOH) at this gene locus, in many forms of human cancer. The mutations tend to occur mainly in the phosphatase domain (Steck et al. 1997; J. Li et al. 1997; W.G. Liu et al. 1997; Guldberg et al. 1997; Cairns et al. 1997; Tashiro et al. 1997; Tang et al. 1997; Kong et al. 1997; Okami et al. 1998; Ali et al. 1999). The seemingly consistent association of inactivating mutations and LOH of PTEN with cancers suggests the possibility that the lipid-signalling pathway could be seriously deregulated in the pathogenesis of neoplasia, and the PTEN gene has been suggested as a putative tumour suppressor gene. This is suggested unambiguously by the ability of PTEN to inhibit rasmediated cell transformation, with a concomitant dephosphorylation of PIP-1,3,4,5tetrakisphosphate (Tolkacheva and Chan, 2000). Germline mutations of PTEN also occur with the pathogenesis of Cowdens syndrome (CS) and the Bannayan-Zonana syndrome (Liaw et al. 1997; Nelen et al. 1997; Tsou et al. 1998; Marsh et al. 1998a, 1988b). CS is an autosomal dominant inherited syndrome. Several benign and malignant neoplastic conditions are associated with this syndrome. Women with CS are believed to be more prone to develop breast cancer, and most of them have been found to develop benign brocystic disease. The CS mutations tend to be in exon 5, which contains the phosphatase core motif (Marsh et al. 1998b; Nelen et al. 1997). Although PTEN germline mutations are frequent in the BannayanZonana syndrome, none of them have been found in exon 5 (Marsh et al. 1998a). An overview of the pattern of mutations suggests that those occurring in tumours could differ in nature from germline mutations (see Ali et al. 1999). Perhaps it is premature to speculate whether the generation of these mutations in different tumours is related to their pathogenesis. On the other hand, Guldberg et al. (1997) have found the same missense mutation in both primary and metastatic melanomas. Although further conrmation of this is essential, this has a considerable implication for metastatic dissemination. Nonetheless, while considering the potential tumour suppressor function of PTEN, it should be borne in mind that in PTEN abnormalities do not occur as a generalised event of tumour development. Yeh et al. (2000) examined human hepatocellular carcinomas for aberrant expression of PTEN, but encountered none. Neither did they nd any abnormalities associated with the gene itself. Furthermore, the PTEN gene does not seem to be involved in the pathogenesis of acute myeloid leukaemia (T.C. Liu et al. 2000). Therefore, it might be prudent to reserve judgment regarding this issue. On
13
the other hand, PTEN overexpression might be a safeguard against progression. In laryngeal papillomas, for example, PTEN is overexpressed and the anti-apoptotic akt kinase is inhibited (P. Zhang and Steinberg, 2000). This should have an overall controlling effect on cell population size, and as a consequence upon progression, because more often than not, tumour progression is related to the rate of tumour expansion.
THE PROTEIN KINASE C PATHWAY

Growth factors and hormones are known to increase the intracellular Ca2+ (Ca [i]), either by releasing Ca2+ from intracellular stores or by promoting Ca2+ inux. Ca2+[i] controls several biological functions, mainly by mediating the phosphorylation of cellular effector proteins. Calcium-binding proteins transduce the calcium signals to specic subcellular compartments. The generation of second messengers such as IP3, 1,2-diacylglycerol (DAG), and cyclic 3,4-adenosine monophosphate (cAMP) is a major mechanism of activation of the Ca2+ signalling pathway. IP3 and DAG are formed through the agency of an inositide specic phospholipase, which is stimulated by biological response modiers. This PLC hydrolyses PIP2 into DAG and IP3. IP3 activates the release of Ca2+ from intracellular stores. The activation of appropriate protein kinases is a downstream event, which occurs in the transduction pathway. The major kinases involved are protein kinase C (PKC), the calcium/calmodulin-dependent protein kinases (CAMPKs) and protein kinase A (PKA). The CAMPKs can in turn activate the cAMP-mediated pathway by activating adenylyl cyclase, resulting in an increase in cAMP levels (Figure 2).
2+
Ca L L
2+
R
PL C PIP 2
R G
AC IP3 DAG Ca [i]
2+
R G
cAMP
PK C
CAMP K
PK A
FIGURE 2 The activation of signal transduction pathways upon binding of a ligand (L) to its appropriate receptor (R). AC, adenylyl cyclase; cAMP, cyclic 3,4-adenosine monophosphate; CAMPK, calcium/calmodulin-dependent protein kinase; DAG, 1,2-diacyglycerol; G, G-protein; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidyl 4,5-bis phosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C.
14
Calcium ions and DAG successively activate PKC and induce its translocation to the plasma membrane or the nucleus. Both intracellular calcium and DAG binding to PKC maintain it in a sustained activated state (Oancea and Meyer, 1998). DAG itself is regulated by phosphorylation by DAG kinase (DAGK). DAG phosphorylation seems to reduce PKC activity (Sakane and Kanoh, 1997) (summarised in Figure 3). It may be noted that as many as eight isoforms of DAGK have been identied. These have the characteristic zinc nger structures and PH domains. PH domains are found in many proteins and consist of approximately 120 amino acid residues. They are electrostatically polarised and show binding to G-protein subunits, PKCs, and phospholipids, thus linking PH domains as a characteristic feature of signal transduction proteins. The presence of these domains in DAGK isoforms suggests that these kinases are a component of the signal transduction network. Besides, DAGK-, -, and - also possess two consecutive EF-hand calcium-binding domains (Sakane et al. 1990; Yamada et al. 1997; Sakane and Kanoh, 1997). Therefore, DAGK isoforms show calcium-dependent activity. This effect is variable, which has been attributed to differences in calcium afnities of and the conformational changes occurring in the EF-hand domains as a response to calcium binding (Yamada et al. 1997). Upon activation, DAG kinases also translocate to the nucleus and remain tightly associated with nuclear matrix (Wada et al. 1996). Some DAGK isoforms, such as DAGK -, -, -, have no EF-hand domains (Sakane et al. 1996; W. Tang et al. 1996; Bunting et al. 1996). So calcium-mediated activation is not a universal phenomenon in the activation of these kinases.
PROTEIN KINASE C AND ITS ISOFORMS IN SIGNAL TRANSDUCTION

DAG, upon being activated by the calcium binding protein DAGK, now activates PKC by enhancing its afnity for Ca2+ and altering its enzymatic activity. This leads to the phosphorylation of substrate proteins, thus effectively transducing the calcium signal into a physiological response. Some CBPs may inhibit PKC-mediated phosphorylation of protein substrates. In other words, CBPs can actively regulate PKC function (see Sherbet, 1987; Sherbet and Lakshmi, 1997a, 1997b, for review). They may also be directly associated with the coupling of calcium signalling with gene regulation. This role is discussed in detail elsewhere in this book. Several isoforms of PKC have been identied and it is believed that they may subserve specic functions in signal transduction. It is well known that PKCs are translocated from cytoplasmic to nuclear location when cells are stimulated by growth factors. In Swiss 3T3 cells, although a number of isoforms are detectable in the cytoplasm, only PKC is found in nuclei isolated from cells that have been exposed to growth factors (Neri et al. 1994). In HL-60 cells that were induced by all-trans-retinoic acid to differentiate, the translocation of PKC and to the nucleus has been observed (Zauli et al. 1996). This differential translocation has been attributed to possible linkage with the nuclear inositol lipid cycle (Neri et al. 1994). Further support for this view comes from the observation by Mizukami et al. (1997) concerning the
15
Ligand binding
Receptor
Calcium spikes
DAG activation
DAG kinase
PKC activation
FIGURE 3 The ow of information of ligand binding to its membrane receptor via intracellular calcium increases and DAG-to-PKC activation and its translocation to specic intracellular sites are illustrated. DAG is in turn regulated by DAG kinases, which are EFhand proteins.
translocation of PKC. They reported that translocation of this PKC isoform to the nucleus could be inhibited by inhibiting phosphatidylinositol-3 kinase. With the plethora of PKC isoforms discovered over the years, their occurrence is being rationalised by the postulate that these isoforms might be specically associated with identiable signal transduction pathways and signal transduction in different physiological functions in a tissue-specic manner. In PC12 cells, the epidermal growth factor (EGF) signal either is mitogenic or induces differentiation. The ow of information and the phenotypic effect appear to depend on the PKC isoform. EGF induces neurite formation and arrests cell proliferation if the cells overexpress PKC-, but not PKC- or . Neurite differentiation is accompanied by phosphorylation of mitogen-activated protein kinase, and inhibition of this phosphorylation event also inhibits neurite differentiation as well as arrest of proliferation. In sharp contrast, overexpression of PKC- and - is strongly associated with the mitogenic function of EGF (Brodie et al. 1999). There are also examples of tissue-specic functioning of PKC isoforms. For instance, calcium-independent vascular smooth muscle contraction seems to involve PKC- rather than PKC-, both calcium-independent isoforms of PKC (Walsh et al. 1996). Such specicity of function might reect the substrate specicity of the kinase. Walsh et al. (1996) suggested the possibility that calponin is the musclespecic protein that might be a substrate of this PKC isoform. DAGKs that activate
16
PKCs show considerable tissue-specic distribution themselves. It may be that the tissue-specic function of PKCs might ow from the distribution of the DAGKs.
INOSITOL PHOSPHATES IN CALCIUM SIGNAL TRANSDUCTION

The transduction of signals imparted to the cell by the binding of extracellular ligands to their respective membrane receptors generates repetitive calcium spikes or oscillations, and these transient increases of intracellular calcium are a result of the production of IP3 by the mediation of PLC (see Figure 3). The repetitive nature of these oscillations is believed to be a consequence of a positive feedback loop in which calcium induces its own release, and a negative feedback effect which stops calcium release. Such calcium oscillations occur in many physiological phenomena. An increase in intracellular calcium is a critical event in the activation of the egg (ovum) in a wide variety of species. Experimental enhancement of intracellular calcium of mouse eggs using calcium ionophores or ethanol results in activation. Conversely, when intracellular calcium levels are reduced egg activation is prevented (Cuthbertson, 1983; Ducibella et al. 1988; Kline and Kline, 1992). That IP3 mediates egg activation is indicated by the demonstration that microinjection of IP3 produces calcium release from intracellular stores and initiates some of the events associated with activation (Kurasawa et al. 1989; Ducibella et al. 1993). IP3 levels are increased by fertilisation of the egg (Kline, 1996). An increase is also known to take place in the number of IP3 receptors (Mehlmann et al.1996). IP3 receptors occur in the anterior acrosomal region of mammalian sperm, and by implication IP3 is involved in the acrosome reaction of mammalian sperm (Walensky and Snyder, 1996). IP3 regulates intracellular Ca2+ levels by mobilising calcium from intracellular ER-associated stores and probably also by stimulating Ca2+ inux into cells. Only a proportion (up to 50%) of the ER Ca2+ pool is IP3 sensitive, and the remainder can be released by calcium ionophores. The IP3-sensitive calcium pool is clearly associated with IP3R function. IP3R is an intracellular calcium release channel on the ER. IP3 stimulates IP3R, which results in calcium mobilisation from intracellular stores. Jayaraman et al. (1995) stably transfected antisense IP3R cDNA into Jurkat T cells. The transfected cells, which then lacked functional IP3R, failed to elevate Ca2+[i] in response to antigen-specic activation. However, the depletion of intracellular calcium stimulated calcium inux, which suggests that IP3R are required for IP3-mediated mobilisation of calcium from intracellular stores, but not for calcium inux. The function of IP3R seems to be regulated by phosphorylation. Both cyclic guanosine monophosphate (cGMP) and cAMP are known to inhibit the mobilisation of intracellular calcium, and this has been found to be a consequence of the phosphorylation of IP3R by cGMP-dependent kinase (Komalavilas and Lincoln, 1994, 1996) (Figure 4). Komalavilas and Lincoln identied serine 1755 of IP3R as the phosphorylation site. Nonreceptor tyrosine kinases have also been shown to generate IP3, activate IP3R, and raise intracellular calcium levels. Jayaraman et al. (1996) demonstrated the association between nonreceptor tyrosine kinase Fyn and IP3R
17
FIGURE 4 Possible mechanisms involved in IP3-mediated mobilisation of intracellular calcium. cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; cGMPK, cGMP kinase; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IP3R-P, phosphorylated forms of IP3R.
following T-cell receptor stimulation. Furthermore, phosphorylation of tyrosine residues was found to be reduced in thymocytes derived from fyn (/) mice. These studies showed that phosphorylation of tyrosine might also regulate IP3R function. It would appear that the phosphorylation of serine and tyrosine might have antagonistic regulatory effects. This situation is somewhat analogous to the activation or inactivation of p34cdc2 kinase, which is involved with the phosphorylation of the retinoblastoma gene product in the regulation of cell cycle progression (see Sherbet and Lakshmi, 1997b). Although the molecular mechanisms by which IP3R raises Ca2+[i] are not fully understood to date, it is obvious that the process of phosphorylation of IP3R is an important event in IP3-mediated calcium signalling. The occurrence of IP3R was recognised many years ago (Supattapone et al. 1988; Snyder and Supattapone, 1989). There are three types of IP3R. Three IP3R genes have been cloned. One of them, the IP3R1, is expressed predominantly in Purkinje neurones of the cerebellum. IP3R1 and IP3R2 genes have been found to be expressed at low levels in several tissues studied to date. The distribution of the mRNA of the three receptor genes appears to display a denable pattern, leading to the suggestion that the different receptor types may have distinct functions (Fujino et al. 1995; T.X. Yang et al. 1995). Indeed, multiple isoforms of IP3R genes may be expressed in cells. Alternatively spliced isoforms could be fullling different cellular requirements, that is, they may be functionally different (Iida and Bourguignon, 1995, 1996; Katusic and Stelter, 1995). IP3R1 may be associated with intracellular calcium mobilisation, because IP3R1-decient T-lymphocytes show a lowered IP3-mediated calcium release by T-cell receptor stimulation (Jayaraman and
18
Marks, 1997). The same study also implicated IP3R1 in apoptosis, by the observation that IP3R1-decient T lymphocytes are resistant to dexamethasone-induced apoptosis. IP3R3 genes have been credited with the capacitative entry of calcium inux, when intracellular Ca2+ is depleted. In Xenopus oocytes IP3R3 was associated with the plasma membrane, which facilitated the inux of calcium (Putney, 1997). The transduction of signals imparted by a variety of biological response modiers occurs via cytoskeletal structures. Compatible with this generalisation, the signal transduction pathway of IP3 has been linked with cytoskeletal dynamics. Ribeiro et al. (1997) found that the disruption of cytoskeletal structures of NIH3T3 cells by treatment with cytochalasin D resulted in the abolition of intracellular calcium mobilisation normally induced by platelet-derived growth factor (PDGF). The depolymerisation of the tubulin network similarly abolished calcium mobilisation. However, PDGF continued to stimulate the generation of IP3 even in the presence of cytochalasin. This suggests that the cytoskeleton is closely involved in the IP3-mediated transduction of the PDGF proliferative signal. It is of interest to note that Bourguignon and Jin (1995) had previously identied a C-terminal amino acid sequence of GGVGDVLRKPS in IP3R that possesses sequence homology to the ankyrin-binding domain of the intercellular adhesion molecule CD44. They further had demonstrated that an 11 amino acid synthetic peptide with that sequence showed specic binding to ankyrin, which is a cytoskeletal protein.
DEREGULATION OF INOSITOL 1,4,5-TRISPHOSPHATE PATHWAY AND ITS CONSEQUENCES

It is hardly surprising that interference with signal transduction pathways and their deregulation should have serious consequences for biological responses in developmental systems as well as in pathogenesis. Ecdysone, a steroid hormone, regulates a variety of developmental processes and metamorphosis in insects. It also inuences neuronal responses, apoptosis, and histiolysis in a variety of larval tissues, binds to nuclear receptors, which show tissue- and stage-specic expression (Kamimura et al. 1997); and regulates ecdysone-responsive genes (No et al. 1996; Truman, 1996). The metamorphic changes involve early and late genes, with the early genes encoding proteins that regulate the expression of late genes (Baehrecke, 1996). Although our knowledge of the mechanics of ecdysone signal transduction is still rudimentary, it is clear that the pathway involves calcium signalling. The prothoracicotropic hormone (PTTH) stimulates the synthesis of ecdysone in the prothoracic gland of Galleria larvae (Birkenbeil, 1996). PTTH increased intracellular Ca2+ levels of prothoracic gland in vitro. This increase could be abolished by removing extracellular calcium and by calcium channel inhibitors. The PTTH-stimulated increase of intracellular calcium was not abolished by agents, which inhibit mobilisation of calcium from intracellular stores. Recently, Venkatesh and Hasan (1997) found that disruption of IP3 receptor gene function in Drosophila larvae grossly affected metamorphosis and ecdysone synthesis and secretion. They generated IP3R gene (itpr) mutants. The mutations delayed moulting and were lethal at early larval stages.
19
Feeding ecdysone to mutant larvae partially counteracted the phenotypic effects of the itpr mutation, and down-regulated ecdysone-inducible genes. General or specic abnormalities of signal transduction pathways (e.g., occurrence of abnormalities in one or more components of the transduction pathway) may be associated with disease processes, including the invasive behaviour and metastatic progression of cancers. Such disruptions of the signal transduction cascade in human cancers has been studied by measuring the levels of three enzymes involved in the generation of IP3, namely, phosphatidylinositol 4-kinase, phosphatidyl 4-phosphate 5 kinase, and PLC. Weber et al. (1996) found that all three enzymes were up-regulated with tumour progression. In rat hepatomas the enzyme levels rose by two- to eight-fold compared with normal liver. A three- to four-fold increase in PIP kinase and PI kinase was found in ovarian epithelial cancers. Weber et al. (1996) found a dramatic 16- and 96-fold increase of these enzymes in human breast carcinoma cells compared with normal breast parenchymal cells. These changes clearly correlated with enhanced IP3 expression and proliferative potential. Further evidence has also been reported by Singhal et al. (1997), who investigated the connection between elevated IP3 and DAG with proliferation of the breast cancer cell line MDA-MB-435 and the ovarian cancer cell line OVCAR-5. They demonstrated that quercetin treatment of the cells inhibited PI kinase and reduced IP3 before any inhibition in cell proliferation was evident. The expression of these kinases seems to be up-regulated by differentiation-inducing agents. Thus Ai et al. (1995) found enhanced expression of PI kinase as well as IP3 in murine erythroleukaemia cells induced toward erythroid differentiation by dimethyl sulphoxide (DMSO) treatment. Another line of circumstantial evidence may be cited. For instance, monoterpenes such as limonene and perillyl alcohol have been reported not only to prevent tumour initiation and promotion, but also to inhibit tumour progression. These monoterpenes appear to inhibit the isoprenylation of G-proteins (Gould, 1997), which are a component of the signal transduction machinery (Figure 5). Similarly, abnormalities in the related pathway involving DAG and PKC may also be associated with cancers, as demonstrated by Hoelting et al. (1997) using the PKC agonist TPA (12-O-tetradecanoyl phorbol 13-acetate). They reported a 15% increase in the invasive ability of a follicular thyroid cancer cell line. In contrast, PKC inhibitors such as staurosporine, chelerythrine and calphostin C reduced invasion by 62%. CAI which inhibits calcium inux into cells, has been reported to inhibit the proliferative and invasive capacity of cell lines derived from human prostate cancer (Wasilenko et al. 1996). The neuroendocrine cells of prostate cancer express bombesin and gastrin-releasing peptide (GRP). In vitro bombesin and GRP signals invoke cell proliferation via bombesin receptors of which there are three subtypes: GRP receptors (GRPr), neuromedin B receptor, and bombesin receptor subtype 3. Transcripts of GRPr only, but not neuromedin B or bombesin receptor subtype 3, have been found in androgen-insensitive prostate cancer cell lines PC-3 and DU-145, suggesting the very important role GRPr play in signal transduction in these cells (Aprikian et al. 1996). In all lines derived from advanced prostate cancer, several agents such as GRP, bombesin, and bradykinin, among others, raised intracellular calcium levels.
20
FIGURE 5 The cAMP/Ca2+-signalling pathway. The binding of extracellular ligands to specic receptors regulates adenylyl cyclase activity via the linking G-proteins. The cAMP generated activates protein kinases that phosphorylate substrate proteins. Another route starts with the raising of intracellular calcium levels, which in turn regulate adenylyl cyclase activity.
However, bombesin did not inuence calcium levels of an immortalised prostate cell line (Wasilenko et al. 1997). The authors have, therefore, suggested not only that there might be multiple receptors that can mediate increases in intracellular calcium levels of androgen-independent prostate cancer cell lines, but also that GRPr expression may be associated with the progression of prostate cancer. Calcitonin-like peptides may also function as neuropeptides in prostate cancer. The peptide binds to high-afnity receptors in the plasma membrane and induces a rapid and marked increase in intracellular calcium. Furthermore, the calcitonin signal is transduced into a clear proliferative response. The pattern of calcium response is biphasic in the form of a spike, and plateau is regarded as indicative of the phospholipid/calciumsignalling system (Shah et al. 1994).
RYANODINE AND RELATED RECEPTORS IN CALCIUM MOBILISATION

Besides IP3R, other receptor-mediated pathways are available to the cell for calcium mobilisation, and the activation and availability of the pathways may be developmentally regulated. In embryonic development, the IP3-dependent calcium signalling is switched to an IP3-independent mode of signal transduction. The ryanodine receptor (RyR) is also capable of regulating intercellular calcium. In the early stages of development, IP3R are ubiquitously expressed. But in subsequent development RyR begin to be expressed. Eventually, although most cell types do continue to
21
express IP3R, in excitable cells including muscle cells, RyR becomes the major Ca2+ release channel (Rosemblit et al. 1999). Ryanodine receptors, of which there are several isoforms, are activated by calcium inux via voltage-gated Ca2+ channels of the plasma membrane. Recently, cyclic ADP-ribose has been identied as an activator of RyR (Petersen and Cancela, 1999). The fatty acid metabolic products, the fatty acyl-CoA esters, can also activate RyR in skeletal muscle (Fulceri et al. 1994; El-Hayek et al. 1993; Dumonteil et al. 1994). This seems to be mediated by the acyl-CoA binding protein (Fulceri et al. 1997). A reference to the multifunctional receptor called megalin would not be out of place here. Megalin is a member of the low-density lipoprotein receptor family, which function as endocytic receptors. The megalin glycoprotein of rat kidney is approximately 330 kDa in size. The 550-kDa human homologue of megalin is found in the luminal surface of cells of the renal proximal tubule and epididymis. It is expressed in mammary epithelia, thyroid follicular cells, and the ciliary body of the eye (Lundgren et al. 1997). It is also found in parathyroid and trophoblast cells. Mackrill et al. (1999) raised antibodies against RyR from rabbit muscle. These antibodies recognised not only skeletal RyR but also another high-molecular-weight protein (k-HMW) in kidney microsomes thought to be a rabbit homologue of megalin. Antibodies raised against k-HMW were unable to recognise RyR. Furthermore, this protein showed partial sequence homology to RyR. Megalin functions as a receptor for thyroglobulin (Marino et al. 1999; G. Zheng et al. 1998), lipoprotein (a) (Niemeier et al. 1999), and insulin (Orlando et al. 1998). It is implicated in the transport of retinol. Megalin-decient mice show greatly increased urinary excretion of retinol and retinol-binding protein (RBP). Furthermore, the uptake of retinol and RBP is partially inhibited by antibodies against megalin (Christensen et al. 1999). Megalin also serves as a receptor for aminoglyocoside antibiotics, such as gentamycin, and mediates the endocytosis and accumulation of the antibiotic in renal tubule cells (Decorti et al. 1999). There is a preliminary report that the nephrotoxicity of gentamycin is related to the loss of megalin receptors (Vaamonde et al. 1996). The megalin receptor has also been attributed with a putative role in calcium homeostasis (Friedman, 1999; Christensen et al. 1998). This seems reasonable in light of its similarity to RyR. The deregulation of this function might be responsible for its apparent involvement in pathogenesis. Megalin might function as an autoimmune antigen in the pathogenesis of membranous glomerulonephritis (Mackrill et al. 1999). Its dysfunction might occur also in neoplasia. Apparently, the expression of megalin is down-regulated in hyperplasia of the parathyroid.
CYCLIC AMP IN CALCIUM SIGNALLING

A majority of hormones, neurotransmitters, and growth factors generate cellular responses by means of a signal transduction machinery that is composed of three elements. These agents bind to specic receptors, which are linked to the effector component by means of G-proteins (Iyengar and Birnbaumer, 1990a, 1990b). Gilman (1989) identied these as components of the machinery regulating adenylyl cyclase
22
activity. A large number of hormones modulate cAMP levels by regulating adenylyl cyclase activity. The G protein family encompasses a number of heterotrimers composed of subunits encoded by three G-protein-type genes, , , and genes. Several G, G, and G genes have been cloned (Iyengar and Birnbaumer, 1990b). The G-protein linker GS mediates stimulatory effects, whereas GI mediates inhibitory effects of hormones. Most G-protein intervention follows the activation of the subunit by binding to GTP, and this activation occurs upon the receptor being bound by the appropriate ligand. Numerous biological responses to extracellular stimuli are mediated by cAMP synthesised by adenylyl cyclase activity, and in turn cAMP activates cAMP-dependent protein kinase, which phosphorylates a variety of cellular substrate proteins (Krebs, 1989). This pathway involves an elevation of intracellular calcium levels either by increased uptake of extracellular calcium or release of calcium from intracellular stores. Calcium can modulate adenylyl cyclase activity. There is a considerable body of evidence that CBPs such as calmodulin may regulate adenylyl cyclase activity. It has been shown that Ca2+/CaM can stimulate adenylyl cyclase activity (Minocherhomjee et al. 1988), but in some tissues the activity of this enzyme is insensitive to Ca2+/CaM. Furthermore, adenylyl cyclase activity may be related to intracellular distribution, and this may or may not be affected by activated GS. The cellular response generated by biological response modiers may depend on the G-protein and the appropriate effector component being expressed by cells (Iyengar and Birnbaumer, 1990b). The control of cellular response to external signalling ligands requires that the levels of the second messengers themselves be regulated strictly. Enzymes such as phosphodiesterases that degrade these molecules regulate cAMP. Besides adenylyl cyclase, phosphodiesterases, protein kinase II, and protein phosphatase are also CaM-dependent enzymes. Receptor proteins and kinases may amplify second messenger signals. Indeed, this mechanism, involving their amplication on the one hand and degradation on the other, maintain the steady state levels of second messengers (see Figure 5). Several biological responses have been identied in which the cAMP/calcium pathway of signal transduction is followed upon the binding of specic extracellular ligands. The processes of aggregation and development and chemotactic response of Dictyostelium discoideum involve the cyclic nucleotides cAMP and cGMP. The chemotactic response appears to be a function of the elevation of intracellular Ca2+, which has been shown to correlate with the association of myosin with the cytoskeleton (Menz et al. 1991). This suggests a close involvement of calcium uptake with the process of cell motility. cAMP has been implicated in sperm motility (Garbers and Kopf, 1980). In fact, high extracellular levels of calcium have been known to increase cAMP levels in sperm cells (Garbers et al. 1982), and possibly Ca2+ could be activating adenylyl cyclase activity. Functionally it seems possible to differentiate between inuxed calcium and Ca2+ released from intracellular stores. This is illustrated by the modulation of S100A4 expression in B16 murine melanoma cells by agents that modulate intracellular calcium levels. Melanocyte-stimulating hormone (MSH) increases calcium inux and cAMP levels. Verapamil is a calcium channel blocker that reduces cAMP levels (Atlas and Adler, 1981; Janis et al. 1987). Thapsigargin is a sesquiterpene lactone that raises intracellular calcium levels by releasing calcium from intracellular
23
stores, by inhibiting the ER-associated Ca2+-ATPase (Takemura et al. 1989; Thastrup et al. 1990). MSH has been reported to up-regulate S100A4, whereas verapamil does the opposite. In contrast, thapsigargin down-regulates its expression despite effecting an increase in intracellular calcium levels (Parker and Sherbet, 1992) (Figure 6). This is compatible with the suggestion by Haverstick et al. (1991) that Ca2+ inux and release from intracellular stores have independent roles. It has also been suggested that a low micromolar range of intracellular calcium levels inhibits adenylyl cyclase, in contrast to calmodulin-stimulated adenylyl cyclase which may be mediated by calcium inux (Cooper, 1991). The release of calcium from intracellular stores may therefore activate specic components of the calcium-signalling pathway (Figure 7). The transcription factor, the cAMP response element binding protein (CREB), is phosphorylated by Ca2+/CaM-dependent protein kinase PKII. The phosphatases PP-2A and -2B (calcineurin) dephosphorylate the phosphatase inhibitor (PI-1), which in turn inhibits the phosphatase PP-1. The inactivation of PI1 by PP-2A or PP-2B may allow the phosphatase PP-1 to dephosphorylate CREB, thus rendering the latter inactive. Calcineurin is reported to have an afnity for CaM two to three magnitudes greater than that of PKII. Therefore, low levels of intracellular calcium, such as those resulting from its release from intracellular stores, might preferentially activate calcineurin.
FIGURE 6 The differential regulation of S100A4 expression by calcium inux into cells as compared with the release of Ca2+ from intracellular stores. Calcium inux up-regulates S100A4. Verapamil, which blocks calcium inux, down-regulates its expression. In sharp contrast, thapsigargin, which raises intracellular calcium levels by releasing Ca2+ from intracellular stores, down-regulates S100A4 expression. This suggests that calcium released from intracellular stores may be functionally differentiated from inuxed calcium and may be activating different components of the calcium signalling system. (Based on Parker and Sherbet, 1992). cAMP cyclic AMP; MSH, melanocyte-stimulating hormone.
24
FIGURE 7 Summary of cAMP and Ca2+/CaM-mediated regulation of immediate early gene expression. cAMP, cyclic AMP; CREBP, cyclic AMP response element binding protein; PI-1, phosphatase inhibitor l; PKA, protein kinase A; PKII protein kinase II; PP-1, protein phosphatase 1; PP2B, calcineurin.
Several extracellular agents are known to activate the cAMP- and Ca2+-signalling pathway. There are also instances where the calcium-signalling pathway is independent of an upstream event of cAMP activation. cAMP and calcium signalling mediate the biological responses of several hormones. Adrenocorticotropic hormone (ACTH) stimulates uptake of Ca2+ and elevates cAMP levels in lymphocytes. Splenic lymphocytes that possess ACTH receptors show ACTH-dependent calcium uptake, but thymocytes, which lack these receptors, do not show enhanced calcium uptake (Clarke et al. 1994). In adrenocortical cells maintained in culture, ACTH exerts a biphasic effect, inhibiting cell proliferation in the initial phase followed by a stimulatory phase of cell differentiation, and synthesis and secretion of corticosterone and 18-OH-deoxycorticosterone. cAMP and 8-bromo-cAMP can induce adrenocortical cells to differentiate and to secrete steroid hormones, but they do not inhibit cell proliferation. This suggests that although cAMP mediates the mitogenic, differentiative, and steroid stimulatory signals of ACTH, it is not involved in the process of inhibition of cell proliferation by ACTH (Arola et al. 1993). Similarly, although the stimulation of bone resorption in foetal limb bud bones by tumour necrosis factor (TNF) appears to involve cAMP, the latter may not be directly associated with TNF signalling (Shankar and Stern, 1993). Calcitonin and calcitonin-like substances induce cell proliferation in prostate cells in culture and possibly also in the genesis of carcinoma of the prostate. Calcitonin induces a dose-dependent enhancement of cAMP and intracellular levels
25
of Ca2+ (Shah et al. 1994). The alterations in intracellular calcium levels in Sertoli cells induced by follicle-stimulating hormone (FSH) involve the generation of endogenous cAMP in response to this hormone treatment (Gorczynska et al. 1994). Hypothalamic neurohormones that control a variety of normal biological processes, such as cell proliferation and differentiation as well as hyperplasia and neoplastic processes, transduce their signals by the cAMP/Ca2+ pathway (Spada et al. 1997). The expression of cytokines in T lymphocytes is a highly regulated feature, involving cAMP. Benbernou et al. (1997) treated activated Jurkat cells with dibutyryl cAMP or pentoxifylline and found that IFN- mRNA expression was inhibited but that of IL-10 was unaffected. Cholera toxin and prostaglandin E (PGE)-2 also inhibited IFN- but greatly enhanced IL-10 mRNA expression. Using immunouorescence techniques to detect intracellular cytokines, these authors conrmed the inhibition of IFN- by dibutyryl cAMP and the enhancement of expression of IL-10 by PGE2. This clearly shows that a differential control is exerted by cAMP at both the transcriptional and the protein levels. Downstream events such as the induction of class II major histocompatibility complex (MHC) genes by IFN-, on the other hand, can be inhibited by agents that trigger the cAMP signal transduction pathway (Ivashkiv et al. 1994). The endpoint of the pathway is obviously the activation of responsive genes. There are certain regulatory regions called cAMP response elements (CREs) that interact with CREB. Besides CREB, the transcription factor known as ATF1 also mediates the transcription of responsive genes upon transduction of the extracellular signal via the cAMP/Ca2+-signalling pathway. Both these transcription factors are phosphorylated by the mediation of cAMP. CREB and ATF may integrate the signals owing from the cAMP/Ca2+ pathway. As stated before, cAMP activation can either inhibit or enhance biological responses. This has been suggested to be a consequence of blocking or activation of CREB. P.Q. Sun et al. (1994) showed by transient transfection studies with the Ca/CaM-dependent protein kinases CAMKIV and CAMKII that the former is a more potent activator of CREB than the latter. This they found was due to the nature of CREB phosphorylation. CAMKIV phosphorylated CREB at serine residue 133, but CAMKII phosphorylated serine 142 in addition to serine 133. The studies also showed that whilst phosphorylation of serine 133 activated CREB, phosphorylation of serine 142 inhibited CREB activation. In ATF1, CAMKII phosphorylates the positive regulatory serine 63, corresponding to serine 133 of CREB, but not serine 72, which corresponds to the negative regulatory serine 144 of CREB (Shimomura et al. 1996). Thus, although CREB and ATF share substantial sequence homology, it may be that these two transcription factors may differentially transactivate the responsive gene (Figure 8).
ARCHITECTURAL ASPECTS OF THE SIGNAL TRANSDUCTION MACHINERY THE ROLE

OF
CAVEOLAE
IN
SIGNAL TRANSDUCTION
The signal transduction machinery contains several elements along which information ows. A wide variety of signals are imparted to the cell and these need to be
26
FIGURE 8 Phosphorylation of the transcription factors CREB (cyclic AMP response element binding protein) and ATF in the negative and positive regulation of genetic activity. The Ca2+/CaM-activated protein kinases CAMKIV and CAMKII phosphorylate specic positive and negative regulatory serine residues of the transcription factors, which therefore differentially transactivate the responsive genes.
targeted and conveyed to specic subcellular compartments for eliciting cellular responses. This inevitably means that there should be an optimal organisation of the various elements to achieve a coordinated functioning of signal transduction. Certain invaginations involving specialised microdomains of the plasma membrane called caveolae were identied in epithelial and endothelial cells many years ago (Simionescu, 1993; Anderson, 1998). Caveolae do occur in many cell types. They have been identied with the endocytosis of macromolecules and in signal transduction and calcium homeostasis. The status of caveolae as a dynamic structural component of signal transduction is underlined by the presence within these membrane microdomains of a variety of signal transduction molecules. They contain G-protein-coupled receptors, ras family proteins, receptor tyrosine kinases (RTKs), and isoforms of PKC (Shaul and Anderson, 1998). Also identied with them are adenylyl cyclase and CBPs such as CaM and annexin. Caveolin, a cholesterol and lipid-binding protein (Murata et al. 1995; Trigatti et al. 1999), of which a number of isoforms are now known, is an integral membrane protein of caveolae. The caveolins are synthesised in the ER and are transported to the plasma membrane. Specic domains of the molecule take part in the movement of caveolins from the ER to the cell surface (Machleidt et al. 2000). The N-terminal cytoplasmic domains of caveolin-1 interact with G-proteins (S. Li et al. 1995), RTKs (Couet et al. 1997), and endothelial nitric oxide synthase (eNOS) (Okamoto et al. 1998).
27
eNOS is also a Ca2+/CaM-dependent enzyme. The increases in intracellular calcium levels induced by extracellular signals and the binding of Ca2+/CaM complex with eNOS seem to lead to its activation. eNOS also appears to be negatively regulated by caveolin-1. The interaction between eNOS and caveolin seems to reduce nitric oxide production (Shah et al. 1999). The vascular endothelial growth factor (VEGF) increases endothelial cell permeability as well as endothelial cell (EC) migration and proliferation. The VEGF signal appears to involve the production of nitric oxide by eNOS (Yiyu et al. 1999), and this mediated by the Flt-1 receptor (Ahmed et al. 1997). The up-regulation could be attributed also to posttranscriptional changes leading to the stability of eNOS mRNA (Bouloumie et al. 1999). In any event, the serine/threonine kinase Akt phosphorylates the serine residue 1179 of eNOS and activates the enzyme (Fulton et al. 1999). The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) inhibits VEGF-mediated EC migration and proliferation (Shizukuda et al. 1999). Nitric oxide is able to inhibit focal adhesion and spreading of endothelial cells and affects the formation of stress bres (Goligorsky et al. 1999). Human tumour cells have been transfected with the NOS gene. The transfectant cells have shown enhanced invasive ability, growth rate, and vascular density, with parallel constitutive expression of NOS (Thomsen and Miles, 1998). The mechanisms involved in the nitric oxidemediated increase in invasive and proliferative capacity have not yet been addressed, but there are suggestions that nitric oxide might up-regulate matrix metalloproteinases and block their endogenous inhibitors (Lala and Orucevic, 1998). IFN- and lipopolysaccharide (LPS) induce nitric oxide synthesis in C3-L5 murine adenocarcinoma cells as well as enhance their invasive ability in vitro. This is also accompanied by an upregulation of matrix metalloproteinase (MMP)-2. Furthermore, endogenous nitric oxide seems to down-regulate TIMP-2 and TIMP-3 (tissue inhibitors of metalloproteinases). This has led to the suggestion that nitric oxide alters the balance between MMPs and their inhibitors (Orucevi et al. 1999). VEGF is a potent inducer of angiogenesis. This angiogenic signal also involves nitric oxide production. L-NAME inhibits angiogenesis in vitro (Pipili-Synetos et al. 1995) as well as tumour-induced angiogenesis in vivo (Jadeski and Lala, 1999). Norrby (1998) supports the view that nitric oxide suppresses angiogenesis, but states that L-NAME did not suppress VEGF-induced angiogenesis. Nitric oxide synthase is also inducible. The inducible form (INOS) is found in large quantities in malignant prostate epithelium and in bladder cancer epithelium, but normal epithelial cells are only weakly positive. eNOS is expressed in the endothelial cells of tumour stroma, but not in stroma of normal bladder tissue (Klotz et al. 1998, 1999). In breast and gastric cancers also INOS occurs predominantly in the stromal component (Thomsen and Miles, 1998). This suggests a possible functional differentiation between INOS and eNOS. A marked up-regulation of NOS isoforms has also been reported in nonsmall cell lung cancers in relation to their progression (Ambs et al. 1998). Also of much interest is the nding by Gallo et al. (1998) that NOS activity in head and neck tumours with lymph node metastasis was higher than in tumours that had shown no metastatic spread. This has received some further support from Thomsen and Miles (1998) who state that NOS activity correlates with tumour grade in breast cancer.
28
It is possible that VEGF might follow two different pathways to increase endothelial permeability, endothelial cell migration, and proliferation, and induce angiogenesis. Whereas nitric oxide production is essential for effecting an increase in endothelial permeability, it appears to negatively regulate the angiogenic signal. This is partly borne out by the demonstration that VEGF reduces the activity of PKC isoforms, especially PKC-, and this could be blocked by pretreatment with LNAME (Shizukuda et al. 1999). Overall, it seems that there is a switching mechanism operating here, which apparently is distinct from the transduction pathway involving eNOS activation. This is likely to be a very rewarding area of investigation in the context of cancer spread and metastasis. The presence of IP3-like receptors (Patel et al. 1999; Fujimoto et al. 1995) and Ca2+-ATPase (Fujimoto, 1993) amply testies to the participation of caveolae in calcium homeostasis. Caveolae seem to sequester extracellular calcium into the vesicles and release it into intracellular locations, presumably by the mediation of IP3 and Ca2+-ATPase. Furthermore, with possible lateral mobility of caveolae one can envisage a regulation of entry and release of calcium to appropriate cellular compartments.
CAVEOLIN EXPRESSION
IN
CANCER
On account of the highly signicant participation of caveolae in signal transduction, the expression of caveolin has been the focus of some investigation in cancer progression. Caveolin expression appears to decrease with tumour progression, which is compatible with the view that there is a serious deregulation of signal transduction in neoplasms. Caveolin-1 and -2 genes, which occur at chromosome 7q31, are frequently deleted in tumours (Fra et al. 1999; Hurlstone et al. 1999). Caveolin expression is lower in breast cancer cell lines as compared with normal epithelial cells of the breast. Furthermore, caveolin expression seems to be inhibitory of growth (S.W. Lee et al. 1998). S.W. Lee et al. (1998) also transfected caveolin cDNA into breast cancer cells that normally show no detectable caveolin and showed that its overexpression in the transfectants resulted in a 50% reduction in growth rate and colony forming ability. Pug et al. (1999) grew LNCaP prostate cancer cells in androgen-depleted culture medium, and from these they derived tumorigenic androgen-independent cell clones. These clones also showed greatly reduced levels of caveolin expression. In contrast, benign prostate epithelial cells express caveolin at high levels. On the face of it, caveolin seems to function as a tumour suppressor. However, metastatic tumours also overexpress caveolin (Thompson, 1998). It would seem therefore that although its overexpression can lead to androgen resistance, its association with metastatic progression could be a secondary event. Caveolin-3, which is believed to be a muscle-specic isoform, is often mutated in patients with limb girdle muscular dystrophy, and the mutations have been suggested to be associated with the pathogenesis of the disease (McNally et al. 1998a; Minetti et al. 1998).
Calcium Binding Proteins and their Natural Classication
Calcium-binding proteins comprise a large family of proteins that can be subdivided into two subfamilies based on their molecular organisation. A large number of these proteins have been identied and characterised, and their physiological functions have been investigated (Tables 2 and 3). The subfamily of CBPs, referred to here as non-EF-hand CBPs, binds to certain phospholipids in a calcium-dependent fashion (Heizmann and Hunziker, 1990). They do not possess the characteristic feature of the other subfamily, popularly described as the EF-hand CBPs. The EF-hand protein subfamily in turn can be divided into two groups (Da Silva and Reinach, 1991). The members of one group function as Ca2+ sensor proteins. These are inactive at calcium concentrations in the low range of 107 to 108 M. They are activated into playing their regulatory role when the Ca2+ concentration increases to around 105 to 106 M. The second group comprises EF-hand proteins that are mainly concerned with calcium buffering and transport. Calcium ions function as second messengers par excellence in many pathways of cellular response. The translation of the calcium signal into biological function is mediated by CBPs that occur ubiquitously in intracellular locations as well as in the extracellular matrix. In order to be able to mediate the transduction of calcium signalling into cellular responses, CBPs need to recognise and interact with downstream target proteins. The binding of Ca2+ to CBPs produces conformational changes in these molecules and these changes appear to endow the CBPs with the ability to recognise and interact with their target molecules. This is discussed in greater detail in a later section. Both EF-hand and non-EF-hand CBPs participate in a host of normal physiological functions and consequently they are also associated with an array of pathological conditions (Tables 2 and 3). Although many EF-hand proteins have been studied for their involvement in normal and aberrant physiology and in pathogenesis, only a small number of them have been studied extensively. Consequently, the information currently available about some EF-hand proteins has tended to be somewhat fragmentary. Nonetheless, there is an indisputable theme that underlies the molecular features of CBPs as associated with their function in normal physiology and in pathogenesis. Their functions appear to be linked closely with the molecular organisation of the EF-hands, and furthermore, to the characteristic conformational changes that they undergo and the new molecular conguration that they assume upon Ca2+ binding.
29
30
TABLE 2 Non-EF-Hand Calcium Binding Proteins

Non-EF-Hand CBP Annexins (individual annexins also known as chromobindin, endonexin, lipocortin, etc.) Function Membrane-related functions; adhesion; fusion; exocytosis; possible cell-cycle-related expression; morphogenesis; differentiation Ref. Martin and Creutz (1987, 1990); Hamman et al. (1988); Martin et al. (1989); Creutz et al. (1992, 1994); further references cited in text Cited in text
Gelsolin family proteins Gelsolin
Severin Villin
Cytoskeletal dynamics; severing and capping of actin laments; cell migration, cancer progression; amyloidosis Sequence homology with gelsolin in actin-interacting domains Regarded as a marker of differentiation; occurs in intestinal and brush border epithelia, and in Barretts metaplasia and adenocarcinoma
Cited in text Cited in text
Cap G Fragmin Calreticulin
ER-associated CBP; molecular chaperone; intracellular calcium storage; cell adhesion; proliferation; autoimmune diseases Muscle homologue of calretinin, intracellular calcium storage Extracellular matrix (ECM) protein; bone metabolism; cell proliferation; differentiation; cancer invasion; osteotropism of cancer metastasis Implicated putatively in cell proliferation; regulation activity of protein phosphatases and Ca2+/CaM-dependent kinases; adenosine triphosphate (ATP)dependent transport of calcium across the plasma membrane
Calsequestrin Osteocalcin
Mishra et al. (1994) Van Ginkel et al. (1998) Constantin et al. (1998) Khanna et al. (1987); Smith et al. (1989); Milner et al. (1991); Heilmann et al. (1993); N. Liu et al. (1993) Cited in text Cited in text
Regucalcin [senescence marker protein-30 (SMP-30)]a
Fujita (1999); M. Yamaguchi (1998); M. Yamaguchi et al. (1996, 1997); T. Murata et al. (1997); Murata and Yamaguchi (1998); Kurota and Yamaguchi (1997); Hanahisa and Yamaguchi (1998); H. Takahashi and Yamaguchi (1997), M. Yamaguchi and Kanayama (1996)
31
TABLE 2 (CONTINUED) Non-EF-Hand Calcium Binding Proteins

Non-EF-Hand CBP Calelectrin Calcimedin Function Ref. Morse and Moore (1988) Moore and Dedman (1984); Moore et al. (1984); Morse and Moore (1988)
A large body of literature about regucalcin, mostly from the laboratory of M. Yamaguchi, has appeared in the past few years. The references cited indicate that it is a non-EF-hand CBP that shows tissuespecic expression mainly in the liver and to a lesser extent in the kidney. It is also found in hepatoma cell lines. The mouse regucalcin gene is described as spanning 18 kbp of the genome and containing seven exons and six introns. The human regucalcin gene has been provisionally mapped to chromosome Xp11.3q11.2.
32
TABLE 3 Functional Grouping of Major EF-Hand Proteins and Their Involvement in Normal and Aberrant Physiology
EF-Hand Protein Function Regulatory Proteins Modulation of enzyme function; cytoskeletal dynamics; binds to various cellular target proteins; control of cell cycle traverse and cell proliferation; apoptosis; intercellular communication, signal transduction modulation of cell shape; motility and invasion; metastasis; ageing; Alzheimers disease Regulation of enzyme activity; inhibition of transcription; cell proliferation; invasion Muscle contraction Muscle contraction Activation of guanylate cyclase; phototransduction Calcium buffering and transport; neuronal function; motor coordination; neuroprotective action Calcium buffering and transport; phosphorylation; cell proliferation and differentiation; tumour marker Photoreceptor-specic CBP Ref.
S100 family members
Cited in text
Calmodulin
Troponin C Myosin light chains Recoverin Calbindins, 9 and 28 kDa
James et al. (1995); Corneliussen et al. (1994) J.D. Potter and Johnson (1982); Wnuk (1988) Stryer (1991); Gorczyca et al. (1995) Bronner et al. (1986); Chard et al. (1993); further references cited in text Rogers (1987); further references cited in text Cited in text
Calretinin
Guanylate cyclase (GC)-activating protein Centrin (caltractin)
Calnuc (nucleobindin) Reticulocalbin family (reticulocalbin, calumenin, ERC-55, Cab-45) DAG kinases , , and
Microtubule dynamics; cell polarity; cell motility; duplication of microtubule organising centre; cell proliferation; response to environmental signals Associated with luminal surface of Golgi membrane ER-associated proteins; membrane trafcking of macromolecules DNA supercoiling (?)
Cited in text
P. Lin et al. (1998, 1999) Cited in text
Phosphorylation of DAG and activation of PKCs
Calpains
Keratin lament aggregation (proteolytic processing of prolaggrin)
Cited in text; Sakane et al. (1990); Sakane and Kanoh (1997); Yamada et al. (1997) Cited in text
33
TABLE 3 (CONTINUED) Functional Grouping of Major EF-Hand Proteins and Their Involvement in Normal and Aberrant Physiology
EF-Hand Protein Osteonectin Function Embryonic development and differentiation; extracellular matrix (ECM) remodelling; modulation of cell adhesion; cell shape; angiogenesis; tumour development and progression Protein phosphatase B regulatory subunit Calcineurin B- and CaM-related 191 amino acids; two C-terminal EF-hands; binds to cytoplasmic domain of platelet integrin II b3; putative regulation of the integrin function Buffer Proteins Lymphocyte maturation Muscle contraction; electrophysiology of neurones Ref. Cited in text
Calcineurin B Calmyrin
Cited in text Naik et al. (1997)
-Parvalbumin (oncomodulin) -Parvalbumin
Brewer et al. (1990) Heizmann (1984); Heizmann and Berchtold (1987); Heizmann and Braun (1990) Bronner et al. (1986); Chard et al. (1993) Rogers (1987)
Calbindins, 9 and 28 kDa Calretinin
Calcium buffering and transport Calcium buffering and transport; phosphorylation
Note: The S100 protein family is separately listed in Table 17. Calretinin has been advisedly included as both a regulatory and buffer protein, for although it was regarded preeminently as a buffer protein, its participation in cell proliferation and differentiation and utility as a tumour marker were considered as providing a strong basis for classifying it with other calcium-binding regulatory proteins. EF-hand proteins have also been identied in prokaryotes. An example of this is the Escherichia coli lytic transglycosylase Slt 35. The EF-hand domain of this enzyme binds Ca2+ as well as Na+, albeit in different molecular congurations. Ca2+ ions are bound preferentially, and this appears to be conducive to the stability of the enzyme (Van Asselt and Dijkstra, 1999). Source: Based on B.W. Schafer and Heizmann (1996) and references cited in the text in the course of discussions relating to their respective physiologic functions.
Non-EF-Hand Calcium Binding Proteins
Several non-EF-hand calcium-binding proteins have been studied in the past few years with regard to their participation in normal physiological processes and their apparent ability to modulate biological behaviour of cells (see Table 2). Of these, annexins, gelsolin, calreticulin, and osteocalcin have been extensively investigated. Although they are grouped together here as non-EF-hand CBPs, the pathways of their approach to the modulation of cell physiology and function are distinctively different. Annexins are calcium-dependent membrane binding proteins that participate in membranerelated processes of fusion, adhesion, exocytosis, and the remodelling of the plasma membrane. Calreticulin also subserves a membrane-associated function (e.g., the intracellular storage of calcium). Osteocalcin is an extracellular matrix protein and is involved in bone metabolism. It is apparently also signicantly implicated in invasive behaviour of cancers, their proliferation, and osteotropic metastasis. In contrast, gelsolin activity is mainly targeted on the cytoskeletal dynamics.
ANNEXINS STRUCTURE
Several non-EF-hand CBPs have been identied and sequenced. Of these, annexins have been studied extensively. Annexins form a family of structurally related and highly conserved proteins that bind phospholipids in a Ca2+-dependent manner. Several annexins have been identied and some of these have been well characterised, e.g., annexins I, IV, V, and XII. A 40-kDa annexin, distinct from annexin XII, has been reported from Hydra vulgaris (Schlaepfer et al. 1992a). Although basically annexins are intracellular proteins, it has been postulated that these proteins may be inserted into the phospholipid bilayer of the plasma membrane. Integration of the molecules into the plasma membrane is essential for their postulated functions of membrane trafcking, fusion, and the formation of ion channels. The crystal structure of annexins is characterised by a common fold that consists of four domains. Each domain has ve helices with a short interconnecting loop. The loops between helices A and B (domain 3) and helices D and E (domain 2) are attributed with the ability to bind phosphatidyl serine in a Ca2+-dependent manner. It has been suggested that annexin XII forms a trimer on the surface of the membrane (Luecke et al. 1995). Furthermore, it appears that an intramolecular Ca2+-binding site participates in the formation of the trimer, and this is quite distinct from the Ca2+ site involved in interaction with phospholipid (Mailliard et al. 1997). Langen et al. (1998) have postulated that, at low pH, annexin is inserted into the membrane as a continuous transmembrane helix that is formed, in a reversible manner, from
35
36
the helixloophelix conguration. They regard this latter conguration as being conducive to ion channel formation. Tyrosine phosphorylation of the tetramer may occur following the interaction of the tetramer with phospholipid. This is suggested by the stimulation of pp60src RTK following the binding of the annexin II tetramer to plasma membrane or phosphatidyl serine vesicles (Bellagamba et al. 1997).
BIOLOGICAL FUNCTIONS
Annexins are regarded generally, although not exclusively, as intracellular proteins with predominantly intracellular functions. Several biological functions have been attributed to annexins, (e.g., membrane fusion, calcium-channel formation, etc.) that involve cAMP as the second messenger in signal transduction. Thus membranebound annexins could be subserving a different class of function. Annexin II, for instance, can be found in both soluble and membrane-bound states. It has been identied on the cell surface, and in this location it could be functioning as a receptor for extracellular signalling ligands (Siever and Erickson, 1997). This argument is further reinforced by the apparent interaction of annexin V with the actin cytoskeleton. Membrane preparations of activated platelets indicate the presence of an 85kDa complex, which contains annexin V. Besides, this complex also contains actin (Tzima et al. 1999). This suggests the presence of a link between the plasma membrane and the actin cytoskeleton, which is potentially of considerable signicance in dening the function of annexins as an early link in the transmembrane signal transduction machinery. Annexins also show a major involvement down-stream in the ow of information in PKC-mediated transduction of growth factor signals. It has been demonstrated that annexin V is able to inhibit PKC and this in turn results in the inhibition of phosphorylation of annexin I (Schlaepfer et al. 1992b, 1992c). Annexin I has phosphorylation sites in the hinge region, which is believed to be important for cell proliferation and differentiation. Annexin XII is also a high-afnity substrate for phosphorylation by PKC (Schlaepfer et al. 1992b). This could be a reason why they show cell proliferation related expression (Schlaepfer and Haigler, 1990). Another aspect of PKC-mediated regulation of annexin function is the phosphorylation of certain amino acid residues that are involved in the interaction with EF-hand proteins of the S100 family. Annexin II is known to interact with S100A10 (p11) protein. There is a serine residue in the N-terminal region of annexin. If this residue is phosphorylated by PKC, annexin II interaction with S100A10 appears to be inhibited. Other amino acid residues have been implicated in similar interactions between annexin I and S100A11 (S100C) (Seemann et al. 1996). Annexin XI-A has been shown to bind to S100A6 (calcyclin) by means of specic hydrophobic residues on the surface of its -helix (Sudo and Hidaka, 1999). It seems, however, that annexin V does not inhibit the phosphorylation of annexin XII by the epidermal growth factor RTK (Schlaepfer et al. 1992c). On the other hand, annexin II, which is also involved with cell proliferation is a substrate for RTKs. Hubaishy et al. (1995) demonstrated that the phosphorylation of annexin II is effected by RTKs. They reported that phosphorylation negatively regulates annexin
37
II function, because it inhibited the binding of annexin II to and the formation of F-actin bundles. The annexins show a denable pattern of intracellular distribution. Barwise and Walker (1996) studied the distribution of annexins I, II, IV, V, and VI in human foreskin broblasts. Annexins IV and V were predominantly located in the cytoplasm and annexin VI was partly associated with the endoplasmic reticulum, whereas annexins I and II were associated with the plasma membrane. Furthermore, annexins I, IV, and V occurred in the nucleus at higher concentrations than in the cytoplasm. When the cells were treated with calcium ionophores and C2+[i] was raised, a marked relocation of the annexins occurred. Annexins II, IV, and V translocate from the nucleoplasm to the nuclear membrane. In MG-63 cells a relocation of intranuclear annexins IV and V to the nuclear membrane occurs within 40 s after treatment (Mohiti et al. 1995). Annexins located in the cytoplasm may be translocated to the plasma membrane. Human osteosarcoma MG-63 cells grown under normal serum conditions show high levels of annexin V in the nucleus. Upon serum starvation, however, the cells lose 72% of the nuclear annexin. When serum levels are restored nuclear annexin levels are also restored (Mohiti et al. 1997). These authors also observed that annexin phosphorylation is essential for the relocation of annexins. The translocation of annexin V appears to be inhibited when tyrosine kinase activity is blocked. Whether PKC-mediated phosphorylation plays any part in the translocation is unclear. PKC itself shows intracellular translocation upon being activated. It would have been of considerable interest also to determine PKC translocation, especially in light of the role PKC plays in the regulation of annexin function. A different pattern of annexin II and V translocation has been described in human neuroblastoma SH-SY5Y cells. Blanchard et al. (1996) found that both annexins were associated with membranes. Annexin II showed a uniform association before elevation of intracellular calcium levels, but upon treatment with calcium ionophores, it was relocated to discrete patches. Annexins take part in the process of calcium-dependent aggregation of liposomes. Annexins I through IV, but not V or VI, seem to mediate liposome aggregation (L. Liu et al. 1996). From this functional viewpoint, there is evidence of cAMP involvement in calcium-dependent aggregation of phosphatidyl serine liposomes and bovine chromafn granules and in calcium channel activity (Cohen et al. 1995). Blanchard et al. (1996) showed that membrane-associated annexin II is reorganised into discrete patches. It seems preferentially to partition into cholesterol-rich domains of the plasma membrane. Furthermore, the transmembrane adhesive glycoprotein CD44 also partitions into the same type of domain, with the result that CD44 clusters at the external membrane surface and annexin II at the cytoplasmic surface (Oliferenko et al. 1999). Whether this seemingly fortuitous partitioning of annexin II has a functional provision in CD44-mediated cell adhesion is unclear at present. Nevertheless, with the demonstration by Tzima et al. (1999) that annexin II might be coupled with the actin cytoskeleton, this is highly suggestive of its involvement in membrane-mediated functions of intercellular adhesion and cell fusion. Several mammalian annexins positively inuence the process of exocytosis (Creutz et al. 1992). Such a function seems to be strengthened by the observation by Oliferenko et al. (1999) that the redistribution of CD44 and annexin II was accompanied by changes in the cytoskeletal organisation. CD44 patching is induced
38
by overexpression of the S100A4 protein, and this appears to relate to the ability of S100A4 to depolymerise cytoskeletal elements (Lakshmi et al. 1993, 1997). Therefore, there is the distinct possibility that annexins and S100A4 might cooperate in generating the membrane-mediated behavioural changes of cells. The intracellular translocation of annexins in the human osteosarcoma cell line MG-63 has been found to be phosphorylation dependent. Serum depletion results in a reduction of nuclear annexin V levels. With serum stimulation these levels increase. Inhibition of tyrosine kinases has been found to reduce nuclear translocation of the annexin (Mohiti et al. 1997), which suggests that the relocation of annexins requires tyrosine phosphorylation.
ANNEXINS
IN
CANCER GROWTH
AND
PROGRESSION
The inuence of annexins on cell growth was recognised some years ago. The expression of annexin VII has been reported to inhibit the growth and viability of some secretory mutants of Saccharomyces cerevisiae at semipermissive temperatures. In contrast, annexin IV mitigated the growth defect of the sec2 secretory mutant (Creutz et al. 1992). Recent studies have in fact elucidated in some detail the signicant part that annexins play in cell proliferation. Raynal et al. (1997) demonstrated convincingly that annexins exhibit a cell cycle-related expression. Apparently, annexins II, V, and VI are, on the whole, uniformly expressed at a low level at all phases of the cell cycle. Annexin IV, on the other hand, showed a 50% increase in the S-phase of the cycle. Annexins I and V decreased by 40% in early G2M phase, although the corresponding gene expression did not vary. These observations show that there are specic patterns of occurrence of annexins and the expression may be regulated at the transcriptional level. The apparent cell cycle-related expression of annexins and the deregulation of PKC-mediated signal transduction with consequent deregulation of proliferation has prompted the investigation of annexins in relation to the proliferative state and progression of human cancers. Masaki et al. (1996) found that annexin I levels were greatly increased, at both the transcriptional and the protein levels, in human hepatocellular carcinomas as compared with normal liver tissue. Annexin II was reported to be overexpressed in human pancreatic carcinomas compared with normal pancreatic tissue (Vishwanatha et al. 1993). Furthermore, these authors found that the expression of annexin II was associated with proliferating ductal adenocarcinomas and especially that its expression colocalised with that of proliferating cell nuclear antigen (PCNA). Roseman et al. (1994) also found some association between levels of annexin II and the BrDU (bromo-deoxyuridine) proliferation index. Compatible with this is the reported histological grade-related expression of annexin II in gliomas (Roseman et al. 1994). They found that annexin II levels were greater in glioblastoma multiforme than in anaplastic astrocytomas, which in turn showed higher levels than did astrocytomas. With variations of annexin levels seemingly in consonance with growth deregulation in human cancers, there has been some effort directed at examining whether variations in expression might relate to the stage of progression of cancers. As stated before, annexin levels have been found to correlate with histological grade of gliomas
39
(Roseman et al. 1994). Annexin I does not occur in ductal luminal cells of normal breast tissue, but it is expressed in a range of breast cancers including noninvasive ductal carcinomas as well as invasive and metastatic breast cancer (Ahn et al. 1997). Annexin VI, in contrast, has been shown previously to inhibit cell proliferation. Theobald et al. (1994) transfected A431 cells with annexin VI. The growth of these cells, in normal serum concentration, was only moderately inhibited by the expression of annexin VI. When grown in low serum, their doubling time seemed to increase. However, Theobald et al. (1994) found that annexin VI-expressing cells grown in low serum conditions showed arrest at the G1 phase of the cell cycle. Furthermore, annexin VI expression inhibited the growth of A431 cells injected subcutaneously into nude mice (Theobald et al. 1995). It appears that the inhibitory effect is exerted only by the larger of the two splice variants of annexin VI. Fleet et al. (1999) found that the larger splice variant inhibited the Ca2+ mobilisation and cell proliferation induced by EGF. The shorter splice variant of annexin VI was not capable of such inhibition. Francia et al. (1996) identied several mRNAs that are differentially expressed in the murine melanoma cells B16F-10 and an immortalised melanocyte cell line called Melan A. Among these was an mRNA that was found to be identical to the 3 region of murine annexin VI. The expression of this mRNA was reduced in B16F-10 melanoma cells. Melan A may not be a proper control cell line with which to compare the mRNA prole of B16 melanomas, and the low metastasis variant B16F1 might have been a more appropriate control cell line. Nonetheless, Francia et al. (1996) followed this up with a study of the expression of annexin VI in human melanomas. In these tumours also a reduced or loss of annexin expression was encountered during progression. The expression of annexin V has been found to be markedly suppressed in carcinomas of the uterine cervix and the endometrium (Karube et al. 1995). Thus, most of the annexins studied to date have related directly, however indecisively, to cell proliferation and progression; however, annexin VI seems to differ from the rest. If conrmed, the reported tumour suppressor function of annexin VI does add a new dimension to the biology of annexins. But clearly, more work needs to be done to examine whether the putative suppressor function is associated with the larger splice variant and whether the second shorter variant of the annexin might possess some compensatory and antagonistic function. Overall, in spite of the cell cycle-related expression of some of the annexins, no clear relationship has emerged between their expression and the neoplastic disease process. Annexin II, which seems to relate to histological grade and differentiation of gliomas, tends to be expressed uniformly in the cell cycle. The expression of annexins I and V seems to change in opposite directions in the context of the cell cycle. Both are down-regulated in their expression at the G2M point in the cell cycle. Perhaps such comparisons are unhelpful because of the differences in the biology and histogenesis of the tumours studied.
ANNEXINS
IN
MORPHOGENESIS
AND
DIFFERENTIATION
The expression patterns of annexins have been studied with respect to cell differentiation. Rahman et al. (1997) reported that annexins V and VI are not detectable in
40
undifferentiated mesenchymal cells of foetal rat limb buds. The progenitor cells of skeletal muscle, which appear in the limb bud on day 14, express annexin VI on the cell surface upon differentiation from myogenic cells into myotubules. Annexins V and VI also are associated with the differentiation of chondrocytes. King et al. (1997) have conrmed that annexin V is required for the formation of the pericellular matrix of chondrocytes.
THE GELSOLIN FAMILY OF CALCIUM-BINDING PROTEINS

Actin-binding proteins constitute a large family of proteins that actively participate in actin bundling, nucleation, actin capping, and severing. Gelsolin, severin, and villin are three important proteins of the gelsolin family of actin-binding proteins that will be discussed here, for they possess the remarkable property of rapidly reorganising the cytoskeleton and also rapidly modulating, in a Ca2+-dependent manner, cell shape, substrate adhesion, and cell locomotion.
GELSOLIN
IN
SEVERING
AND
CAPPING
OF
ACTIN FILAMENTS
Gelsolin was identied as an 82-kDa actin-binding protein that is involved in Ca2+dependent severing of actin lament and capping of laments. The kinetics of polymerisation of actin laments, namely, the association and dissociation of monomeric subunits, essentially involves F-actin elongation by the association of ATPbound monomers with the barbed end of the lament and a slower loss of ADPactin from the pointed end (Sheterline and Sparrow, 1994). Actin polymerisation is altered by the sequestration of monomeric elements and by lament capping, which prevents the addition of subunits. The exchange of subunits at the lament ends is controlled by gelsolin and gelsolin-related proteins. They bind to the laments and inhibit the addition of actin subunits. Several proteins that participate in the regulation of cytoskeletal organisation have been identied (A. Schafer and Cooper, 1995). A large number (>60) of actin-binding proteins are known (Pollard et al. 1994) and the new ones are continually being identied and reported. A group of proteins composed of the gelsolin family and of tensin, and prolin bind to barbed ends. Others bind to pointed lament ends, whereas some proteins bind alongside the lament and yet successfully inuence lament assembly. Certain isoforms of tropomyosin have been found to protect actin laments from gelsolin-mediated severing. This protective function may be further enhanced by caldesmon (Ishikawa et al. 1989a). Tropomyosins appear to be able to anneal the actin fragments and again caldesmon is said to accentuate this process (Ishikawa et al. 1989b). The Drosophila ightless-I gene and the homologous human FLII gene encode gelsolin-like actin-binding proteins (H.D. Campbell et al. 1997). Constantin et al. (1998) have isolated a gelsolin-like protein from Physarum polycephalum that affects cytoskeletal integrity when introduced into mammalian cells. Tropomodulin and spectrin bind to pointed lament ends. Two isoforms of gelsolin had been identied: a cytoplasmic form and a secreted isoform called plasma gelsolin. A third isoform has now been reported; all three isoforms arise by alternative splicing of the gelsolin
41
transcript (Vouyiouklis and Brophy, 1997). The FLII maps to chromosome 17p11.2 (Chen et al. 1995), in the region that is consistently deleted, and is associated with Smith-Magenis Syndrome (SMS). Cytoplasmic-free calcium and inositol 4,5-bisphosphate regulate the function of gelsolin family proteins. Gelsolin binding to PIP2 has been found to be modulated by Ca2+ (K.M. Lin et al. 1997). Several functional domains that regulate actin lament length have been identied in the gelsolin molecule. The N-terminal domain (S1) is able to inhibit actin polymerisation. The inhibitory activity has been attributed to a small peptide sequence. The interaction of the C-terminal domains (S46) with actin laments occurring during the severing process has been localised with residues 112 to 120 of actin subdomain 1 (Feinberg et al. 1997a, 1997b). By deletion mutagenesis, residues close to the S2 domain have also been implicated in actin lament binding, capping and as well as interaction with phosphoinositides (H.Q. Sun et al. 1994). Colin is another protein that binds actin and promotes its depolymerisation. There are important sequence homologies between colin and gelsolin. The actin-binding peptide of colin has been found to compete with gelsolin segment S2 for binding to actin, suggesting that they share a binding site on the actin lament (Van Troys et al. 1997). Interaction with gelsolin induces conformational changes in actin, and nucleation of actin polymerisation may be promoted by these conformational changes (Khaitlina and Hinssen, 1997).
GELSOLIN
IN
EMBRYONIC DEVELOPMENT
AND
MORPHOGENESIS
Gelsolin and related proteins have been shown to be actively involved in normal embryonic development and morphogenesis as well as in pathogenesis of diseases such as amyloidosis and cancer. The gelsolin gene has been mapped to the chromosome 17p11.2 region, which is a critical region deleted in SMS which encompasses short stature, brachydactyly, dysmorphic features, retarded development, and behavioural problems. Gelsolin may also participate in the process of programmed cell death or apoptosis. Gelsolin shows a denable pattern of expression in the developing CNS. It is found predominantly in oligodendrocytes and Schwann cells. It is also found in the myelin sheath. In oligodendrocytes, gelsolin is found in the soma and in cultured cells it is detectable in the branched cell processes (Lena et al. 1994). The protein is detectable in the brain of newborn rats. The expression of gelsolin gradually increases at 8 to 10 days after birth, reaching a maximum at 20 to 30 days when myelin formation is actively occurring. However, subsequently, its levels decrease even though myelin basic protein levels continue to rise until 6 months after birth. In Schwann cells gelsolin is found in the cytoplasm and in compact myelin (J. Tanaka and Sobue, 1994). It would appear therefore that gelsolin may be involved in the wrapping of myelin sheaths around the axons by promoting a process of motility generated by means of its effects on the cytoskeleton (J. Tanaka and Sobue, 1994; Lena et al. 1994). The gelsolin-like protein, fragmin, which has been isolated from Physarum, has been shown to interact with actin in a Ca2+-dependent fashion, when introduced into PtK2, CV1, and NIH3T3 cells. This has been reported to cause cytoskeletal disruption and bring about changes in cellular morphology (Constantin
42
et al. 1998). Fibroblast migration has been found to be dependent on the actinsevering activity of gelsolin (P.D. Arora and McCulloch, 1996). Furthermore, gelsolin has been implicated in cytoskeleton-mediated transduction of EGF signal and EGF-induced cell motility (P. Chen et al. 1996). The Drosophila melanogaster ightless-I gene product is a gelsolin-like protein that is also involved in embryonic development and in the structural organisation of the indirect ight muscle. However, it should be pointed out that gelsolin-null transgenic mice appear to develop normally, albeit associated with certain abnormalities such as prolongation of bleeding time caused by abnormalities of platelet shape, and also inhibition of neutrophil migration. The transgenic mice also show increased stress bre formation in dermal broblasts together with reduced motility and increased contractility (Witke et al. 1995).
GELSOLIN EXPRESSION
IN
AMYLOIDOSIS
The strong association of gelsolin with CNS components has initiated studies of possible alterations in its expression in CNS-associated abnormalities. CNS abnormalities in patients with familial amyloidosis may conceivably be related to gelsolin expression (Kiura et al. 1995). Paunio et al. (1997) recently measured the mRNA levels of both intracellular and secreted gelsolin isoforms in tissues of human subjects. Intracellular gelsolin mRNA has been found to be a major component in all tissues. Most adult tissues can show mRNAs of both isoforms, with muscle and skin tissues showing especially high levels of expression. The high levels of skin gelsolin may be related to skin amyloidosis. The levels of expression may differ between adult and infant tissues. Patients with gelsolin-related amyloidosis had higher serum levels of gelsolin but did not show increased gene expression.
GELSOLIN
IN
CANCER
A major feature of cancer development is the perceived derangement of the cell signalling system and the inappropriate acquisition of cell motility. The process of actin lament severing and capping brings in its wake alterations in cellular motility. The involvement of gelsolin in the remodelling of the cytoskeleton has naturally led to the investigation of the expression and possible involvement of gelsolin and related proteins in cancer development and progression. Neoplastic transformation alters cell behaviour, morphology, and pattern of growth. These changes are indeed attributable to the loss of stress bres and focal adhesion. Accompanying cell transformation, modulation of the expression of cytoskeleton-associated proteins is observed, and among them is gelsolin. Van de Kerckhove et al. (1990) showed some time ago that gelsolin expression is down-regulated in transformed human broblasts and epithelial cells. Transformed cells also show a down-regulation of tropomyosin. Upon restoration of tropomyosin status, cells return to the normal modes of spreading and organisation of stress bres. These events are far from being coincidental, for the higher molecular weight forms of tropomyosin appear to protect actin laments from the severing activity of gelsolin (Matsumura et al. 1985; Ishikawa et al. 1989a).
43
Although only a few studies have been carried out to date, gelsolin appears to be consistently down-regulated in cancers. Moriya et al. (1994) found that gelsolin expression was down-regulated in several tissue culture cell lines derived from human gastric carcinomas. Gelsolin levels were found to be markedly reduced in human breast cancer cell lines as compared with normal breast epithelial cells and cell lines derived from benign breast diseases. Gelsolin loss was also found in 70% of 30 sporadic invasive breast carcinomas as well as in all chemically induced murine and rat mammary tumours (Asch et al. 1996). In human transitional cell bladder carcinoma cell lines as well as tumour tissues (14 out of 18 samples) gelsolin was not detectable or detectable only at low levels compared with normal bladder epithelium (M. Tanaka et al. 1995). M. Tanaka et al. (1995) also transfected gelsolin cDNA into the human bladder cancer cell line UMUC-2. The transfectants showed a greatly reduced colony forming ability as well as tumorigenicity. One can see from these studies the emergence of the concept of tumour suppression by gelsolin. However, it is paradoxical that gelsolin on the one hand can enhance cellular motility and on the other suppress tumorigenicity. Tumorigenicity and cellular motility can be dissociated temporally, and therefore one can envisage a situation where the loss of gelsolin could lead to tumour development and be involved in a positive way at a later stage of tumour progression by conferring invasive properties on tumour cells by altering cytoskeletal dynamics. There is little doubt that this is a fruitful avenue to explore. One needs to investigate the pattern of gelsolin alteration at various stages of tumour progression. The colon carcinoma model immediately comes to mind. The progression of colon carcinoma has been described in clearly identiable phenotypic phases, and there is considerable evidence that different genetic changes are associated with the different phases of progression and that the development of carcinoma is a cumulative effect of these genetic alterations (Sherbet and Lakshmi, 1997b). It would be interesting to see whether alterations of gelsolin or related gene alterations have a place in this picture. There is also the need to glean more information about the intracellular localisation of these proteins in the context of the alterations in cytoskeletal dynamics occurring in proliferating and transformed cells. Other questions can be raised. For example, are gelsolin and related proteins associated with cell proliferation and apoptosis? We have noted earlier that EGFinduced cell motility involves gelsolin. The alteration of cytoskeletal dynamics is an essential ingredient of cell proliferation. There is compelling evidence that other Ca2+-binding proteins such as the S100A4 (18A2/mts1), which also causes perturbation of cytoskeletal dynamics (Lakshmi et al. 1993), can control cell cycle progression by interfering with G1S and G2M checkpoint control exerted by the p53 suppressor gene and stathmin (Parker et al. 1994a, 1994b; Cajone and Sherbet, 2000). Therefore, if the gelsolin story is to be comprehended it is of the utmost importance to determine whether gelsolin is associated with cell proliferation. There are indications that gelsolin may indeed inuence cell population dynamics by inhibiting apoptotic death of cells (Ohtsu et al. 1997).
44

AND
SEVERIN
CYTOSKELETAL REORGANISATION
Severin is a 40-kDa Ca2+-activated actin-binding protein that was isolated from the amoeba Dictyostelium discoideum, and of this, mammalian homologues have also been identied. Severin severs F-actin, nucleates actin assembly, and caps actin laments. It is a gelsolin-related protein and shares sequence homology with gelsolin and prolin in certain conserved functional domains that are involved with actin interaction (Schnuchel et al. 1995). Severin possesses two actin-binding sites located next to each other, and these participate in both severing and nucleation functions. The third actinbinding site, situated near the N-terminus, sufces for the lament capping function (Eichinger et al. 1991). Eichinger et al. (1998) recovered a protein kinase from cytosolic extracts of D. discoideum. This kinase phosphorylates severin, but phosphorylation is reduced in the presence of calcium. Eichinger et al. (1998) have therefore suggested that phosphorylation might be another regulatory mechanism. Severin is known to alter rapidly the organisation of the cytoskeleton and as a consequence inuence cell adhesion to the substratum, pseudopodial activity, and cell motility. Early work had suggested that mutants decient in severin did not affect the motility of Dictyostelium (Andre et al. 1989). But Schindl et al. (1995), who studied mutants that were decient in severin, found the protrusion of pseudopodia was slower and the pseudopodia were shorter in the mutants. As noted in the previous section, the loss of gelsolin has been associated by some with cell motility, especially in neoplastic transformation. Folger et al. (1999) have reported that severin replaces gelsolin in murine Lewis lung carcinoma (LL2) cells. No severin is detected in normal epithelial lung tissue, but gelsolin is expressed at high levels. Folger et al. (1999) suggested tentatively that severin takes over from gelsolin the function of actin fragmentation in transformed cells. In light of the paradoxical effects of gelsolin of suppressing tumorigenicity on the one hand, and stimulating invasive behaviour on the other, further work on the expression of gelsolin and severin in a temporal relationship to tumour progression seems warranted.
VILLIN
IN
DIFFERENTIATION
AND
NEOPLASIA
Villin is another actin-binding protein that shows sequence homology with gelsolin with respect to certain conserved sites involved in actin-binding. It occurs in intestinal and kidney brush border epithelia. Villin is known to take part in actin bundling, nucleation, lament capping, and severing in a Ca2+-dependent manner, in vitro. Villin cDNA has been transfected into cells that do not synthesise the protein, and it has been found that this induces the growth of microvilli and the reorganisation of the submembranous actin cytoskeleton of the transfectant cells. This process is inhibited by cytochalasin (Friederich et al. 1993). Friederich et al. (1999) have provided further experimental evidence for the involvement of villin in actin bundling. Villin not only induces the growth of microvilli, but also in parallel induces actin bundling. This actin bundling ability seems to depend on the KKEK motif
45
occurring at the C-terminal end of the F-actin-binding site. However, some uncertainty about these events has arisen with the report by Ferrary et al. (1999) that in villin-null mice there is no disruption or disorganisation of microvilli, nor are there any changes noticed in the localisation of actin-binding and membrane proteins of the intestinal brush border in these null animals. Nevertheless, because the expression of villin appears to be a marker of differentiation, it has been investigated as a potential marker of tumour progression. Villin has been employed as a means of differentiating between primary pulmonary adenocarcinomas and pulmonary metastases of neoplasms of the bronchial or digestive tract. Nambu et al. (1998) examined 57 primary pulmonary adenocarcinomas for villin expression and found that 31% of the cancers expressed villin. Of the positives, 10.5% showed a diffuse cytoplasmic distribution of villin and 21% showed cytoplasmic distribution with minor brush border staining for villin. In contrast, metastatic lung tumours showed mainly primary brush border localisation that is characteristic of villin. The ndings of J.Y. Tan et al. (1998) are similar. These authors found that villin, together with cytokeratin 7 and 20, is useful in differentiating between primary pulmonary adenocarcinomas and pulmonary metastases of colon carcinomas. Villin is expressed in Barretts metaplasia as well as in Barretts adenocarcinoma. According to Regalado et al. (1998), all metaplasias and 93% (30) of adenocarcinomas were villin positive. Furthermore, Northern analyses revealed the presence of both 3.5- and 2.7-kb villin mRNAs. Thus, overall, villin seems to be expressed in most adenocarcinomas, metaplasias, as well as other oesophageal tumours. Villin also has been investigated as a possible marker of neuroendocrine tumours of the gastrointestinal tract and also of hepatic tumours (P.J. Zhang et al. 1997; Velazquez et al. 1998), but denitive reports of these studies have not been published. Another actin-regulatory protein called Cap G, which belongs to the gelsolin family, has been investigated somewhat supercially. Cap G is an actin-capping but not -severing protein. The Cap G gene is located on the short arm of chromosome 2 and consists of ten exons and nine introns. The open reading frame has nine exons and eight introns (Mishra et al. 1994). Van Ginkel et al. (1998) found that this gene is differentially expressed between normal uveal melanocytes and uveal melanomas and cell lines derived from these tumours. In summary, there seems to be ample evidence that villin gene function is involved in the organisation of the brush border cytoskeletal assembly and in the formation of microvilli. Therefore, the use of this protein as an indicator of brush border differentiation, with an implicit relationship to neoplastic transformation, seems fully justied. However, as a marker, villin will need to perform more stringently and with greater specicity than merely providing a means of differentiating between primary tumour of the lung and tumours metastatic to the lung. There is much scope for combining villin expression with other proteins such as ezrin and moesin, which link the plasma membrane with the cytoskeleton. These proteins not only localise with the actin cytoskeleton, but they are also specically associated with the formation of microvilli and membrane rufes.
46
CALRETICULIN AND ITS FUNCTIONAL DIVERSITY STRUCTURE

AND
MOLECULAR FEATURES
OF
CALRETICULIN
Two types of CBPs are associated with the endoplasmic reticulum (ER), namely reticulocalbin (an EF-hand CBP whose localisation and function are discussed in Chapter 12 and calreticulin). Calreticulin is a non-EF-hand CBP that is ubiquitously associated with the ER of a variety of tissues. It is regarded as the nonmuscle equivalent of calsequestrin, which is associated with the sarcoplasmic reticulum (SR). Calreticulin, which was identied as a CBP and isolated from SR (Ostwald and MacLennan, 1974), is believed to be a homologue of calnexin, which is a 64kDa transmembrane protein (Hammond and Helenius, 1995). It is a highly conserved protein. Calreticulin obtained from rat liver has a molecular size of 60 kDa. In some cell-free systems a 62-kDa protein has been detected, which is probably processed into 60-kDa calreticulin (Denning et al. 1997). Hershberger and Tuan (1998) have cloned a full-length cDNA for calreticulin from mouse trophoblast coding for a 57kDa protein showing a high degree of sequence homology to calreticulin. Yamamoto and Nakamura (1996) isolated and sequenced the ER-associated calnexin from Rana rugosa, which shared 77% sequence homology with calreticulin. Two distinct isoforms of calretinin have been isolated from spinach leaves and from the pollen of Liriodendron tulipifera L. and Ginkgo biloba L. (Navazio et al. 1995, 1998; Nardi et al. 1998). These are reported to show very low sequence homology with animal calreticulin. However, maize calreticulin is said to be a 48-kDa protein (as deduced from its cDNA sequence), highly acidic in nature, sharing 77 to 92% sequence homology with other plant calreticulins, and have approximately 50% homology with animal calreticulin (Dresselhaus et al. 1996). Calreticulin consists of 400 amino acid residues. It has three distinct domains: an N-terminal domain containing 180 amino acid residues, a C-terminal domain with acidic residues and lysines, and a middle domain (P-domain) that is composed of three repeats of a 17 amino acid motif. The C-domain binds calcium with high capacity but low afnity. In contrast, the P-domain binds calcium with low capacity but high afnity. Overall, calreticulin may be seen as a high-capacity but low-afnity CBP. The mouse calreticulin gene spans 4.2 kbp of genomic DNA and contains nine exons and eight introns (Waser et al. 1997). In the mouse the gene is located on chromosome 8 (Rooke et al. 1997). It should be pointed out here that P. Lin et al. (1998) state that nucleobindin, a mammalian protein showing a high degree of homology to calreticulin, is an EF-hand CBP. This protein is found in the cytosol and is associated with membranes, in the latter case mainly with the luminal surface of Golgi membranes.
REGULATION
OF
CALRETICULIN EXPRESSION
The expression of calreticulin appears to be regulated by intracellular calcium levels. The promoter region of the calreticulin gene contains elements responsive to the calcium ionophore A23187 and to agents such as thapsigargin, which can raise intracellular calcium levels by releasing calcium from intracellular stores. Both A23187 and thapsigargin have been shown to increase transcription of the calreticulin gene. It is not
47
affected by changes in extracellular or cytoplasmic levels of calcium. This has led to the suggestion that loss of calcium levels in the intracellular stores could induce gene transcription (Waser et al. 1997). Calreticulin gene expression is also up-regulated by other stimuli such as heat shock and heavy metals like zinc and cadmium (Nguyen et al. 1996; Szewczenko-Pawlikowski et al. 1997). A number of heavy metal ions, e.g., Ni2+, Zn2+, Cu2+, and La3+, stimulate the release of calcium from intracellular stores (McNulty and Taylor, 1999). Hyperthermia also raises intracellular calcium levels by calcium mobilisation from intracellular stores as well as by simulating its inux into the cell (Itagaki et al. 1998). Therefore, the up-regulation of calreticulin gene expression might, again, be mediated by the stress response of depletion of calcium held in the intracellular stores. This is in sharp contrast with the effects of hyperthermia and thapsigargin on the expression of S100A4. Both treatments have been shown to downregulate S100A4 expression (Parker and Sherbet, 1992; Albertazzi et al. 1998a). This might suggest a different mode of regulation from that of calreticulin. The thesis that the calreticulin gene is regulated by androgen has been advocated vigorously by N. Zhu et al. (1998). This is based on the nding that androgen ablation down-regulates and its restoration up-regulates both calreticulin mRNA and protein expression in the prostate. Calreticulin is expressed at higher levels in the prostate than in the seminal vesicle and other organs and muscle. The regulation by androgen occurs only in the prostate and seminal vesicles. In vitro, the induction of calreticulin by androgen is not inhibited by inhibitors of protein synthesis, hence the suggestion that the calreticulin gene might be regulated by androgen. Furthermore, in androgen-sensitive LNCaP prostate cancer cell lines, androgen is able to block apoptosis induced by the calcium ionophore A23187. This effect of androgen can be negated by antisense calreticulin oligonucleotides (N. Zhu et al. 1999).
PHOSPHORYLATION
OF
CALRETICULIN
Calreticulin phosphorylation occurs under different physiological conditions. However, it is unclear at present whether this is a physiological mechanism by which calreticulin activity is regulated. A phosphorylated form of calreticulin is detectable in cells infected with rubella virus (RV). It has been reported that the binding of phosphorylated calreticulin to the 3-end of RV genomic RNA is necessary for initiating RNA replication (Singh et al. 1994). Phosphorylation is a requirement also in the binding of calreticulin to Leischmania donovani RNA (Joshi et al. 1996). Calreticulin from spinach leaves is phosphorylated by protein kinase (casein kinase) CK2, but those of animal origin are not substrates for this kinase. It appears that this is due to structural differences between the two types of calreticulin (Baldan et al. 1996). CK2 was identied as an ER-associated kinase involved in the phosphorylation of calreticulin (Ou et al. 1992). CK2 might be localised in the lumen of the ER (N.G. Chen et al. 1996). The in vitro phosphorylation of calreticulin and other proteins in plasma membrane-enriched fractions has been attributed also to other protein kinases susceptible to inhibition by the PKC inhibitor staurosporine (Droillard et al. 1997). CK2 also phosphorylates calnexin in vitro as well as in vivo. Serine residues that occur in the C-terminal half of the cytosolic domain of calnexin were exclusively phosphorylated (Wong et al. 1998). Sphingosine-dependent kinases
48
(SDKs) are another kinase variety that have been shown recently to phosphorylate calreticulin. SDK1 has been found specically to phosphorylate calreticulin and protein disulphide isomerase (PDI) (Megidish et al. 1999). Heat shock proteins are also substrates of SDKs. Calreticulin, heat shock proteins, and PDI have a common function as molecular chaperones. Furthermore, calreticulin and PDI both occur in the ER. Therefore, these observations might be the closest researchers have come to suggesting a relationship between calreticulin function and phosphorylation. In spite of these various ndings, how phosphorylation of these molecules regulates their participation in their apparently diverse physiological functions is yet to be elucidated. At present, no changes in the state of calreticulin phosphorylation have been found to correlate with a specic function. Furthermore, it has been suggested that calreticulin could occur in a constitutively phosphorylated form (Cala, 1999). This would deny the process of phosphorylation any regulatory control over calreticulin function. Nevertheless, the induction of apoptosis of HL-60 cells by geranylgeraniol has been shown to be accompanied by a decrease in calreticulin as well as a decrease in the phosphorylation of another protein of 36 kDa molecular size. These reductions occur prior to DNA fragmentation (Nakajo et al. 1996). The occurrence of these events in parallel suggests that calreticulin may inuence the phosphorylation of the 36-kDa protein.
INTRACELLULAR DISTRIBUTION
OF
CALRETICULIN
As stated earlier, calreticulin shows a predominant association with the intracellular membrane system, the ER. The molecule contains KDEL/HDEL sequence at the Cterminal, which is the targeting signal for its localisation at the ER. Calreticulin occurs also in the SR and membranes of the Golgi bodies. In protoplasts of plants, it is localised in the ER (Opas et al. 1996b). In animal as well as in some plant cells, calreticulin is found in the lumen of the ER, nucleus, and nuclear membrane, on the surface of the cells, and in the cytoplasm (Opas et al. 1991; Dedhar, 1994; White et al. 1995; Dresselhaus et al. 1996).
CALRETICULIN
IN INTRACELLULAR
CALCIUM STORAGE
Calreticulin is prominently associated with the endoplasmic reticulum, as its nomenclature implies. Over the past few years, it has been found to participate in several biological phenomena. With its occurrence in the ER, calreticulin has been linked empirically with the cellular faculty of intracellular storage of calcium. This role has been conrmed by a variety of experimental data. There are several lines of evidence on this score. Using reporter gene constructs carrying the promoter of calreticulin gene, Waser et al. (1997) showed that the promoter can be transactivated by agents such as bradykinin and thapsigargin, which are known to release calcium held in intracellular stores. They have identied two regions of the promoter that are responsive to thapsigargin as well as the calcium ionophore A23187. Furthermore, both these agents have been shown to be able to enhance the transcription of calreticulin gene in NIH3T3 cells.
49
Mery et al. (1996) transfected a mouse broblast cell line with an expression vector carrying a calreticulin cDNA insert. In the transfectant cells, a 1.6-fold overexpression of calreticulin was found, and concurrently, intracellular calcium levels rose to nearly two-fold. Furthermore, most of this calcium originated from intracellular stores, as indicated by thapsigargin sensitivity. A study of the intracellular distribution of calreticulin in motor neurones of rat spinal cord has revealed that it is localised not only in the ER but also in coated vesicles (Copray et al. 1996). Copray et al. (1996) suggested that these vesicles may be counterparts of calciosomes, which are calcium storage vesicles found in liver cells and in cerebellar Purkinje cells. It ought to be pointed out, nonetheless, that Coppolino et al. (1996) found that the intracellular calcium stores were unaffected in cells in which the calreticulin gene had been knocked out.
CALRETICULIN
AND
CALNEXIN
AS
MOLECULAR CHAPERONES
A second function attributed to calreticulin is that of a molecular chaperone. The assembly of the T-cell antigen receptor (TCR) complex involves several genes, including calreticulin. Calnexin, which shares considerable sequence homology with calreticulin, is known to be involved with the chaperoning of newly synthesised TCR proteins. Similarly, calreticulin seems to be involved in the chaperoning of nascent TCR- and - proteins (Van Leeuwen and Kearse, 1996). Indeed, on the one hand, both calnexin and calreticulin seem to bind to and promote correct folding of proteins. They prevent aggregation, delay oligomerisation, and suppress degradation (Herbert et al. 1996). On the other hand, they possess the ability to bind to glycoproteins of the ER. Calnexin was shown to interact with and be involved in the assembly of class I MHC (major histocompatibility complex) molecules (Degen and Williams, 1991). Calreticulin has also been shown to interact with class I MHC molecules and probably with greater specicity than calnexin (Zhang and Salter, 1998; Harris et al. 1998). Both calnexin and calreticulin are lectin-like proteins and are able to interact with newly synthesised glycoproteins that have been partially trimmed of the N-linked oligosaccharides and assist in their folding and assembly (Helenius et al. 1997; Zhang and Salter, 1998). Receptors for insulin and insulinlike growth factor (IGF)-1 are receptor tyrosine kinases that have to dimerise before they are exported to the ER. The receptor monomers form dimers through disulphide bonding, when the newly synthesised monomers are associated with calreticulin or calnexin (Bass et al. 1998). Some of these studies implicate oligosaccharide-dependent binding to calnexin and calreticulin in a direct way in protein folding, assembly, and secretion. However, there are instances in which a lack of binding to these proteins has been of little consequence. It is possible that the chaperoning function of calreticulin might have implications for gene function. Calreticulin-decient cells show an inability to import transcription factors belonging to the NF-AT (nuclear factor of activated T cells) family into the nucleus (Mesaeli et al. 1999). This could be a reection of the part played by calreticulin-mediated chaperoning of the transcription factors. The NFAT transcription factors are substrates for calcineurin, and thus calreticulin might indirectly inuence the expression of genes regulated by these factors. (See Figure
50
19.) This view is compatible with the intracellular distribution of calreticulin. An overall increase in the levels of calreticulin has often been associated with cancers. Yoon et al. (2000) have reported that the total calreticulin content of hepatocellular carcinomas is similar to that of corresponding normal tissues, but carcinomas differ from normal tissues in their intracellular distribution. Calreticulin occurs more abundantly in the nuclear matrix of hepatocellular carcinomas as compared with controls. Whether this might reect an enhanced transport of substances such as transcription factors into the nucleus of cancer cells is an interesting subject for speculation. Such altered chaperoning of materials into the nucleus could greatly alter the pattern of gene expression.
CALRETICULIN
IN
CELL PROLIFERATION
AND
DIFFERENTIATION
There is very little evidence at present that calreticulin is involved in cell proliferation and differentiation. A retinoblastoma susceptibility (rb) regulatory element has been identied in the promoter region of the calreticulin gene (Valente et al. 1996). The rb gene is a negative regulator of cell cycle progression (see Sherbet and Lakshmi, 1997b). This observation provides some basis for implicating the calreticulin gene in cell proliferation. Dresselhaus et al. (1996) found an enhancement in the expression of calreticulin cDNA in the maize zygote during cell division. It has also been reported that calreticulin reduces intimal hyperplasia following arterial injury (Dai et al. 1997). There is considerably more information with regard to the involvement of calreticulin in cell adhesion and morphology, which, by implication, may be deemed to support the view that it might inuence also cell proliferation. However, its expression has been related to differentiation in NG108-15 neuroblastoma/glioma hybrid cells (N. Liu et al. 1996; Johnson et al. 1998). Higher levels of calreticulin expression were observed in these cells, when induced to differentiate by exposure to dibutyryl cAMP (db-cAMP). Although there could be a true effect on cell proliferation, calreticulin, in general, does not seem to affect the proliferative capacity of the cell. However, cell numbers might increase merely by the ability of calreticulin to protect cells from apoptosis (see below).
CALRETICULIN
IN
CELL ADHESION
Two other examples of the physiological involvement of calreticulin are in the process of cell adhesion and cell spreading and in disease states engendered by autoimmune conditions. The inuence exerted by calreticulin on cell adhesion to substratum was appreciated from early experiments of White et al. (1995). They demonstrated that anticalreticulin antibodies inhibited the binding of murine B16 melanoma cells to a substratum covered with laminin. They also showed the antibodies bound to the cell surface. The interaction of cells with the substratum also appeared to involve mannoside residues. Further, cell spreading could be inhibited by adding puried calreticulin to the medium, which presumably competed with cell surface calreticulin and denied the cells interactive binding with the substratum. Incidentally, this also conrms the lectin-like properties of calreticulin.
51
An exciting avenue has opened in the continued search for potential functions of calreticulin. It seems to possess the ability to inuence cell adhesion mediated by integrins. The walk along this avenue is exciting because integrins are a class of transmembrane heterodimeric glycoproteins that function as receptors for adhesion mediating macromolecules. The integrin family of cell surface receptors has been extensively studied and it has been established that their mediation of signal transduction as well as cell adhesion is accomplished by linking up with the cytoskeletal machinery. Vinculin, -actinin, and talin provide the linkage to the actin cytoskeleton. This involves a specic interlinking of the three proteins as follows: integrin/talin/vinculin:-actinin/actin. There is considerable evidence that adhesiondependent phenomena such as invasion and metastatic deposition are modulated by macromolecules that make up the extracellular matrix (Sherbet, 1982, 1987; Sherbet and Lakshmi, 1997b). In the rst place, there is much evidence that implicates integrins in the modulation of cell adhesion by calreticulin. Embryonic stem (ES) cells from calreticulin knock-out mice, which are decient in calreticulin, and broblasts derived from calretinin mutant mice have been reported to show severe reductions in integrinmediated cell adhesion. Transfecting ES cells with calreticulin cDNA restored this faculty (Coppolino et al. 1997). The cytoplasmic domain of the subunit of integrins contains the amino acid sequence KXGFFKR to which calreticulin is known to bind (Shago et al. 1997). Cell adhesion is modulated by a mechanism in which the expression of the linking proteins is regulated. Overexpression of calreticulin in Lbroblasts is accompanied by increased cellsubstratum and cellcell adhesion, and this overexpression also enhances cell spreading and decreases motility. Fibroblast cells overexpressing calreticulin also overexpress vinculin protein as well as its mRNA, and reduced levels of vinculin correlate with a down-regulation of calreticulin expression (Opas et al. 1996a). It is intriguing to note that another link in the system, namely talin, is unaffected by calreticulin overexpression. Nevertheless, calreticulin does not seem to affect the expression of -catenin. The latter reputedly links other CBPs such as cadherin to the cytoskeleton (Sherbet and Lakshmi, 1997a, 1997b). Shago et al. (1997) have shown that calreticulin can interfere with signal transduction mediated by retinoic acid receptors that contain an amino acid sequence similar to that found in the subunit of integrins. Retinoids are known to be able to inhibit invasive ability (Fazely and Ledinko, 1990). Furthermore, they can inuence the ow of information from ligand binding to integrins. Therefore, this possible complication of the perceived events should be borne in mind when interpreting the effects of calreticulin overexpression on cell adhesion.
CALRETICULIN
IN
NEOPLASIA
Although the above discussion indicates a signicant involvement of calreticulin in cell adhesion and possibly also in cell proliferation, only a few studies have dealt with its occurrence in tumours. Of the multiple functions attributed to calreticulin, its perceived effects on cell proliferation are the most relevant in relation to cancer. Calreticulin is one of several peptides of human breast cancers studied by Franzen et al. (1997). They state that it is expressed at higher levels in carcinomas than in
52
nonmalignant conditions of the breast, conrming an earlier study in which calreticulin expression in ductal carcinomas of the breast was compared with normal breast tissue (Bini et al. 1997). In the latter study, the enhancement of expression of calreticulin, and a number of other peptides, appeared to be specic for epithelial neoplasms. It would be premature to speculate on the nature of these correlative observations, but it has been suggested that calreticulin might control tumour progression by initiating a loss of apoptotic potential in tumours (Bruchovsky et al. 1996). Calreticulin levels increased in NG 108-15 neuroblastoma/glioma hybrid cells when induced to differentiate by treatment with db-cAMP (Johnson et al. 1998). Interestingly, however, antisense oligonucleotides for calreticulin do induce cell death in undifferentiated cells but not in differentiated cells. When the apoptotic pathway is experimentally induced in HL-69 cells by exposing them to geranylgeraniol, the expression of calreticulin is inhibited (Nakajo et al. 1996). Similarly, antisense calreticulin oligonucleotides seem to protect prostate cancer cell lines from calcium ionophore A23187-induced apoptosis. In this experimental system both androgen and calreticulin seem to be involved in the control of apoptotic events. A23187 induces apoptosis in both LNCaP and PC-3 cells. However, androgen is able to block apoptosis only in the androgen-sensitive LNCaP cells and not in the androgeninsensitive PC-3 cells. Furthermore, the inhibition of apoptosis of LNCaP cells by androgen is reversible by antisense calreticulin oligonucleotides (Zhu and Wang, 1999). One should recall here that in experimentally induced apoptosis of HL-60 cells, changes were also found in another protein species together with a downregulation of calreticulin expression (Nakajo et al. 1996). There was a decrease in the phosphorylation of a 36-kDa protein species. Thus it might be premature to link loss of calreticulin expression exclusively with apoptosis. Nevertheless, these observations suggest that calreticulin might protect cells against apoptosis. It is possible that deregulation of apoptosis is involved in both the growth of primary tumours as well as their metastatic deposits. Deregulation of growth control as well as inhibition of apoptosis can lead to cell population expansion in tumours. The phenomenon of metastatic dormancy may imply that cell mass is maintained by apoptosis-mediated regulation. Protection of cells from apoptotic death may lead to the appearance of overt metastasis. Most of these arguments are speculative. It would be interesting to see how the apoptosis regulating genes are affected by calreticulin expression.
IMMUNOLOGICAL IMPLICATIONS
OF
CALRETICULIN FUNCTION
Calreticulin has been implicated in the pathogenesis of some autoimmune disease conditions. In systemic lupus erythematosus (SLE), calreticulin might support the formation of the Ro/SS-A autoantigen complex (reviewed by Eggleton et al. 1997), and this could be related to its response to heat shock and stress factors. An interesting aspect of calreticulin in autoimmune diseases that has recently come to light is the amino acid sequence identity between calreticulin and the C1q receptor (Malhotra et al. 1993). C1q is a subunit of the rst component of complement C1. C1q recognises and binds to circulating immune complexes (N.R. Cooper, 1985). A consequence of this binding is that C1q is released from the complex (Sim
53
et al. 1979). It can now interact with the surface receptors on the various types of blood cells and endothelial cells (Tenner and Cooper, 1981; Malhotra and Sim, 1989; Peerschke and Ghebrehiwet, 1987; Peerschke et al. 1993). The binding of C1q to the receptor elicits a variety of immunological responses. The binding site of C1q to the C1q receptor or calreticulin has been identied (Stuart et al. 1996). Calreticulin occurs predominantly in association with immune complexes and C1q in sera of patients with SLE (Kishore et al. 1997). Kishore et al. (1997) suggest that inammatory episodes occurring in autoimmune conditions could be attributed to the release of calreticulin from leukocytes, leading to an antigenic reaction, and further by virtue of its identity with C1q receptor, to an interference with C1q-mediated inammatory effects. It is unclear at present why calreticulin should function as an autoantigen, but one reason could be that some parasitic organisms contain proteins that possess partial sequence homology with calreticulin. The inhibition of expression of steroid hormone-regulated genes is one of the postulated functions of calreticulin. As Coppolino and Dedhar (1998) have stated, it is truly remarkable that a single protein can have such a diversity of function. Clearly more work needs to be done on the multiple functions attributed to this CBP. Calreticulin has been highly conserved in the evolutionary process. Its conservation in a structural sense is probably less stringent than its functional conservation in signal transduction involving Ca2+ as a second messenger. Also it should be pointed out that calreticulin may not be the only CBP with multiple functions. The S100 family protein S100A4 has been described to participate in a wide spectrum of functions (Sherbet and Lakshmi, 1997b, 1998).
CALSEQUESTRIN AND INTRACELLULAR CALCIUM STORAGE

Calsequestrin is a high-capacity, low-afnity CBP and is the muscle equivalent of calreticulin. Like calreticulin, calsequestrin is associated with specialised regions of the SR and participates in intracellular calcium homeostasis. Its major function seems to be intraluminal calcium storage. When intracellular calcium channels are activated, calcium is released from the intracellular pools leading to increases in cytosolic calcium levels, and this is an important ingredient of sustained muscle contraction. The release of calcium requires the activation of ryanodine receptor/Ca2+ channels of SR (Berridge, 1993). These ion channels are formed by tetrameric complexes consisting of 565-kDa monomers of the ryanodine receptor (Sutko and Airey, 1996). Three isoforms of ryanodine receptor are known. The type I isoform is restricted to skeletal muscle and is involved in voltage-gated Ca2+ release. The distribution of types II and III is less restricted. Type II is the main mediator of Ca2+induced Ca2+ release in cardiac cells (Lai et al. 1988; Gianini et al. 1992). Ryanodine receptors resemble IP3 receptors, which are another mediator of Ca2+ release, both in respect of their structure and function (Sutko and Airey, 1996). The calsequestrin molecule binds to and releases 4 to 50 calcium ions. Calsequestrin must be linked with the ryanodine receptor/calcium release channel, and indeed a close physical relationship does exist between them (B.E. Murray and
54
Ohlendieck, 1998). The anchoring of calsequestrin to the SR has been found to involve a family of transmembrane proteins. One such transmembrane protein is triadin. Triadin seems to anchor calsequestrin to junctional regions of SR and couples it with the ryanodine receptor/Ca2+ release channel (W. Guo and Campbell, 1995). A 26-kDa protein identied by Mitchell et al. (1988) as a calsequestrin binding protein was puried and characterised by L.R. Jones et al. (1995). This protein, now called junctin, bears signicant sequence homology to triadin and aspartyl -hydroxylase. Junctin contains a transmembrane domain and a short N-terminal portion extending into the cytoplasm. But a major portion of this molecule extends into the lumen of the SR (L.R. Jones et al. 1995). Junctin is distributed with ryanodine receptors and calsequestrin in the SR, which suggests that it may also be involved in calcium release from intracellular stores. The binding interactions are attributed to specic amino acid motifs that occur in the luminal portion of both triadin and junctin. L. Zhang et al. (1997) have suggested that calsequestrin, junctin, and triadin might form a quaternary complex with the ryanodine receptor to achieve calcium release. It seems possible that calsequestrin might regulate the calcium release mechanism. This is suggested by the nding that in transgenic mice overexpressing calsequestrin, the expression of junctin, triadin, and ryanodine receptor is downregulated (L.R. Jones et al. 1998). Very little is known about the involvement of calsequestrin in pathogenesis. In a recent report the sera of some patients with ocular myesthenia gravis showed the presence of antibodies that react against calsequestrin (Gunji et al. 1998). This suggests that an autoimmune mechanism might be operating in the pathogenesis of this disease.
OSTEOCALCIN IN BONE METABOLISM AND OSTEOTROPISM OF CANCER THE BIOLOGY

OF
OSTEOCALCIN
Osteocalcin is a noncollagenous protein. It is the most abundant matrix protein of bone and dentine (Price, 1992). It is regarded as a marker of bone turnover and metabolism, where bone resorption and formation are coupled. Osteocalcin is synthesised exclusively by osteoblasts and secreted into the extracellular matrix (ECM) during bone mineralisation. It is released also during osteoclastic degradation. Therefore, it is considered to be a marker of bone formation when formation and resorption of bone are uncoupled (J.P. Brown et al. 1984; Delmas et al. 1985, 1986; R.H. Christenson, 1997). Osteocalcin expression in developing chick and rat embryos coincides with the onset of mineralisation of the bone and hence it is considered to play a role in this process (Hauschka et al. 1989). Osteocalcin functions as a chemoattractant for osteoblast progenitor cells (Mundy and Poser, 1983). Also, bone resorption is poor under conditions of osteocalcin deciency (Lian et al. 1984). These observations have supported a role for osteocalcin in bone resorption. Metastatic spread to the bone might activate bone metabolism, and therefore osteocalcin has been investigated as a potential marker of metastatic progression in certain forms of cancer, such as breast cancer and carcinoma of the prostate, which tend to show osteotropic secondary spread. Osteocalcin has turned out to be a good marker for
55
bone turnover in osteoporosis (Delmas et al. 1983; Eastell et al. 1993). Mutations of the gla protein, a member of the osteocalcin family of proteins, have been suggested as a causal factor in the autosomal recessive Keutel syndrome, which is characterised by abnormal calcication of the cartilage (Munroe et al. 1999).
CALCIUM-BINDING PROPERTIES
OF
OSTEOCALCIN
Osteocalcin is a non-EF-hand CBP and binds 10 Ca2+ per mole protein at pH 7.5. The binding is virtually halved when pH is lowered to 5.0 (Kobayashi et al. 1998). Kobayashi et al. (1998) produced, by proteolysis, peptides of less than 8 amino acids from the N- and C-terminal ends of osteocalcin. The middle fragment bound 4 to 5 Ca2+ per mole protein. A proline-rich segment and three -carboxyglutamic acid residues are believed to participate in calcium binding (Heizmann, 1996).
OSTEOCALCIN GENE STRUCTURE
AND
FUNCTION
The osteocalcin gene has been mapped to human chromosome 1q25q31 (Barille et al. 1996). The gene is regulated developmentally and in a tissue-specic manner. The tissue-specic expression seems to be due to the fact that the osteocalcin promoter functions only in cells of the osteoblastic lineage. This is suggested by the work of Ko et al. (1996), who constructed a virus vector carrying the thymidine kinase (TK) gene under the control of osteocalcin promoter. When this vector was introduced into osteoblastic cells TK gene was expressed, but no expression was detected in NIH3T3 or in a cell line derived from a transitional cell carcinoma of the bladder. The regulation of the osteocalcin gene is mediated by several transcription factors. Sequences that can bind the general transcriptional promoter AP1 have been identied in the promoter region of the osteocalcin gene (Goldberg et al. 1996; Lian et al. 1996). Lian et al. (1996) have also described two highly conserved regulatory motifs, which are, somewhat surprisingly, reported to relate to the cell type-specic expression of osteocalcin, albeit being functional in osteoblastic as well as nonosteoblastic cell lines. The modulation of expression of osteocalcin from the proliferative state to the differentiated state accompanying mineralisation of the ECM involves the function of a silencer element. An osteonectin silencer element (OSE) has been identied in the human osteocalcin gene, constituting a 7-bp (+29 to +35) sequence TGGCCT of the rst exon of the osteocalcin gene (Y.P. Li et al. 1995). In proliferating cells, OSE is activated by the binding of an OSE-binding protein (OSE-bp) and consequently osteocalcin is either not expressed or is found in very low levels. When OSE-bp expression is downregulated OSE is inactivated, which leads to enhanced osteocalcin expression and the onset of ECM mineralisation. A silencer element also occurs in the rst intron of the rat osteocalcin gene. Mutation of this suppressor element seems to inactivate the suppressor function (Goto et al. 1996). Other genes such as Dix-5 and MsX-2 have been shown to regulate the expression of osteocalcin during osteoblast differentiation. Dix-5 is believed to repress osteocalcin gene transcription (Ryoo et al. 1997). Interestingly, there is an increase in the expression of Dix-5 with osteoblast differentiation, which suggests an important regulatory role for this gene in the control of ECM mineralisation.
56
Among other regulatory elements that have been identied is a short motif called the OSCARE-2. OSCARE-2 is reported to bind a number of proteins including AP1. Furthermore, this element appears to be able to bind also nuclear proteins that are induced by vitamin D3 (VD3; 1, 25-dihydroxyvitamin D3) (Goldberg et al. 1996). As described below, VD3 receptor response elements have also been identied in the osteocalcin promoter region. This provides a molecular basis for the cooperation of VD3 and osteocalcin in the regulation of several processes such as calcium homeostasis, osteoblast differentiation, and cell proliferation and apoptosis.
REGULATION
OF
OSTEOCALCIN
BY
VITAMIN D3
Vitamin D3 (VD3) is an important regulator of calcium homeostasis. It plays a major role in bone mineralisation of type I collagen matrix, the transport of calcium and phosphate. VD3 inuences several important biological processes such as cell differentiation, apoptosis, inhibition of cell proliferation, and cell signalling. It is known to suppress proliferation of T cells and their ability to produce cytokines. The function of VD3 is mediated by the VD3 receptor (VDR). VDR is a phosphoprotein transcription factor that functions in the form of a heterodimer with the retinoid X receptor (RXR). VD3 induces the formation of a heterodimeric complex between VDR and RXR (Carlsberg and Polly, 1998). VDR has a number of identiable domains that participate in DNA binding, binding of the ligand, receptor dimerisation, and gene transactivation, as well as the C-terminal activation function (AF-2) domain required for co-factor interaction (reviewed by Issa et al. 1998). RXR seems to enhance the DNA-binding afnity of VDR as well as its specicity. VDR/RXR transcription factor regulates transcription of target genes by zinc nger-mediated DNA binding and proteinprotein interaction. However, the RXR ligand 9-cis retinoic acid (RA) prevents RXR from forming heterodimers with VDR and promotes the formation of RXRRXR homodimers. This effectively negates the transcription of VDR responsive genes. On the other hand, once VDRRXR is generated, these seem to block the formation of RXR homodimers (Haussler et al. 1997). The genes that encode the major bone matrix proteins, namely, osteocalcin, osteopontin, and 3-integrin, are among the target genes activated by VDR. A VD response element (VDRE), which consists of hexanucleotide repeats with intervening trinucleotide spacers, has been identied in the promoter regions of these genes. VD3 induces the synthesis of osteocalcin by osteoblast cells in both in vitro and in vivo conditions (Price and Baukol, 1980). VD3 not only increases the transcription of the osteocalcin gene but also seems to increase the half-life of osteocalcin mRNA (Mosavin and Mellon, 1996). However, species differences might exist in VD3-induced osteocalcin expression. Transgenic mice carrying human osteocalcin gene do respond to VD3 and show increased serum levels of human osteocalcin, but there is no increase in the endogenous mouse osteocalcin. These species-specic differences are believed to be due to possible differences in the regulatory sequences of the mouse and human osteocalcin genes (Clemens et al. 1997; Sims et al. 1997).
57
OSTEOCALCIN
IN
CELL PROLIFERATION
AND
DIFFERENTIATION
The osteocalcin gene is expressed in consonance with the inhibition of cell proliferation and the onset of cell differentiation and ECM mineralisation (Y.P. Li et al. 1995). VD3, as discussed above, induces osteocalcin expression, but on the other hand, it inhibits cell proliferation, apparently with the mediation of cdk inhibitors. M.J. Campbell et al. (1997) synthesised a number of VD3 analogues and demonstrated that these were capable of inhibiting proliferation of the prostate cancer cell lines LNCaP, PC-3, and DU145. The inhibition of cell proliferation was accompanied by an up-regulation of the expression of the cdk inhibitors p21waf1 and p27kip1. An up-regulation of p21waf1 expression has also been encountered in VD3-induced differentiation of human MG-63 osteosarcoma cells, and this has been shown to be independent of p53 function (Matsumoto et al. 1998). Therefore, these observations seem to dene a direct and novel pathway of inhibition of cell cycle progression by VD3. Other hormones, such as thyroid hormones, that regulate the differentiation of osteoblasts also seem to function through the mediation of osteocalcin. Triiodothyronine (T3) has been found to inhibit the proliferation of the osteoblast-type MC3T3E1 cells and, in parallel, induce the expression of osteocalcin mRNA and protein and alkaline phosphatase activity (Varga et al. 1997; Luegmayr et al. 1998). Oestrogen has been reported to increase cell proliferation in the early stages of in vitro culture of osteoblasts derived from mouse bone marrow. The effects of oestrogen seem to involve the osteoblast-specic transcription factor osf2 (cbfa1) (SasakiIwaoka et al. 1999). Oestrogen increases the expression of osteocalcin, alkaline phosphatase, osteopontin, and transforming growth factor (TGF)-1 as well as collagen type 1. Furthermore, exposure to oestrogen also increased the formation of bone nodules. Anti-oestrogens (Qu et al. 1998) blocked all these effects. Postmenopausal breast cancer patients treated with the anti-oestrogen tamoxifen have reduced (22%) levels of osteocalcin in serum (Marttunen et al. 1998). Fibronectin (FN) is a component of the ECM that has been implicated in several biological activities. Thus FN inuences cell adhesion to substratum, spreading, and modulation of cell shape. It inuences membrane rufing and cell motility and also is associated with cell differentiation (Sherbet, 1987). The pattern of FN expression in osteoblast differentiation has attracted some attention. Although there is very little information about its role in vivo, there seems to be some correlation between the expression of FN and osteocalcin, in relation to the state of proliferation and differentiation in vitro. In the initial period of growth of osteoblasts, derived from foetal rat calvaria, on a collagen-coated substratum, a 50 to 70% reduction of FN occurs, but the expression of osteocalcin, osteonectin, and osteopontin is up-regulated severalfold (Lynch et al. 1995). Similarly, in chicken osteoblast cell cultures at 6 to 18 days, FN is associated with the cell membrane, but subsequently it remains associated with the ECM in a brillary form. Overall, FN increases in the early periods of cell culture and its levels are then maintained. In contrast, the major bone matrix ECM markers show increased expression with the onset of differentiation (Winnard et al. 1995). There is no suggestion, however, that these events are necessarily related,
58
beyond the recognition that collagen type I could be involved in the signal transduction pathway. Possibly, in the initial stages where it is bound to the membrane, FN could be mediating extracellular signals through the occupancy of its specic integrin receptors. The subsequent association with ECM in a brillary form is clearly a postsignalling event. This suggestion seems to be upheld by the experiments described by Moursi et al. (1996). These authors found that anti-FN antibodies inhibited the formation of bone-like nodules and the expression of osteocalcin and alkaline phosphatase genes. Generally compatible with this is the ability of VD3 to regulate FN expression at the transcriptional level. A VDRE has been identied in the FN gene (Polly et al. 1996). Interestingly, osteoblast differentiation requires more than the RGD domain of FN that is functional in cell adhesion. Certainly, the experiments of Moursi et al. (1996) indicate that FN regulates the differentiation of osteoblasts. There is now general acceptance that integrin receptors form an important link in the transduction of signals generated by ECM components (Sherbet and Lakshmi, 1997a). Integrin receptors are actively involved in the recognition of and interaction with ECM ligands occurring in the process of osteoblast differentiation (Uemura et al. 1997). The integrin 51 has been identied as the critical component in FN interaction with osteoblasts (Moursi et al. 1997). The murine MC 3T3 cell line responds to ascorbic acid treatment by synthesising a collagen matrix, and collagen type 1 ligand seems to be essential for the subsequent expression of osteoblast markers as well as the activation of the osteocalcin promoter element, OSE2. OSE2 is recognised by the osteoblast-specic transcription factor osf2 (also known as cbfa1, AML3, PEBP2, and alpha A). The latter is expressed only in osteoblastic cell lines, such as MG63, ROS 17/2.8, and MC3T3-E1, but not in cell lines of nonosteoblastic origin (Sasaki-Iwaoka et al. 1999). When the collagen type 1 receptor 2subunit is blocked, the binding of osf2 to OSE2 is also blocked, with consequent inhibition of transcription of the osteocalcin gene (Xiao et al. 1998). These experiments indicate the importance of the interaction between collagen type 1 and its integrin receptor for transduction of the signal that can elicit osteocalcin gene activation. Much effort needs to be directed toward a dissection of the pathway of signal transduction in order to provide the crucial evidence that can link these events in a coherent fashion. Intracellular adhesion as well as cellsubstratum adhesion is determined by the components of the ECM. Their temporal and spatial expression is invariably associated with the alterations in the adhesive interactions, as well as changes in cell motility or migration on a substratum or invasive behaviour of cancers. The faculty of migration could be an important feature in bone resorption. Osteoclast precursors, for example, need to target sites of bone resorption and they do possess the ability of diapedesis across capillary endothelia. The modulation of certain ECM components such as FN, in conjunction with osteoblast differentiation and osteocalcin expression, has inevitably led to investigations of a potential association of osteocalcin with cell migration. Stringa et al. (1995) set up osteoblast cultures from 7day-old rat tibia fragments. They found that when the cells were exposed to parathyroid hormone they synthesised and secreted large amounts of osteocalcin
59
together with collagen type III. The conditioned medium from these cultures stimulated the migration of EA HY-926 endothelial cells in vitro. Osteoblast cells obtained from rat calvaria adhere and show migratory behaviour upon plating on three-dimensional matrices. These migrating cells expressed osteocalcin as indicated by immunohistochemical staining (Attawia et al. 1995). TGF-1 mRNA expression in regenerating tissue in distraction osteogenesis is said to coincide with osteoblast migration and ECM mineralisation (Mehrara et al. 1999). However, there is also much evidence that TGF-1 can inhibit osteonectin expression. Hence, it is conceivable that, in this experimental model, this promotion of migration by TGF-1 could be an effect mediated by means other than involving osteocalcin. TGF-1 is a highly versatile cytokine that can elicit a wide-ranging cellular response. Not least among these is that it can induce the expression of S100A4 protein (Okada et al. 1997), which has itself been implicated in the induction of cell motility. The identity of the ECM component that might be instrumental in osteoblast migration is uncertain at present. The two major adhesion mediating glycoproteins FN and laminin both regulate adhesive interactions involving osteoblasts. As seen earlier, FN expression does vary with the state of cell proliferation and differentiation, but it has not been directly implicated in the motile behaviour of osteoblasts. Laminin, on the other hand, does seem to mediate the adhesion to substratum as well as migration of osteoclasts. There is also a suggestion that there might be some form of cooperative functioning of laminin and FN in osteoblast migration. It has been suggested, for instance, that FN might be secreted in response to laminin-2mediated adhesion (Colucci et al. 1996). This postulate needs to be tested further. There are a number of possibilities that can be tested, e.g., whether there is de novo synthesis of FN, whether there is a deletion of FN into the medium, whether there is a modulation in the expression of FN receptors, etc. But Colucci et al. (1996) have shown that osteocalcin induces the migration of osteoclasts on laminin-2- but not collagen-coated substratum. One could concede easily that osteocalcin can promote migration involving ECM components and their particular integrin receptors. However, it is unclear at present how one can envisage a physical mechanism that transduces the osteocalcin signal to the cytoskeletal machinery to bring about cell locomotion. Also relevant in this context is the question of whether other biological macromolecules, such as the cadherins, might be involved too. Cadherin has been studied extensively for its ability to suppress invasion by cancer cells and, indeed, it has often been described as an invasion suppressor gene. Now there is evidence that VD3 analogues up-regulate the expression of E-cadherin in prostate cancer cell lines (H.D. Campbell et al.1997). In osteoblastic cell lines, VD3 analogues up-regulate osteocalcin expression. It would be of much interest to examine the status of cadherin expression in osteoblasts and determine what effects VD3 exerts on cadherin. Besides its effects on cell motility, TGF is also a powerful modulator of growth and differentiation. TGF peptides are known to inhibit proliferation of a number of cell types. More than coincidental is that the mechanism by which TGF brings about growth inhibition involves cdk inhibitors, e.g., p27kip1 (see Sherbet and Lakshmi, 1997b for references). TGF isoforms subserve many functions, including bone turnover. Banerjee et al. (1996) found that TGF-1 down-regulated osteocalcin
60
expression in ROS 17/2.8 osteosarcoma cells. Similar effects have also been described in foetal mouse long bone cultures (Staal et al. 1998). Thus, although it would appear that TGF could be regulating bone metabolism by inhibiting osteocalcin expression, its effects on cell proliferation are achieved by a different route involving cyclin/cdks (Figure 9). This conclusion is supported by the suggestion made by J.H. Liu et al. (1999) that TGF might be inhibiting G1-S arrest partly by inactivating cyclin B/cdc2 kinase. TGF treatment results in the phosphorylation of the cdc2 component in the TGF receptor IIcyclin B2cdc2 complex and downregulates its kinase activity. Despite this, it should be recognised that there is much obvious coordination in the functioning of osteocalcin, VD3, and TGF in the inhibition of cell proliferation and induction of cell differentiation.
FIGURE 9 A graphical illustration of how some of the functions of osteocalcin, VD3, and TGF overlap, and of the putative mechanisms involved. AP1, jun/fos transcription factor; cdk, cyclin-dependent protein kinase; FN; bronectin; RA, retinoic acid; RXR, retinoid X receptor; TGF; transforming growth factor ; VD3; vitamin D3; VDR; VD3 receptor; VDRE; VD response element. (Based on references cited in the text.)
OSTEOTROPISM
OF
METASTATIC DISSEMINATION
Metastatic spread of cancer is a nonrandom process. The perceived organ specicity of metastasis can be attributed to a variety of factors intrinsic to the cancer cell as well as to the target organs (see Sherbet, 1982 for review). Metastatic spread to the bone is a common occurrence in certain forms of cancer. It has been argued often that bone metastases can activate the processes of bone metabolism. For that reason
61
osteocalcin has been regarded as a potential surrogate method for detecting metastatic spread as well as for the purpose of monitoring the outcome of therapy on metastatic bone lesions. Several markers of bone metabolism have been employed in studies of this kind. Among those employed are osteocalcin, C-terminal peptide of type I procollagen (PICP), N-terminal peptide of type III procollagen (PIIINP), pyridinoline crosslinked C-terminal peptide of procollagen I (ICTP), and bone-specic alkaline phosphatase (BA-1p). Bloomqvist et al. (1996) found that ICTP and PICP levels correlated with that of osteocalcin, but not with urinary or serum calcium. All three markers correlated with the number of metastases detected by bone scans. The aminobisphosphonate ibandronate has marked osteoclast inhibitory activity and has been investigated as a treatment modality for metastatic bone disease and cancer-induced hypercalcemia. In combination with taxol/taxotere, ibandronate appears to be able to inhibit invasion of the bone by the human breast cancer cells MDA-MB231 (Magnetto et al. 1999) and the development of bone lesions in animals injected with myeloma cells (Dallas et al. 1999). Ibandronate markedly affects bone resorption in metastatic bone disease (Coleman et al. 1999). Osteocalcin, PICP, and BA-1p have been found to be reliable dose-dependent markers in a phase II clinical trial with ibandronate treatment of metastatic breast cancer. They were also found to be suitable for monitoring the effects of treatment of osteoporosis (Schlosser and Scigalla, 1997). However, Bombardieri et al. (1997) seem to disagree that any of these markers can replace bone scans. So far as prostate cancer is concerned, ICTP has been reported to reect bone metastasis more accurately than other markers, including PSA (prostate-specic antigen). Osteocalcin showed no correlation with metastatic spread (Maeda et al. 1997). In another study, PICP and BA-1p were found to increase with progression as indicated by bone scans. A slight increase in osteocalcin was also reported in patients with remission of metastatic bone lesions, but not related to progression (Koizumi et al. 1995). Obviously more clinical trials are needed to arrive at any rm conclusions. This need is underlined by laboratory work on the differentiation of osteoblastic cells, using conditioned media of the human prostatic carcinoma PC-3 cells and cell extracts. The effects of the conditioned medium have been studied on two osteoblastic cell lines, namely, a primary cell line derived from foetal rat calvaria and a rat osteosarcoma cell line ROS 17/2.8. The conditioned medium inhibited bone nodule formation in both cell lines without affecting cell proliferation. The conditioned medium also inhibited osteocalcin mRNA but not that of osteopontin (Kido et al. 1997). These data suggest there might be other factors involved in the formation of bone metastasis. A development with much promise is the use of toxic gene therapy for cancer, with osteocalcin promoter for targeting the expression of the toxic gene in the treatment of tumours of osteoblastic lineage and metastatic tumours. Ko (1996) made an adenovirus construct with the TK gene under the control of an osteocalcin promoter (Ad-OC/TK). When this viral construct was introduced into cells of osteoblastic lineage, e.g., murine ROS cells and human MG-63 cells, the TK gene was expressed. In contrast, no TK expression occurred in nonosteoblastic cell lines. The addition of acyclovir (ACV) caused cell death in vitro. Similar growth inhibition
62
and cytotoxicity were encountered in vivo when the adenovirus construct was injected into murine ROS osteosarcoma followed by intraperitoneal injection of ACV. (Cheon et al. 1997) has extended its ndings and has reported that the administration of the Ad-OC/TK construct coupled with methotrexate was highly efcacious in the treatment of osteosarcoma. Shirakawa et al. (1998) adopted this strategy in another experimental tumour model. They introduced ROS rat osteosarcoma cells into nude mice by intravenous route. These cells formed tumour nodules in the lung. They then injected the Ad-OC/TK construct into the tail vein with subsequent intraperitoneal ACV treatment. This treatment markedly reduced the number of tumour nodules in the lungs and signicantly enhanced survival. Furthermore, the cell-type specicity of the functioning of this construct was also demonstrated by Shirakawa et al. (1998). They constructed the adenovirus vector with RSV (Rous sarcoma virus) promoter, rather than by osteocalcin promoter, and placed the E. coli -galactosidase gene under its control. When this construct was used, there was no osteoblast-specic expression of the -galactosidase gene, but it was expressed nonspecically in lung parenchyma. This constitutes a rather elegant demonstration of cell-type-specic targeting of gene therapy coupled with cytotoxic drugs and certainly deserves further testing using experimental models of spontaneous metastasis.
The EF-Hand Calcium Binding Proteins
MOLECULAR ORGANISATION OF CALCIUM-BINDING EF-HAND PROTEINS

The members of the EF-hand CBPs share a general structural feature of possessing variable numbers of domains that bind to Ca2+ with high specicity and afnity. The EF-hand structural motif was rst reported from the crystal structure of carp parvalbumin (Kretsinger and Kockolds, 1973; Kretsinger, 1980). Specic structural features have been dened as being necessary for calcium binding. The EFhand consists of a consensus sequence of 12 amino acid residues in a helixloop helix conguration that can ligate Ca2+ (Linse and Forsen, 1995). The Kretsinger principle states that calcium binding is coordinated by ve oxygen-containing amino acid residues and a conserved glycine residue, which causes the bending of the loop. The calcium-binding afnities of these proteins range from Kd 104 to 109 M, and this is dependent on the amino acid sequence of the EF-hand loop. EF-hand loops occur as a pair with antiparallel -sheet interaction between the two loops. A high degree of cooperativity exists between calcium-binding loops (M. Zhang et al. 1995; Ames et al. 1995). The regulatory (sensor) and calcium buffer functions of EF-hand CBPs are closely related to the conformational reorganisation that occurs as a consequence of calcium binding. Conformational changes are essential for the regulatory function of EF-hand proteins, whereas for their calcium buffer function only a small conformational change appears to sufce (Ikura, 1996). A pair of EF-hands may form a globular domain and the domains within a protein may be functionally different, i.e., they may be either a regulatory domain or a buffer domain. Thus calmodulin has a regulatory domain at the Nand C-terminal regions of the protein. Troponin C and recoverin have an N-terminal regulatory domain and a C-terminal buffer or structural domain. In contrast, 9kDa calbindin has only one buffer domain at the N-terminal end. The regulatory domains undergo conformational changes upon binding to calcium and participate in the activation of target proteins (Ikura, 1996). The remarkable similarity in the genetic organisation of the genes coding for the EF-hand proteins has led to the formulation of an evolutionary prole of the EFhand protein family. As shown in Table 4, three classes of EF-hand proteins can be identied. Type I proteins include calmodulin, myosin light chains (MLCs), and spec I. The coding sequences of these are interrupted by ve introns, of which four occur at identical sites. The type II proteins, the S100 and related proteins typically possess two EF-hands and the corresponding genes have two introns, one of them
63
64
TABLE 4 EF-Hand Calcium Binding Proteins and Their Genetic Characteristics

Protein Class Type I Number of EF Domains 3 4 4 4 4 2
Name Parvalbumin Calmodulin Myosin light chains Centrin (caltractin) Spec I (sea urchin) S100 proteins
Genetic Organisation 5 introns interrupting EF-hand coding sequences
Type II
2 introns: one in the 5 untranslated region and the other between the two EF-hand domains In S100A8/A9, one EF-hand occurs at each terminal region
Type III
Calcyclin S100A8 (MRP-8, myeloidrelated protein) S100A9 (MRP-14) Calbindin, 9 kDa Calbindin, 28 kDa Calretinin
2 6 6
2.5 kbp; 3 exons and 2 introns; exons 2 and 3 coding for EF-hand domains 10 introns; 7 within the EF-hand coding domains 8 introns; distribution coinciding with their distribution found in calbindin
Source: Based on Heizmann and Hunziker (1990) and Ikura (1996) and other references cited in the text.
in the 5 untranslated region and the second separating the two coding domains. Type III proteins, such as the 28-kDa calbindin and calretinin, have six EF-hands. The corresponding genes have ten introns that occur with the EF-hand coding domain and they occur at identical sites in the 28-kDa calbindin and calretinin genes (see Heizmann and Hunziker, 1990 for a full review) (see Table 4). Kawasaki et al. (1998) have proposed a more elaborate classication of EF-hand CBPs based on the congruence of EF-hand domains. They have identied as many as 43 subfamilies on this basis. Of these, a group composed of 13 subfamilies is thought to have evolved and diversied, by gene duplication and fusion, from a progenitor domain. The remaining subfamilies do not show this probable evolutionary relationship and, indeed, some EF-hand CBPs do not appear to lend themselves to forming natural groups of subfamilies. The evolutionary relationship of EF-hand CBPs is also highlighted by the similarity that odd and even numbered domains of one protein show to the corresponding odd-and even-domains of another protein. Nonetheless, the framework for discussion adopted in the present work is based on the functional, rather than structural, homology of the CBPs.
65
CALCIUM BINDING AND THE MOLECULAR CONFIGURATION OF CALCIUM-BINDING PROTEINS

A characteristic feature of the binding of Ca2+ to CBPs is the conformational changes that these proteins undergo as a result. These changes in their molecular conguration have a signicant impact on their function. The calcium-induced switch between active and inactive forms of target proteins is often a consequence of changes in molecular conformation. Ca2+ can induce conformational changes in both non-EFhand and EF-hand CBPs. Calcium-mediated modulation of conformational changes in actin gures prominently in actin dynamics. For instance, following calcium binding, actin seems to take a conformation that is conducive to strong binding with MLC (Avrova et al. 1998). The gelsolin family proteins are regulated by calcium, interact with actin, and induce conformational changes. These conformational changes seem to be essential in actin dynamics. Cross-linking of actin is mediated by actin-binding proteins such as mbrin. The function of mbrin itself is regulated by conformational changes induced by the binding of calcium ions to mbrin. The EF-hand CBPs contain one to six EF-hand calcium-binding domains. The EF domain consists of a loop of 12 amino acid residues anked by -helical domains. Calcium binding to these domains brings about changes in the conformation of these domains, the major changes being the relative orientation of EF-hands. The EFhands open upon Ca2+ binding and assume a closed conformation state in the absence of Ca2+ (M.R. Nelson and Chazin, 1998). The open conguration is conducive to interaction with target proteins. However, CBPs might differ in the assumption of the closed position of EF-hands in the absence of Ca2+. Thus the C-terminal EFhand of CaM shows a closed position, whereas the C-terminal EF-hand of MLC would show a semi-open position. This would in effect mean a differential response of these proteins to the presence of calcium. In other words, the interaction of some CBPs with their target molecules might not be calcium sensitive. Sastry et al. (1998) have studied the three-dimensional structure of calcyclin and other EF-hand proteins such as calbindin D-9K (CBD9K) and reported the occurrence of major differences in the calcium-induced changes between these proteins. The exibility and dynamics of the EF-hand are very much dependent on the amino acid sequence of the entire EF-hand (Malmendal et al. 1998). The degree as well as the nature of the various changes are probably characteristic of individual EF-hand proteins, as in the case of S100 proteins. Furthermore, a combination of changes might endow individual S100 proteins with specic functional properties. Conformational changes can affect protein function in many ways. For instance, the activation of calpains, which possess protease activity, seems to be a consequence of conformational changes in the molecule that result in the re-orientation of the protease domain to produce a functional active site of the enzyme (Hoseld et al. 1999). A classical example of the effects of another facet of molecular conguration is provided by CaM. The changes undergone by CaM seem to enable the protein to bind to a wide spectrum of target proteins and bring about their activation. Guanylate cyclase-activating protein (GCAP), on the other hand, seems to be inactivated by calcium binding to EF-hands 3 and 4. Mutations of EF-hand 3 sufce to inactivate
66
GCAP. Possibly, this suggests that calcium binding to EF-hand 4 is a secondary, albeit important, event. An intriguing example would be a CBP that at least putatively contains domains that are involved in diametrically opposed functions. Osteonectin is a prime example of this. Two subdomains have been identied in the osteonectin molecule, of which one is involved in the inhibition of endothelial cell proliferation and the other stimulates endothelial cell proliferation in vitro. In a similar fashion, osteonectin is able to exert differential effects on cell adhesion and spreading. Possibly, all these effects are regulated in a tissue-specic manner. The process of regulation could involve a mechanism in which molecular conformation might conceivably play a prominent part. One could postulate that changes in molecular conguration might change the relative orientation of one functional domain to the other. One can cite another example taken from the signal transduction system. As suggested in relation to osteonectin, conformational changes occurring upon Ca2+ binding might be linked closely with function. Only nonmuscle and cytoskeletal isoforms of -actinin are capable of binding Ca2+, and only these isoforms are functional in the interaction with the cytoskeleton. This interaction is essential for successful signal transduction. Posttranslational changes of CBPs can inuence calcium binding and in this way also inuence molecular conguration and function. Several S100 proteins are modied at the posttranslational level. S100A8 and S100A9 are often found in the phosphorylated form. Phosphorylation can affect the functions of these proteins and can be a positive or negative regulator. It can inuence the binding of Ca2+ and other ions and the conguration that molecules undergo as a consequence. These changes in molecular conguration are an essential feature of the process of target protein recognition. Phosphorylation can also target S100 proteins to specic subcompartments of the cell. For instance, S100A8 and S100A9 translocate to the cell membrane, following their phosphorylation (Guignard et al. 1996). Recoverin is myristoylated at its N-terminus. It has been suggested that this modication induces cooperative calcium binding. More precisely, calcium binding to EF-hand 3 of RCN is believed to produce reorientation of the molecule to enable EF-hand 2 to bind calcium. The myristoylation of recoverin might be involved functionally in the targeting of the molecule to the appropriate cellular compartment. Some CBPs might inuence molecular changes in other CBPs merely by altering intracellular calcium homeostasis. It has been shown, for instance, that calretinin deciency markedly alters intracellular calcium levels in calretinin-null Purkinje cells. As a result, calbindin D-28K (CBD) is saturated with calcium and undergoes conformational changes. Obviously, in this case calretinin functions as a calcium buffer and thereby inuences or controls the molecular conguration and function of another CBP. Calcium binding also alters the quaternary structure and consequent oligomerisation of these molecules. Oligomerisation is essential for the binding of heat shock proteins to their targets and their oligomerisation is also known to be regulated by calcium. Such changes could alter the pattern of target recognition and in this way confer on S100 proteins the ability to interact with diverse target molecules and thus dene the specicity of their function. Besides calcium, the biological functions of CBPs might be regulated by other divalent cations such as Zn2+ and Cu2+ (Heizmann
67
and Cox, 1998). EF-hand proteins can bind other divalent ions such as Mg2+ and Zn2+. Indeed, a subgroup of S100 proteins that binds Zn2+ with high afnity has been identied. These bind two to four Zn2+ ions per protein monomer (Fohr et al. 1995; Fritz et al. 1998). As with Ca2+ binding, Zn2+ ion binding also occurs with much cooperativity. In S100A3, for instance, the binding of the rst Zn2+ ion facilitates the binding of the second Zn2+ ion. This cooperative binding appears to be associated with and, indeed, requires structural reorganisation of the molecule (Fritz et al. 1998). Whether these conformational changes also have functional signicance is still unclear. S100 proteins are known to form dimers readily in both intracellular and extracellular environments. Both homo- and heterodimers are formed by means of disulphide linkage, or the monomers may be held together by ionic bonds, depending on the environment and conditions. The relevance of the formation of dimers or tetrameric complexes to their function is yet uncertain, except probably in the case of S100A8 and S100A9. These are translocated from the cytosol to the plasma membrane. They seem to be able to form tetrameric complexes (A82/A92). Some recent work has brought to light the possibility that, at least in some CBPs, both Ca2+ and Zn2+ ions might be involved in the regulation of their function. For instance, the heterodimeric complexes of S100A8 and S100A9 bind arachidonic acid and transport it to its target site where it is metabolised. Kerkhoff et al. (1999c) have found that Ca2+ induces the binding of arachidonic acid by the S100A8/A9 complex, but this is reversed by Zn2+. This is an interesting joint functional involvement of these divalent cations, which contrasts with the demonstrations hitherto that they act on their own in bringing about changes in molecular conguration in the context of their function. Precise knowledge of the changes in conformation occurring here is still lacking. It would appear that S100A8 and S100A9 can form heterodimers in the absence of Ca2+. Possibly, Ca2+ induces a primary change in the molecular conguration of the constituent monomer to expose Zn2+-binding sites. In the wake of binding of the zinc ions a secondary molecular reorganisation could be occurring, which is incompatible, by reason of spatial constraint, with the continued association of arachidonic acid with the S100A8/A9 complex. It would be essential, therefore, to know the disposition of Zn2+-binding sites relative to the terminal calcium-binding EF-hand domains. Fritz et al. (1998) pointed out that Zn2+-binding cysteine residues are clustered in the C-terminal part of the S100A3 molecule that is said to be involved in the recognition of target proteins. Transcription factors recognise and bind to specic sequences in the promoter regions of genes, which initiates gene transcription. The binding of transcription factors to the DNA has been attributed to specic structural motifs that are present in the transcription factors. Many transcription factors possess Zn2+-binding domains in the form of protrusions and these are described as zinc ngers. The zinc nger motif binds to specic elements in the promoter. Zinc ngers, rst identied in the transcription factor TFIIIA derived from Xenopus, are known to be present in a large family of proteins that includes the thyroid hormone receptor, vitamin D3 receptor, and retinoic acid receptors, among others. Four types of zinc nger motifs have been identied. The TFIIIA zinc nger is regarded as the classical type or the prototype, which binds one Zn2+ atom via two cysteine and two histidine
68
conserved residues and to DNA as a monomer with high afnity. The zinc atoms seem to stabilise the structure of the zinc nger (Miller et al. 1985; Berg, 1990). The TFIIIA shows dual specicity in that it binds to DNA (5S RNA gene) as well as to 5S RNA (Pelham and Brown, 1980). The second type of zinc nger motif occurs in the steroid/nuclear receptor. Here the zinc nger binds two Zn2+ atoms binding to four cysteine residues each, and these receptors bind to the DNA as a dimer (Hard et al. 1990; Schwabe and Rhodes, 1991). The third type of motif is found in retroviral proteins and is characterised by 2 cysteine, a histidine, and a cysteine residues (Summers et al. 1990). The GAL4-type zinc nger from yeast binds two Zn2+ atoms, with six cysteine ligands to each Zn2+ (Keegan et al. 1986; Tao and Coleman, 1990; Pan and Coleman, 1991). Fritz et al. (1998) found that S100A3 binds two Zn2+ atoms. Of these, one zinc atom bound four cysteines and the second one bound four cysteines and one histidine residue. There is therefore some similarity between S100A3 and the zinc nger organisation of GAL4 transcription factor. These observations underline the potential importance of Zn2+ binding and the related changes in the molecular conguration in the function of S100A3. Gribenko and Makhatadze (1998) have proposed an attractive model in which molecular properties are postulated to change dependent upon relative the concentrations of Ca2+ and Mg2+. Calcium binding may not produce conformational changes in some proteins. S100A7 (psoriasin) has been reported to show very little change in its conformation upon calcium binding (Brodersen et al. 1998), but there were earlier reports of signicant changes in conformation of S100A7 upon binding of Ca2+, Zn2+, and Mg2+ (Vorum et al. 1996). It is possible that in this particular case Ca2+-mediated changes may not be functionally relevant.
THE STRUCTURE AND ORGANISATION OF S100 FAMILY GENES

The members of the S100 gene family have a shared intronexon organisation. S100 has three exons and two introns. The rst exon codes for the 5 untranslated region and the second and third exons each code for an EF-hand (Allore et al. 1990). Similarly, the S100A4 gene (h-mts1) has three exons interspersed with two introns. The rst exon is a noncoding exon and exons 2 and 3 contain the coding sequences for the mts1 protein (Ambartsumian et al. 1995).
ALTERNATIVELY SPLICED VARIANTS OF S100A4

Ambartsumian et al. (1995) also reported the occurrence of a splice variant of the h-mts1 cDNA. In the generation of this variant, alternative splicing seems to have occurred within the 5 untranslated region of the original cDNA transcript, and a second noncoding exon is inserted in the variant form. This insertion, nonetheless, retains the main open reading frame (ORF), without creating a new longer ORF. In a recent study of the expression of S100A4 (h-mts1) in carcinomas of the breast, Albertazzi et al. (1998c) discovered the presence of another variant form, which has been designated as h-mts1v. This variant is a shorter version of the original h-mts1
69
message. They have deduced the molecular organisation of the h-mts1v by reverse transcriptase polymerase chain reaction (RT-PCR). These studies have suggested that a loss of exon 1 (1a as well as 1b) sequences corresponding to one or more primers used for PCR may have occurred in the variant cDNA. The organisation of the h-mts1 gene and that of the variant cDNA is presented in Figure 10. The only and striking difference is the loss of exon 1 (1a as well as 1b) in the shorter splice variant, which is present in the larger variant reported by Ambartsumian et al. (1995).
FIGURE 10 Molecular organisation of the h-mts1 (S100A4) and that of the variant h-mts1v cDNA. Exon 1 (1a and 1b) of h-mts1 is not translated. This is spliced out in h-mts1v. Exons 2 and 3 encode the protein. (From Albertazzi et al. 1998c.) Reprinted by permission of the publisher Mary Ann Liebert Inc.
FUNCTIONAL SIGNIFICANCE OF ALTERNATIVELY SPLICED ISOFORMS

Alternative splicing of pre-mRNAs is a ubiquitous phenomenon in higher eukaryotes. However, the signicance of the occurrence of splice variants is poorly understood. Alternative splicing has been regarded as a mechanism of regulation of gene function. Splice variants may have different functions and different intracellular locations, and may indeed subserve opposing functions (Sherbet and Lakshmi, 1997b). Alternative splicing of pre-mRNA can generate isoforms of biologically active molecules and of membrane receptors for extracellular ligands functioning as biological response modiers (Gunthert et al. 1991; Kim and Yamada, 1997; Shi et al. 1994). This results in changes in the mode of function. Splicing out of the transmembrane domain of the IL-6 receptor appears to cause its deletion from the cell membrane (Horiuchi et al. 1994). The generation of splice variants of the kinases and phosphatases might be a common mechanism associated with the regulation of cell cycle progression. The PITSLRE protein kinases are members of the p34 (cdc2) kinase family, and are so named on the basis of the amino acid sequence of an important regulatory region. Several splice variant isoforms of the PITSLRE protein kinases have been described
70
(Xiang et al. 1994). They may be expressed differentially in different cell types and subserve different functions (Lahti et al. 1995). Similarly, the phosphatase cdc25B, which is involved with the function of the cyclin/cdk complex in cell cycle regulation, occurs in the form of several splice variants, which may be involved together with the wild-type phosphatase in the regulation of the cell cycle (Forrest et al. 1999). Another striking example is provided by the splice variants of annexin VI. Annexin VI has marked effects on cell proliferation. In A431 cells, it inhibits the mobilisation of Ca2+ induced by EGF. But this effect is produced only by the larger isoform. The shorter isoform does not affect Ca2+ mobilisation nor does it inuence cell growth (Fleet et al. 1999). Alternative splicing could also generate protein isoforms that may be unable to interact with target proteins or, as in alternatively spliced p53, to bind to DNA (Bayle et al. 1995). Possibly, the 5 region is most prone to splice modication, and the stability and translation efciency could be affected in these splice variants (N. Roy et al. 1992; Bingham et al. 1988). There is the further possibility that the 5 untranslated region may have a role in the regulation of the mRNA translation, as demonstrated for the alternatively spliced variants of the muscle-specic enolase gene (Oliva et al. 1995). On similar lines, the noncoding exons 1 and 2 of the angiotensin II receptor gene have been shown to be able to regulate the translation of the transcripts (Curnow et al. 1995). The 3-untranslated region seems to inhibit the expression of the cell membrane receptor of luteinising hormone. The inhibition has been attributed to a decrease in the half-life of the receptor mRNA (Nair and Menon, 2000). As stated before, Ambartsumian et al. (1995) reported that the occurrence of the larger splice variant of h-mts1 that contained an additional noncoding exon. It has been argued that splice variants may subserve different, often opposing, functions. If this were the case, one would expect to nd a differential expression of splice variants in tissues (Sherbet and Lakshmi, 1997b), which appears to be the case with respect to the h-mts1 splice variants (Ambartsumian et al. 1995). Ambartsumian et al. (1995) have stated that this splice variant showed marked variations in the level of expression in different human tissues. This large isoform was not found in the breast cancers, which Albertazzi et al. (1998b) investigated. However, there are indications that the expression of the h-mts1v isoform described here may have some bearing upon nodal spread of the disease (Albertazzi et al. 1998c), although h-mts1v did not appear to inuence nodal spread as decisively as h-mts. It would be premature to speculate on the signicance or relevance of h-mts1v expression to the state of the disease, because the deleted exon 1a/1b is not transcribed and exons 2 and 3 that encode the functional protein are intact. It may be argued that the loss of the 5 untranslated exon could be affecting the secondary structure of the transcript and its stability and translatability as a consequence. Possibly, the integrity of the S100A4 protein and its molecular properties might be affected by the splicing process, and this might impair the ability of S100A4 to promote metastatic spread.
71
REGULATION OF EXPRESSION OF S100 FAMILY GENES

The widespread involvement of S100 genes in diverse physiological function, and the demonstration that abnormal expression of many of these genes is associated with aberrant physiological events and the pathogenesis of several diseases, has inevitably led to the investigation of how the expression of S100 genes is regulated. Two modes of gene regulation have been investigated: transcriptional regulation and epigenetic regulation.
TRANSCRIPTIONAL REGULATION
OF
S100 GENES
The promoter regions of several S100 genes have now been characterised. The functional promoter of S100 has been localised to a region 168 to +697 and contains 168 bp upstream of the transcription start site. The activity of this promoter has been identied in several cell types. Furthermore, there are both positive and negative regulatory elements in the promoter region. There are positive regulatory elements in 788 to 391 and 1012 to 788. Further upstream, in the region 4437 to 1012 and in 1012 to 788, occur negative regulatory elements. The regulatory element in 4437 to 1012 has been found to suppress promoter activity in many cell types (Castets et al. 1997). Several potential regulatory elements, such as the CRE and AP2, have been identied in the promoter region of S100 (Allore et al. 1990). AP1-like binding sites are found also in the promoter region of the calcyclin gene, but the latter shows no binding by transcription factors such as AP-2, AP-3, or NF1 (Bottini et al. 1994). The murine homologue of S100A4 has been investigated by Tulchinsky et al. (1990, 1992), who have found homology between the 5 region of S100A4 and the promoter regions of rat brinogen and human prothrombin, and also with certain enhancer elements of SV40. Tulchinsky et al. (1992) found no cis-acting control elements in the 5 region of S100A4. Subsequently, a 16-nucleotide cis-acting transcription regulatory element has been identied in the rst intron of the gene, and this element has been found to bear a high degree of sequence homology to the enhancer element of the CD delta gene. The S100A4 promoter region also has been reported to contain motifs that bear a high degree of homology to p53 binding negative regulatory elements and AP1-like enhancer elements in the 3 region (Parker et al. 1994a). Okada et al. (1998) have identied a cis-acting element stretching from 187 to 88, upstream of the rst intron, that they believe species the transcription of the S100A4 (FSP1) gene in broblasts. They have also reported the occurrence of an enhancer element in the rst intron. This does not show cell type specicity. Okada et al. (1998) have reported further that a 5-bp domain TTGAT at 177 to 173 interacts specically with nuclear extracts derived from broblasts. This broblast-specic expression is not a typical feature of S100A4, which is indeed expressed to a variable degree in a variety of normal and neoplastic tissues. Thus, there are clear suggestions that the function of S100 genes may be controlled by a variety of transcription factors.
72

OF
REGULATION
GENE EXPRESSION
BY
DNA METHYLATION
DNA methylation is an epigenetic mechanism that regulates gene expression and gene imprinting. The CpG dinucleotide sites or islands that occur within and around genes are the targets of methylation (Ng and Bird, 1999). The process of methylation alters the conformational state of the DNA and it has been suggested therefore that methylation may play a role also in genomic stability. The association of the state of DNA methylation with gene expression has been known for many years (Kass et al. 1997). Hypomethylation is associated with constitutive gene expression. Demethylation of promoter regions of a gene, e.g., by treatment of cells with 5-azacytidine, increases the level of its expression (S. Lu and Davies, 1997). The regulation of tissue-specic expression of a tyrosine hydroxylase gene is dependent on the methylation status of the cytosine bases occurring around the CRE of the promoter (Okuse et al. 1997). Similarly, the expression of the manganese superoxide dismutase gene is suppressed when certain cytosines present in the 5 anking region are methylated (Y.H. Huang et al. 1997). Methylation of regulatory elements may interfere with the binding of transcription factors, as shown by Ryhanen et al. (1997). These authors found that AP-1 transcription factor bound to unmethylated response elements with far greater afnity than to methylated ones. The pattern of DNA methylation has been studied extensively. Methylation patterns may be somatically inherited, but probably not through the germ line. Epigenetic DNA methylation may be linked with genetic changes. However, in contrast with the generally held view that DNA methylation is a heritable and stable phenomenon, Ramchandani et al. (1999) have postulated that it is indeed a reversible signal. This postulate makes it easier to appreciate how DNA methylation might be involved in developmental processes as well as in neoplastic progression, both inherently associated with changes in the patterns of gene expression. Wachsman (1997) has argued that DNA damage, e.g., by alkylation of bases, oxidative lesions, etc., can interfere with the methylation of CpG dinucleotides by DNA methyltransferases (DNA-MTases) and alter the distribution of 5-methylcytosine (5-MC). The presence of 5-MC results in an increased risk of mutagenesis. CpG islands are hot spots for mutations with the presence of 5-MC and DNA-MTases. Furthermore, the levels of DNA-MTases correlate with the state of DNA methylation. Quite clearly, epigenetic changes of DNA methylation and mutations are interrelated and also are associated with carcinogenesis.
DNA METHYLATION
IN
CANCER
It has been known for many years that changes in methylation patterns are associated with normal developmental processes (E. Li et al. 1992; Neumann and Barlow, 1996) as well as human cancers (P.A. Jones, 1986; P.A. Jones and Chandler, 1986). Whether abnormal methylation status is associated with neoplastic transformation has been the subject of many investigations. Tumorigenic and non-tumorigenic cell lines differ markedly in their DNA-MTase levels (Kautiainen and Jones, 1986), and human tumours show a range of 5-MC levels (Gamasosa et al. 1983). The expression of DNA-MTase is said to be deregulated in colonic carcinomas, when compared with
73
normal colonic epithelia and adenomatous lesions. The carcinomas have also been reported to be highly heterogeneous with regard to DNA-MTase expression (De Marzo et al. 1999). However, because cancers tend to be intrinsically heterogeneous, the heterogeneity of DNA-MTase might not be sufcient grounds for postulating an association between possible deregulation of its expression and the process of carcinogenesis. The overexpression of certain oncogenes in animal tumour models has been associated with their demethylation (Wainfan and Poirier, 1992). Indeed, the oncogene ras was reported many years ago to be hypomethylated in human cancers (A.P. Feinberg and Vogelstein, 1983). Using an experimental tumour model, Counts et al. (1997) have demonstrated that hypomethylation of Ha-ras and raf oncogenes is related to tumour promotion, and further that carcinomas were more hypomethylated than adenomas. In breast carcinomas, marked hypomethylation has been reported in the vicinity of the promoter region of the calcitonin gene (Hakkarainen et al. 1996). pS2 is a protein associated with oestrogen-induced breast cancers. The expression of this protein has been found to be associated with hypomethylation of a CCGG site within the promoter/enhancer region of the pS2 gene (V. Martin et al. 1997). In contrast with oncogenes, tumour suppressor genes appear to be hypermethylated and inactivated in association with tumorigenesis (Zingg and Jones, 1997; Denissenko et al. 1997). The suppressor gene p53 is mutated in a large number of human tumours. Not only does a large number of somatic mutations occur at methylated CpG dinucleotide sites, but also these might be preferred targets for mutagenic agents (Denissenko et al. 1997; M.S. Tang et al. 1999). There is a generalised DNA hypermethylation in chronic lymphocytic leukaemia. More specically, two tumour suppressor genes cyclin-dependent kinase inhibitor genes CDKN2A and CDKN2B have been reported to be methylated. Apparently their expression is suppressed thereby in B-cell chronic lymphocytic leukaemia (Martel et al. 1997), and also in many other forms of human cancer, such as carcinoma of the breast, prostate, and kidney (Herman et al. 1995). The candidate suppressor gene HIC1 is relatively unmethylated or hypomethylated in peripheral blood or in acute myelogenous leukaemia as compared with recurrent acute lymphocytic leukaemia and blast crisis chronic myelogenous leukaemia (Issa et al. 1998). The inactivation or silencing of other tumour suppressor genes has also been reported, e.g., the MUC2 gene in colorectal cancer (Hanski et al. 1977) and the Wilms tumour gene (Kleymenova et al. 1998). The promoter region of the E-cadherin gene, which is regarded as a putative tumour and invasion suppressor, often has been found to be hypermethylated in gastric carcinomas. This has been associated with the reduced expression of E-cadherin in these tumours, and it has been suggested that hypermethylation of the promoter might occur as an early event in gastric neoplasia (G. Tamura et al. 2000). In prostate cancer, there is a decreased expression of endothelin receptor as compared with normal prostate epithelium. This appears to be due to methylation of a 5 CpG island covering the transcriptional regulatory region of the endothelin receptor gene (J.B. Nelson et al. 1997).
74

OF
REGULATION
S100 GENE TRANSCRIPTION
BY
METHYLATION
The expression of S100 genes may also be regulated by methylation. This has been suggested by Tulchinsky et al. (1995) for S100A4. They have implicated the methylation of the rst exon and the rst intron in the repression of transcription of S100A4. Subsequently, Tulchinsky et al. (1996) showed that the rst intron of this gene contains a methylation-dependent AP1 binding site. They further stated that the transcription of the gene is related to hypomethylation of the rst intron in murine adenocarcinoma cells. In human colonic adenocarcinoma cell lines, the expression of S100A4 closely correlates with hypomethylation of the second intron of the gene. Also, where S100A4 expression was low, this could be increased by treating the cells with 5-Aza-2-deoxycytidine (N. Nakamura and Takenaga, 1998). Indeed, the pattern of methylation of the gene could be a reason why S100A4 often shows a differential pattern of expression (D.S. Chen et al. 1999). S100A2, on the other hand, may be regarded as a tumour suppressor because it is expressed in normal breast tissue but it is down-regulated in the progression of breast cancer. Wicki et al. (1997) demonstrated the presence of an enhancer element 1.2 kb upstream of the transcription start site. Here they identied two AP1-like binding sites. The same enhancer element regulates the expression of S100A2 in normal as well as neoplastic breast epithelia suggesting the involvement of an epigenetic mechanism of methylation. Although their observations are not conclusive, it would appear that an element proximal to the transcription start site shows differential states of methylation in normal cells, tumorigenic cells, and cells derived from a breast tumour biopsy. Nonetheless, they have demonstrated that site-specic in vitro methylation of S100A4 gene in normal cells leads to a down-regulation of its expression. Perhaps it is unhelpful to overemphasise the signicance of the regulation of gene expression at the transcriptional level. Ambartsumian et al. (1999) have reported that, in S100A4 transgenic mice, S100A4 mRNA was expressed in all organs. The mRNA was expressed at a higher level in some organs. However, they have made the crucial observation that the S100A4 protein was down-regulated in the organs that did not express the gene in the wild-type animal. It is obvious from this that there must exist a complex mechanism that regulates the expression of the protein at the translational level or regulates the decay of the protein. This is not unique to S100A4, and the expression of other S100 proteins in common with a wide variety of cellular proteins might also be controlled at the posttranscriptional level. For instance, S1001 protein and its mRNA are differentially expressed in differentiating neuronal and skeletal muscle cells (Zimmer and Landar, 1995).
The Calmodulin Family of Calcium Binding Proteins
A number of calcium regulatory proteins may be treated as belonging to the calmodulin family of CBPs. In addition to calmodulin, troponins, calbindins, recoverin, and calretinin can be assigned to this family. The regulatory function of these CBPs in normal as well as aberrant physiology will be discussed in the following pages.
CALMODULIN AND ITS PHYSIOLOGICAL FUNCTION STRUCTURE

AND
MODE
OF
ACTION
OF
CALMODULIN
Calmodulin (CaM) is a 17-kDa CBP. Structurally it may be described as a dumbbellshaped molecule with two lobes each containing two Ca2+-binding domains. The two lobes are joined by an -helix comprising 28 amino acid residues. A conformational change occurs in the CaM molecule in the presence of calcium and as result CaM can bind to a large number of target molecules. The conformational change involves the exposure of two methionine-rich hydrophobic areas, one in each globular domain, and these take part in the interaction with target proteins and in their activation. CaM can activate a large number of proteins, and this ability has been attributed to the highly conserved methionine residues (Kincaid et al. 1987; Perrino et al. 1992; Milan et al. 1994), the mutation of which might affect activation of target proteins. The -helix linker also plays a crucial role in this process of activation. Tabernaro et al. (1997) studied a mutant form of CaM in which the amino acid residues Thr 79 and Asp 80 are deleted from the -helix. This deletion appears to alter the relative orientation of the globular domains, which results in the hydrophobic patches coming closer together and consequently becoming less accessible to interaction with target proteins. The CaM binding domains of target proteins occur in short stretches of 15 to 25 amino acid residues (ONeill and De Grado, 1990; Ikura et al. 1992; Crivici and Ikura, 1995). CaM appears to recognise certain conserved structural features, which might differ from one target protein to another with respect to their topographical arrangement. CaM orientates its globular domains by virtue of the exible nature of the linker. An interaction with CaM is essential for the activation of several of the target proteins (Kretsinger, 1992). Specicity of activation of the target enzymes seems to be achieved by the phosphorylation of CaM (Williams et al. 1994; Sacks et al. 1995; Quadroni et al. 1994; Saville and Houslay, 1994). Several kinases phosphorylate
75
76
CaM, among them the myosin light chain kinase (MLCK) (Davis et al. 1996); casein kinase II (Sacks et al. 1992), and receptor tyrosine kinases (Graves et al. 1986; San Jose et al. 1992). Ubiquitination of calmodulin also has been suggested as a possible regulatory mechanism (Laub and Jennissen, 1991). The occurrence of numerous target proteins indicates the broad spectrum of physiological function in which CaM participates. CaM mediates the function of enzymes that are involved in cyclic nucleotide metabolism, phosphorylation and dephosphorylation, and smooth muscle contraction (Means, 1988; Harrison et al. 1988; MacNeil et al. 1985; G. Li et al. 1989).
CALMODULIN-MEDIATED SIGNAL TRANSDUCTION

There is currently a large body of evidence demonstrating the important role calmodulin plays in the transduction of signals of cell proliferation and growth control, and in cell locomotion and invasion. The signalling pathways that are initiated by the activation of cell membrane receptors, as alluded to earlier, involve the raising of intracellular calcium levels and the subsequent phosphorylation of target proteins. Because Ca2+/CaM interacts with and activates target enzymes, CaM has been deemed as an important component of the signalling pathway by mediating protein phosphorylation via CAMKs. CaM kinases subserve a variety of functions. Several examples can be cited for the purposes of discussion and illustration. Thus T-cell activation, cell migration, and proliferation will serve amply to underline the significance of CaM as a signalling molecule. The activation of the T-cell receptor not only increases intracellular calcium but also activates CaM-dependent kinases, which results in the regulation of phosphatases (Ostergard and Trowbridge, 1991). CAMKs are also involved in cell proliferation. CAMK II has been shown to be involved in the induction of S-phase delay in broblasts exposed to -irradiation. The exposure to radiation also induces CAMK II activity. This induction does not occur if the cells are pretreated with CAMK II antagonists (Famulski and Paterson, 1999). An outstanding example of CaM-mediated signal transduction is the transduction of angiotensin signals. The hormone angiotensin II, derived from the decapeptide angiotensin I, generates many cellular responses. Angiotensin II possesses cytokinelike activity. It functions as a cardiovascular growth factor and is mitogenic to cardiac broblasts. It has also been attributed with the ability to induce angiogenesis. Not surprisingly, therefore, angiotensin signal transduction has many similarities to that of growth factors. Some of the effects of angiotensin are indeed mediated by growth factors such as PDGF, EGF, basic broblast growth factor (bFGF), and TGF. The transduction of the angiotensin signal involves two types of angiotensin receptors: type 1 (AT-1) and type 2 (AT-2). The binding of angiotensin to AT-1 triggers a cascade of signalling events and the activation of transduction pathways (Thomas, 1999). The C-terminal domain of the AT-1, close to the inner membrane, interacts with CaM in a Ca2+-dependent fashion. This interaction seems to be essential for the transduction of angiotensin binding to AT-1A (Thomas et al. 1999). In cardiac broblasts, AT-1-induced signals appear to transactivate epidermal growth
77
factor receptors (EGFr) and eventually result in DNA synthesis. This effect is inhibited by CAMK inhibitors (Murasawa et al. 1998). Angiotensin is able to induce the synthesis of TGF and the type II TGF receptor (Wolf et al. 1999). TGF and its receptor activity have potentially profound effects on the composition and properties of the ECM. Furthermore, angiotensin and TGF can both induce cytoskeletal changes, such as the formation of actin stress bres and the phosphorylation of focal adhesion proteins (Riedy et al. 1999). There can be little doubt that the cytoskeletal modulations and the remodelling of the ECM will inuence cell migration as well as cell proliferation and differentiation. Cell migration is another cellular activity that demonstrably involves CaM in signal transduction. Cell migration is believed by some to involve activation of the pathway that includes members of a family of serine/threonine kinases called extracellular signal-regulated receptor kinases (ERK), which are also known as the MAPK pathway. In PC12 cell, calcium inux activates ERK. This activation is inhibited by the CaM inhibitor W13, which indicates that CaM is involved in this pathway (Egea et al. 1998). Egea et al. (1998) also showed that other receptors, e.g., trk A and EGFr, are not involved in the activation of the ERK pathway. In gastric epithelial cultures also CaM appears to be involved in the transduction of the cell migration signal (Ranta-Knuuttila et al. 1998). Of late, much evidence has emerged that links CaM with G-protein-mediated signal transduction. There is a marked parallelism between angiotensin signal transduction and the transduction of growth factor signals. It has been recognised, therefore, that G-proteins might be involved in angiotensin signal transduction. Angiotensin II has been known to bind to a high-afnity G-protein receptor (T.J. Murphy et al. 1991; Sasaki et al. 1991; Sandberg et al. 1992). Recently, CaM has been reported to enhance the Ca2+-dependent binding of guanosine triphosphate (GTP) to the ras-related protein Ral-A (Wang and Roufogalis, 1999). Fischer et al. (1998) found that the PI3K, an enzyme that functions downstream of G-proteins, possesses high-afnity binding sites for CaM. The association of CaM with G-proteins is not restricted to animal cell systems. CaM appears to be closely involved with G-proteins in pollen germination and the growth of pollen tubes (Ma et al. 1999).
CALMODULIN
AND
CELL PROLIFERATION
As stated earlier, among the targets of CaM are the cytoskeletal proteins MAP-2 and the tau protein. The conclusion is inescapable, therefore, that CaM might be involved in cytoskeletal reorganisation. Both cell proliferation and cell motility have been regarded as the natural foci of CaM involvement. CaM seems to be actively associated with cell proliferation, as amply demonstrated by several investigators. Indeed, it is regarded as an essential ingredient of cell proliferation and the progression of the cell cycle (Means, 1994). The exit of cells from the cell cycle has been reported to be accompanied by a decrease in CaM levels (Christenson and Means, 1993). CaM levels increase as cells progress into the mitotic phase, and there is an elegant demonstration that both Ca2+ and CaM are essential for this process. When either of these is reduced cells undergo G2 arrest (K.P. Lu et al. 1993).
78
Some investigators have examined the levels of CaM expression in relation to growth responses. Exaggerated growth responses have been recorded in cardiomyocytes resulting from an overexpression of CaM (Gruver et al. 1993). Recently, however, Prostko et al. (1997) found no effects on growth responses arising from an overexpression of CaM in C6 glioma cells in culture, but reduction of CaM expression was found to inhibit their growth. The antiproliferative effects exerted by CaM inhibitors have also provided a substantial body of evidence that suggests an association of CaM with growth responses. Several CaM inhibitors have been tested to date. Schuller et al. (1991) found that B859-35, which is a dihydropyridine derivative, markedly inhibited proliferation of three human lung cancer cell lines. Hait et al. (1994) reported that several phenothiazine antipsychotic drugs inhibit CaM and also the proliferation of C6 glioma cells. They also found that the antiproliferative effects corresponded with the inhibition of CaM-sensitive phosphodiesterases. Further work from the same laboratory has shown that KS-501 and KS-502 similarly affect cell proliferation, not by direct action on the enzymes but by interfering with their function of activating CaM (Hait et al. 1995). In other words, the failure to activate CaM appears to lead to an inhibition of cell proliferation. Glass-Marmor et al. (1996) and Glass-Marmor and Beitner (1997) have investigated the effects of another class of CaM inhibitors, which reduce intracellular levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, and ATP and also detach glycolytic enzymes bound to the cytoskeleton. Four antagonists tested thioridazine, CGS 9343B, clotrimazole, and bifonazole brought about a marked reduction in cell viability (Glass-Marmor et al. 1996). The detachment of glycolytic enzymes associated with the cytoskeleton can also affect the function of the latter in cytokinesis. CaM cDNA has been transfected in sense as well as in the antisense orientation into C6 glioma cells. Transfection with sense-cDNA has been found to produce more clones than transfection with antisense constructs. The DNA content of cells has been reported to correlate with CaM levels (Liu G.X. et al. 1996). This is compatible with the nding that cells experience a delay in DNA synthesis in the presence of CaM inhibitors W7 and W13 and the CaM-dependent protein kinase inhibitor KN-62 (Mirzayans et al. 1995). CaM can inuence the transduction of growth factor signals via the receptor kinases by modulating their phosphorylation by means of CaM-dependent protein kinases. Thus one can visualise several ways in which CaM could regulate physiological processes.
CALMODULIN
IN
NEOPLASIA
An enhancement of CaM has been recognised as a feature of cell transformation and of malignant cells. CaM levels of human lung cancer cells are higher than that of benign tumours of the lung or normal lung tissue (Liu G.X. et al. 1996). These authors also describe a correlation between tumour grade and TNM stage and levels of CaM. However, Edelman et al. (1994) have described a CaM-like protein that is apparently restricted to epithelial cells. This CaM-like protein was identical in size and largely homologous to CaM, but, unlike CaM, its expression seems to be signicantly lower in malignant cells.
79
The CaM antagonist J8 inhibits the invasive behaviour of the cutaneous melanoma cell line A-375SM and uveal melanoma cells (Dewhurst et al. 1997). Tamoxifen, its metabolites N-desmethyltamoxifen, and 17-oestradiol also inhibit invasion in the absence of oestrogen receptors, which suggests that the inhibition produced by these anti-oestrogens was mediated by mechanisms other than receptor binding, e.g., CaM inhibition. A deregulation of intracellular calcium resulting in cell death is produced by tamoxifen at high concentrations where its effects are believed to be not mediated by oestrogen receptors (Jain and Trump, 1997). CaM antagonists could inhibit invasion by starving the cytoskeletal machinery of local ATP generation. Glass-Marmor and Beitner (1997) found that the CaM inhibitors, which they had previously shown to reduce the levels of certain glycolytic enzymes and ATP and cause loss of viability (Glass-Marmor et al. 1996), also detach these enzymes from their association with the cytoskeleton. It is possible that CaM could be inuencing cell invasion by altering the expression of ECM-associated enzymes. Some years ago A. Ito et al. (1991) suggested that CaM could be differentially modulating the expression of TIMP and prometalloproteinases 1 and 3 in broblasts derived from human cervical carcinoma. The expression of these enzymes is known to markedly alter the invasive behaviour of cancers (Sherbet and Lakshmi, 1997b). Possibly, therefore, modication of the expression of ECM-associated enzymes and the properties of the ECM as a means of modulating cell behaviour, ought to be seriously considered. S100A4 does seem to operate through such a mechanism (Merzak et al. 1994b; Lakshmi et al. 1997). It would seem, therefore, that CaM might involve more than one target enzyme in the modulation of invasive behaviour.
RECOVERIN SUBFAMILY OF NEURAL CALCIUM BINDING PROTEINS AND THEIR FUNCTION

Recoverin (RCN) and related proteins belong to a four-EF-hand recoverin family of neural calcium-binding proteins (NCBPs) (Table 5). Two types of NCBP can be distinguished. Type A proteins, e.g., recoverin, have two canonical EF-hands. Type B proteins, e.g., VILIP (visinin-like protein) and NCS-1, possess three regular EFhands. Flaherty et al. (1993) have elucidated the three-dimensional structure of RCN. According to them, the four EF-hands are arranged in a compact array. The tertiary structure and arrangement of the EF-hand may control calcium binding. Although generally described as NCBPs, individual NCBPs do show characteristic differences in their localisation within the neural tissue and the retinal component itself.
THE G-PROTEIN SIGNALLING
PATHWAY
Many extracellular modiers of biological response transduce their signals via heterotrimeric guanine nucleotide regulatory proteins (G-proteins), which consist of , , and subunits (Koelle, 1997; Dohlman and Thorner, 1997; Berman and Gilman, 1998). These G-proteins are coupled to cell surface receptors. Several agents, such as epinephrine, norepinephrine, cytokines, and nonsteroid hormones, activate Gprotein receptors (Hein and Kobilka, 1997; Watson et al. 1996). Upon activation
80
TABLE 5 Recoverin Family of Neural Calcium Binding Proteins

Protein Designation Recoverin (S-modulin) Rem-1 Visinin s26 VILIP NCS-1 Occurrence Photoreceptor cells; mammalian bipolar cells Retinal cells; haematopoietic cells; gut cells Photoreceptor cells Photoreceptor cells (frog retina) Inner retina Photoreceptor inner segments; inner plexiform layer; ganglion cells Ref. De Raad et al. (1995) Kraut et al. (1995)
Kawamura et al. (1996) De Raad et al. (1995) De Raad et al. (1995)
these receptors promote guanine nucleotide exchange of guanosine diphosphate (GDP) to GTP on the G. This in turn leads to the dissociation of G complex from G. These then regulate the activity of the target protein, leading eventually to the mobilisation of second messengers. A family of proteins called RGS (regulators of G-protein signalling) can preferentially bind to activated G. RGS proteins appear to function as guanosine triphosphatase (GTPase) activating proteins (GAP) (Berman et al. 1996; Watson et al. 1996; Hunt et al. 1996) and attenuate or block the signalling pathway (Popov et al. 1997; Tesmer et al. 1997; Hepler et al. 1997). The G-protein-dependent and calcium-signalling pathways are often closely allied. For example, neurotransmitters inhibit calcium channel current, which seems to be regulated by the photoreceptor G-protein called transducin (Jeong et al. 1999; also, see below). Similarly, dopamine-coupled receptors inhibit voltage-activated calcium channels (Wolfe and Morris, 1999). Dolphin et al. (1999) have identied the calcium channel-protein domains that might be involved in the modulation of calcium channel currents by G-proteins. In the signalling events associated with the acrosome reaction in spermatozoa, G-proteins require calcium together with PLA2 to induce acrosomal reaction (Dominguez et al. 1999).
RECOVERIN
AND ITS
FUNCTION
The transduction of the extracellular sensory signal involves the interaction of activated rhodopsin with the photoreceptor G-protein, transducin. The rhodopsin intermediate meta II activates transducin by catalysing the exchange of GDP to GTP. The RGS protein down-regulates this signalling pathway by promoting G GTPase activity and additionally by down-regulating cGMP phosphodiesterase, which effectively enhances GTPase activity. RCN (a homologue of this from the frog is known as S-modulin) is a 23-kDa protein that plays a specic role of phototransduction in mammalian retinal photoreceptors. The transduction of visual signals requires rhodopsin to be activated by phosphorylation. Upon illumination rhodopsin is phosphorylated by rhodopsin kinase at several serine and threonine residues. The activated rhodopsin is dephosphorylated by phosphatases to return it to the basal state.
81
RCN is involved in the process of light and dark adaptation by rod cells by regulating rhodopsin phosphorylation, thereby controlling photoreceptor light sensitivity, which is Ca2+ dependent. Several mutant forms of RCN, with mutations in EF-hands 2 and 3, and others with mutation of EF-hand 4, have been isolated recently. Of these, EFhand 4 RCN mutants were found to be able to inhibit rhodopsin kinase more effectively than could wild-type RCN (Alekseev et al. 1998). Apparently, a decrease in cytoplasmic calcium levels is necessary for light adaptation (Figure 11). Besides RCN, other calcium-binding proteins are involved in this process. Among them are the photoreceptor-specic GCAP and calmodulin, together with their targets, namely rhodopsin kinase, guanylate cyclase, cGMP-gated channel, and nitric oxide synthase (Koch, 1995; Gorczyca et al. 1995).
FIGURE 11 The pathway of sensory signal transduction involving recoverin and RGS (regulator of G-protein signalling) protein.
The RCN gene has been mapped to human chromosome 17p13.1 (J.F. McGinnis et al. 1995). Interestingly, the autosomal-dominant progressive cone dystrophy (CORD5) gene maps to chromosome 17p12p13. The genes, coding for retinal guanylyl cyclase and pigment epithelium-derived factor, and the retinitis pigmentosa (RP) genes also occur in the RCN region (Balciuniene et al. 1995). Recoverin occurs predominantly in mammalian photoreceptor cells. It may be found in other retinal cell types and may therefore subserve other functions besides phototransduction (McGinnis et al. 1997). RCN appears to be a highly conserved protein, as suggested by its occurrence in the photoreceptor cells of the lamprey (Dalil-Thiney et al. 1998). Its expression may be developmentally regulated (Yan and Wiechmann, 1997). It is found to be transiently expressed in developing follicular and parafollicular pinealocytes in the developing chick embryo (Bastianelli and Pochet, 1994). Four isoforms of RCN occur and each of these shows N-terminal myristoylation. RCN not only participates in phototransduction, a role suggested by its localisation in photoreceptor cells, but it is also associated with the pathogenesis
82
of the autoimmune state of cancer-associated retinopathy and uveoretinitis. However, it is uncertain whether RCN is involved with autosomal recessive RP.
MODE
OF
ACTION
OF
RECOVERIN
Recoverin regulates photoreceptor response by inhibiting rhodopsin phosphorylation. Rhodopsin kinase is active in the absence of RCN (Gorodovikova et al. 1994a). Rhodopsin phosphorylation and consequent cGMP hydrolysis are Ca2+- and ATPdependent processes. When the free Ca2+ level is raised, phosphorylation of rhodopsin is reduced and there is an increase in the lifetime of phosphodiesterase. This Ca2+ effect is negated by anti-RCN antibodies, which has been interpreted as suggesting that the calcium effects observed are a result of the inhibition of rhodopsin kinase (Gorodovikova et al. 1994b). Upon addition of RCN, rhodopsin kinase becomes sensitive to free Ca2+. Calcium-dependent interaction between RCN and rhodopsin kinase is indeed necessary for the inhibition of rhodopsin phosphorylation by RCN (C.K. Chen et al. 1995). All four isoforms of RCN inhibit rhodopsin phosphorylation in the same free calcium range (0.3 to 0.8 M), but they differ with respect to the magnitude of inhibition achieved, which appears to be related to their hydrophobicity (Sanada et al. 1995).
POST-TRANSLATIONAL MODIFICATION
OF
RECOVERIN
Four isoforms of RCN have been identied; all appear to be posttranslationally modied at the N-terminal glycine residue with myristic acid or related lipids. Myristoylation is believed to be essential for the function of RCN. Senin et al. (1995) compared the inhibitory effect on rhodopsin phosphorylation of myristoylated and nonmyristoylated forms of recombinant RCN. They found that both forms of RCN inhibit rhodopsin kinase in the presence of Ca2+, but myristoylated RCN was more efcient in inhibiting the kinase. However, others believe that myristoylation is not necessary for the kinase inhibitory effect of RCN and that it only induces a cooperative Ca2+ dependence of the process (Kawamura et al. 1994; Calvert et al. 1995). Ames et al. (1994) have suggested, on the basis of the exibility of the N-terminal helix in myristoylated calcium-free and nonmyristoylated calcium-bound form, that calcium binding to the EF-hand 3 domain induces EF-hand 2 to adopt a conformation that promotes calcium binding to RCN. Covalent attachment of a myristoyl or related N-acyl group to the N-terminal glycine appears to promote the binding of RCN to the optic disc membrane when free Ca2+ is raised. The so-called calciummyristoylation switch is believed to play an essential role in the targeting of RCN to the cell membrane. The addition of the myristoyl group has been found to reduce the calcium afnity of RCN and induce cooperative calcium binding. Two conformational states have been recognised viz. the T and R states. In the T state the myristoyl group is sequestered inside the protein, whereas in the R state it is exposed, and furthermore, calcium binds to the R state several thousand-fold more strongly than to the T state (Ames et al. 1995). Calcium binding to the myristoylated RCN induces its translocation to the membrane. The
83
nonmyristoylated RCN is not translocated in this way (Tanaka et al. 1995). It would appear, therefore, that posttranslational modication of RCN is required for targeting to and interaction of RCN with the membrane. The RCN family protein VILIP also shows calcium-dependent targeting to the cell membrane. It has been found to interact with the actin component of the cytoskeleton in its recruitment to the cell membrane (Lenz et al. 1996). Braunewell et al. (1997) have conrmed that myristoylated VILIP can be shown to be associated with membranes, whereas nonmyristoylated VILIP is not. Membrane association may stabilise the RCNrhodopsin kinase complex, as suggested by experimental demonstration of a preferential association of RCN with cell membrane with concomitant increase in rhodopsin kinase inhibition (Sanada et al. 1996). However, Johnson et al. (1997) found that conformational changes and distribution of RCN in the cell were not inuenced greatly by calcium concentration and suggest, therefore, that the calciummyristoylation switch may not be the only mechanism involved in the targeting of RCN to the cell membrane.
RECOVERIN
AND
CANCER-ASSOCIATED RETINOPATHY
Recoverin and Cancer-Associated Retinopathy in Small Cell Lung Cancer A degeneration of the retina is infrequently associated as a paraneoplastic condition with some forms of cancer. This condition, often described as cancer-associated retinopathy (CAR), is an autoimmune syndrome involving the degeneration of the photoreceptor cells of the eyes. Injection of RCN had previously been shown to induce degeneration of photoreceptor cells of the eyes of Lewis rats, and this was correlated with high titres of RCN antibodies in the circulation (Adamus et al. 1994). There are several reports of incidence of CAR in patients with small cell lung carcinoma (SCLC) (Polans et al. 1995; Matsubara et al. 1996). Polans et al. (1995) showed RCN is expressed in lung tumour biopsies of patients who had CAR but not in tumour samples of patients without CAR. They further identied two regions of RCN that were associated with its immunogenicity. Amino acid residues 6470 and 4852 were the major determinants. Immunisation of Lewis rats with peptide 6470 caused photoreceptor degeneration in the animals. This 6470 region is in close proximity to the EF-hand 2 calcium-binding domain. The autoimmune reactivity has been found to depend on changes in the conformation of the 6470 stretch of amino acid residues induced by calcium binding to the EF-hand 2 present in the neighbourhood (Adamus and Amundson, 1996). Murphy et al. (1997) have encountered high titres of antibodies to a 60-kDa retinal protein, as well as high titres of RCN antibodies, in association with SCLC. The titres of both declined with treatment and Murphy et al. (1997) have therefore suggested that the detection of RCN antibodies alone should not be used as the sole criterion for diagnosing CAR. A recent report described a patient with non-SCLC with recoverin antibodies in the serum (Salgia et al. 1998).
84
Retinopathy Associated with Other Forms of Human Cancer Retinopathy is associated also with other neoplasms such as breast and cervical tumours (Holz et al. 1997) and metastatic melanoma (Kirati et al. 1997). Again, this has been attributed to autoimmune responses to retinal proteins. RCN is expressed in cell cultures derived from retinoblastoma. Using antibodies raised against recombinant RCN, Weichman (1996) demonstrated that RCN is expressed in the cytoplasm of retinoblastoma cell line Y79. The expression was greatly increased by treating cells with 2 mM butyrate, and to a lesser degree by db-cAMP, together with the formation of neurite-like cellular processes indicating a state of induced differentiation. This possible link-up between RCN expression and differentiation is worthy of further investigation. In the sera of patients with CAR, heat shock protein (HSP) 70 has been detected in addition to recoverin (Ohguro et al. 1999). The involvement of bacterial HSPs in the induction of autoimmunity has been recognised with the demonstration that HSP65 from the tubercle bacillus mycobacterium can induce autoimmune disease in certain animal tumour models. Certain peptides derived from mycobacterial HSP65 and their homologous peptide obtained from patients with Behcets uveitis or iridocyclitis induce uveitis in Lewis rats. High antibody titres against these peptides are found in rats that developed uveitis as compared with those that had not (Uchio et al. 1998). This suggests that HSPs do function as autoantigens. Therefore, the detection of HSP70, together with recoverin, might suggest that both these antigens stimulate humoral autoimmunity in the pathogenesis of CAR. This view would be in line with the implication of HSPs in other autoimmune diseases such as rheumatoid arthritis (RA) (Wineld, 1989) and SLE (Minota et al. 1988; Conroy et al. 1994). Antibodies against human HSP60 and E. coli HSP60 have been found in patients with RA and SLE and in Reiters syndrome which combines arthritis with conjunctivitis, but the antibody titres are far higher than the titres of antibodies against mycobacterium HSP65 (Handley et al. 1996). This seems to suggest that the immune system recognises certain epitopes of these HSPs (Van der Zee et al. 1998). Although these observations suggest that HSPs might play a role in autoimmune conditions, they do not explain the coexpression of RCN with HSPs. It may be that some other HSP function, such as protein folding, may be involved here. As we have seen above, certain critical conformational changes are required in the proper functioning of RCN. Although, in the realms of speculation, the possibility that HSPs inuence RCN conformation in such a way that sequestered autoimmune epitopes might be exposed cannot be excluded. It is unclear whether RCN forms complexes with HSPs. This is an interesting avenue to approach; one can envisage a situation where HSPs regulate RCN function by forming a complex. HSPs are known to form complexes with important biological macromolecules such as p53, rb proteins, and S100A4, and possibly inuence their function as regulators of the cell cycle (Sherbet and Lakshmi, 1997b).
85
Is Recoverin Involved in Retinitis Pigmentosa? Retinitis pigmentosa (RP) is an autosomal-recessive disease involving degeneration of photoreceptor cells. RP has been attributed to mutations in the genes coding for rhodopsin and for the rod cGMP-gated channel. Naturally, therefore, the possibility of RCN implication in RP has received some attention. However, mutations in the RCN gene have been ruled out in 42 Spanish families with autosomal-recessive RP. Furthermore, the study by Parminder et al. (1997) did not encounter any mutations in the RCN gene in RP or allied heritable retinal diseases.
GUANYLATE CYCLASE-ACTIVATING PROTEINS

The mammalian retina contains two GCAPs: GCAP1 and GCAP2. They are photoreceptor-specic calcium binding protein. GCAP1 is expressed at a high level in the outer segments of rods and cones. GCAP2 occurs mainly in the cone inner segments and is less intensely expressed in inner retinal neurons. Therefore, whereas GCAP1 has a phototransduction function, GCAP2 may not (Otto-Bruc et al. 1997). Furthermore, the expression of GCAP1 and 2 gene transcripts is substantially reduced in retinal degeneration (rd/rd) mutant chicken. Here, GCAP2 protein seemed to be normal, but the expression of GCAP1 proteins was down-regulated by more than 90% and this seems to be consistent with the loss of phototransduction (SempleRowland et al. 1996). GCAP1 has four EF-hand motifs. When not bound to calcium, GCAP is able to activate GC. Calcium binding inactivates GCAP. This seems to be a consequence of Ca2+ binding by EF-hands 3 and 4 and the conformational changes in the molecule (Rudnicka-Nawrot et al. 1998). The involvement of EF-hand 3 is also indicated by an inactivating mutation A G in codon 99, exon 2 of the GCAP1 gene (Payne et al. 1998). This mutation is reported to occur in the GCAP1 gene in a family with autosomal-dominant cone dystrophy.
The Structure of Contractile Proteins
THE ACTIN COMPONENT OF CONTRACTILE MACHINERY OF THE CELL

The physical alterations of the shape of the cell, its structure, motility and contractile ability are an attribute of the structure and organisation of the cytoskeletal machinery, of which the intermediate laments (IF) (10 nm diameter), actin microlaments (7 nm diameter), and microtubules are the important components. Actin microlaments are polymers of F- and G-actin. These laments are exible, double-helical polymeric structures made up of F-actin strands. The G-actin subunits participate in binding to the myosin globular heads. G-actin also possesses ATP- or ADP-binding domains. Actin laments have a dened polarity, which ows from the fact that the monomers themselves show a distinct polarity. In association with actin-binding proteins, the actin laments form specialised structures. To these organised structures the cell owes its faculty of modulation of shape, motility, and other adhesionmediated phenomena. The reorganisation of actin cytoskeleton is also involved with plant cell division, differentiation, and light-induced plastid migration, among other cellular features (Staiger et al. 1997).
ACTIN ISOFORMS
Four actin isoforms have been identied, of which two, the and isoforms, are smooth muscle specic and the other two are the cytoplasmic isoforms. The transfection of mutated -actin genes in normal diploid cells alters their morphology and growth characteristics, and at the same time mutated -actin protein is seen to be incorporated into the cytoskeleton (Leavitt et al. 1987a). Leavitt et al. (1987b) have further shown that the transfectant cells that expressed mutant actin possessed greater tumorigenic potential than those with lower levels of mutant actin. Cells derived from these tumours had a shorter latency period for tumour formation as compared with the original transfectant cells. None of these interesting studies seem to have been followed up to examine motility, invasiveness, and metastatic potential, which, in retrospect, seems highly desirable in light of the changes in cell morphology and growth features engendered by the expression of mutant actin. A natural extension would be to investigate whether wildtype -actin possesses tumour suppressor function. The suppressor function of actin has been demonstrated recently. The rat broblast cell line 3Y1 expresses -actin, but this is down-regulated when the cells are transformed by RSV. In
87
88
contrast, transfection of -actin into RSV-transformed broblasts resulted in the reduction of tumour growth and invasiveness (Okamoto-Inoue et al. 1999).
REGULATION
OF
ACTIN DYNAMICS
Actin polymerisation and depolymerisation occurring in the dense actin cytoskeleton at the leading edge of a cell is instrumental in achieving locomotion (Tilney et al. 1983; Zigmon, 1993). The polymerisation of actin provides the protrusive power that allows the formation of lamellipodia or lopodia in the direction of cell movement. There is net polymerisation at the front edge while net depolymerisation occurs at the rear of the lamella. The relative rates of polymerisation and depolymerisation are at the basis of cell motility regulation. Actin dynamics are affected and may, indeed, be regulated by many cellular proteins. The actin-binding proteins regulate cell motility by inuencing the rates of actin polymerisation and depolymerisation. Actin-binding proteins such as gelsolin thymosin, and prolin are cytosolic proteins that inhibit the process of actin polymerisation and profoundly inuence cell morphology and motility. It follows, therefore, that these proteins could be indirect determinants of the altered cell motility and metastasis that often accompany neoplastic transformation. Among other notable cellular proteins that affect cytoskeletal dynamics is the S100A4 calcium-binding protein. S100A4 has been shown to be closely associated with the invasive and metastatic behaviour of cancer cells, and also to promote depolymerisation of the cytoskeletal structures (Lakshmi et al. 1993, 1997). It would be worthwhile, therefore, to discuss the role played by thymosin, colin, prolin, and mbrin in altering cytoskeletal dynamics and assess their impact on cell behaviour. The currently held view is that the actin-binding proteins alter the ratio of monomeric G-actin and F-actin in cells. G-actin-binding proteins such as thymosin 4 (T4) and prolin sequester G-actin from the cell pool and thereby prevent the process of polymerisation, whereas F-actin-stabilising factors such as myosin subfragment I and phalloidin can promote repolymerisation from the T4/G-actin complex (Ballweber et al. 1994) (Figure 12). In continuous cellular locomotion or inherent invasive ability, the cell has to maintain a net high level of actin polymerisation and a constant F-actin content. Actin subunits derived from depolymerisation occur at the rear of the lamella, are assembled into laments at the front of the lamella (Y.L. Wang, 1985; Small 1995). It would be of considerable interest to review how the expression of these actin-binding proteins affects cell behaviour and especially if any one of them shows any correlation with the aberrant behavioural properties manifested by neoplastic cells.
COFILIN
IN THE
REGULATION
OF
ACTIN DYNAMICS
Recently it has been shown that the phosphoprotein colin, also known as the actin depolymerisation factor, is actively involved with actin depolymerisation and in cellular processes that require cyclical changes in the actin cytoskeleton (Abe et al. 1996; Lappalainen and Drubin, 1997; Theriot, 1995). Colin is a 20-kDa protein found in muscle as well as nonmuscle cells. Two isoforms of this protein occur in
89
T4
Profilin
G-actin F-actin
Sequestration of G-actin
T4/G-actin complex
Profilin/G-actin
Myosin Fragment I Phalloidin Prevention of polymerisation
Repolymerisation
FIGURE 12 Schema of the participation of actin-binding proteins in the regulation of actin dynamics T4, thymosin 4.
mammals: the muscle (M) type and nonmuscle (NM) type. M-type colin expression is up-regulated in the myogenesis of C2 cells in vitro (Ono et al. 1994). Goode et al. (1998) identied a 37-kDa protein in budding yeast, and they named it twinlin because it contains two colin-like regions. Twinlin is able to sequester actin monomers, but it does not inhibit actin polymerisation. Homologues of this protein may occur in man, mouse, and Caenorhabditis elegans, as sequence data searches have revealed, and Goode et al. (1998) suggest twinlin may be highly conserved in evolution. Colin regulates actin polymerisation in a pH-dependent manner. Rapid cycles of assembly and disassembly of actin depend on this protein (Lappalainen and Drubin, 1997). Overexpression of colin leads to disorganisation of actin laments (Ono et al. 1996). Its intracellular distribution as well as its interaction with actin are regulated by phosphorylation (Obinata et al. 1997). Colin is phosphorylated by LIM kinases (LIMK-1 and -2) (Arber et al. 1998; Yang et al. 1998; Sumi et al. 1999). Phosphorylated colin loses its normal function of depolymerising actin laments. Cellular response in the form of alterations in cellular morphology, membrane rufing, and neuronal outgrowths results from the growth factors being able to activate colin. In the case of neurite extension, the dephosphorylation of colin
90
has been found to correlate with its translocation to the membrane (Meberg et al. 1998). Actin-binding peptide derived from colin has been reported to compete with gelsolin segments 2 and 3 for actin-binding. These binding sites of colin and gelsolin have structural homologies and therefore they possibly share the same binding domain on the actin lament (Van Troys et al. 1997). How this structural relationship between colin and gelsolin translates in functional terms is yet uncertain. However, Van Troys et al. (1999) seem to have made a beginning by providing a framework based on the structure of the actin-binding modules that occur in actinbinding proteins, the similarities and differences between these binding domains, and how, overall, they might impinge on actin dynamics.
PROFILIN
IN THE
REGULATION
OF
ACTIN DYNAMICS
Prolin is another protein that participates in and regulates actin dynamics in cellular responses to external as well as internal signals (Staiger et al. 1997). It is a 14 to 15-kDa protein that has been found to sequester monomeric actin, to promote nucleotide exchange occurring on actin monomers, and to be capable of inducing actin polymerisation when barbed ends of actin laments are free. Prolin also binds to poly-l-proline and phosphoinositol lipids. In mammalian cells two isoforms of prolin have been described and these also may be differentially expressed in different tissues (Witke et al. 1998). Prolin forms a high-afnity complex with actin (Di Nubile and Huang, 1997). The importance of this complex formation in the cellular function of actin laments is demonstrated by the effects of deleting residue proline 96 and threonine 97 that occur near the major actin-binding site. Deletion of these amino acids reduces the ability of prolin to bind to actin and its ability to promote nucleotide exchange on actin monomers. When injected into Swiss 3T3 broblasts, the mutant prolin failed to affect microlament organisation, which the wild-type was capable of achieving (Hajkova et al. 1997). A diminution of prolin expression has been shown to result in abnormalities of cytokinesis and the formation of multinucleate cells (Cao et al. 1997). Prolin is also a strong competitor against T4 in binding to actin (Ballweber et al. 1998; also, see below). It would be reasonable to accept that prolin is directly involved in actin polymerisation and in this way inuences morphological features and biological behaviour of cells.
RHO GTPASES
IN
ACTIN DYNAMICS
AND
SIGNAL TRANSDUCTION
The Rho family of GTPases are ras-related GTPases. In the yeast Saccharomyces cerevisiae, certain members of the family are known to control the process of budding and the determination of cell polarity (Johnson and Pringle, 1990). The mammalian Rho proteins have been implicated in cytoskeletal remodelling, and colin could be the terminal target that brings this about. Rho proteins also have been associated with the control of microlament assembly in vitro and the formation of cytoskeletal structures such as lopodia, lamellipodia, adhesion plaques, and intercellular junctions (Ridley and Hall, 1992; Nobes and Hall, 1995, 1999; Peterson et al. 1990; Van Aelst and DSouza-Schory, 1997). Intercellular adhesion is another cellular faculty in which Rho proteins are involved (Morii et al. 1992; Tominaga et al. 1993). The
91
processes of cell spreading and alterations in cell morphology and the transduction of growth factor signals also require the participation of and are regulated by Rho proteins (G.A. Murphy et al. 1999). At the fundamental level in all these cell functions is the contractile ability of the actin cytoskeleton, which has been attributed to the activation of Rho GTPases in response to microtubule depolymerisation (B.P. Liu et al. 1998). The Rho GTPases seem to activate a host of signalling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. These kinases phosphorylate downstream target proteins, directly or indirectly via the phosphorylation of other kinases. Rho and Cdc42 induce the reorganisation of the actin cytoskeleton and promote the formation of stress bres. This process appears to be related to the activation of LIMK-2. LIMK-2 phosphorylates colin and inhibits its normal function of cytoskeleton depolymerisation (Sumi et al. 1999). The Rho-activated serine/threonine kinases (ROCK) seem to be important elements in cancer invasion. Itoh et al. (1999) transfected a dominant active mutant of ROCK cDNA into rat MMI hepatoma cells. These transfectant cells were found to possess greatly enhanced invasive ability, in comparison with cells that had been transfected with a dominant negative ROCK cDNA. The MLCK of endothelial cells is another kinase that seems to be regulated by Rho (Garcia et al. 1999). The possibility that Rho proteins might regulate the contractile apparatus of endothelial cells and alter the permeability of endothelia as a consequence has serious implications for cancer cell dissemination, in which the permeation of the endothelial barrier is an essential event. The induction of membrane rufes has been attributed to Rac, a GTPase that belongs to the Rho family. Rho GTPases have been implicated in Rho kinasemediated phosphorylation of transmembrane adhesion proteins, such as the CD44 splice variants, and their interaction with the cytoskeletal protein, ankyrin (Bourguignon et al. 1999). This interaction between CD44 and ankyrin is manifested as changes in membrane activity in the form of rufing. In this particular instance, Rho A GTPase stimulates ROCK, which phosphorylates CD44. This leads to an enhancement of CD44ankyrin interaction. Bourguignon et al. (1999) also demonstrated that the induction of CD44-associated membrane rufing can be achieved by injecting the catalytic domain of ROCK and, furthermore, that the membrane activity is inhibited by anti-CD44 antibodies. A more indirect route might be adopted in the Rho-mediated reorganisation of the cytoskeleton involving colin. Here ROCK phosphorylates and activates LIMK, which then phosphorylates colin (Maekawa et al. 1999). Some members of the S100 protein family are known to be able to modulate and possibly regulate actin dynamics. Among them is S100B, which has recently been shown to bind and activate the serine/threonine kinase Ndr (Millward et al. 1998). The Ndr kinase has structural and possibly also functional similarities with ROCK. It is suggested, therefore, that there might exist a general and common mechanism by which S100 proteins could regulate actin dynamics and the cellular processes and functions upon which the modulation of actin dynamics and cytoskeletal organisation might strongly impinge. A family of proteins called WASP [Wiskott-Aldrich syndrome (WAS) protein] is regarded as the regulators of actin reorganisation. WAS is an X-linked condition
92
that affects male children. The syndrome is generally described as including immunodeciency, thrombocytopenia, and eczema, but the actual phenotype may be variable. The WAS gene is mutated in patients and the type of mutation seems to relate to the phenotype of the disease. WASPs are cytoplasmic proteins. One WASP isoform is found only in the lymphocytes. Another isoform called the N-WASP, which was isolated from neural cells, shows a more ubiquitous distribution. These proteins are known to bind to several cellular proteins and kinases and also to Rho GTPases, such as Cdc42 and Rac. They interact directly or indirectly with the actin cytoskeleton. The haemopoietic cells derived from patients with WAS also have been reported to show abnormalities of the actin cytoskeleton. These observations suggest that WASPs play an important part in the transduction of extracellular signals to the cytoskeleton (OSullivan et al. 1999). They are believed to function downstream of Rac. The association of WASP with the induction of membrane activity has been demonstrated recently. Castellano et al. (1999) showed that experimentally induced recruitment of activated Cdc42, or its downstream effector WASP, to the cell surface results in actin polymerisation and the formation of lopodia. Other actin-binding proteins, such as prolin, that are associated with actin dynamics may therefore be expected to enter into this picture of WASP- and Rhomediated regulation and organisation of the actin cytoskeleton. Suetsugu et al. (1998) generated a mutant prolin, H119E, that is defective in actin-binding. This mutant prolin suppressed actin polymerisation induced by N-WASP. Under normal circumstances prolin associates with N-WASP, and is essential for a rapidly polymerising actin by N-WASP; this cannot happen with mutated prolin.
INTERACTION
OF
FORMIN
WITH
PROFILIN
AND
RHO GTPASES
The formin family of proteins constitutes another component of the actin regulatory system. Formin and related proteins, which are expressed ubiquitously in several organisms, seem to knit together prolin and Rho GTPases in their function of regulating cytoskeletal dynamics. Members of the formin family share many structural features such as the coiled-coil molecular organisation, the occurrence of homology sequences known as formin homology (FH) 1 and FH2 domains, the collagen-like domain, nuclear localisation signals, and phosphorylation sites. The formin gene was identied by mutations in the gene associated with recessive limb bud deformity (Maas et al. 1990, 1991; Chan et al. 1995). The gene has 24 exons encompassing 400 kb of genomic DNA (C.C. Wang et al. 1997). The formins are predominantly nuclear proteins participating in cytokinesis and cell polarisation (Zeller et al. 1999). They are essential for the correct orientation and alignment of the cell division spindle (L. Lee et al. 1999). Formin also participates in morphogenesis, e.g., in the yeast budding process. The importance of their role in morphogenesis is also indicated by the fact that mutation in the formin gene leads to disruption of epithelialmesenchymal interactions, which in turn leads to abnormal limb development. This has suggested the possibility that it may participate in the transduction of morphogenetic signals (Zeller et al. 1999). Formin has several ligands. Prominent among them is prolin (Mittermann et al. 1998). Rho GTPase is a downstream target of formin (Watanabe et al. 1997). It seems, therefore, that
93
formin is an important element in prolin/Rho-dependent actin polymerisation of cytoskeletal dynamics and in this way might regulate cytokinesis, cell morphology, and adhesion-dependent processes such as cell motility and invasion.
THE ROLE OF THYMOSIN FAMILY ACTIN-BINDING PROTEINS IN ACTIN DYNAMICS SEQUESTRATION

OF
ACTIN
BY
THYMOSINS
The beta thymosins constitute an important family of actin-binding proteins, which are recognised as major participants in the regulation of actin dynamics. Among its members are T4, T10, and T15, which are the most intensively investigated forms of thymosin. T4 is a 4.5-kDa protein consisting of 43 amino acid residues. T14 is another protein isolated from the sea urchin, that has 40 amino acid residues and a molecular size of 4.53 kDa and bears a high degree of sequence homology to T4 (Stoeva et al. 1997). T15 is a 5.3-kDa protein. Thymosins seem to occur ubiquitously. Actin-binding proteins showing a high degree of homology to mammalian thymosins have been isolated from invertebrates. The thymosins from invertebrates have 40 amino acid residues, but they appear to differ with respect to their afnity for binding to rabbit muscle actin (Safer and Chowrashi, 1997). The thymosins function by sequestering monomeric actin and thereby diminishing actin polymerisation (Safer, 1992). T4 binds to actin monomers by means of its N-terminal 5 to 20 amino acid residue sequence (Van Compernolle et al. 1992; Czisch et al. 1993; Van Troys et al. 1996). In vitro this peptide adopts a folded conformation for achieving high-afnity interactions with actin (Feinberg et al. 1996a). Feinberg et al. (1996b) have discovered homologous short sequences in T4 and gelsolin that bind to the C-terminal region of actin. They have also commented on the ability of these short sequences to form secondary structures and its relationship to their biological function. However, Safer et al. (1997) found that T4 crosslinks to both barbed and pointed ends of actin, and this requires that the C-terminal domain, which participates in the binding, is in an extended conformation.
EFFECTS
OF
THYMOSINS
ON
CELL PROLIFERATION
The ability of T4 to inhibit actin polymerisation is well documented. Also adequately demonstrated is the involvement of thymosins in proliferation, motility, cell differentiation, and angiogenesis, all of them important features of embryonic development and cancer development and progression. Recently, Sanger et al. (1995) experimentally enhanced the intracellular levels of T4 and studied the structural integrity of actin bundles of stress bres and cytokinetic furrows. They microinjected a viral vector carrying T4 cDNA, transfected the vector into PTK2 cells, or injected puried T4 protein into these cells. Both microinjection and transfection of cDNA produced a disassembly of stress bres. The effects of injection of the pure protein were even more marked; a disassembly of stress bres occurred within 10 min after injection, resulting in delayed cytokinesis. However, although T4 reduces actin
94
polymerisation by sequestering monomeric G-protein, at higher concentrations its ability to depolymerise F-actin appears to decrease (Carlier et al. 1996). The effects of thymosins on cell proliferation are therefore inevitable. Sanger et al. (1995) demonstrated that an overexpression of T4 delays cell division. The N-terminal part of the protein contains an acetylated tetrapeptide sequence of AcNSerAspLysPro that is known to inhibit haemopoiesis. This peptide has been shown to inhibit the growth of normal bone marrow progenitor cells. It markedly reduces the growth of granulomacrophagic and erythroid progenitor cells and the size of their S-phase fraction (Bonnet et al. 1996). However, Bonnet et al. (1996) also found that although the inhibitory effect of the T4 is similar to that of the tetrapeptide, T4 from which this sequence is deleted retains the inhibitory effect.
THYMOSINS
AND
CELL MOTILITY
AND
DIFFERENTIATION
Needless to say, the amply conrmed effects of thymosins on the structural integrity of the cytoskeleton are bound to nd expression in altered cell motility. T4 has been shown to enhance strongly the migratory behaviour of an established cell line of endothelial cells derived from human umbilical vein (Malinda et al. 1997). Of the primary human cell lines that Malinda et al. (1997) examined, the migration of only human coronary artery cells seemed to respond to T4. They also reported an increase in the production of metalloproteinases upon treatment with the thymosin. These enzymes can remodel the extracellular matrix and aid migration by altering cell adhesion to the substratum. NIH3T3 cells induced to overexpress T4 have been reported to be more adherent than corresponding control cells (Golla et al. 1997). This enhanced adhesion appears to be due to adhesion-mediating proteins such as -5 integrin as well as the transmembrane proteins such as vinculin that link cell membrane proteins to the cytoskeleton. There are also important implications of thymosin overexpression for cell migration in the context of cancer invasion, because thymosins have been reported to be overexpressed in neoplastic cells (see below). Especially signicant are the reported specicity of response by endothelial cells to T4 and the associated overexpression of metalloproteinases. Metalloproteinases have been closely associated with breaching of the endothelium and aiding the diapedesis of cancer cells into the vascular compartment (Sherbet and Lakshmi, 1997b). T4 is reported to be highly expressed in the intrinsically highly motile embryonic mesenchymal cells (Carpintero et al. 1996). The expression of T15 seems to be up-regulated in the highly motile Dunning rat prostate cancer cell lines. Besides, the transfection of antisense constructs of T15 gene apparently alters the motility of these cells (Bao et al. 1996).
EXPRESSION
OF
THYMOSINS
IN
EMBRYONIC DEVELOPMENT
Differential cell adhesion and migration patterns are an integral feature of cell differentiation and morphogenesis. The expression of T4 and T10 shows some relationship to specic developmental stages (Carpintero et al. 1996), although there are as yet no indications that it is developmentally regulated. Carpintero et al. (1996) found a marked increase in T10 mRNA in early postimplantation mouse embryos.
95
This mRNA is also found in certain tissues, including mesenchymal tissues and the spinal cord, in 9.5 to 12.5-day old embryos. These authors have also encountered differences in the distribution of T4 and T10 mRNAs, which could conceivably suggest differences in their function in embryonic development. T4 has been detected in early chick embryo cells but not in adult skeletal muscle cells. Colin also is associated with muscle development, but its relative contribution may change during muscle development (Nagaoka et al. 1996). These observations are consistent with the notion of development-related expression of thymosins. Gomez-Marquez et al. (1996) showed that T4 mRNA occurs in mouse embryonic stem cells, and further that T4 is able to induce embryonal P19 cells to differentiate into neuronal- and glial-type cells or into cardiac and skeletal muscletype cells. Furthermore, T4 mRNA was demonstrable around blood vessels and in heart tissues.
POTENTIAL ROLE
OF
THYMOSINS
IN
CANCER PROGRESSION
As stated before, the putative association of overexpression of T4 with increased production of metalloproteinases in normal cells together with the relationship between T4 and high invasive ability of the Dunning rat prostate cancer cell lines point to the relevance of thymosins in cancer invasion and metastasis. Both T10 and T15 have been investigated for their possible relationship with cancer progression. T10 protein was reported to occur at high levels in the malignant cell rather than the normal tissue component of human breast cancer, and the level of expression increased with tumour grade (Verghese-Nikolakaki et al. 1996). The expression of T10 gene was higher in ve thyroid carcinoma cell lines as compared with normal thyroid-derived primary cells. Expression of the gene was higher in anaplastic tumour tissue (Califano et al. 1998). T15 levels have been examined in several tumour cell lines of various histological origins. An up-regulation of the gene has been found in highly metastatic mouse lung and human breast cancer cell lines. This study also involved an examination of the levels of T15 protein in human breast and prostate cancer tissues. Higher expression of T15 has been correlated with the Gleeson grade in prostate carcinomas (Bao et al. 1996). In breast cancer, higher levels of the protein correlated with greater metastatic potential (Bao et al. 1998). This is in contrast with their earlier studies (Gold et al. 1997). Gold et al. (1997) had encountered higher T15 expression in nonmetastatic breast cancer, which suggested the involvement of thymosins in the early stages of breast cancer development rather than in the late stages of the disease. Although much attention has been focused on -thymosins, Tsitsilonis et al. (1998) found -thymosins to be of prognostic value in breast cancer. They determined levels of prothymosin and parathymosin in breast carcinoma tissue as well as in tissues from benign breast disease, and found that levels of both are much higher in carcinomas than in benign tumour tissue and normal breast tissue. They also reported that these levels correlated with overall survival of patients. As stated before, T4 expression has been found to be very high in Dunning rat prostate carcinoma cells, which possess high motility. When T4 antisense constructs were transfected into these cells the invasive ability was suppressed,
96
suggesting that the high levels of T4 expression might be associated with high invasive ability. Contrary to this, transfection of adenovirus E1A and E1B genes has been carried out in a highly metastatic human melanoma cell line by Van Groningen et al. (1996) who reported that a number of markers for tumour progression were down-regulated as a consequence. Among them was T10. Admittedly, E1A gene products can inhibit oncogene-mediated cell transformation as well as invasion and metastasis in certain animal models. E1A protein is known to be able to induce and stabilise p53 phosphoprotein, which controls the G1-S checkpoint transition of cells, and this is accompanied by apoptic loss of cells. On the other hand, E1B protein can bring p53 levels to normality (Sherbet and Lakshmi, 1997b). However, the raft of changes produced by the transfection of the adenovirus genes makes it a rather inappropriate model for the study of tumour progression. Although the transfected cells may have shown a reduction in tumorigenicity upon implantation into compatible hosts, under these circumstances it would be difcult, even unacceptable, to try to extrapolate as to the signicance of the suppression of individual markers in relation to tumorigenicity. Some of the difculties are inherent in the interpretation of data obtained from experimental studies such as gene transfer in vitro and on tumour tissues from patients. Therefore, it is imperative that further studies be undertaken to assess the potential value of thymosins in determining the state of tumour progression.
THE FIMBRIN FAMILY OF ACTIN-BINDING PROTEINS MOLECULAR FEATURES

OF
FIMBRIN
Fimbrins are EF-hand calcium-binding proteins that actively participate in binding to and bundling of actin. In actin laments, one molecule of mbrin might bind eight actin monomers under optimal conditions (Namba et al. 1992). Fimbrins are highly conserved in evolution, with regard to both their structure and function. Fimbrin from diverse origins, e.g., from Dictyostelium discoideum to humans, share structural and biochemical properties. Fimbrin-like proteins have also been isolated from plants, such as wheat (Triticum aestivum) and Arabidopsis thaliana (CruzOrtega et al. 1997). The Dictyostelium mbrin is a 67-kDa protein containing two EF-hands that are followed by two actin-binding sites (Prassler et al. 1997). The Nterminal actin-binding domain is a highly conserved domain that mbrin shares with other actin-binding proteins. This N-terminal domain contains two tandem calponin homology (CH) domains which are implicated in the binding of F-actin (Goldsmith et al. 1997). Fimbrin-mediated cross-linking and bundling of actin is regulated by free calcium, which it binds with high afnity and selectivity. However, the optimal concentration for cross-linking of actin was determined to be below 0.15 M. This process was progressively inhibited at higher free calcium concentrations and halfmaximal inhibition of cross-linking occurred at 1.6 M Ca2+ concentration (Pacaud and Derancourt, 1993). Pacaud and Derancourt (1993) also noted that calciumbinding of mbrin, in the concentration range of 0.15 to 1.5 M, produced conformational changes in the molecule, to which they attribute the calcium-mediated regulation of mbrin function. It may be that the conformational alterations actuated
97
by calcium at different concentrations could render actin cross-linking reversible. A further possibility has arisen from the studies of Hanein et al. (1997a) that mbrin might induce conformational changes in actin itself. Both types of change will inevitably contribute to cell behaviour. Fimbrins form a large family of proteins. Four genes encode mbrin in Salmonella (Collinson et al. 1996). Three isoforms of mbrin have been identied and designated as I-mbrin, L-mbrin, and T-mbrin. I- and L-mbrin are characteristically associated with intestinal and kidney epithelia, leukocytes, and tumours, whereas T-mbrin shows a more general distribution in a variety of cells and tissues (Chafel, 1995).
FUNCTION
OF
FIMBRIN
IN
CYTOSKELETAL ORGANISATION
A major function of mbrin appears to be in the assembly of actin laments. Fimbrin as well as actin capping protein (CP) are required for proper assembly of these laments in the yeast Saccharomyces cerevisiae. There is a reduced lament assembly and mbrin in the CP mutants of the yeast. Actin obtained from CP mutants shows defects in polymerisation as well as in its binding to mbrin (Karpova et al. 1995). In mammalian cells, Rho GTPases may mediate actin lament assembly and bundling. A constitutive expression of the Rho GTPase Cdc42Hs causes impairment of cytokinesis of HeLa cells. This seems to be a consequence of a reorganisation of F-actin with which, among other actin-binding proteins, T-mbrin is associated (Dutartre et al. 1996). Functional conservation of mbrin isoforms has been amply demonstrated by Adams et al. (1995), who found that human T- and L-mbrin can substitute for the yeast mbrin called Sac6p. T- and L-mbrin were both able to complement the temperature-sensitive growth defect that is seen in sac6 null mutants, and they could also restore normal cytoskeletal organisation and cell shape in these mutants. The null mutants show defective sporulation, which is restored by human T- and L-mbrin (Adams et al. 1995). Fimbrin isoforms show not only a tissue-specic distribution, but the specicity appears also to extend to differentiation and morphogenesis. Fimbrin shows a denable temporal and spatial pattern of expression in the course of the development of the cochlea and may be involved in the formation of the inner and outer stereocilia of the hair cell (Zine et al. 1995). The differentiation of intestinal epithelium is associated with the expression of T-mbrin in the apical area of the cell and Lmbrin in the basal area, until 14.5 days of development. Both isoforms are said to be totally down-regulated in expression by day 16.5, but instead, I-mbrin appears on day 14.5 of development to give the epithelium a brush border-like localisation. These ndings, reported by Chafel et al. (1995), suggest possible differences in their function in the course of differentiation. A number of the events occurring in morphogenesis and differentiation require changing patterns of cell adhesion, for which mbrin seems to be ideally placed. Babb et al. (1997) studied the localisation of mbrin in mature as well as differentiating osteoclasts. Fimbrin is a component of the osteoclast adhesion complexes called podosomes. During migration podosomes are found at the cell periphery. Microlament organisation and podosome assembly is important in the rapid modulation of adhesion to the substratum, and
98
in the motility of cells. T- and L-mbrin, but not I-mbrin occurred as an integral component of the core of the podosomes throughout the process of monocyte-derived osteoclast differentiation. The levels of T-mbrin increased with and appeared to be related to the formation of podosomes. The involvement of mbrin in podosomes is compatible with the association of mbrin with the adhesive organelles called mbriae that have been described in bacteria. Fimbriae are bre-like structures that mediate the attachment of bacteria to host cells. These are assembled from mbrin subunits (Smyth et al. 1996). Frederick et al. (1996) investigated the adhesive interactions between lymphokineactivated killer (LAK) cells and came to the conclusion that mbrin might be an important factor in intercellular adhesive contacts. They also showed that contact between LAK cells and target tumour cells, namely SK-Mel-1 human melanoma cells and Raji lymphoma cells, leads to the phosphorylation of L-mbrin of the LAK cells. S.L. Jones et al. (1998) have also suggested that induction of mbrin phosphorylation is an important step in mbrin function. However, as discussed below, the question of whether phosphorylation leads to mbrin activation must be regarded as sub judice at present. Podosomes that participate in cell adhesion and locomotion have been found to contain other cytoskeletal linking proteins such as talin and vinculin, besides mbrin. L-mbrin has been shown to regulate integrin-mediated adhesion of leukocytes (S.L. Jones et al. 1998). It is possible that mbrin is instrumental in the organisation of physical pathways, such as mbriae and podosomes, by which extracellular signals for intercellular and cellsubstratum adhesion are transduced to the cell. Another membrane organelle with which mbrin might be associated in organising a signal transduction machinery is the caveola. Caveolae are plasma membrane invaginations, of 50 to 100 nm dimension, that occur in many cell types. Caveolae have been attributed with many functions, notably transport of molecules across endothelia and signal transduction (Lisanti et al. 1995). A major component of caveolae is a 21- to 24-kDa protein called caveolin. Caveolin is said to function as a scaffolding protein that organises the signalling molecules in the caveolae. That caveolae contain essential components of the signal transduction machinery may be deemed to be rmly established. Thus caveolin has been shown to interact directly with signal transduction molecules such G-protein subunits and the ras protein (Lisanti et al. 1995; Song et al. 1996). The receptors for PDGF and EGF are located in caveolin-rich microdomains, and caveolin has been shown to interact directly with the EGF receptor (G.X. Liu et al. 1996; Couet et al. 1997). The interaction of caveolin negatively regulates RTK activities associated with activated EGFr and cerbB2 (Couet et al. 1997), and in mammary tumours expressing c-erbB2, caveolin expression is down-regulated (Engelman et al. 1998). Therefore, this interaction between growth factor receptors and caveolin may deregulate the transduction of the growth factor signals. Compatible with this view are recent reports that overexpression of caveolin results in growth inhibition of tumour cells (S.W. Lee et al. 1998; T. Suzuki et al. 1998). On the other hand, caveolin has been associated with cancer progression. It is reported to be overexpressed in inltrating ductal carcinoma of the breast and prostate. In prostate cancer, caveolin is expressed in both primary and metastatic tumours (N. Yang et al. 1998). These two sets of data may appear to
99
contradict each other, but it is possible to envisage caveolin functioning as a growth inhibitor in the early stages of tumour development. Caveolin might function in later stages of tumour progression merely by virtue of its ability to promote cellcell and cellsubstratum adhesion, which are essential requisites for a successful transition of the cancer cell along the metastatic cascade. In this connection, it would be worthwhile to note that T-cadherin, an adhesion-mediating molecule, also occurs in caveolin-rich plasma membrane microdomains (Philippova et al. 1998). After a seemingly long digression, it should also be noted that mbrin is a component of the caveolae. Fimbrin occurs together with other proteins, e.g., src and ezrin, in cell membranes that are rich in caveolin (Mirre et al. 1996). The available evidence, albeit circumstantial in nature, may be interpreted as implicating mbrin in tumorigenesis and tumour progression by deregulating signal transduction pathways and cell adhesion mechanisms.
REGULATION
OF
FIMBRIN EXPRESSION
The apparent tissue-specic distribution of mbrin isoforms has led inevitably to investigations directed toward understanding the mechanisms of mbrin expression and whether there is any tissue-specic regulation of its expression. C.S. Lin et al. (1997) have characterised the promoters of human and murine L-mbrin. They described considerable similarity in the organisation and found that both promoters functioned with equal efciency in most cell types. Therefore, the apparent tissuespecic and differentiation-related expression of mbrin could be due to posttranscriptional modication of mbrin rather than to its regulation at the transcriptional level. Phosphorylation could be a mechanism of regulation of mbrin function, as demonstrated for L-mbrin. The serine residues of the head-piece region of L-mbrin are phosphorylated (Messier et al. 1993), although earlier, Namba et al. (1992) found unphosphorylated L-mbrin of human T cells to be quite effective in F-actin bundling. Shinomiya et al. (1995) reported that LPS stimulated the phosphorylation of L-mbrin in macrophages. Phosphorylation occurred on amino acid residues at the N-terminal region close to the rst Ca2+-binding domain. Shinomiya et al. (1995) also noticed that the phosphorylated region contained motifs that are phosphorylated by CK II, protein kinase A (PKA), and PKC, but not motifs specic for MAPK. Frederick et al. (1996) also have recently implicated PKC in mbrin phosphorylation and have conrmed further that only serine, not tyrosine, residue is phosphorylated. The possible linkage of mbrin function with phosphorylation is indicated by the ability of cytokines and phorbol esters, among other agents, to induce mbrin phosphorylation. Polymorphonuclear leukocytes (PMN) stimulated by IL-8, IL-1, neutrophil-activating proteins, monocyte-derived neutrophil chemotactic factor, and TNF have been reported to stimulate the phosphorylation of I-mbrin. Phosphorylation was also inuenced by phorbol 12-myristate 13-acetate (PMA) (Shiroo et al. 1988; Shibata et al. 1993a, 1993b). Fimbrin of T cells is phosphorylated in response to IL-2 (Zu et al. 1990). The adhesion of neutrophils to immune complexes induces L-mbrin phosphorylation. Furthermore, it appears that phosphorylation-mediated regulation may be distinct from calcium-mediated regulation of mbrin (Jones and Brown, 1996). It seems likely that the transduction of extracellular signals involves
100
this important component of the membrane cytoskeleton. However, despite the clear demonstration of a mbrin phosphorylation response to extracellular signals, there is little direct evidence that these features are functionally related. Messier et al. (1993) provide circumstantial evidence for this. They found phosphorylated mbrin was mainly associated with the insoluble cytoskeleton. When this is read with their observation that the serine residues of the head piece were specically phosphorylated, one might be justied in concluding that phosphorylation could regulate the process of actin-binding and bundling by mbrin. Whether the isoforms have different functions in the regulation of cell morphology is a question that has been addressed, albeit supercially, by Arpin et al. (1994). In CV1 broblast-like cells, both T- and L-mbrin cause change of cell shape and reorganisation of the actin stress bres, and only L-mbrin is associated with microlaments. In epithelial cells such as LLC-PK1 cells, T-mbrin remains associated with actin laments of microvilli and produces shape changes in them. Lmbrin has no effect on these structures. These observations might suggest functional differences between the isoforms. The invasion of Shigella exneri, a bacterium that causes dysentery in humans, involves the formation of heavy actin polymerisation and actin bundling near the site of host cell contact with the bacterium. These bundles form protrusions with which the bacterium coalesces. Adam et al. (1995) experimentally overexpressed T- and L-mbrin in HeLa cells and demonstrated that Tmbrin might be preferentially recruited to the zone of bacterial entry.
IS FIMBRIN INVOLVED
IN
CANCER?
The potential of mbrin to modulate cell behaviour is obviously quite considerable. There are references in the literature since the early 1980s that mbrin expression increases with cell transformation, especially in broblast cells. However, little incisive investigation seems to have been carried out. More recent investigations have pursued the worthwhile aim of establishing a role for mbrin in neoplastic transformation and tumour progression. The expression of L-mbrin has been investigated in cell lines derived from carcinoma of the prostate. L-mbrin has been found in many carcinoma cell lines, but not in normal epithelial cell lines derived from the prostate. L-mbrin occurs at higher levels in prostate cancer tissue as compared with normal prostate tissue. Immunohistochemical staining has suggested that the increase of mbrin occurs predominantly in the glandular epithelial cells of the carcinoma, whereas in normal prostate tissue, it is found mainly in the bromuscular stroma. It would have been interesting to explore the mbrin expression pattern of benign prostatic hyperplasia (BPH). Nonetheless, the differences shown to exist between normal prostatic epithelium and carcinomatous glandular epithelium are persuasive enough to accept that mbrin expression may be related to the carcinomatous changes. Interestingly, mbrin expression was also reported to be rapidly up-regulated in LNCaP cells by dihydrotestosterone and oestradiol (J.P. Zheng et al. 1997). This observation does not per se contribute much to the argument that mbrin is involved in the transformation of normal epithelium to carcinoma. Testicular hormones are the major factors that regulate the growth and development of the prostate. It is
101
possible that changes in hormonal milieu are responsible for the development of BPH and onward to the development of prostatic carcinoma. Androgen receptors (ARs), oestrogen receptors (ERs), and progesterone receptors (PgR) occur in BPH and in prostate cancer (Srinivasan et al.1995). Indeed, AR occurs in all histological types and all clinical stages of prostate gland cancer. Because androgens are involved in the development of the prostate itself, it would be reasonable to suppose that AR occurs in the developing prostate as well. Therefore, one would expect that the androgen-sensitive cell line LNCaP would respond by an altered level of mbrin. What is crucially important is to nd out whether normal epithelial cells respond differently from carcinomas. Testicular hormones bind to the appropriate receptors and stimulate the transcription of androgen-responsive genes, including those that regulate the growth of prostate cells. It should be borne in mind, further, that the progression of prostate cancer is associated with a change from androgen-dependent to androgen-independent state. The mbrin gene may be a steroid-regulated gene, and it is important to determine whether this is the case, and whether androgen differentially stimulates mbrin transcription in normal prostatic epithelium and prostatic carcinoma cells. Another isolated observation, which may, nonetheless, be signicant in the context of drug resistance of tumours, is the relationship discovered by Hisano et al. (1996) between cisplatin resistance and mbrin expression. They observed that cisplatin-resistant cells possessed severalfold greater levels of T-mbrin compared with sensitive cells. They transfected T-mbrin antisense cDNA into cisplatin-resistant cells and showed that reduction of mbrin expression resulted in increased sensitivity to the drug.
MODULATION OF ACTIN DYNAMICS DISSEMINATION
AND
CANCER CELL
Conceptually there can be no difculties in accepting the proposition that the modulation of actin dynamics should be involved in some way with tumour dissemination. A reasonable body of experimental evidence, in the form of changes in the expression of gelsolin, thymosins, and mbrin in cancer progression, has been adduced in support of this concept. In this context of actin-interacting proteins, the vasodilator-stimulated phosphoprotein (VASP) deserves a mention. VASP is approximately 45 to 50 kDa in size and occurs in domains of the plasma membrane that are involved in the formation of lamellipodia (Reinhard et al. 1992, 1995b). Together with the Drosophila- and murine-enabled (Ena) proteins, VASP belongs to a family of actin-binding proteins that are actively engaged in the regulation of the actin cytoskeleton. VASP has been shown to interact with actin stress bres via its Cterminal region (Huttelmaier et al. 1999). VASP as well as Ena can interact with the actin CP prolin (see below) (Reinhard et al. 1995a) and with focal adhesion proteins (Reinhard et al. 1995b, Brindle et al. 1996; Lanier et al. 1999; AhernDjamali et al. 1998). This suggests that the VASP family members might be involved in signal transduction (Huttelmaier et al. 1998). Besides, during in vitro morphogenesis of human umbilical endothelial cells, VASP, prolin, and gelsolin are markedly up-regulated (Salazar et al. 1999). In view of the close association of all three
102
proteins in actin dynamics, Salazar et al. (1999) have postulated that reorganisation of the actin cytoskeleton is required for the alignment of the endothelial cells during capillary morphogenesis. The expression of prolin or VASP in neoplastic transformation has not been studied in great detail. NIH3T3 broblasts that have been made VASP decient by experimental manipulation have been reported to be able to form tumours in nude mice. In vitro, these cells show loss of contact inhibition and a deregulation of cell proliferation (Liu K.Y. et al. 1999). With the demonstration that these proteins are associated with capillary morphogenesis, it would be of much interest to see whether VASP and/or prolin are down-regulated in neoplastic transformation. One can envisage that perturbation of the actin cytoskeleton could make the cell membrane malleable and the cell more deformable and in this way aid invasion. Mechanistic perturbations in endothelial cell alignment could generate abnormal fenestration in tumour-associated microvasculature that might serve as ports of entry into the vascular compartment for tumour dissemination. These observations highlight the potential importance of CBPs in cancer invasion, and also provide a fertile ground for extracting information that might be highly relevant in assessing the degree of malignancy of cancers and in cancer management.
-ACTININ
The actin cytoskeleton is regulated by several actin-binding and cross-linking proteins that are themselves regulated by free calcium. -actinin is an EF-hand protein approximately 110 kDa in size and it forms a major component of the cytoskeleton in many cell types. It forms antiparallel, highly stable homodimers. Actinin isoforms can also form heterodimers, both in vivo and in vitro (Y.M. Chan et al. 1998). Y.M. Chan et al. (1998) suggest the possibility that heterodimers formed by different isoforms could potentially have new functional characteristics. The dimerisation of smooth muscle actinin is believed to be mediated by a segment of the C-terminal region of the molecule (Baron et al. 1987; Imamura et al. 1988; Kahana and Gratzer, 1991; A.P. Gilmore et al. 1994). This segment has two cross-linking sites (A and B), and the actinin molecules form cross-links in an antiparallel fashion by the binding of an A site of one molecule with the B site of the second molecule (Imamura et al. 1988). Several isoforms of -actinin have been identied from vertebrate cytoskeletal, skeletal, and smooth muscle sources (Duhaiman and Bamburg, 1984; Imamura and Masaki, 1992; Landon et al. 1985; J.P. Bennett et al. 1984). Isoforms also have been isolated from invertebrate sources such as Dictyostelium discoideum (Noegel et al. 1987) and Drosophila melanogaster (Fyrberg et al. 1990). A cDNA has been cloned from the nematode Caenorhabditis elegans. The sequence of this clone predicts that it codes for an -actinin, which possesses a high degree of sequence homology to actinin from other sources (Barstead et al. 1991).
MOLECULAR STRUCTURE
OF
-ACTININ
-actinin is an EF-hand calcium-binding protein. Two EF-hands are located at the Cterminal region of the molecule. An actin-binding domain occurs at the N-terminal
103
end, followed by the rod domain, which consists of four spectrin-like repeat elements, followed by the EF-hand to the C-terminus (A. Blanchard et al. 1989; Flood et al. 1995). The rod domain is required for stable dimerisation of the molecule, and dimerisation is reduced markedly if either of terminal repeats 1 and 4 is deleted (Flood et al. 1995). Although all the isoforms possess EF-hands, these have been found to be in a functional state only in the nonmuscle or cytoskeletal isoforms of -actinin (Burridge and Feramisco, 1981; Duhaiman and Bamburg, 1984). Their signicance has been elucidated by introducing point mutations to make them nonfunctional. The rst EF-hand seems to regulate the Ca2+-dependent cross-linking activity of -actinin (Janssen et al. 1996). Nonmuscle isoforms might themselves differ in calcium-binding ability (Imamura et al. 1994). The EF-hands of -actinin isoforms 2 and 3 of human skeletal muscle are not capable of binding calcium, and therefore the binding of these isoforms to actin might not be calcium sensitive (Beggs et al. 1992). Calcium sensitivity or the lack of it can be viewed from a functional viewpoint. The EF-hands appear to undergo conformational changes from a closed position in the absence of calcium, to an open position when calcium is present (Trave et al. 1995). The open conformation may be conducive to proteinprotein interactions, which are required for the transduction of signals via the actin cytoskeleton.
-ACTININ ISOFORMS
Two skeletal isoforms of -actinin have been designated as -actinin-2 and -3 and two nonmuscle isoforms as -actinin-1 and -4 (Millake et al. 1989; Beggs et al. 1992, 1994; Honda et al. 1998). The skeletal muscle isoform -actinin-2 is found in both human skeletal and cardiac muscle, but -actinin-3 occurs only in limb skeletal muscle (Beggs et al. 1992). The isoforms -actinin-1 and -4 occur in the actin microlament bundles and at the adherens junctions. In skeletal, cardiac, and smooth muscle, -actinin is localised to the Z-discs and dense bodies, and it participates in the anchoring of the actin thin laments and giant titin molecules to the Z-disc (Endo and Masaki 1984; Geiger et al. 1990). -actinin has been shown to interact with an N-terminal domain of titin in vitro (Ohtsuka et al. 1997). In the Zdisc, the assembly of the actinintitin complex seems to involve two types of interaction between titin and -actinin. In the other regions of the Z-disc, titin binds by means of a single binding site with the outermost pair of -actinin molecules, but in the middle of the Z-disc the titin binds several -actinin molecules through binding sites at their C-terminal region (Young et al. 1998). Thus -actinin seems to play a major part in anchoring actin thin laments of the two halves of the sarcomere at the Z-disc (see Figure 15). Abnormal expression of -actinin and other thin lament-associated proteins are known to interfere with the assembly of Zdiscs, which could lead to the formation of so-called nemaline bodies. Two loci are known to be involved in nemaline myopathy: the tropomyosin-3 locus and the nebulin locus on 2q21.2q22. Mutations of both genes have been implicated in this form of myopathy (Laing et al. 1995; Pelin et al. 1999). The formation of the complex of tropomyosin, titin, nebulin, -actinin, and actin can conceivably be affected by these mutations, leading to abnormalities in Z-disc assembly and the formation of nemaline bodies.
104

OF
FUNCTION
-ACTININ
-actinin functions predominantly in F-actin bundling and anchoring of the laments to specic sites within the cell as well as to the cell membrane and in the linking up of the cytoskeletal machinery to the ECM. -actinin might form a bridge between the actin cytoskeleton and integrin receptors then function as a receptor for components of the ECM (Burridge et al. 1990; Geiger et al. 1990). The cytoplasmic tail of integrins and intercellular adhesion molecule-I (ICAM-I) interact with -actinin via binding sites occurring in the rod domain. Furthermore, -actinin appears to link the cytoskeleton to the cell membrane (Baron et al. 1987). Kahana and Gratzer (1991) have identied binding sites for long-chain fatty acids in the spectrin-like repeats of the -actinin domain rod. Han et al. (1997) recently have shown that bundling of actin laments occurs if diacylglycerol is present in the membrane. It follows, therefore, that -actinin would be an important component of the signal transduction pathway as well as an important link in the cytoskeleton-mediated changes in cell shape and motility. Miyamoto et al. (1995) have delineated the pathway of integrin-mediated signal transduction. They reported that integrin aggregation induced the accumulation of several signal transduction molecules, such as Rho A, Rac1, ras, raf, and others, of cytoskeletal components, such as vinculin, talin, and -actinin. The tyrosine kinase inhibitor genistein inhibits accumulation of both the signal transduction molecules and the cytoskeletal signal transduction components.
ACTININS IN CELL ADHESION, MOTILITY, AND SIGNAL TRANSDUCTION

The cross-linking of actin laments increases lament elasticity and viscosity and may be expected to affect the structural properties of actin laments. It follows from this that actinin might, in this fashion, change the properties of the cell membrane, such as intercellular and cellsubstratum adhesion, which in turn would be reected in alterations in cell shape and motility. These changes will have serious implications for cancer invasion and secondary spread. Furthermore, there are strong indications that actin cross-linking in endothelial cells might result in metabolite transport and permeability. Although yet to be demonstrated, one would expect that alterations in the organisation of adherens junctions might effectively alter endothelial integrity and lead to enhanced tumour cell diapedesis across the endothelium. Changes in the structural properties of the actin cytoskeleton would impugn the link-up between it and the ECM. Such changes would affect seriously the physical pathway of signal transduction, and effectively lead to its deregulation, and eventually to the deregulation of intercellular adhesion and communication and of cell proliferation and growth.
THE CADHERINCATENIN COMPLEX CELL ADHESION
IN
SIGNAL TRANSDUCTION
AND
A signal transduction complex that occurs in the cell membrane, consisting of the transmembrane glycoprotein cadherin and other components such as -actinin, catenin, and the adenomatous polyposis coli (APC) protein, has assumed considerable signicance by virtue of its apparent dual function, namely in signal transduction
105
and cellular adhesion. Intercellular adhesion mediated by cadherin is dependent on the integrity of the link-up with the actin cytoskeleton (Sherbet and Lakshmi, 1997b) (Figure 13). -catenin subserves two functions, viz. as a signal transduction molecule and in the formation of adherens-type junctions. Its participation in signal transduction has become apparent with the demonstration that plakoglobin and -catenin are homologues of the armadillo protein of Drosophila that is involved in segment polarity.
N-terminus
Extracellular Ca
2+
binding domains
Plasma membrane -catenin -catenin -actinin Actin filaments
Adherens junction protein APC Protein
FIGURE 13 Schematic representation of the interaction of the transmembrane adhesion protein cadherin with the actin cytoskeleton, via -catenin, -catenin, and -actinin. (Based on van Roy [1992] and Sherbet and Lakshmi [1997b]). Reprinted by permission of the publisher Academic Press, from The Genetics of Cancer, (Sherbet and Lakshmi, 1997b).
The armadillo (arm) protein is a component of the signal transduction pathway of the wg (wingless) molecule (Riggleman et al. 1990). The arm family of proteins, e.g., -catenin, plakoglobin, and the p120ctn protein, are characterised by a central arm repeat domain (Riggleman et al. 1989). The p120ctn gene potentially can code for a large number of isoforms that are generated by alternative splicing, and these have been regarded as constituting a subfamily of signalling proteins (Keirsebilck et al. 1998b). Jou et al. (1995) have shown that -catenin binds to a C-terminal 25 amino acid region in the cytoplasmic domain of E-cadherin and to the N-terminal domain of -catenin. The p120ctn protein, on the other hand, seems to bind to a juxtamembrane domain of the cadherin cytoplasmic tail (Yap et al. 1998). The armadillo and wg proteins exert similar effects on embryonic development (Peifer et al. 1991). A family of wnt proteins has been identied. The wnt protein is a secreted glycoprotein signalling factor. It is a vertebrate homologue of wg and has been shown to be a regulator of morphogenesis of Xenopus (Gumbiner, 1996; Miller and Moon, 1996). In a similar vein, -catenin and plakoglobin are components of the signal transduction pathway of the wnt gene as well as being involved in the formation of adherens junctions and Ca2+-mediated cellcell adhesion (Hinck et al. 1994; Peifer, 1995). Cadherin, as a part of this signalling complex, not only regulates intercellular adhesion, but also seems to negatively regulate the signalling function of -catenin. These two functions can be dissociated. Fagotto et al. (1996) noticed that -catenin deletion mutants affect cadherin-mediated adhesion but not its signalling function. Sanson et al. (1996) showed that a full length E-cadherin and a
106
truncated form of cadherin that have opposite effects on cadherin-dependent adhesion, nonetheless function effectively in wg signalling. Two further elements are involved in the signal transduction complex involving E-cadherin and catenins. One is the APC protein and the other component is axin or conductin. -Catenin forms complexes with both axin and APC. The APC protein is known to compete with cadherin in its binding to the internal repeats of -catenin. The -catenin/APC complex is phosphorylated by the glycogen synthase kinase GSK3. A consequence of this phosphorylation is that the degradation of -catenin is greatly enhanced (Munemitsu et al. 1995; Hayashi et al. 1997; Hart et al. 1998). Axin also induces -catenin degradation and is suggested to function downstream of APC (Behrens et al. 1998). The GSK3-mediated phosphorylation is inhibited when the wnt signal transduction pathway is active. This results in the accumulation of -catenin in the cytoplasm (Hinck et al. 1994; Giarre et al. 1998; Papkoff and Aikawa, 1998) and leads to the formation of a complex with the T-cell factor/lymphoid enhancer factor (Tcf/Lef). The binding between them occurs via the arm repeats. This complex functions as a transcription factor in the wnt/wg signalling pathway (Behrens et al. 1996; Van de Wetering et al. 1997). The pathway might be deregulated by inhibition of -catenin degradation, leading to a constitutive activation of the transcription complex (Figure 14).
Axin/conductin Wnt/Wg
APC/-catenin
GSK3b
APC-P/-catenin-P
-catenin Cell adhesion
-catenin degradation
-catenin/Tcf-Lef Transcription complex
FIGURE 14 The wnt signal transduction pathway and cell adhesion signal involving catenin, APC, and cadherin. (Based on references cited in the text.)
The deregulation that accompanies neoplastic changes seems to be more frequently due to mutation of -catenin or APC protein than to inactivating mutations of E-cadherin (Morin et al. 1997; Efstathiou et al. 1999). These mutations seem to stabilise -catenin. They occur at the phosphorylation sites that are essential in the ubiquitination and degradation of the protein. -catenin mutations occur in approximately 50% of colorectal tumours. Again, these tend to occur in the serine/threonine phosphorylation sites. They seem to occur more frequently in small adenomas than
107
in larger adenomatous lesions. Furthermore, a majority of the mutations have been found in adenomas rather than in carcinomas (Samowitz et al. 1999). A high rate (61% of 31 patients) of somatic mutation of -catenin has been encountered in human anaplastic thyroid carcinoma (Garcia-Rostan et al. 1999). In a murine hepatocarcinoma model, mutations have been reported to occur in the carcinomas but not in adenomas. These ndings suggest that deregulation of signal transduction occurs as an early event in the pathogenesis and progression of tumours. The catenin mutations in colorectal tumours reported by Samowitz et al. (1999) were not accompanied by APC mutations. The suggestion has been made, therefore, that mutations of APC and -catenin might be functionally different. Mutations of APC leading to the loss of its suppressor function have been identied with the familial adenomatous polyposis, and are also closely related to the progression of the disease. Furthermore, mutated APC seems to lack the ability to regulate -catenin levels. Therefore, APC may be regarded ipso facto as functioning upstream of -catenin. The adhesion function of -catenin ows from its linkage to cadherin, which spans across the cell membrane, on the one hand, and to the cytoskeleton, on the other, by means of two other elements, namely -catenin and -actinin. In broblasts, this linkage has been demonstrated by Knudsen et al. (1995), who also noted that -actinin specically immunoprecipitates with -catenin and cadherin. Niesset et al. (1997) have identied the binding sites involved in the interaction of -catenin with -catenin on the one hand and with -actinin on the other. In normal thyroid epithelial cells, the intercellular adhesion junctions show the presence of cadherin together with the catenins. In contrast, CGTHW-2 cells derived from thyroid carcinoma show a marked alteration in the pattern of distribution of these linking proteins in the foci of intercellular adhesion. Further evidence in support of the importance of the integrity of the cadherincatenin complex in intercellular adhesion comes from the apparent involvement of the Rho GTPase family in the regulation of cadherin-mediated cell adhesion. IQGAP1, which is an effector of two members of the family, namely Cdc42 and Rad, has been shown to be able to dissociate -catenin from the cadherin/-catenin/-catenin complex. This results in the disruption of intercellular adhesion. Both Cdc42 and Rad counteract this process (Kaibuchi et al. 1999). The inhibition of Rho and Rac (another member of the Rho GTPase family) has been reported to lead to a disruption of E-cadherin localisation at keratinocytekeratinocyte cell junctions. However, when the GTPases themselves are inhibited, the cadherin-mediated adhesive contacts are reestablished (Braga et al. 1999). Rho protein, when microinjected into four-cell blastomere-stage embryos, disrupts cortical microlaments and reduces interblastomere adhesion. Cdc42 in the same way also disrupts the cortical cytoskeleton and interblastomere contacts (Clayton et al. 1999). But the modes of involvement of Rho and Cdc42 are clearly distinguishable in NIH3T3 cells transformed by the dbl oncogene, which codes for a supposed exchange factor for RhoA and Cdc42. The transformed cells respond to adhesion to bronectin substratum by changing cell shape. This involves the activation of RhoA and the associated ROCK and CRIK, but not Cdc42. In nontransformed cells, however, change of cell shape seems to require Cdc42 activation (Olivo et al. 2000). Rho GTPases may also be involved in the regulation of endothelial cell adhesion involving cadherins. Rho
108
GTPases are also involved in the regulation of the function of other adhesionmediating glycoproteins such as CD44. Therefore, prima facie there is a reasonable basis for investigating their role in maintaining the integrity of the vascular endothelia and possible implications for metastatic spread of cancer. Other proteins that link the plasma membrane with the actin cytoskeleton may disrupt cadherin-mediated adhesion. The proteins ezrin, radixin, moesin, and merlin subserve such a linking function. Hiscox and Jiang (1999) used antisense nucleotides to inhibit ezrin expression in colon carcinoma cells and noticed that this resulted in a loss of intercellular adhesion and acquisition of motility. They further noticed that ezrin co-precipitated with E-cadherin and -catenin. In cells treated with antisense ezrin nucleotides, this could result in a reduced interaction between ezrin and the cadherin complex. Such a reduction indeed occurs when ezrin is phosphorylated or when the cells are treated with hepatocyte growth factor. Huang et al. (1998) found no cadherin or -catenin at the adhesion junctions, and - and -catenins were distributed diffusely in the cytoplasm of most cells. However, -catenin, when detected at intercellular junctions, was found to colocalise with -actinin. Huang et al. (1998) have therefore suggested that the loss of intercellular adhesion could be due to an incorrect assembly of the linking components. Implicit also in this is the suggestion that such an incorrect assembly might lead to the acquisition of invasive ability. Although this is an attractive hypothesis, it should be mentioned here that Honda et al. (1998) have reported the occurrence of a novel actinin, namely actinin-4, whose expression is said to be upregulated with enhanced cellular migration. This new isoform is said to occur in the cytoplasm. It is reported to be associated with cytoplasmic extensions and is found in peripheral migrating cells of cell clusters. Actinin-4 appears to be translocated from the cytoplasm to the nucleus, when actin is depolymerised. Honda et al. (1998) have also examined the expression of this novel actinin in breast cancers. It is expressed in inltrating breast carcinomas, and the expression was found to correlate with poor prognosis. Mamura and Masaki (1996) identied a 115-kDa -actinin in vascular endothelial cells. This actinin differed from other known actinins of muscle or nonmuscle origin in its sensitivity to calcium. However, this isoform of actinin did occur in heart tissue, the pectoralis muscle, and the gizzard. What could be signicant about this actinin in relation to cancer progression is its apparent location in the vascular endothelium. It is well known that several inammatory agents induce a reversible change in endothelial cell shape that results in the formation of intercellular gaps. If cancer cells can reduce the endothelial integrity in this way, that could form ports of entry into the vascular system for cancer cells. There is a view that invading cancer cells take advantage of naturally occurring fenestration of the endothelium. There is no demonstration to date that they might secrete substances that could induce the formation of ports of entry. From this point of view it would be interesting to see if cancer cells induce any changes in the expression of actinins of the endothelial barrier. A new facet that can be added to the story of actinins in invasion and metastasis is their apparent relationship to the expression of metalloproteinases. It was shown some years ago that the production of these enzymes often paralleled the invasive
109
and metastatic abilities of cancers, which has suggested the possibility that metalloproteinases might reconstruct the ECM and provide cancer cells with ECM properties conducive to invasion (Sherbet and Lakshmi, 1997b). Crawford et al. (1999) have made some interesting observations on the expression of matrilysin and catenin. They found that -catenin and matrilysin mRNAs were expressed in parallel in murine intestinal adenomas. A further observation of considerable importance is that -catenin signicantly up-regulated the matrilysin promoter. Because -catenin is a structural component of signalling pathway and intercellular adhesion machinery, these experiments bring together, in a unique fashion, transduction of an extracellular signal that can putatively remodel the ECM and change the invasive behaviour of cells. Other cell adhesion molecules such as the ICAM similarly require interaction with the actin cytoskeleton. A peptide containing a cytoplasmic sequence of ICAM2 has been shown to interact with -actinin in vitro. Indeed, interaction occurs between several sites of -actinin and ICAM-2. Besides, ICAM-2 co-localises with -actinin (Heiska et al. 1996). The epithelial ICAM (Ep-ICAM), an adhesion molecule regarded as specic for epithelial cell adhesion, has a cytoplasmic domain that regulates the cellular adhesion function. It appears that Ep-ICAM achieves this via -actinin, with which Ep-ICAM appears to interact directly by means of specic binding sites (Balzar et al. 1998). Shigella exneri, which causes bacillary dysentery, is known to induce changes in the cytoskeleton of epithelial cells. These changes result in the production of membrane protrusions that engulf the bacterium. The bacterium secretes a protein called IipaA. The cytoskeletal reorganisation, which is essential for this process of bacterial invasion, involves the recruitment by IipaA of the linking protein vinculin as well as -actinin (Van Nhieu et al. 1997). The effects of the loss of -actinin on cell shape, aggregation, and motility have been studied in Dictyostelium. Rivero et al. (1996) generated mutants lacking actinin as well as the gelation factor and these mutants presented a rounded shape rather than the typical polarised morphology of aggregating cells. The mutants also showed considerable loss of motility. In rat bladder carcinoma cells, the experimentally induced loss of motility was associated with the reorganisation of F-actin and -actinin (Morton and Tchao, 1994). The transmembrane integrin 21 functions as the receptor for the ECM components collagen and laminin. This integrin as well as -actinin have been implicated in the invasive ability of melanoma cells in vitro. L.M. Duncan et al. (1996) reported that -actinin was not detectable in benign melanocytic naevi and in laterally spreading lesions, but it occurred in all nodular melanomas and in metastatic melanomas. It would have been of much interest whether there was an association between -actinin expression and the vertical growth of melanoma. EGF is known to alter cell morphology as well as promote cell invasion in vitro (Shiozaki et al. 1995). These cellular responses seem to originate from the linkage of the activated EGFr to the cytoskeletal system (Roy et al. 1989, 1991; Van Bergen en Henegouwen et al. 1989). There is some recent evidence that EGFr regulates intercellular adhesion by interfering with the formation of a complex between the invasive suppressor transmembrane protein E-cadherin and the actin cytoskeletal
110
elements, including -actinin and -catenin (Hazan and Norton, 1998; see also Figure 13). As alluded to earlier, the proliferative signal imparted to the cell by EGF is mediated by PKC. N.R. Murray et al. (1999) generated transgenic mice that express PKC-II at high levels. The colonic epithelium showed high cell proliferation. Besides, the epithelium was more prone to preneoplastic changes. They found that these effects were also accompanied by increased expression of -catenin and a reduction in glycogen synthase kinase 3 activity. These studies suggest, therefore, that EGFr-mediated signal follows the wnt/APC pathway of transduction leading to cell proliferation. This pathway also may be utilised in neoplastic changes of epithelia. Overall, these data now provide a physical basis for previous observations that high EGFr expression correlated with poor prognosis, and that EGFr status is a powerful marker of clinical aggressiveness and metastatic potential of tumours (Sherbet and Lakshmi, 1997b).
MYOSIN FILAMENTS
Myosin is a lament protein found in association with actin in both muscle and nonmuscle cells. Like actin, myosin is a bipolar protein. It possesses a globular head region (subfragment S1), which interacts with actin, and a brous region, which promotes aggregation into laments. The laments consist of two myosin heavy chain (MHC) subunits and four myosin light chain (MLC) subunits. The class II skeletal myosin has two heads whereas other myosins have only one head. The N-terminal globular domain comprises the motor domain. The heavy chain subunit possesses the ATPase activity that provides the energy for the contractile force. The process of muscle contraction is driven by a cyclical interaction between actin and myosins. Myosins have been described as mechanoenzymes that catalyse the hydrolysis of ATP and release of energy, which is converted into mechanical force (Hasson and Moosekar, 1995). Energy is transduced by a series of interactions between myosin heads and hydrolytic changes of ATP. This is believed to deform the protein, and this change is transmitted to actin as a mechanical effect. The mechanical displacement and force generated in this way has been measured. The force generated by double-headed myosin is nearly twice as large as that produced by single-headed myosins (Tyska et al. 1999). In the skeletal muscle, the two heads of myosin interact when it is bound to lamentous actin (Rayment et al. 1993). The smooth muscle myosin heads interact in the presence of lament actin and in the absence of ATP (Onishi et al. 1992). Such headhead interaction also is required in the phosphorylation-dependent regulation of smooth muscle myosin (Matsuura and Ikebe, 1995). The processes of muscle contraction and cell motility are usually evaluated by an in vitro technique in which actin laments are slid over myosin and measured using an electron microscope. Actin sliding as well as actin activation of myosin-ATPase is dependent on phosphorylation when myosin is dimerised, but not while myosin is in the monomeric form. Furthermore, the sliding velocity of the dimer is twice as large as that of the monomer (Sata et al. 1996).
111
MYOSIN HEAVY CHAIN (MHC) ISOFORMS

A large number of MHC isoforms are known to exist (Table 6). There are preliminary reports that the pattern of their expression is modulated in embryonic development and also in association with certain disease processes. For instance, thyroid hormone is reported to alter the pattern of MHC isoform expression in embryonic development (Maruyama et al. 1995). During metamorphosis of Xenopus tadpoles, thyroid hormone has been reported to down-regulate the expression of embryonic but upregulate adult isoforms (Sachs et al. 1997). Modulations in MHC isoform expression have been reported in the progression of human atherosclerosis (Aikawa et al. 1995). Similar changes have also been described in tumour-associated brosis and in neoplastic transformation of breast epithelium (Chiavegato et al. 1995).
TABLE 6 Myosin Heavy Chain (MHC) Isoforms

MHC Isoform Myosin I slow MHC 1, Myosin II fast MHC II a, b, x MCH 3 II c MHC1, MCH 9 Myosin II SM1 (204 kDa); SM2 (200 kDa) Nonmuscle MIIA, B1, B2 Unconventional myosins V, VI Myosin VIIa Tissue Skeletal, cardiac, foetal Skeletal Foetal Skeletal, immature bres Smooth muscle Fibroblasts, endothelial cells, macrophages Intestinal brush border, Va in a variety of cell types Cochlea, retina, testes, lung, kidney
Source: Based on several sources including Kelley and Adelstein (1994); Heintzelman et al. (1994); A. Kimura et al. (1995); Chiavegato et al. (1996); Galler et al. (1997); Hasson et al. (1997); Wu et al. (1998).
The metastasis-associated S100A4 (mts1), which is itself a CBP, has been found to bind to the myosin rod in the light meromyosin region and could be destabilising the myosin lament. Besides, S100A4 binding has been found to inhibit the actinactivated ATPase of myosin (Ford et al. 1997). These interactions could conceivably alter the faculty of motility, which has been associated with cancer invasion, and this is especially associated with high expression of S100A4. It would be of interest to note here that the unconventional myosin V could be involved in the mechanisms associated with lopodial extension of neuronal growth cones. When myosin V is inactivated, cones of dorsal root ganglia show a rapid retraction of the lopodia (F.S. Wang et al. 1996). The distribution of the nonmuscle isoforms of myosin II (A and B) shows a pattern that is compatible with their involvement in cell locomotion. These myosins occur in association with lamellipodia and around the cell nucleus. However, myosin IIA shows a preferential localisation along the leading edge, but
112
myosin IIB occurs along the trailing edge of cells in locomotion (Kolega, 1998). It is signicant that, when injected into the cells, these isoforms show the same pattern of localisation as the endogenous variety. This has led to the suggestion that their localisation pattern is an intrinsic property of locomoting cells (Kolega, 1998). The apparent specicity of localisation may not be a consequence of their synthesis at specied sites, but could indicate that they are being targeted to those locations. HSPs are believed to behave like molecular chaperones for S100A4. HSPs might target S100A4 to the plasma membrane to be secreted into the ECM, and in this way endow the cells with invasive ability. Therefore, further effort into the possible mechanisms involved in the perceived specicity of myosin IIA and B localisation in the locomoting cell would be eminently worthwhile. Among other myosin-driven biological phenomena that may be cited here is cytokinesis in cell division. The myo2 gene of Schizosaccharomyces pombe encodes a protein called myo2, which shows a high degree of homology to myosin type II heavy chain. A contractile ring composed of actin and myo2 is formed before cytokinesis. Disruption of myo2 reportedly leads to an inhibition of cell proliferation (Kitayama et al. 1997). In Dictyostelium the formation of the contractile ring requires phosphorylation of myosin (Sabry et al. 1997). The unconventional myosin Va has been localised to the microtubule organising centre of interphase cells and to the mitotic apparatus in dividing cells. Using specic antibodies against myosin Va, Wu et al. (1998) have been able to demonstrate its association with microtubules in a number of cell types. The specicity of this association was obvious from experiments using cells that were derived from mice with null (dilute gene /) phenotype, in which no association with microtubules could be detected.
ACTOMYOSIN ASSEMBLY
Myosin thick laments are composed of two MHCs of 220 kDa and four MLCs of 20 kDa molecular size. The N-terminal end of MHC forms the globular head. The tail region of the lament is an -helical coiled coil that constitutes part of the long rigid tail, of which the part proximal to the head region is composed of the Cterminal half of MHC and the light chains form the distal end of the myosin tail. The light chains are of two classes: a phosphorylatable form found in heart muscle and the CNS, but not in skeletal muscle, and a nonphosphorylatable form. Indeed, the N-terminal section of MHC together with two light chains, one of each class, form the globular head. MLCs bind Ca2+ with great afnity and regulate myosinATPase activity. The conformation of the myosin globular heads is regulated by the phosphorylation of the MLC by MLCK, which binds to actin via residues 242 of the N-terminal end of the kinase (Gallagher and Stull, 1997). This phosphorylation is believed to be a Ca/CaM-dependent process. Although reversible phosphorylation of MLC is generally regarded as the regulatory event, other proteins, e.g., caldesmon and calponin, which are capable of binding to the actinmyosin assembly, could subserve important functions. The skeletal and heart muscle cells possess a characteristic striated pattern. The striations are due to individual brils within the muscle cells. The striations are produced because the brils are arranged in register, which accentuates the striation.
113
FIGURE 15 A sarcomere with its bands. The pattern is a result of the arrangement of actin, myosin, and titin. (Based on Gaub and Fernandez, 1998.)
The striations form three distinct zones: the dark and birefringent A-band with a lighter zone in the middle called the H-band, which in turn is divided by the Mline; the I-band, which is lighter than the A-band; and the Z-line, which is a dark line in the middle of the I-band (Figure 15). The contractile, regulatory, and structural components of these zones are summarised in Table 7. The A-band is composed of
114
TABLE 7 Contractile, Regulatory, and Structural Component Proteins of Myobrils of Vertebrate Skeletal Muscle
Location Band Content % wt. Molecular Weight (kDa)
Protein Contractile Myosin Actin Regulatory elements Troponin Tropomyosin M-protein C-protein -actinin Structural component Titin Nebulin
A I I I M-line A Z-line AI I
43 22 5 5 2 2 2 10 5
ca. 500 42 70
ca. 110 3000 600800
Source: The data in this table have been collated from references cited in the text.
thick myosin laments. These are arranged in a parallel array and in register and therefore they determine the length of the A-band. Thin actin microlaments form the lighter I bands. However, the thick myosin laments form bridges with the microlaments, especially in the region of overlap of A and I bands. In transverse section of the muscle, the organisation of the laments can be described as a lattice. Each thick myosin lament has six thin actin microlaments and each actin lament has two neighbouring myosin laments. In the thin laments, tropomyosin occurs in a head-to-tail orientation in one groove of actin helix by means of seven actinbinding sites. It is believed that molecular elasticity is regulated by a species of giant rod-like proteins called titin or connectin. Human titin is a ca. 3000-kDa protein consisting of variable numbers of immunoglobulin (Ig)- and FN-like repeat domains and a P (proline), E (glutamate), V (valine), and K (lysine)-rich region of variable length. Titin overlaps the A-band and the I-band, extending from the Z-line to the M-line (see Figure 15). It is believed that the part of titin that overlaps the I-band forms the elastic region of the molecule (Linke et al. 1998). Alternative splicing of the I-band titin is said to result in changes in the component Ig and FN modules and in the length of the PEVK region (K. Wang et al. 1991; Labeit and Kolmerer, 1995a). Another large protein called nebulin, of approximately 600 to 800 kDa molecular size, spans the whole length of the thin laments of the I-band. Indeed, nebulin is regarded as the determinant of the length of the thin laments (Kruger et al. 1991; Jin and Wang, 1991; Labeit et al. 1991; Trinick, 1994). The assembly of mature Z-discs occurs from precursors called I-Z-I bodies, which contain titin,
115
nebulin, actin, and -actinin (Holtzer et al. 1997). Tropomyosin seems to be involved in nemaline myopathy. Two loci involved in this disease are the tropomyosin-3 locus and the nebulin locus on 2q21.2q22 (Laing et al. 1995; Pelin et al. 1999). Mutations of both genes have been implicated. The formation of the complex of tropomyosin, titin, nebulin, -actinin, and actin can conceivably be affected by these mutations, leading to abnormalities in Z-disc assembly and the formation of nemaline. The C-terminal region of nebulin contains an SH3 domain, which may anchor the nebulin lament system to the Z-disc (Labeit and Kolmerer, 1995b). This SH3 domain has been found to interact with actin, tropomyosin, troponin and calmodulin. This could suggest that nebulin, together with tropomyosin and troponin, might form a complex that is calcium-regulated (K. Wang et al. 1996). In cardiac muscle, a nebulin-related protein called nebulette substitutes for nebulin (Millevoi et al. 1998). Nebulette is similar to skeletal muscle nebulin in its organisation and contains four domains: an N-terminal domain followed by a domain that contains nebulin-like repeats, a linker domain connecting the nebulin repeats to the C-terminal SH3 domain. The nebulin repeats possess actin-binding ability and the linker zone appears to target the protein to the Z-line. This is suggested by the binding of recombinant nebulette fragments to actin, tropomyosin, and -actinin in vitro (Moncman and Wang, 1999).
MYOSIN LIGHT CHAIN (MLC) PHOSPHORYLATION
AND
FUNCTION
As stated earlier, there are two classes of myosin light chain (MLC), viz. a nonphosphorylatable and a phosphorylatable regulatory chain. The reversible phosphorylation of the regulatory MLC is regarded as the key event in the regulation of myosin-ATPase and the generation of the contractile force. The phosphorylation of MLC is carried out by the MLCK. MLCK is a Ca2+/CaMdependent kinase (Stull et al. 1993) and is itself subject to regulation by kinases and phosphatases (Quadroni et al. 1998). MLCK is known to be able to bind to actin and thereby regulate actinmyosin interaction (Kohama et al. 1992; see also below). Naturally this will inuence the function of the ATPase. On the other hand, binding of calponin and caldesmon to actin may inhibit myosin-ATPase and this inhibition may be reversed by calcium-binding S100 proteins and calmodulin (see Fattoum, 1997). Rho kinase also seems to be able to phosphorylate MLC, together with inactivation of myosin phosphatases (Fukata et al. 1999) (Figure 16). The regulation of the contractile machinery of the cell has obvious implications for cell motility and diapedesis of cells. The ability of activated PMN to traverse the endothelium appears to be related to the state of MLCs phosphorylation of endothelial cells. Hixenbaugh et al. (1997) found that activated PMNs increase phosphorylation of serine 19 and threonine 18 of MLCs of endothelial cells. Garcia et al. (1998) have conrmed the enhanced phosphorylation of MLC in endothelial cells and have further shown that inhibitors of MLCK signicantly inhibit the diapedesis of activated PMNs across the endothelium. Phosphorylation of MLCK by protein kinase C also inhibits MLC phosphorylation and causes enhanced
116
Muscle contraction
Actomyosin Mg-ATPase
Caldesmon Calponin S100/S100A4 Caltropin Ca 2+ /Calmodulin
Myosin
Actin RMLC +P PKC +P MLCK
Ca2+/CaM +P Casein Kinase II -P PPIA/PPIB
FIGURE 16 Control of regulatory myosin light chain (MLC) function in muscle contraction. CaM, calmodulin; MLCK, MLC kinase; PKC, protein kinase C; PP1A/PPiB: protein phosphatases; RMLC, regulatory MLC.
epithelial resistance (Angle et al. 1998). The association with actin and the assembly of myosin II into the cytoskeleton is dependent on the phosphorylation of the regulatory chain. MLC associated with the cytoskeleton of cultured endothelial cells has been reported to show a vefold greater phosphorylation than MLC in soluble form. The phosphorylation of serine 19 and threonine 18 appears to regulate the assembly of MLC in vitro. In contradistinction, soluble MLC seems to be phosphorylated at threonine 9 (Kolega and Kumar, 1999). It would seem that the phosphorylation of the cytoskeletal machinery could increase the intercellular gaps thus allowing cell penetration. It would be worthwhile to investigate whether, like activated PMNs, cancer cells affect the state of MLC phosphorylation of endothelial cells. Unquestionably, vascular permeability is an important determinant of metastatic spread of cancer. However, invasive ability is an intrinsic property of the cancer cell. Gillespie et al. (1999) have recently shown that the MLCK inhibitors, ML7 and KT5926, inhibit the invasive ability of glioma cells. MLC phosphorylation in both the invading tumour cell and the endothelial cell barrier might determine the degree of successful entry of the tumour cell into the vascular compartment (Figure 17). Some studies have implicated MLC phosphorylation in the regulation of certain other biological features such as cell growth and apoptosis. Mills et al. (1998) have reported that membrane blebbing associated with apoptotic cell death could be reduced by MLCK inhibitors, and, further, that there was increased phosphorylation
117
Diapedesis
MLC-P +P
Inhibitors
MLCK +P MLC-P
+P
MLCK kinases (PKC)
Cancer Cell Invasion

FIGURE 17 Possible involvement of MLC phosphorylation in the migration of cells across the endothelial membrane as well as with the intrinsic invasive potential of the tumour cell. MLC, myosin light chain; MLCK, MLC kinase; PKC, protein kinase C.
of MLCs in membrane blebbing. There are also suggestions based on preliminary ndings that MLC phosphorylation may also be associated with cell proliferation (Bresnick et al. 1996; Yamakita et al. 1996). MLCK may affect actin reorganisation by a mechanism that is independent of its kinase activity. As stated earlier in this section, MLCK is known to be able to bind to actin and inuence actinmyosin interaction. Another aspect of MLCK function is that it can bind to and result in actin bundling into laments (Hayakawa et al. 1994). Two sites on MLCK have been identied and characterised; one of them is Ca2+/CaM sensitive and the other is Ca2+/CaM insensitive (Ye et al. 1997). These participate in actin lament assembly in vitro (Hayakawa et al. 1999). By inference from and in common with the properties of other actin-binding and bundling proteins, it would be reasonable to suggest that MLCK might inuence cell shape and exibility and thereby affect cell migration and diapedesis, quite independently of its kinase activity.
118
TROPONINS AND TROPOMYOSINS IN THE REGULATION OF MUSCLE CELL CONTRACTION THE REGULATORY ROLE MUSCLE CONTRACTION
OF
TROPONINS
AND
TROPOMYOSINS
IN
Troponins are CBPs that play a major regulatory role in cardiac and skeletal muscle contraction in consort with tropomyosins. Three troponin (Tn) molecules TnT, TnI, and TnC bind to each tropomyosin molecule. Many tropomyosin isoforms with a range of molecular sizes have been identied (see below). The high molecular weight isoforms bind actin more strongly than the others (Matsumura et al. 1985). There is a complex and specic form of association between the troponins and tropomyosin that produces conformational changes in actin that lead to muscle relaxation or contraction. TnT binds to the C-terminal region of tropomyosin and forms a link between TnI and TnC, which both bind to tropomyosin. The structural domain C and regulatory domain N of tropomyosin interact with specic amino acid sequence motifs of TnI (Pearlstone et al. 1997). Calcium binding to the N domain of TnC can alter the afnity of the N- and C-termini of TnI for TnC. In other words, calcium binding can provide a switch between these two sites for binding with TnC (Tripet et al. 1997). At any rate, the whole complex interacts with actin via TnI. TnI is the myosin-ATPase inhibitory protein. TnC is the calcium binding subunit. In the absence of Ca2+ binding to TnC, the conformation of actin lament is such that its interaction with myosin heads is weak and the muscle bres are in a relaxed state. The ATP that is bound to myosin is hydrolysed by a myosin-catalysed ATPase. In the absence of calcium, the troponin/tropomyosin complex inhibits myosin-ATPase. But when intracellular calcium levels are raised, Ca2+ binds to TnC and myosinATPase inhibition is released, ATP hydrolysis occurs and muscle contraction ensues. The contracted state is maintained when Ca2+ and ATP levels are maintained at high levels (Figure 18). The SR around myobrils stores Ca2+ as well as the membraneATPase that is required for the release of Ca2+ and raises intracellular levels. There is therefore a homeostatic mechanism at work, maintaining a 10-7 M Ca2+ level in the resting muscle, and the increase of this by an order of magnitude produces the cascade of events leading to muscle contraction.
Tropomyosin Muscle contraction
Tnl
TnT
TnC
Myosin-ATPase
Ca
2+
Actin
conformational change
FIGURE 18
Cascade of events leading to muscle contraction upon Ca2+ binding to TnC.
119
The calciumTnC trigger leads to the contraction of cardiac and skeletal muscles. However, different mechanisms may be involved in the activation of myolaments of the heart and skeletal muscles. In heart myolaments, Ca2+ binds to one regulatory site on TnC but to two sites in skeletal myolaments. Besides, there are also differences with regard to Ca2+-induced conformational changes between cardiac and skeletal muscle (Spyracopoulos et al. 1997). In the smooth muscle, i.e., involuntary muscle under CNS control and muscle tissue of invertebrates, there is no SR. In these, intracellular Ca2+ alterations tend to be slower than those found in striated muscle. Furthermore, Ca2+ regulation of contraction involves MLCs, which have a high afnity for Ca2+. At low calcium levels the interaction of myosin head with actin is weak, but when the light chains bind Ca2+, the myosin headactin interaction activates myosin-ATPase leading to muscle contraction.
TROPOMYOSIN ISOFORMS
IN
BENIGN
AND
MALIGNANT CELLS
Tropomyosin isoforms are expressed in cells of muscle as well as nonmuscle origin. They are found in many organisms from unicellular ones to mammals (Pittenger et al. 1994). In human broblasts, eight isoforms have been identied and characterised. They are products of four genes (Novy et al. 1993a, 1993b). The isoforms of tropomyosin that have been identied range in molecular size from 32 to 40 kDa (J.J.C. Lin et al. 1985; Matsumura et al. 1983; Novy et al. 1993; Pittenger et al. 1994; Warren et al. 1985). The isoforms seem to differ with respect to the strength of their association with actin and may also differ in their function (Matsumura et al. 1985; Novy et al. 1993a; Gunning et al. 1997). The possible functional differences between the isoforms has been emphasised by the nding that they appear to be spatially sorted out or compartmentalised in neuronal development (Gunning et al. 1997). The localisation of tropomyosin isoforms shows a distinctive pattern in the kidney epithelial cells LLC-PK1. Temm-Grove et al. (1998) found that these cells expressed both high and low molecular weight isoforms of tropomyosin. The high molecular weight isoforms were associated with stress bres but not with adhesion belts. In contrast, the low molecular weight isoforms were found on adhesion belts. These differences in localisation were also encountered when these isoforms were introduced into cells by microinjection or transfection. These observations support the view that the different isoforms might subserve different functions. The loss of cell shape, cell adhesion to the substratum, and the loss of stress bres in cellular transformation has focused attention on the expression of tropomyosins in benign and malignant cells and tissues. The expression of tropomyosins has been reported to be frequently down-regulated in transformed cells (Cooper et al. 1985; Hendricks and Weintraub, 1981, 1984; Leavitt et al. 1986; J.J.C. Lin et al. 1985; Matsumura et al. 1983). In chick embryo broblasts transformed by RSV, a differential repression of tropomyosins has been reported to occur at the transcriptional level (Hendricks and Weintraub, 1984). The normal human broblast KD cells express four tropomyosin mRNAs. The expression of two of these of 1.1 and 3.0 kb was found to be markedly reduced in transformed cells and in a cell line derived from pancreatic cancer (Novy et al. 1993b). Leavitt et al. (1986) have reported alterations of tropomyosin isoforms that are expressed in human broblasts trans-
120
formed by chemical carcinogens. In these cells, the high molecular weight tropomyosins appeared to be down-regulated. However, there was a slight down-regulation of one and an up-regulation of another high molecular weight isoform in nontumorigenic immortalised cells. A change in pattern of isoform expression has also been reported to occur upon cell transformation by viruses (Matsumura et al. 1982, 1983). Oncogenic retroviral genes indeed have been shown to suppress tropomyosin synthesis (H.L. Cooper et al. 1985). Furthermore, the suppression of the tropomyosins appears to have a bearing on cellular morphology. Prasad et al. (1999) transfected v-src-transformed cells, which express the TM1 isoform at very low levels, with TM1 cDNA. This resulted in higher levels of TM1 expression in the transfected cells, which also showed a reduction in cell growth and enhanced cell spreading. These effects also were accompanied by alterations in the architecture of microlaments. Therefore, differential expression of the isoforms could be seen as suggesting differences in their cell function. A loss of high molecular weight tropomyosin isoforms and MLCs has been reported in tumorigenic cell lines as compared with nontumorigenic cells derived from prostate epithelium transformed by SV40 or X-irradiation (Prasad et al. 1997). However, TPA, which is a tumour promoter, is able to induce a high molecular weight isoform in human melanocytes (Vogt et al. 1997). Ovarian carcinomas reportedly have lower levels of a high molecular weight isoform of tropomyosin as compared with benign tumours (Alaiya et al. 1997). Franzen et al. (1996) found that the average levels of high molecular weight isoforms were far lower in breast carcinomas and nonmalignant tumours. The level of expression of one of these isoforms seemed to relate to the presence of tumour in the axillary lymph nodes, with the primary tumours showing 1.7-fold greater expression as compared with those that showed no nodal dissemination. The same high molecular weight isoform was found to be expressed at higher levels in H-ras-transformed broblast cell lines that possessed high metastatic ability than in transformed cells with lower metastatic ability. It may be pointed out that this implies that the more malignant the cell the greater is the level of expression of the high molecular weight isoform. This contradicts the relationship subsisting between the normal cell and its transformed counterpart, where transformed cells show a down-regulation of tropomyosin expression. Nonetheless, such a comparison might lead to oversimplied conclusions. It should be recognised that viral transformation of cells does not always lead to metastatic spread. The criterion of tumorigenicity does not contain any element of metastasis. For instance, tumours formed by SV40-transformed 3T3 broblasts rarely, if at all, metastasise. Furthermore, tumours are too heterogeneous and therefore association of metastatic ability with specic isoforms requires a detailed investigation of specic subpopulations of the tumour. Also desirable would be an investigation of primary tumours and the corresponding metastatic tumours. Hashimoto et al. (1996) compared K-1735 murine melanoma cells with differing metastatic potential. They reported that the isoform of tropomyosin was expressed only in the low metastasis variant of K-1735 melanoma. The differential expression of the high and low molecular weight isoforms in adhesion belts and stress bres (Temm-Grove et al. 1998) underwrites these efforts at exploring differential expression of tropomyosin isoforms in the progression of tumours to the metastatic state.
121
Cellcell and cellsubstratum adhesion are important factors in tumour cell dissemination and their adhesion to metastatic target tissues. However signicant might be the association between tropomyosins and neoplasia, it would be worth recalling here that TnI, which is the inhibitory component of the troponin/tropomyosin complex, might itself be associated with metastatic progression of cancer. As shown by Moses et al. (1999), TnI can inhibit angiogenesis both in vitro and in vivo and also inhibit metastatic dissemination.
THE REGULATORY ROLE
OF
CALDESMON
The cyclical interaction of the globular head domain myosin with thin actin laments coupled with the breakdown of ATP drives muscle contraction. Although reversible phosphorylation of MLC is regarded as an important regulatory event, there is general recognition that not all the events in muscle contraction can be explained by invoking this mechanism alone. Additional mechanisms have been sought. Caldesmon and calponin are two major proteins, both capable of binding to the major components such as actin, myosin, tropomyosin, and phospholipids (see Fattoum, 1997; Childs et al. 1992; Szymanski and Tao, 1997; Bogatcheva and Gusev, 1995), and both have been seen as offering alternative modes of regulation. Furthermore, both proteins interact with calmodulin in the presence of Ca2+ (Medvedev et al. 1996; Zhang and Vogel, 1997; Graether et al. 1997). Whether caldesmon and calponin have individual roles in the regulation of smooth muscle contraction is being debated. Some argue that the two proteins interact with each other. Graceffa et al. (1996) showed that a strong binding occurred between calponin and the carboxyl domain of caldesmon. They have suggested that this interaction could lead to the stabilisation of the complex. However, Czurylo et al. (1997) subscribe to the view that these two proteins have independent roles in muscle contraction. Caldesmon is found in both smooth muscle and nonmuscle cells. It is a regulatory protein that occurs in the groove of the actin helix. Caldesmon and calponin inhibit actin-mediated activation of myosin-ATPase in smooth muscle bres. Several segments of the C-terminal region of the caldesmon molecule may be involved in binding to actin and in its inhibitory function (Heubach et al. 1997). Whereas the C-terminal end binds to actin, the N-terminal end appears to be dissociated from but present in the close vicinity of actin and seems to mediate the link between myosin and actin laments (Graceffa, 1995, 1997). In the C-terminal domain encompassing residues 663-763, three regions have been identied that take part in myosinATPase inhibition: a central segment encompassing residues 747767, a segment that is N-terminal, and another segment C-terminal to this central segment. The central segment is essential but not sufcient to produce myosin-ATPase inhibition (I.D.C. Fraser et al. 1997). Two isoforms of caldesmon arising from alternative splicing of pre-mRNA are known. The high molecular weight (8993 kDa) isoform is restricted to adult and fully differentiated smooth muscle cells, whereas the nonmuscle low molecular weight isoform is found in de-differentiated nonmuscle cells. There are several conserved domains in the caldesmon molecule, which determine its interaction with and binding to actin, calmodulin, myosin, and phospholipids. All the isoforms are
122
powerful inhibitors of myosin-ATPase, but the specic nature of their distribution suggests possible differences in function. In nonmuscle cells, caldesmon could be stabilising the microlament network and in smooth muscle, it may be involved in the inhibition of muscle contraction (see Huber, 1997). The function of caldesmon in the regulation of cytoskeletal organisation and contractile processes is not limited only to animal cells. A caldesmon-like protein occurs in higher plants. A 107-kDa protein with properties similar and immunologically related to caldesmon has been isolated from extracts of pollen tubes of Ornithogalum virens. This protein binds to actin in a CaM/Ca2+-dependent manner (Krauze et al. 1998). This is consistent with the view that actomyosin cytoskeleton participates in the growth and elongation of pollen tubes. Pollen tube elongation is a calcium-dependent process (Masserli and Robinson, 1997). Caldesmon also can bind to S100 and proteins. The S100 binding site appears to be in the C-terminal region. Interestingly, the cross-linking of caldesmon via cysteine residues with S100 seems to reduce the inhibitory effect of caldesmon on myosin-ATPase activity, and S100 seems to be as effective as calmodulin in this respect (Polyakov et al. 1998). There are no implications, as yet, that the different isoforms of caldesmon differ functionally, because there is extensive sequence homology between isoforms occurring in different species (Novy et al. 1991). However, as stated above, the expression of some isoforms has been related to the state of differentiation. Furthermore, early studies by Novy et al. (1991) have shown a two- to four-fold reduction in the expression of caldesmon in certain transformed cell lines.
CALPONIN: ITS FUNCTION
AND
REGULATION
Calponin is a 33-kDa protein highly expressed in smooth muscle cells. The human smooth muscle calponin gene contains seven exons encompassing >11 kb and it has been assigned to chromosome 19p13.2 (Miano et al. 1997). Calponin is a thin lament-associated TnT-like protein. It is strongly implicated in smooth muscle contraction. As with caldesmon, calponin binds to actin, myosin, tropomyosin, and CBPs such as calmodulin. Actin possesses two calponin-binding sites (Kolakowski et al. 1997; Mino et al. 1998). The interaction between calponin and myosin occurs via dened regions of the calponin molecule (Szymanski and Tao, 1997). Three isoforms of calponin have been described: the basic h1 isoform, the neutral isoform h2, and the acidic isoform. Two distinct genes encode the isoforms h1 and h2. They are not generated by alternative splicing of pre-mRNA (Fukui et al. 1997). All three isoforms contain the calponin-homology (CH) actinbinding domain in the N-terminal region of the protein. The CH domain comprises a sequence motif containing approximately 100 amino acid residues. Some actincross-linking proteins may contain two distinct CH domains and have contested the rather generalised concept that a single CH domain is sufcient to confer actinbinding properties on these proteins (Stradal et al. 1998). CH domains also occur in other actin-interacting proteins. Fimbrin is an actin-cross-linking protein. Its association with F-actin is similar to that of calponin (Hanein et al. 1997b) and, furthermore, it contains two CH domains. The proto-oncogene vav, which is
123
expressed in haematopoietic cells, codes for a signalling molecule that contains a CH domain (Castresana and Saraste, 1995; Romero and Fischer, 1996). Nonmuscle isoforms of calponin have been reported in brain tissue. The h2 isoform was identied by Fukui et al. (1997) from human skin tissue and human keratinocytes in culture using antibodies raised against smooth muscle calponin. Calponin generally has been attributed with the ability to inhibit myosin-ATPase. In vivo, calponin occurs in the ratio of approximately one calponin molecule to seven actin monomers (Lehman and Kaminer, 1984; Takahashi et al. 1986; Lehman, 1991), but there is a view that higher levels of calponin may be required for an effective inhibition of myosin-ATPase (Gimona and Small, 1996). Exogenous calponin has been shown to inhibit Ca2+-responsive muscle contraction (Horowitz et al. 1996; Naka et al. 1998). Calponin serves as a substrate for several kinases and phosphatases, and its function seems to be regulated by phosphorylation. Pohl et al. (1997) have shown that unphosphorylated calponin is able to inhibit actin sliding, which is demonstrable using in vitro motility assays, but when calponin is phosphorylated by PKC this effect is inhibited. In vivo, calponin puried from tracheal smooth muscle stimulated with carbachol (carbamyl choline chloride), which is a Ca2+-mediated stimulator of phosphorylation, showed two-fold greater phosphorylation than unstimulated muscle. Naka et al. (1998) also demonstrated a correlation between calponin phosphorylation and contraction of porcine coronary artery. However, as discussed below mechanisms other than phosphorylation have been adduced for calponin regulation, e.g., one involving caltropin. Aside from being regulated by phosphorylation, it has been reported that calponin co-precipitates with MAPK, but there is no obvious phosphorylation of calponin. The latter also co-precipitated with PKC-. A translocation of calponin to the cell membrane occurs in cells that are stimulated with phenylephrine, when MAPK and PKC- are also translocated to the cell membrane. On the basis of these ndings, Menice et al. (1997) have implicated calponin in signal transduction. Indeed, the targeting of these various components to the cell membrane might be a consequence of the tight binding of calponin to cytoplasmic -actin (Parker et al. 1998). Calponin seems also to be able to regulate the amount of free actin available for cytoskeletal organisation. The h2 isoform, for instance, appears to be localised in the cytoplasm of basal cells in situ, whereas in cultured keratinocytes it occurs along the cellcell contact areas, which suggests that it might play a part in the organisation of the cytoskeleton. Tang et al. (1997) believe that calponin, being polycationic in nature, could promote the formation of F-actin bundles by reducing the polyanionic repulsive interaction between actin laments. Calponin can also bind to tubulin, which decreases with increases in Ca2+ concentration and ionic strength (Fujii et al. 1997). These authors also state that calponin has distinct sites for binding actin and tubulin. Furthermore, calponin seems to bind strongly to desmin and is incorporated into IF and, therefore, it is possible that it might mediate the association of desmin IF with actin (Mabuchi et al. 1997). The ability of calponin to promote cytoskeletal organisation may be reected in its ability also to inhibit cell growth (Z. Jiang et al. 1997). It also has been reported recently that the expression of calponin and smooth muscle MHC expression can distinguish between in situ and invasive carcinoma of the breast. Antibodies against
124
MHC and calponin stain epithelial cells of ducts and acini of normal breast tissue and carcinomas in situ, but these antibodies hardly show any reactivity in invasive carcinomas (Wang et al. 1997). Calponin is prominently expressed in myobrils of leiomyosarcomas (Meyer and Brinck, 1997). Overall, calponin seems to show a generally reduced expression in human leiomyosarcomas. Compatible with the lack of its expression in invasive breast carcinomas as compared with in situ tumours are the recent ndings of Horiuchi et al. (1999). These authors transfected human calponin h1 into leiomyosarcoma cells. The transfectants showed a one third reduction in cell proliferation, together with reduction in anchorage-independent growth as well as tumorigenicity. Nonetheless, much further work is required before one can attribute a tumour suppressor function to calponin.
CALTROPIN-MEDIATED REVERSAL OF MYOSIN-ATPASE INHIBITION BY CALDESMON AND CALPONIN

It is obvious from the above discussion that caldesmon and calponin both inhibit myosin-ATPase and inuence muscle contraction. The binding of caldesmon and calponin to actin appears to produce this inhibition and this effect is reversible by calmodulin. Another CBP, i.e., caltropin, appears to be involved in the regulation of the inhibitory effects of caldesmon and calponin on myosin-ATPase. Caltropin is an 11-kDa protein derived from smooth muscle. It occurs as a dimer in the native form (Willis et al. 1994). Some years ago, Mani et al. (1992) demonstrated that caltropin, like CaM, was able to reverse the inhibition of myosin-ATPase by caldesmon in the presence of Ca2+. They found that caltropin directly interacted with caldesmon. This seemed to inuence the interaction of caldesmon with the actin component of heavy meromyosin (Mani and Kay, 1993, 1995a). In vitro, caltropin seems to inhibit the polymerisation of G-actin (Mani and Kay, 1995b), thus providing further conrmation of interference by caltropin in caldesmonactin interactions. Although Mani et al. (1992) state that caltropin is as effective as CaM in reversing caldesmon-mediated inhibition of myosin-ATPase, there is evidence that the binding afnity of caltropin to caldesmon is far greater than that of CaM (Zhuang et al. 1995). In consonance with these observations are the ndings of Willis et al. (1994) that caltropin also is able to form a complex with calponin in a calcium-dependent fashion in the ratio of 2:1 and reverse the inhibitory action of caldesmon. Caltropin has been described as being more efcient than CaM. Thus there seems to be a reasonable body of evidence, albeit from the same laboratory, that caltropin has considerable implications in the regulation of myosin-ATPase function.
Structure and Biology of Calbindin
Calbindins D-28K (CBD) and D-9K (CBD-9K) are EF-hand proteins that are regarded as calcium buffer proteins together with parvalbumins (PV). CBD is a type III EF-hand protein possessing six EF-hands (see Table 4), all of which seem to be packed in one globular domain, but the folding and packing of individual domains within the globular domain appears to be specic (Linse et al. 1997). The rat CBD9K gene consists of three exons and two introns. Exon 2 contains sequences encoding the rst EF-hand and exon 3 for the second EF-hand domain.
CALBINDIN IN NEURONAL POPULATIONS

CBD is a calcium-binding protein that is expressed abundantly in a number of cell types, such as neuronal cells, mammalian and avian kidney, brain, and pancreas, and in avian intestines. Wasserman and Taylor (1966) isolated this calcium-binding EFhand protein from the intestine of chicken later found to be a component of the nervous system of a wide variety of species (Celio, 1990; Reifergerste et al. 1993). The occurrence of CBD is predominent in long-axon as well as in short-axon neurones (see Baimbridge et al. 1992; Celio 1990; Frantz and Tobin, 1994; Sequier et al. 1990). By virtue of its calcium buffering ability as well as its ability to modulate calcium channel activity, CBD could be regulating calcium homeostasis in neurones (Albritton et al. 1992). CBD also occurs in the neuroendocrine cells of adult and foetal lung of hamsters. This has been demonstrated by immunochemical staining together with neuroendocrine markers. In contrast, other CBPs, such as PV and calretinin, are not detectable (T. Ito et al. 1998). Calretinin is a protein that bears a high degree of homology with CBD and shows similar calcium-binding properties (Cheung et al. 1993). The occurrence of CBD and calretinin are often mutually exclusive, although many cells may express both proteins at the same time (Rogers and Resibois, 1992). In light of the sequence similarities, it may be suggested by inference that these proteins subserve similar functions and could dene similar neuronal properties.
NEURAL CELL LINEAGE AND THE REGULATION OF CALBINDIN EXPRESSION

It has been argued that clonally related cortical neurones do not express the same complement of CBPs, and therefore their expression may be controlled and inuenced by extracellular factors rather than be genetically endowed. Pappas and Parnavelas (1997) exposed cultures of developing brain cortex from rat embryos to bFGF and the neurotropins NGF, brain-derived neurotropic factor (BDNF), and
125
126
neurotropin-3 (NT3). They found an enhanced morphological differentiation of large GABA-containing neurones together with the expression of calbindin, the effects being dependent on age and maturation of the subpopulations. As stated before, calbindin is highly expressed particularly in Purkinje cells of the cerebellum, but it also occurs in other neuronal cell types in other regions of the CNS. The specic expression in Purkinje cells seems to be regulated by a 40-bp element, occurring in the promoter region of the calbindin gene. This regulatory element called PCE1 is present also in the promoter of calmodulin II and, obviously, can regulate the expression of other CBPs as well (Arnold and Heintz, 1997). Cell type-specic transcriptional regulation mediated by CBPs may underlie this phenomenon, and it follows from this that different extraneous factors might differentially affect the expression of these proteins depending on the neuronal subpopulation. These CBPs in turn may regulate the expression of transcription factors that induce the expression of other responsive genes. This could rationalise the presence of calbindin in a wide variety of tissues and their responses to a variety of extracellular stimuli. It should be recognised, nevertheless, that other mechanisms such as enhanced stability of transcripts of target genes might also be involved.
CALBINDIN EXPRESSION IN EMBRYONIC DEVELOPMENT AND AGEING

Calbindin expression changes during embryonic development to develop into an adult expression pattern. In the cerebellum, calbindin gene transcripts as well as the protein increase to reach a peak at 2 weeks postnatal stage (Iacopino et al. 1990). CBD is transiently expressed during embryonic development (Andressen et al. 1993). In mouse embryos, cerebellar Purkinje cells appear from 11 to 13 days of gestation (Miale and Sidman, 1961), and soon after, they begin to express CBD (Enderlin et al. 1987). CBD is the major component cellular protein of mature Purkinje cells, and the high protein levels maintained in adult life decrease in ageing animals (Baimbridge et al. 1982; Lledo et al. 1992). In developing human cerebellum, CBD immunoreactivity is rst encountered at 14 weeks of gestation, with CBD expression being detectable in a variety of cell types. However, the number of CBD staining cells and the intensity of staining decreases with development. At 21 to 31 weeks of gestation, CBD reactivity is restricted to Purkinje and basket cells of the cerebellar cortex (Yew et al. 1997). A developmental pattern is also discernible in the appearance of CBD in neuroendocrine cells of foetal hamster lung from days 15 and 16 of gestation, but endocrine cells appear on day 13 (Ito et al. 1998). In the hamster brain, CBD transcripts have been reported to decrease markedly (50 to 68%) in 19 to 24 month-old hamster as compared with 4- and 9-month-old animals, but, in contrast, calretinin and PV do not show any changes with ageing (Kishimoto et al. 1998). Calbindin immunoreactivity changes in the cerebellum of frog tadpoles induced to metamorphose by treatment with thyroxin (Uray et al. 1998). The potential role of calbindin in cell differentiation has not received much attention to date. Together with PV, calbindin has been shown to be associated with phenotypic conversion of supporting cells into hair cells (Steyger et al. 1997). However, the extent of their individual contribution to the nonmitotic regeneration
127
of hair cells is unclear at present. At any rate, the information available is scanty, when compared with the massive evidence available about the effects of S100 proteins on cell growth and differentiation.
PHYSIOLOGICAL FUNCTION OF CALBINDIN

Calbindin expression is modulated in a variety of tissues by several extracellular signals and in an apparently tissue-specic manner. There are strong pointers that calbindin may have several functions in normal cellular physiology and that its expression in these tissues may be regulated by specic extracellular ligands by different mechanisms resulting in specic outcomes. Therefore, calbindins could perform tissue-specic functions (Table 8). Among the functions attributed to these CBPs are facilitation of calcium ux, protection of neurones from degeneration, and as a calcium buffer in restricting calcium signals in nerve synapses and hair cells. Neuronal ring patterns may be determined by calbindin (Chard et al. 1995), and in hippocampal slices it can modify synaptic interactions (Heizmann and Braun, 1992).
TABLE 8 Extracellular Biological Signals Inuencing Expression of Calbindins

Extracellular Signal 1,25-dihydroxy vitamin D3 Oestrogen Adrenal steroids (corticosterone) Growth hormone Target Tissue Intestine, kidney Kidney Hypothalamic tissue Intestine Comments Not brain tissue Independently of vitamin D3 During prenatal development Calbindin D-9K mRNA, also induced in parallel, mRNAs for vitamin D3 receptor and insulin-like growth factor (IGF)-1 Parallel production of insulin and its secretion Ref. Christakos et al. (1989) Criddle et al. (1997) Lephart et al. (1997a) Salih et al. (1997)
Butyrate
Rat insulinoma cell line
S. Lee et al. (1994)
The regulation of calbindin expression by VD3 in the kidney and the intestine has been well documented. In contrast, VD3 does not affect calbindin expression in brain tissue. Absorption of calcium and lead in the intestines is greatly affected by serum VD3. Conversely, serum levels of VD3 increase markedly with lead ingestion and calcium deciency. Also apparent, under these conditions, are changes in the expression of calbindin, which suggests a possible mediation of calcium and lead absorption by calbindin (Fullmer, 1997). In MadinDarby bovine kidney (MDBK) cells, TPA has been found to enhance CBD expression, and this is preceded by the activation and translocation of PKC-. Furthermore, PKC phosphorylates CBD in
128
a calcium- and phospholipid-dependent manner. VD3 also increases CBD expression as well as up-regulates the expression of PKC. PKC-mediated phosphorylation of two threonine residues of CBD could lead to the functional activation of CBD, which is consistent with the transduction of calcium signal by PKC mediation (Gagnon and Welsh, 1997). Calbindin seems to be involved with insulin secretion by pancreatic cells. Lee et al. (1994) found that butyrate is able to induce CBD mRNA as well as protein levels in the rat insulinoma cell line RIN 1046-38. This treatment also enhanced insulin production and its secretion. The involvement of CBD in insulin secretion has been conrmed in further experiments by Reddy et al. (1997), who transfected the insulinoma cell line with the CBD gene. In the transfected cells a >20-fold increase in CBD mRNA was found, and at the same time insulin mRNA showed a >20-fold enhancement. Insulin production and release also increased approximately six-fold. It seems possible that this effect of CBD is also mediated by PKC, because activation of PKC can inuence insulin secretion. Both CBD and CBD-9K appear to be capable of regulating genetic transcription (Reddy et al. 1997; Fukushima et al. 1998). Possibly, calbindin inuences the process of calcium-mediated regulation of transcription factors (see Reddy et al. 1997). It is to be expected that the expression of calbindins themselves would be subject to regulation at the transcriptional level. That the specicity of their expression might be linked with denable transcription pathways is an interesting thought that has been explored experimentally. Arnold and Heintz (1997) have identied a 40-bp element, the PCE1, that is necessary for the expression of the CBD in Purkinje cells. This element seems to regulate transcription of CBD gene. PCE1 also occurs in calmodulin II. Both CBD and calmodulin II are expressed abundantly in Purkinje cells, and PCE1 might be a common component in the regulation of expression of these CBPs in Purkinje cells. CBD and calmodulin II are expressed in other cell types too. Transcription elements other than PCE1 may be involved in the regulation of the genes expression at these sites (Arnold and Heintz, 1997). Oestrogen-mediated induction of CBD expression is attributed to two regions (1075/702 and 175/78) of the mouse CBD promoter (Gill and Christakos, 1995). Similarly, oestrogen can regulate specic regulatory sequences of CBD-9K (Romagnolo et al. 1996). The levels of expression of CBD may be regulated by other means, as indicated by Wang and Christakos (1995), who found that retinoic acid (RA) increased, by 10- to 15fold, both CBD protein and CBD mRNA in the medulloblastoma cell line D283. The RA effect was mediated by RA-specic receptors, which ordinarily would have indicated the regulation of transcription of the target gene by RA (see Redfern, 1997). However, the regulation in this case did not appear to occur at the transcriptional level, but the increased expression of CBD and its mRNA seemed to be due to an increase in the stability of the mRNA. CBD seems to play a role in renal reabsorption of calcium. Ovariectomy reduces the expression of CBD gene transcripts, and this is reversed by the administration of 17-oestradiol in the ovariectomised animals (Criddle et al. 1997). These alterations in CBD expression show no relation to serum levels of VD3. Thus the role played by CBD in renal absorption may be distinguished from that in intestinal tissue.
129
NEUROPROTECTIVE FUNCTION OF CALBINDIN

CBD is generally regarded, in common with PV and calretinin, as an intracellular calcium buffer because it has not been found to inuence enzyme activity or calcium ion channels (see Baimbridge et al. 1992). Nevertheless, buffer function may modulate intracellular calcium levels and in this way regulate neuronal function. Thus, distinct physiological functions may be identied that are affected by alterations in the intracellular levels as well as the conformational state of CBD. For instance, calretinin deciency seems to lead to an impairment of motor coordination and marked abnormalities in the ring behaviour of Purkinje cells in vivo. In calretinin null (cr /) Purkinje cells, CBD is detected by anticalretinin antisera. This has been attributed to the fact that these antisera detect the presence of CBD that has undergone conformational changes upon being saturated with Ca2+ (Schiffmann et al. 1999). These events clearly implicate an impairment of calcium homeostasis in the cr (/) cells in vivo. The pattern of CBD occurrence in ageing neurones has led to the postulate that it may function as a neuroprotective agent. Lally et al. (1997) found that the size and number of CBD-immunoreactive neurones were reduced in Alzheimers disease, and this has been suggested to be a consequence of cellular degeneration related to the reduced CBD. Alzheimers nerve cells containing CBD are believed to be less susceptible to degeneration than those that have greatly reduced amounts of CBD or no CBD at all. This is supported by experiments with PC12 cells into which CBD cDNA was transfected. These cells were far less susceptible to degeneration caused by serum withdrawal, glutamate, and the neurotoxin 1-methyl-4-phenylpyridinium. However, it would appear that CBD cannot protect these cells from degeneration caused by calcium ionophores (McMahon et al. 1998). Calbindin-null mutant mice show severe impairment of motor coordination (Airaksinen et al. 1997). However, these null mutants do develop normally without any disturbances in calcium metabolism and, therefore, Airaksinen et al. (1997) believe that CBD expression in peripheral organs may not be crucial for normal development.
CALBINDIN EXPRESSION AND THE METASTATIC PHENOTYPE

The ability of steroids to inuence the expression of calbindins has led to investigations aimed at examining their potential involvement in the development and growth of tumours, their progression, and prognosis. Watanabe et al. (1994) investigated CBD levels of lung cancers. The levels of CBD were low in normal lung tissue, but its expression was higher in lung cancers, with SCLC tissue expressing greater amounts than non-SCLC (NSCLC). Furthermore, CBD levels correlated with progression of NSCLC to advanced stages of the disease involving metastatic involvement of the lymph nodes. Upon investigating cell lines derived from these tumours, Watanabe et al. (1994) noticed that CBD expression might also relate to the expression of neuroendocrine-related paraneoplastic properties of these tumours. They found that the CBD could be used to differentiate classical SCLC from variant
130
SCLC. The specicity and sensitivity with which this could be achieved was comparable to the neuroendocrine marker neurone-specic enolase. Signicant differences also have been noted in the expression of CBD in human colonic cell lines derived from primary tumours and those derived from metastatic tumours. Sampson-Johannes et al. (1996) found that SW480 cells obtained from primary colorectal tumours did not express CBD, but in sharp contrast, SW620 cells derived from metastatic tumours did. They also demonstrated that SW480 cells were not capable of colonising human foetal lung tissue transplanted into SCID-hu mice, but SW620 cells colonised the grafts upon intravenous introduction into the animals. Furthermore, SW620 cells expressed sialyl Lewis-x and Lewis-a antigens which are conducive to metastatic spread at far higher levels than SW480, which were unable to colonise the foetal lung grafts. On the basis of these results, the authors suggest that the appearance of the metastatic ability could be associated with the acquisition of CBD expression. It ought to be recognised, however, that this experimental model is too remote from the spontaneous metastasis. The differences in the abilities of the two cell types to localise in foetal lung grafts may simply reect their adhesive capabilities, rather than CBD expression. Some preliminary work has been reported (Castro et al. 1998) that relates the value of CBD expression to prognosis of lung cancer.
Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker
CALRETININ AND ITS ALTERNATIVELY SPLICED ISOFORMS

Calretinin is a neuronal calcium-binding protein. It shows marked homology with CBD, and presumably on account of this, calretinin and CBD show mutually exclusive expression in neuronal populations, although in some cell types both may be expressed simultaneously (Rogers and Resibois, 1992). The pattern of their expression and marked similarities between them with respect to calcium-binding properties (Cheung et al. 1993) could indicate that they perform similar functions in neurones. Thus, calretinin seems to share with CBD certain features such as regulation of expression by growth factors and involvement in cell proliferation, differentiation, and neoplastic transformation. Also, calretinin may possess, in common with CBD, a neuroprotective property. On the other hand, the apparent mutually exclusive nature of their expression might be a reection of the cooperative effects between calretinin and CBD. The calcium-buffering function of calretinin might result in CBD not being detected by specic antisera. As stated in the previous section, in cr (/) Purkinje cells, there is an impairment of calcium homeostasis. As a result, CBD is oversaturated with calcium. This brings about changes in the conformational state of CBD. As a consequence, CBD becomes detectable by specic antisera (Schiffmann et al. 1999). These conformational changes seem to translate into alterations in cell function. Therefore, the experiments of Schiffmann et al. (1999) might suggest a cooperation between calretinin and CBD in function. Calretinin is expressed as two splice variant isoforms, encoding two proteins of 20 and 22 kDa molecular size, truncated at the C-terminal region. The 20-kDa isoform is a result of splicing of exon 7 to exon 9, and the 22-kDa isoform results from splicing of exon 7 to exon 10. Both alternatively spliced messages show frame shift and contain stop codons in exon 9 (in calretinin-20K) and exon 10 (in calretinin22K) (Schwaller et al. 1995). Calretinin-22K contains the rst 178 amino acid residues of unspliced calretinin, and this sequence is followed by a C-terminal 14 amino acid sequence that is not found in the unspliced calretinin (Gander et al. 1996). Antibodies raised against this 14-amino acid sequence do not recognise fulllength calretinin, which indicates the novel nature of the C-terminus of calretinin22K. However, in spite of their variant secondary structure, the calcium-binding
131
132
properties of calretinin-20K and -22K appear to be unaffected (Schwaller et al. 1995). Calretinin-22K occurs in many colon carcinoma cell lines. However, the signicance of the expression of splice variants remains unclear, especially in view of the small numbers of cell lines studied and the absence of information on the stability of the variant isoforms and their incidence in vivo.
REGULATION OF CALRETININ EXPRESSION

The expression of calretinin is regulated by a number of neurotropic agents and growth factors and correlates with the appearance of phenotypic features of differentiation. Farkas et al. (1997) found that glial cell-derived neurotropic factor (GDNF) increased the number of neurones expressing calretinin in cultures of embryonic striatal neurones. The numbers of calretinin-expressing cells also is increased by bFGF, alone or in combination with retinoic acid (Pappas and Parnavelas, 1998). The neurotropic factor BDNF induces the extension of neuronal dendrites of hippocampal sections maintained in culture. The subpopulations that respond to BDNF appear to be those that express calbindin and calretinin (Marty et al. 1996). There are also indications that there could be some specicity in the morphological response to the neurotropic factor. No response to BDNF is observed in neurones that express PV. Furthermore, the calretinin content of CajalRetzius neuronal cells is regulated by BDNF together with induction of differentiation (Marty et al. 1996), but the latter is not essential for their survival. Calretinin expression in medial basal hypothalamic regions of male rats is reported to be greater than that of female rats at 19 and 20 days of gestation, suggesting a developmental and hormonal regulation of calretinin (Lephart et al. 1997b). There is no question that expression is developmentally regulated as shown by Jiang and Swann (1997), in the evolution of the neuronal populations and the emergence of cells expressing calretinin in the mature hippocampus of the rat. The development and maturation of neurones of the chick dorsal root ganglion are closely related to the expression of calretinin. The ganglia become calretinin positive after 9 days of incubation, and the number of calretininexpressing cells increases with the length of incubation (Kiraly and Celio, 1992). Calretinin-staining clusters of cells are also detected in the medullary epithelium of the thymus at day 11 of incubation, coinciding with the functional maturation of the thymus (Kiraly and Celio, 1993). It is of interest that PV is detected in the epithelial cells at day 9, but CBD is not detectable. This would suggest that at least in some developing organ systems, calretinin and CBD may be individually associated with functional maturation, in spite of their molecular homology to each other. The expression of calretinin and CBD in the oxytocin (OT)- and vasopressin (VP)-containing neurones of the supraoptic nucleus has been the subject of a detailed investigation by Miyata et al. (1998). In both types of neurones, there is a marked differential distribution of calretinin and CBD positivity. In OT neurones, 72 to 84% of cells were CBD immunoreactive as compared with ca. 50% staining for calretinin. In VP neurones, no calretinin staining cells were detectable and only 25% of neurones stained for CBD. Whether this can form a functional differentiation between the two CBPs is uncertain. However, because calretinin and CBD do not differ
Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker
133
substantially with respect to their calcium-buffering ability, the possibility that they may yet be functionally different in other respects needs to be explored further.
CALRETININ EXPRESSION IN CELL PROLIFERATION AND DIFFERENTIATION

The relationship between calretinin expression and cell proliferation and the state of differentiation also has been reported in nonneuronal cell types. Indeed, studies of several tumour cell lines have provided evidence supporting such a relationship. Gotzos et al. (1996a) found that 8 of 10 colonic carcinoma cell lines expressed calretinin, and the protein was found in rapidly proliferating cells. Upon being induced to differentiate by treatment with sodium butyrate and hexamethylene bisacetamide, WiDR colon adenocarcinoma cells showed marked reductions in cell proliferation as well as expression of full-length calretinin and its splice variant isoform calretinin-22K (Schwaller and Herrmann, 1997). HT29-18 colon adenocarcinoma cells have been induced to differentiate into enterocytes by using a different strategy glucose starvation. Again, a marked reduction in calretinin mRNA was found in the differentiated cells (Cargnello et al. 1996). These authors read between the lines and suggest that calretinin may maintain an undifferentiated proliferative state characteristic of neoplastic transformation.
CALRETININ AND ITS POSSIBLE NEUROPROTECTIVE PROPERTY

In common with CBD, calretinin may be neuroprotective. In Alzheimers disease, large pyramidal neuronal cells show a differential susceptibility to degeneration, and specic subpopulations, which express calretinin, might be resistant to degeneration (Hof et al. 1993). The presence of calretinin also seems to provide some protection against serum deprivation of rat cerebral cortex organ cultures (Weisenhorn et al. 1996). A study of the expression of calretinin, together with CBD and PV mRNA, in hamster brain in relation to ageing, has also helped to focus attention on differences between calretinin and other CBPs. Kishimoto et al. (1998) found that whereas CBD transcript levels fell by 50 to 68%, calretinin and PV transcripts remained unchanged. Implicit in these ndings is the suggestion that the down-regulation of CBD expression associated with the ageing process might reect the neuroprotective action of CBD, and further, by inference, that calretinin may not function in this way. However, it ought to be recognised that most of the available evidence is too preliminary in nature to warrant conclusive interpretation.
CALRETININ AS A POTENTIAL TUMOUR MARKER

The expression of calretinin in a number of colonic carcinoma cell lines and the loss of its expression upon differentiation, which has been discussed in the previous sections, has inevitably led to the suggestion that it may serve as a potential maker
134
of human malignancies. Gotzos et al. (1996b) examined a series of mesotheliomas for the expression of calretinin, PV, and CBD. Calretinin was found in mesotheliomas of the epithelial type and in the epithelial component of mixed tumours. Sarcomatoid mesotheliomas and the sarcomatoid component of mixed tumours did not express calretinin. Neither were PV and CBD expressed in these tumours or in normal lung tissue. Furthermore, none of the three CBPs was detected in adenocarcinoma of the lung. Therefore, Gotzos et al. (1996b) suggest that calretinin expression could provide a useful means of differentiating epithelial-type mesotheliomas from metastatic adenocarcinoma of the lung. Doglioni et al. (1996) have claimed a diagnostic sensitivity of 100% in a series of 44 mesotheliomas investigated. Leers et al. (1998) have advocated a combination of cadherin and calretinin to distinguish between metastatic carcinomas and mesotheliomas and regard this combination of markers as a powerful tool. Calretinin has been proposed as a marker for serous carcinoma of the ovary (Folpe and Gown, 1997). Also, high levels of calretinin-22K have been found in the serum of many cancer patients. Serum levels of calretinin in breast and colon cancer have been reported to be very high. The protein is detectable in epithelial cells, nerve bres, connective tissue, and mesothelial cells. Calretinin also has been found in ischemic necrosis of the gut (Schwaller et al. 1998). Needless to say, there is much scope for investigating the clinical value of serum calretinin determination in tumour typing. There is virtually no information available about its relevance in cancer prognosis.
10
Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading
MOLECULAR FEATURES OF CALCINEURIN

Among more than 30 target proteins activated by CaM is calcineurin. Calcineurin is a serine/threonine phosphatase. It is also known as protein phosphatase (PP)-2B. Together with PP-1, PP-2A, and PP-2C, calcineurin forms an important group of phosphatases that play a critical role in the phosphorylation/dephosphorylation cycles associated with regulation of the activity of a variety of enzymes. Calcineurin is highly conserved as well as widely distributed. Nevertheless, it has restricted substrate specicity. Its major substrates are the NF-AT and other members of the NF-AT family of transcription factors, N-methyl-D-aspartate (NMDA) receptors (Barford, 1996; Tong et al. 1995; also see below), and IP3 receptors (Cameron et al. 1995; Cunningham, 1995). Microtubule-associated proteins MAP-2 and the tau protein (Ferreira et al. 1993; see below for further references) are also among its substrates. The biochemical and pharmacological significance of these substrates have been discussed below in the context of the various physiological functions performed by calcineurin. Calcineurin is a major protein of brain tissue, accounting for approximately 1% of its total protein. In brain tissue it is associated with the cytoskeletal structures or is bound to the plasma membrane. It occurs as a major component of neurones, especially of the neurostriatum and cerebellum. Lymphocytes also contain large amounts of calcineurin (Kincaid et al. 1987). Calcineurin is composed of two subunits: a catalytic subunit known as calcineurin A and a regulatory subunit called calcineurin B. Calcineurin is usually isolated with bound iron and zinc ions, but it still requires the binding of exogenous metal ions to achieve maximum activity. Calcineurin possesses low catalytic activity in the absence of exogenous metal ions. It can be activated by Mn2+, Mg2+, and Ni2+. The activation by Mn2+ seems to involve the catalytic subunit calcineurin A. The calcineurin B sequence is highly conserved, as compared with calcineurin A. Nevertheless, in the latter the catalytic domain is highly conserved in evolution. Calcineurin A is 59 kDa in size and binds CaM. The regulatory 19-kDa calcineurin B contains four EF-hand calcium-binding domains. It is encoded by a single gene and
135
136
contains 163 amino acid residues (reviewed by Klee et al. 1988). However, alternative splicing might result in a number of variant forms of calcineurin B (C.D. Chang et al. 1994). Three isoforms (, , and ) of calcineurin A and two of calcineurin B have been described (Tumlin, 1997). Calcineurin A- has been mapped to human chromosome 10q2122, and calcineurin B to chromosome 2p1516. The calcineurin A- occurs on chromosome 4 (M.G. Wang et al. 1996).
CALCINEURIN IN CELL PROLIFERATION AND ADHESION-RELATED PHENOMENA

Calcineurin has been implicated in a number of physiological events, such as cell proliferation, cell death, and signal transduction, and in immunosuppression. It also has been implicated in certain functions of the nervous system, e.g., in neurotransmission. The discussion here will be restricted mainly to areas pertinent to the biological behaviour of cancers, although some reference will be made to Alzheimers disease, in which cytoskeletal abnormalities occur prominently. The regulation of physiological activity of a large number of biological macromolecules is dependent on phosphorylation. It is to be expected that, as a phosphatase, calcineurin would be a key component in the phosphorylation of some of these molecules. From this it should follow, therefore, that calcineurin would impinge signicantly on the biological behaviour of cancers and in other disease states.
PUTATIVE ROLE
OF
CALCINEURIN
IN
CELL CYCLE PROGRESSION
There is a large body of evidence that points to the importance of calcineurin in the progression of the cell cycle. Calcineurin A gene seems to be essential for the G1S transition of Aspergillus nidulans cells. A disruption of the gene by homologous recombination results in the arrest of the cell cycle in its early phase. Calcineurin A mRNA accumulates in the G1 phase well before the G1S transition point (Rasmussen et al. 1994). The induction of DNA synthesis in response to growth factor stimulation of cells has been shown to depend on Ca2+ uptake and on the activation of calcineurin (Tomono et al. 1996). A suggested modus operandi is by the regulation of specic cyclins. The initiation of DNA synthesis and mitosis is controlled by two key factors: (1) cyclin-dependent protein kinases (cdk) composed of a cyclin regulatory subunit and a cell division control (cdc2) family kinase and (2) CDK inhibitors (see Sherbet and Lakshmi, 1997b for further information). The involvement of calcineurin in the progression of the cell cycle is demonstrated by the fact that the inhibition of calcineurin leads to an inhibition of broblast growth factor FGF-induced expression cyclins A and E in Swiss 3T3 cells (Tomono et al. 1998). Calcineurin seems to be associated also with determining the length of the G2 phase of the cell cycle and entry of the cell into the mitotic phase. The G2M transition, in Saccharomyces pombe for instance, is dependent on the activation by dephosphorylation of a complex of G2specic cyclin with cdc2 kinase. This process of activation is inhibited by Swe-1 (Wee1 homologue) tyrosine kinase. The transcription of Swe-1 is regulated by Zds-1. Calcineurin and Mpk-1 seem to regulate the transcription and posttranslational modication of Swe-1, respectively (Mizunuma et al. 1998).
137
It would be of considerable interest, therefore, to examine whether some of the effects of calcineurin could be explained by its putative participation in the phosphorylation events associated with cell cycle progression. A number of cyclins and cdk are expressed in a cell cycle-related fashion. The cyclin D/cdk-4 complexes are activated by cdk-activating kinases. This activated complex now phosphorylates rb protein and enables the cell to transit into the S phase. The cyclin B/p34cdc2 complex is associated with the G2M transition of cells. The phosphorylation of p34cddc2 at tyrosine 161 is believed to activate it, which enables the cell to make the G2M transition. Phosphorylation of threonine 14/tyrosine 15 is believed to produce a negative regulatory effect on cell G2M transition (see Sherbet and Lakshmi, 1997b for references). Calcineurin, being a threonine phosphatase, could conceivably affect this negative regulation of the G2M transition and allow cells to enter the mitotic phase. It is also possible that calcineurin functions by means other than altering the cyclins associated with the G1S transition. Tomono et al. (1998) found that cyclosporin A, at calcineurin-inhibiting concentration, inhibited cyclins A and E, but not DNA synthesis. An interesting aside to this story is that carbachol, a Ca2+-mediated stimulator of tyrosine phosphorylation, induces the formation of actin stress bres and markedly increases the F-actin/G-actin ratio in smooth muscle cells in vitro. Tyrosine kinase inhibitors have been found to inhibit the formation of stress bres in the cell system (Togashi et al. 1998). The opposite effects exerted by the phosphorylation of serine/threonine and tyrosine residues could conceivably suggest a regulatory mechanism of stress bre formation by a process of differential phosphorylation. Such a mechanism is encountered in the control cell cycle progression, where cyclin function is regulated by differential phosphorylation. As alluded to above, the phosphorylation of the threonine residues and inactivation of p34 has a negative regulatory effect on cell cycle progression. In the light of the crucial role played by the cytoskeleton in cell division, further studies of the potential involvement of calcineurin in the cell division cycle and allied areas are clearly warranted and may turn out to be highly fruitful. The progression of the cell cycle is controlled at both G1S and G2M transition checkpoints by the phosphoprotein p53. The biochemical basis of p53 function is its ability to regulate transcription by binding to DNA. Calcineurin appears to be able to modulate p53 binding to the long terminal repeats (LTR) of human immunodeciency virus (HIV-1). The compound PD 144795 inhibits HIV-1 transcription, which correlates with the inhibition of the phosphatase activity of calcineurin (Gualberto et al. 1998). This demonstration of p53-mediated transactivation provides another putative mechanism by which calcineurin could inuence cell cycle progression. Calcineurin is known to modulate the function of other transcription factors, e.g., NF-AT3 and GATA-4, by dephosphorylation (Molkentin et al. 1998). We know from the work of Cai et al. (1996) that calcineurin might be involved also in the transcription of the IL-2 gene. They showed that inactivation of calcineurin leads to an inhibition of the transcription of the IL-2 gene. The expression of bre-type specic genes, of slow- and fast-twitch myobrils of adult skeletal muscles, seems to be regulated by calcineurin. The transcription of these genes occurs by the
138
mediation of NF-AT and MEF2 transcription factors (Chin et al. 1998). The calcineurin inhibitor FK506 reversibly inhibits insulin secretion by HIT-T15 cells in culture, and in parallel, intracellular levels of insulin mRNA and insulin also decrease. The inhibition of insulin gene transcription has also been conrmed using human insulin/CAT reporter gene constructs (Redmon et al. 1996). Recent reports show that IGF, which is involved in muscle growth, regeneration, and hypertrophy, also appears to follow the calcineurin NF-AT, GATA pathway in activating the expression of the appropriate genes. The transfection of IGF-1 gene into skeletal myocytes and its expression at the postmitotic stage induce the expression of calcineurin mRNA transcripts and the protein. The latter becomes localised in the nucleus. IGF-1 as well as activated calcineurin also induce GATA-2 expression. This transcription factor associates with calcineurin and NF-Atc1 (Musaro et al. 1999). In parallel studies, Semsarian et al. (1999) have shown that treatment of skeletal muscle cells with IGF-1 or dexamethasone activates calcineurin and induces the translocation of NF-Atc1 to the nucleus. These observations clearly implicate the calcineurin/NF-AT signalling pathway in the cellular responses generated by these growth factors. Finally, the ubiquitous HSPs come into this picture as well. HSPs have gained much coverage as stress-induced proteins. Besides, they are of exceptional importance to the life of the cell, for they take part in processing of nascent protein, transport, and targeting of proteins to various cellular subcompartments. Furthermore, not only are HSPs expressed in a cell cycle-related manner, but they also interact with many cellular proteins that are involved in cell cycle regulation. It is of much interest, therefore, to cite here some recent work that seems to strengthen the association of both HSPs and calcineurin in the progression of the cell cycle. Someren et al. (1999) have shown that HSP70 and HSP90 both activate calcineurin in the presence of CaM. The requirement of CaM seems to be absolute for the activation by HSP70. Activation of calcineurin does not occur in the absence of CaM. Someren et al. (1999) have also demonstrated that calcineurin co-precipitates with HSP. However, in light of much evidence to be discussed in a later section, it is difcult to establish the precise pathway by which either HSPs or calcineurin might inuence the progression of the cell cycle.
THE EFFECTS
OF
CALCINEURIN
ON
CELL ADHESION
AND
MOTILITY
The expression of calcineurin A as well as B is up-regulated in HL-60 cells induced to differentiate by treatment with all-trans RA. A progressive increase in calcineurin phosphatase activity occurs in parallel with granulocytic differentiation and inhibition of cell proliferation (Kihara et al. 1998). Therefore, there may be another pathway by which calcineurin inuences cell proliferation. RA is a well-known modulator of cell adhesion and inhibitor of cell motility and the invasive behaviour of cancer cells (Fazely and Ledinko, 1990; Edward and Mackie, 1990; Edward et al. 1989, 1992; Wood et al. 1990). Hendey and Maxeld (1993) found that the motility of neutrophils on vitronectin-coated substratum could be inhibited by using inhibitors of calcineurin. The formation of lopodia and the motility of neurones of chick dorsal root ganglia are
139
said to depend on calcineurin. The calcineurin inhibitors cyclosporin A and FK506 delay the formation of neurites and inhibit neurite extension. A targeted and focal inactivation of calcineurin in regions of growth cones affects lopodial retraction and direction of its subsequent outgrowth (Chang et al. 1995). FK506 has also been found to inhibit the transendothelial migration of human lymphoma cells, Nalm-6, in both in vitro and in vivo situations (Tsuzuki et al. 1998). These authors have suggested that the inhibition of migration might be related to an inhibition of vascular cell adhesion molecule (VCAM)-1 and its 41 integrin receptor VLA-4. The VCAM-1/VLA-4 system is known to provide the recognition mechanism and adhesive facility for certain tumour cell types to the endothelium. VCAM expression seems to relate to the progression of melanomas (Denton et al. 1992), but not of breast cancer (Fox et al 1995). Overall, the evidence for any general relationship between the expression of VCAM and its receptor and metastatic potential is not compelling. However, calcineurin can stimulate the transcription factor NF-B and enhance its DNA-binding property (Franz et al. 1994). This transcription factor is also involved in the induction of cell adhesion molecules, and anti-NF-B reagents are able to block cancer cell adhesion to endothelial cells (Tozawa et al. 1995). Therefore, the effects of FK506 on transendothelial migration described by Tsuzuki et al. (1998) could be interpreted as indicating that calcineurin might itself be inuencing the diapedesis by cancer cells. Mohri et al. (1998) have recently provided some direct evidence that shows that calcineurin affects cell adhesion to substratum and, by implication, also modulates the invasive behaviour of cells. They used the colon cancer cell line Colo201 and demonstrated that the protein kinase inhibitors K252a and KT5720 enhanced cell adhesion to and their spreading on substratum. This process was accompanied by the formation of actin stress bres. The adhesion appeared to be mediated by integrin 2 and 1, as indicated by their accumulation at the sites of focal adhesion. The higher adhesion mediated by K252a and KT5720 could be blocked by cyclosporin and FK506, which also inhibit calcineurin. An increase in the expression of TGF1 in peripheral blood mononuclear cells obtained from patients treated with cyclosporin has been reported (Shin et al. 1998). Other cell types, such as A549 cells, seem to respond to cyclosporin in a similar fashion. The increased expression of TGF is associated with enhanced cell motility, invasive behaviour in vitro, and enhanced metastatic behaviour, as indicated by inhibition of the cyclosporin-induced effects of anti-TGF antibodies (Hojo et al. 1999). It would be worthwhile to point out here that TGF produces a variety of effects. Among these are induction of angiogenesis, inhibition of cyclin-dependent kinases, and induction of inhibitors of metalloproteinases (see Sherbet and Lakshmi, 1997b for references). The invasive behaviour of trophoblast cells is believed to be controlled by the induction of TIMPs by TGF (Lala and Graham, 1990). These cellular faculties of adhesion to substratum, cell spreading, and cell motility are closely related to cell proliferation, besides being dependent on cytoskeletal organisation. Therefore, one should consider whether calcineurin could be altering cytoskeletal dynamics by altering the phosphorylation status of cytoskeletal components.
140
Calcineurin and the CaM-dependent protein kinase II have been reported to regulate the phosphorylation levels of neurolament subunits and -tubulin elements of rat cerebellar cytoskeleton (De Mattos-Dutra et al. 1998). The tau protein has been shown to be an important requirement for neurite outgrowth and growth cone motility. The inactivation of tau seems to lead to an inhibition of neurite outgrowth, and other microtubule-associated proteins are unable to compensate for the inactivated tau (C.W.A. Liu et al. 1999). In common with many other proteins, the biological function of tau is regulated by phosphorylation. tau promotes microtubule assembly and inhibits depolymerisation (Drechsel et al. 1992). Phosphorylated tau has a reduced capacity to bind to microtubules (G.V.W. Johnson, 1992). The tau protein of Alzheimers disease paired helical laments (PHF) has been reported to contain 21 phosphorylation sites, of which some are serine/threonineproline residues (Morishima-Kawashima et al. 1995). The phosphorylation of threonine 231, serine 235, and serine 262 is essential for maximal inhibition of binding of tau protein to microtubules (Sengupta et al. 1998). Mandelkow et al. (1996) believe that the single serine 262 phosphorylation determines the binding of tau to microtubules. This abolishes taumicrotubule binding and greatly affects the ability of tau to stabilise microtubule dynamics. Because calcineurin is a serine/threonine phosphatase, it may be expected to control tau phosphorylation and in this way also regulate its interaction with microtubules. The dephosphorylation of the tau element of the cytoskeleton of PC12 cells appears to be mediated by calcineurin (H.Q. Xie and Johnson, 1998). Cyclosporin A consistently inhibits the phosphatase activity of calcineurin and inhibits axonal extension (Ferreira et al. 1993). Also, tau protein is found in a hyperphosphorylated form in calcineurin A- knock-out (/) mice (Kayyali et al. 1997). Furthermore, calcineurin strongly inuences F-actin stability in cultured hippocampal neurones. The exposure of these cells to NMDA produces a rapid loss of dendritic spines together with a loss of actin laments. This is prevented by actin-stabilising agents and calcineurin inhibitors (Halpain et al. 1998).
CALCINEURIN IN ALZHEIMERS DISEASE

A characteristic feature of Alzheimers disease is the formation of neurobrillary tangles (NFTs). NFTs are intraneuronal and consist mainly of PHF. It has been postulated that the assembly of PHF involves initially a process of antiparallel dimerisation of tau monomers. The dimers then form PHF (Mandelkow et al. 1996). The tau protein is a major component of PHF. Tau is phosphorylated to an abnormally high degree in Alzheimers disease. The phosphorylation is three- to four-fold greater in Alzheimers tau protein than in the native protein (Mandelkow et al. 1996; Grundke-Iqbal et al. 1986a, 1986b). This abnormally phosphorylated state is regarded as being responsible for the detachment of tau from microtubules and the consequent microtubule instability. Iqbal and Grundke-Iqbal (1996) state that abnormal phosphorylation might precede the polymerisation of tau into PHF. Furthermore, they claim that dephosphorylation of abnormal tau can restore the ability of tau to interact with and promote microtubule assembly. However, others believe that no such causal relationship exists between tau phosphorylation and the formation of
141
PHF (Goedert, 1996). Another factor that might be envisaged is genomic mutations. Mutations in the exons as well as introns of the tau gene have been reported in cases of familial dementia and Parkinsons disease. Mutations in the exons seem to affect the ability of tau to promote microtubule assembly (Hasegawa et al. 1998). Nonetheless, this abnormal phosphorylation state has been attributed to the generation of an imbalance between protein kinases and phosphatases, engendered by a reduction in the levels of phosphatases (Iqbal and Grundke-Iqbal, 1996). More specically, the high state of tau phosphorylation has been related to calcineurin dysfunction. This is suggested by the ndings of Kayyali et al. (1997) that calcineurin A- (/) knock-out mice show hyperphosphorylation of tau. The abnormal tau accumulates, and these mice also exhibit cytoskeletal changes that might affect neuronal function. The neurodegenerative condition of amyotrophic lateral sclerosis (ALS) is characterised by a loss of motor neurones and neurones of the corticospinal tract. This neuronal loss has been attributed to a deregulation of NMDA receptors (Krieger et al. 1993; Plaitakis, 1990). Glutamate-induced apoptosis and necrosis of cerebellar granule cells is prevented by the calcineurin inhibitors FK506 and cyclosporin (Ankarcrona et al. 1996). Abnormal levels of calcineurin have been reported to occur in ALS. This is believed to result in abnormal phosphorylation of NMDA receptors, which in turn has been suggested to result in the pathogenesis of ALS (Wagey et al. 1997). Alzheimers disease and Downs syndrome also are characterised by extracellular occurrence of amyloid deposits in the form of senile plaques and deposits associated with the microvasculature. The brillary amyloid is composed of the amyloid- protein (ABP) with 39 to 43 amino acids. ABP is derived from the integral membrane glycoprotein, the -amyloid precursor protein (APP) The latter shows an abnormally high accumulation in the brain of people with Alzheimers disease. Calcineurin seems to be involved in the formation of this peptide (Desdouits et al. 1996).
CALCINEURIN IN IMMUNOSUPPRESSION
Calcineurin plays a key role in immunosuppression. There has been an unequivocal demonstration over the past few years that the drugs cyclosporin A, FK506, and rapamycin achieve immune suppression by inhibiting the function of calcineurin. That calcineurin was the target of some of these immunosuppressants was established some years ago. OKeefe et al. (1992) transfected the catalytic calcineurin A into cells and showed that this raises the IC50 inhibitory concentration of cyclosporin and FK506. Besides, Jurkat cells that overexpressed calcineurin were resistant to the action of cyclosporin and FK506 and showed enhanced gene transcription that depended on NF-AT and related transcription factors (Clipstone and Crabtree, 1992). These immunosuppressant drugs interact with specic intracellular immunophilin receptors, which results in the formation of a drugimmunophilin complex that is capable of blocking the function of its target, which, in this instance, is calcineurin (J. Liu et al. 1991; Ho et al. 1996). When T cells are activated by mitogenic signals or by antigen binding, they proliferate and switch on the expression of cytokine genes. Antigen binding to the TCR produces a cascade of signalling events (Figure 19), which have been elucidated in some detail.
142
FIGURE 19 The involvement of calcineurin in a key position in the TCR-signalling cascade. See text for details.
Calcineurin participates in this signalling cascade as a key enzyme. It is responsible for dephosphorylating the transcription factor called NF-AT and this factor then translocates to the nucleus. NF-AT1 contains a regulatory domain, which lies N-terminal to its DNA-binding region, that binds to calcineurin (C. Luo et al. 1996). In this way calcineurin seems to be targeted to NF-AT. The maintenance of NF-AT in the nucleus requires high and sustained levels, not transients, of calcium (Timmerman et al. 1996), probably reecting its continual calcineurin-dependent activation and translocation. The NF-AT proteins are expressed in many cell types of the immune system, and these constitute a family of proteins that possess this conserved regulatory domain involved in their binding to calcineurin. NF-AT proteins resemble the Rel family of proteins with respect to their ability to bind to the regulatory elements of certain genes coding for cytokines (Rao et al. 1997). In the nucleus, NF-AT forms a complex with the transcription complex AP1 (jun/fos). The AP1/NFAT complex then initiates the transcription of a number of genes, such as genes coding for the lymphokine, IL-2, IFN-, and TNF-, among others. The inhibition of calcineurin by the immunosuppressants inhibits the dephosphorylation of NF-AT and thereby inhibits its translocation to the nucleus and all the downstream events. Batiuk et al. (1997) have fully correlated the effects of cyclosporin A with the various
143
FIGURE 20 The multiple functions of calcineurin, on the cell division cycle, in the modulation of cell shape, adhesion and motility, and in the regulation of gene transcription. Also indicated are its participation in the functioning of immunosuppressants and its possible association with abnormal phosphorylation of tau in Alzheimers disease.
events of the cascade, namely the dephosphorylation of NF-AT, its translocation, binding of the transcription complex to the DNA, and the eventual activation of gene transcription. They also demonstrated the relationship between the extent of calcineurin inhibition, the inhibition of lymphocyte proliferation, and the degree of IFN- production in vitro. It would not be out of place to briey mention here that calcineurin is involved in the autoimmune condition SLE. SLE affects women of child-bearing age. The menstrual cycle and pregnancy seem to affect disease activity (Lahita, 1993; Jungers et al. 1985; Mund et al. 1963). Rider et al. (1998) have shown that T cells from SLE patients respond to oestradiol by an up-regulation of calcineurin mRNA expression, in a dose-dependent fashion. The effect seems to be specic for oestradiol, because progesterone and dexamethasone fail to elicit a similar response. The increase in mRNA expression corresponds with calcineurin phosphatase activity in oestradiol-treated T-cell extracts. As Rider et al. (1998) pointed out, the mechanism by which oestrogen inuences calcineurin expression is unclear, because the latter has no identiable oestrogen response elements in its promoter region. However, they suggest that oestrogen could be exerting control at the level of calcineurin transcription. They cite the ndings of H. Becker et al. (1995) that there is an increased NF-AT binding to IL-2 promoter in SLE patients as compared with control subjects. As discussed earlier in this section and shown in Figure 19, calcineurin
144
plays a key role in the translocation of the NF-AT transcription factor to the nucleus. Therefore, it is conceivable that oestrogen inuences the T-cell signal transduction cascade in SLE. One is fully justied in concluding at the end of this chapter that calcineurin is a highly versatile phosphatase, and its functional repercussions, shown in Figure 20, are discernible in many facets of biological behaviour.
11
Centrins (Caltractins) and Their Biological Functions
Centrin (also known as caltractin) is an EF-hand calcium-binding protein of approximately 20 kDa molecular size. Errabolu et al. (1994) have cloned human centrin cDNA, which contains an ORF of 516 bp and a predicted 172 amino acid residues in the protein. Sequence analysis shows that human centrin is closely related to centrins from plants, protozoa, algae, and Xenopus, and to CDC31 of Saccharomyces. The sequence also suggests the occurrence of four calcium-binding EF-hands. The centrin gene has been mapped to chromosome Xp28 (Chatterjee et al. 1995). However, the identication of several isoforms of centrin suggests the possibility of the existence of a family of centrin genes. Centrin is a component of spindle pole bodies, basal bodies, and centrosomes. Centrin and -tubulin are common components of these organelles. A major physiological function of centrin is the organisation of microtubules. The contractile bres associated with centromeres, for instance, show calcium mediated contraction, and lament contraction occurs independently of ATP (Chiebel and Bornens, 1995). Using immunouorescence techniques, researchers have shown that centrin localises in the centrosome of interphase cells. The centrosome duplicates during this phase of the cell cycle and centrin then redistributes between the spindle and the polar bodies. Quite obviously, therefore, it plays an important role in the separation of centrosomes during mitosis (Errabolu et al. 1994). Other functions also have been attributed to centrin. It is associated in the yeast with the duplication of the spindle pole body. Vallen et al. (1994) showed that the CDC31 of Saccharomyces cerevisiae, a protein that is closely related to centrin, interacts with the KAR1 gene product in the duplication of the microtubule organising centre. Centrin interacts with KAR1 protein with great afnity. This interaction is regulated by changes in cellular calcium levels, which, in their turn, seem to be inuenced by certain amino acid residues within the KAR protein (Geier et al. 1996). In fact, antibodies to the centrin expressed by the agellate Naegleria gruberi can recognise CDC31 of yeast (Levy et al. 1996). Centrin and related proteins are involved in agellar severance and the retraction of ciliary apparatus. In Chlamydomonas reinhardtii the excision of agella occurs by a process of microtubule severance caused by contraction of centrin-containing bres (Sanders and Salisbury, 1994). The molecular mechanisms involved in the function of centrins are still poorly understood, but centrosome fractions prepared from haematopoietic cells have been
145
146
found to contain active Ca2+/CaM-dependent protein kinase II. In vitro, this enzyme has been shown to phosphorylate several centrosome-associated proteins (Pietromonaco et al. 1995). However, there is no evidence at present of centrin phosphorylation in relation to its function, apart from the observation that centrin is phosphorylated during prophase and metaphases of mitosis (Lutz et al. 1995). The H6 gene of the plant Atriplex nummularia encodes a protein with a high degree of sequence homology to algal centrin. Three transcripts of this gene, of 1.3, 2.2, and 2.4 kb size, are expressed, and it would appear that these transcripts are differentially expressed in relation to mitotic activity and developmental and extracellular signals. The 1.3-kb transcript is induced by tactile and hyperthermic signals. Expression of the 2.2-kb transcript is related to the state of mitotic activity. The 2.4kb transcript is expressed in response to heat shock (J.K. Zhu et al. 1996). Centrin has been found in the form of a complex with the high molecular weight HSP70 and HSP90 in Xenopus oocytes that are arrested by exposure to cytostatic factors. When the oocytes are activated by electric shock or by means of ionophores, the centrinHSP70 complexes appear to undergo dissociation (Uzawa et al. 1995). The functional signicance of the formation of these complexes is unclear, although the work of Uzawa et al. (1995) appears to suggest a relationship with activation and division of the oocytes. It ought to be pointed out that high molecular weight HSPs may not be involved in the process of cell proliferation, but they may inuence the size of the steady-state cell population by protecting cells from apoptosis. In contrast, the lower molecular weight forms such as HSP28 are indeed so involved. HSP28 inhibits cell proliferation. It may form complexes with EF-hand proteins such as S100A4 (see Albertazzi et al. 1998a). HSP70 has been reported to show a cell cyclerelated pattern of expression. Milarsky and Morimoto (1986) found that it is expressed at the G1S boundary. It can bind also to the cell cycle regulatory p53 phosphoprotein (Pinhasi-Kimhi et al. 1986; Finlay et al. 1988). So, it is conceivable that it is involved in the regulation of the cell cycle. On the other hand, HSPs participate prominently in protein folding and transport. Whether the formation of a complex between HSP and centrin alters the molecular folding of the latter is not known, but such a modulation of molecular shape has the potential to alter the accessibility and binding of calcium to centrin and thus affect its function. Conversely, complex formation with HSPs might be an aspect of centrin function. The formation of the complex could itself depend upon the conformation adopted by centrin upon calcium-binding. Centrin also may be involved, together with other components of the cytoskeletal system, in maintaining cell polarity and motility (Lingle and Salisbury, 1997). Centrins have been found in nonmotile ciliary structures. Wolfrum (1995) has found centrin in photoreceptor cells. There is some preliminary evidence suggesting that the central helix of centrin may be important for its biological function. A study of the mutations of the Chlamydomonas v2 centrin gene has revealed that mutation of the glycine residue at 101 to lysine results in the loss of centrin function. However, this suppressor effect is reversed with additional mutations at amino acid residues 96 or 104, and all three residues occur in the central helix of the protein (Taillon and Jarvik, 1995).
Centrins (Caltractins) and Their Biological Functions
147
Centrin is highly conserved in evolution. Bhattacharya et al. (1993) studied the molecular organisation of centrins of several lower organisms. From the phylogenetic point of view, the EF-hand domains of several centrins show congruence and may have arisen by gene duplication from an ancestral EF-hand domain. Furthermore, the domains of centrin are congruent with those of CaM. These observations provide strong indicators that centrin and related proteins may have evolved from a common ancestral form by means of gene duplication. A similar conclusion can be reached from the pattern of expression of centrin and related proteins in association with a phylogenetically evolving contractile system (Levy et al. 1996). In mammals, three centrin genes have been described: HsCEN1, HsCEN2, and HsCEN3 (Middendorp et al. 1997). These authors found that HsCEN1 was related to the yeast centrinencoding gene CDC31 and further, that HsCEN2 and HsCEN3 possessed a greater degree of identity to algal centrin than did HsCEN1. This is interpreted as suggesting a divergence in the evolution of centrins.
12
Reticulocalbin Family of EF-Hand Proteins
There are two species of calcium binding proteins that are associated with the endoplasmic reticulum. One of them is a non-EF-hand protein, calreticulin. A family of EF-hand proteins, with variable number of EF-hand domains, has been identied over the past few years (Table 9). Among them are reticulocalbin, calumenin, ERC55 and ERC-fc, and Cab-45, and these may be described as belonging to the reticulocalbin family of CBPs. These proteins are expressed ubiquitously in a variety of tissues. Human calumenin occurs at high levels in the heart, placenta, and skeletal muscle, and at lower levels in the lung, kidney, and pancreas. Its levels are very low in the brain and liver (Vorum et al. 1998). Scherer et al. (1996) have isolated Cab45 from mouse adipocytes.
TABLE 9 The Reticulocalbin Family of EF-Hand Calcium Binding Proteins

Protein Nomenclature Reticulocalbin Molecular Features 44-kDa protein; C-terminal HDEL sequence; 6 EF-hands; gene 13 kbp and 6 exons 11p13 (WAGR locus) 55-kDa protein; 6 EF-hands 40-kDa; 6 EF-hands 45-kDa cytoplasmic, 30-kDa nuclear 315 amino acid residues; 7 EF-hands 45 kDa; 6 EF-hands; Golgi location; no signal for membrane anchoring 50-kDa protein; 1 EF-hand motif (?) 315 amino acid residues; 6 calciumbinding domains; binds to phospholipase A2 Ref. Ozawa and Muramatsu (1993); Ozawa (1995a,b) Kent et al. (1997) Weis et al. (1994) La Greca et al. (1997) M. Kobayashi et al. (1998) Vorum et al. (1998); Yabe et al. (1997) Scherer et al. (1996) Hseu et al. (1997) Hseu et al. (1999)
ERC-55 ERC-pf DNA-SCFa Calumenin (human) Cab-45 (murine) CBP-50 Crocalbin
DNA supercoiling factor, found to show homology to reticulocalbin.
149
150
MOLECULAR FEATURES OF RETICULOCALBIN HOMOLOGUES

Reticulocalbin and other members of the family possess a variable number of EFhand domains. Also variable is their afnity for calcium ions. The EF-hand domains of reticulocalbin bind calcium with low afnity (Vorum et al. 1998). According to Tachikui et al. (1997), only EF-hands 1, 4, 5, and 6 bind calcium. The EF-hands 2 and 3 do not bind Ca2+. Calcium binding produces conformational changes in reticulocalbin. The reticulocalbins are approximately 40 to 45 kDa in size and show a high degree of homology among themselves, mostly with respect to sequences outside the EF-hand domains. As the name of the family denotes, these CBPs are associated with the ER. Cab-45 occurs mainly in the Golgi complex. These proteins contain a membrane localisation and anchoring sequence HDEL of 4 amino acid residues at the C-terminal region (Ozawa and Muramatsu, 1993; Vorum et al. 1998; Yabe et al. 1997). The deletion of these residues from ERC-55 has been shown to result in loss of anchorage to the membrane and secretion of the protein (Weis et al. 1994). Reticulocalbin also contains an N-terminal signal for its transfer to the lumen of the ER (Ozawa and Muramatsu, 1993). Cab-45, which occurs in the lumen of the Golgi bodies, does not possess the membrane-anchoring signal (Scherer et al. 1996). The mouse reticulocalbin gene spans more than 13 kbp of the genome. It contains six exons. The human reticulocalbin shows more than 95% amino acid sequence homology with the murine protein, in both EF-hand and non-EF-hand regions of the protein. Both human and murine proteins contain six EF-hand motifs. Whereas human reticulocalbin possesses the HDEL sequence, the mouse homologue contains the KDEL sequence at the C-terminal end (Ozawa, 1995a, 1995b). The human reticulocalbin gene has been mapped to chromosome 11p13 in the WAGR locus, between the Wilms tumour gene wt1 and the aniridia PAX gene (Kent et al. 1997).
PUTATIVE FUNCTIONS OF RETICULOCALBIN AND ITS HOMOLOGUES

There has not been sustained effort to study the functions of the reticulocalbin family of proteins. Three putative functions can be identied. Their association with ER and the evolutionarily conserved ER anchoring sequence suggests that one function may be protein trafcking. M. Kobayashi et al. (1998) have identied two cDNA clones of 1.6 and 1.8 kb, expressed in Drosophila melanogaster. The 1.8-kb mRNA contained an open reading frame (ORF) that showed marked homology to mouse reticulocalbin. The shorter mRNA encoded a protein in which an N-terminal sequence was deleted. Antibodies against the DNA supercoiling factor identied a cytoplasmic 45-kDa protein and a 30-kDa protein in the nucleus. They also found that the 30-kDa nuclear protein interacted with topoisomerase II, which suggests that this protein is the functional form of the supercoiling factor. In early developmental stages, such as the blastoderm stage of embryonic development, the antibodies stain interphase nuclei. M. Kobayashi et al. (1998) have also described the pattern
Reticulocalbin Family of EF-Hand Proteins
151
of immunostaining of the polytene chromosomes of Drosophila, which are essentially interphase chromosomes. The polytene chromosomes have characteristic condensed bands harbouring specic genes. These form puffs when the genes are transcriptionally active. M. Kobayashi et al. (1998) found that immunostaining for the supercoiling factor was associated with naturally occurring puffs or those induced by ecdysone. These data seem to associate the supercoiling factor with gene expression. There is a further suggestion that reticulocalbin may be associated with the invasive behaviour of tumours. It is reported to be overexpressed in breast cancer cell lines with high invasive ability, but not in cell lines that are weakly invasive (Z.D. Liu et al. 1997). Although these authors have carried out in vitro invasion assays in parallel, it is difcult to tie the invasive behaviour specically to the expression of reticulocalbin. The MBA and MCF7 breast cancer cell lines that they have used are known to differ markedly in the expression of S100A4, growth factor receptors, and plasminogen activator, among others, which are known to affect the invasive ability of these cell lines. Perhaps some gene transfer experiments are warranted before any evidence can be regarded as conclusive. Even then, conceptually, it is difcult to visualise a mode of function at the present stage in our knowledge of the participation of reticulocalbin homologues in cell physiology.
13
Calpains in Normal and Aberrant Cell Physiology
THE CALPAIN FAMILY OF CALCIUM-BINDING PROTEINS

Calpains constitute a family of calcium-dependent proteases showing ubiquitous distribution in a wide spectrum of animal species. They are intracellular cysteine proteases. They cleave substrate proteins in a manner that might constitute a mechanism of regulation of the protein substrates (Croall and De Martino, 1991; K. Suzuki et al. 1992). Therefore, they have been regarded here as regulatory CBPs. Mammalian calpains were known, on the basis of activity, as - and m-calpains (referred to herein with their gene designation as capn1 and capn2). These are the ubiquitously occurring isoforms (Table 10). Other isoforms showing tissue-specic distribution also are known, and these have been designated as capn3 and capn4. Capn5 and capn6 have been discovered recently and, despite their amino acid sequence homology with other calpains, appear to be distinctive in that they do not possess the CaM-like calcium-binding domain (Dear et al. 1997). The tra-3 gene, which encodes the calpain of nematode worms, shows the highest degree of homology to capn5 and capn6. Thus, in spite of the sequence similarities with other isoforms, capn5, capn6, and tra-3 are not dependent on calcium for their activity and may indeed be devoid of all proteolytic activity (Dear et al. 1997; Barnes and Hodgkin, 1996). The human homologue of tra-3, known as the htra-3 gene, has been cloned. This encodes a protein that is highly homologous to the calpain 80-kDa subunit. Furthermore, like tra-3, the htra-3 peptide lacks the CaM-like calcium binding motifs (Mugita et al. 1997). It would seem, therefore, that calpains might have diverse functions that may be related to the existence of some mechanism by which they might be activated. This is compatible with the discovery of a family of calpain activator proteins, which not only resemble one another but also possess chaperonin-like properties (Melloni et al. 1998a, 1998b). The 30-kDa activator described by Melloni et al. (1998a) has been reported to show a rigorous specicity for capn1 and to be totally ineffective on capn2. This activator protein binds to the 80-kDa subunit of calpain and promotes its dissociation from the smaller subunit. The process of activation then takes the form of autoproteolysis, leading to the formation of two forms of calpain of 78 and 75 kDa molecular size. The activator described by Melloni et al. (1998a, 1998b) bears a high degree of sequence homology with another calpain activator that has
153
154
TABLE 10 Calpain Family Isoforms

Gene/Protein Designation capn1 (m-calpain) capn2 (m-calpain) capn3 (p94; nCL-1) Lp82 (splice variant of capn3) capn4 (nCL-2) nCL-4 capn5 capn6 capn8 tra-3 htra-3 (human homologue of tra-3) 11q14d Chromosome Location Chromosome 11q13a Chromosome 1 Chromosome 15q15.1q21.1b Characteristic Features Active at micromolar concentration Active at millimolar concentration Skeletal muscle specic Rat lens Stomach specic Stomach, intestine, but not uterus Chromosome 11q13.5q14c X chromosome
Expressed only in the placenta Murine calpain, expressed in brain, kidney, and the digestive tract Caenorhabditis elegans sex determination High expression in colon, small intestine, and testis tissues
Note: Chromosomal location data from (a) Pang et al. (1997); (b) Richard et al. (1995); (c) Ollendorf et al. (1992); (d) Mugita et al. (1997). Source: Data collated from Ohno et al. (1990); Richard et al. (1995); Schuler et al. (1996); Matena et al. (1998); H.J. Lee et al. (1998); Ma et al. (1999). Isolation of the new capn8 was recently reported by Braun et al. (1999b).
been isolated from goat liver and called UK114 (Ceciliani et al. 1996a, 1996b). UK114 has been regarded as a cancer marker. However, whether an inappropriate activation of calpains can lead to transformation and acquisition of properties that characterise cancer cells would be a fruitful avenue of approach. The possibility that calpains might, however indirectly, control gene transcription certainly makes an exploration of this avenue highly worthwhile.
MOLECULAR ORGANISATION OF CALPAINS

The calpain molecule consists of two subunits: an 80-kDa and a smaller 30-kDa subunit. The gene encoding the large subunit of rat capn2 contains 22 exons, of which exons 3 to 21 encompass 33 kb of the rat genome. The corresponding cDNA of 3.2-kb size codes for a protein consisting of 700 amino acids. This protein shows a high degree (93%) of sequence homology with human capn2 and 61% homology with human capn1. The cDNA, when tagged to an expression vector, generated an 80-kDa protein that was found to be identical to human capn2 (De Luca et al. 1993). The 80-kDa subunit has four domains. Domain II is the cysteine protease domain and domain IV is the Ca2+-binding domain. The smaller subunit has two domains:
155
domain V, the N-terminal glycine-clustering hydrophobic domain, and the Ca2+binding domain VI (Nishimura and Goll, 1991; Crawford et al. 1993; Minami et al. 1988). The calcium-binding domains (IV and VI of the two subunits) possess sequence similarities to CaM (Ohno et al. 1984). These domains contain four EFhands each (EF-hands 25). Two additional EF-hands occur in the 80-kDa subunit, one at the junction between domains II and III and the second one at a position Nterminal to EF-hand 2 (Theopold et al. 1995; Andresen et al. 1991). The association of the subunits occurs through domains IV and VI, and EF-hand 5 may play an important part in maintaining the dimeric organisation (Y. Minami et al. 1988; Kretsinger, 1997).
REGULATION OF PHYSIOLOGICAL EVENTS BY PROTEOLYTIC FUNCTION

Calpains are not wide-spectrum proteases. Importantly, the limited number of substrates that they cleave are involved with the activation of specic cellular systems. Notable among their substrates are PKC-, the actin-binding proteins, -fodrin and the homologous - and -spectrin, and growth factor receptor proteins (Lofvenberg and Backman, 1999; Saido et al. 1994; Croall and De Martino, 1991; Martin et al. 1995). It is to be expected that calpains would be involved in physiological events merely by virtue of their proteolytic activity. Several such events can be cited. One of these is keratinocyte differentiation. Calpains are actively involved in the proteolytic cleavage of prolaggrin into laggrin in the process of keratin lament aggregation (Figure 25). They also may regulate the levels of MAPs during neuronal differentiation. During early brain development MAPs occur as MAP-1B and MAP2. MAP-1B occurs as two isoforms differing in the level of phosphorylation, and MAP-2 occurs as two isoforms with different molecular weights. The isoforms of MAP-1B as well as MAP-2 show equal sensitivity to calpain, but overall, MAP-2 is more susceptible to the proteolytic action of calpain than MAP-1B (I. Fischer et al. 1991). Calpain has been shown to be able to induce apoptotic cell death, and this is associated with the cleavage of substrate proteins such as -fodrin (spectrin), Ca/CaM-dependent protein kinase IV, etc. Neurobromatosis type 2 (NF2) is an autosomal dominantly inherited condition characterised by a disposition to develop certain intracranial tumours such as vestibular schwannoma and meningioma. The NF2 gene codes for a protein called merlin, also known as schwannomin, and it is believed to function as a tumour suppressor. Mutations of merlin have been found in a signicant proportion of NF2-associated tumours and to a lesser extent in sporadic schwannomas and meningiomas. However, NF2 tumours that show no mutations of the NF2 gene also have been found. Kimura et al. (1998), therefore, examined the possibility that the suppressor protein may be inactivated by mechanisms other than mutation. These authors found that a marked activation of calpain occurs in sporadic meningiomas. Merlin is cleaved by calpain, and inactivation of calpain restores merlin in meningioma cells in culture. It would appear, therefore, that proteolytic control by calpain might be a mechanism by which the tumour suppressor function of merlin is regulated. Much of this evidence is
156
correlative, but admittedly proteolytic pathways are involved in the regulation of expression of several cellular regulatory proteins such as p53 (see below) and the mdm2 protein (L.H. Chen et al. 1997), which negatively regulates p53 function. Indeed, the interaction of mdm2 with p53 reduces p53 protein levels by targeting it to ubiquitin-dependent degradation (Kabbutat et al. 1997). The phosphoprotein p53 is intricately involved in the checkpoint control at the G1-M as well as at the G2-M transition of cells. p53 has a short half-life. Its degradation has been attributed to ubiquitination as well as to proteolysis by calpain. The PEST (Pro, Asp/Glu, Ser, and Thr) motif is generally regarded as a signal in the substrate that is required for its recognition by an intracellular protease. p53 contains the PEST sequence, which is required for proteolysis by calpain. Ubiquitination of certain substrates, e.g., STE3 receptor of yeast, has been attributed to a PEST-like sequence (Roth et al. 1998), although we do not know if this is the case with p53 ubiquitination. Mutated p53, which has an increased half-life, does not contain these sequences. However, in some instances, p53 may be degraded by calpain in the absence of the PEST sequences. Wild-type p53 is also stabilised when cells expressing it are treated with inhibitors of calpain. However, the presence of the PEST motif may not be an absolute requirement for calpain function (Carillo et al. 1996; Van Antwerp and Verma, 1996). The human papilloma virus (HPV) E6 protein promotes p53 degradation through the ubiquitin-dependent pathway, and this is unaffected by inhibitors of calpain. Overall this suggests that the stability of p53 might be controlled by the proteolytic action of calpain (Kabbutat and Vousden, 1997). Similarly, cyclin D1, another regulator of cell cycle progression, has been reported to be a target of calpain proteolysis. Choi et al. (1997) showed that cyclin D1 is rapidly proteolysed in NIH3T3 cells when deprived of serum, but calpain inhibitors reversed this. These inhibitors also raise the half-life of cyclin D1. Proteolytic cleavage of the cytoplasmic domain of the cytokine receptor -chain (-c) by calpain may represent an important regulatory or control event in -cmediated signalling. Noguchi et al. (1997) demonstrated that the small subunit of calpain cleaves -c, which contains the hydrophilic amino acid PEST sequence, but not when the PEST sequence is mutated. Calpain inhibitors inhibited -c cleavage in TCR-stimulated murine thymocytes. Furthermore, in these cells, anti-CD3 cleaved -c, but calpain inhibitors inhibited this. Calpain inhibitors enhance the proliferative effect of anti-CD3 antibodies, and this is effectively prevented by -c antibodies. Cytokines and growth factors transduce their signals by using the Janus tyrosine kinase (Jak) and STAT (signal transducer and activator of transcription factors) pathway (OShea, 1997; Darnell, 1997). The binding of these ligands to the appropriate receptors results in the activation of the Jak tyrosine kinase and the activation of latent transcription factors called STAT proteins that occur in the cytoplasm. In fact, STAT-3 appears to regulate G1S transition of cells in response to cytokine stimulation (Fukada et al. 1998). Calpain inhibitors have been shown to inuence cytokine-mediated cell proliferation and STAT protein synthesis (Noguchi et al. 1997). It would appear, therefore, that the proteolytic function of calpain could constitute an intricate part of signal transduction pathways. These are prime examples of specic modes of regulatory function performed by calpain, in which a specic target is hydrolysed by calpain to bring about the
157
regulation of a particular physiological event. In addition, one should bear in mind the nding that calpain can degrade transcription factors such as AP1. Hirai et al. (1991) reported that the c-jun and c-fos gene products, which form the AP1 transcription factor, contain the PEST sequence that is the target sequence of calpain action. Calpain inhibitors increase the expression of AP1 (Zhu et al. 1995). Therefore, one could expect calpain would have a generalised effect on the transcription of a variety of genes, merely by virtue of its ability to regulate the function of the transcription factor itself.
INVOLVEMENT OF CALPAINS IN DEVELOPMENT AND DIFFERENTIATION

There have been no signicant investigations into the involvement of calpains in developmental processes. However, there is some evidence that could be construed as providing a partial burden of proof. For instance, the tra-3 gene has been implicated in the process of sex determination during early development of the nematode C. elegans. tra-3 lacks the CaM-like calcium-binding sites (of domain IV), but EF-hand 6 is conserved. Its involvement in sex determination has been suggested to be a consequence of potentiating the function of another gene, e.g., tra-2 (Barnes and Hodgkin, 1996). The conservation of EF-hand 6 in tra-3 might suggest that the latter is required in calcium signalling in the sex determination cascade. Calpain homologues of Drosophila may be associated with developmental abnormalities. Mutations of the sol gene of Drosophila result in neuronal defects that cause behavioural abnormalities. Delaney et al. (1991) cloned two alternatively spliced transcripts of the sol locus. The predicted sequences of the proteins encoded by them showed similarities to different regions of calpain. The carboxylic region of the larger protein showed similarities to the catalytic domain of calpain, and the N-terminal region contained several zinc nger-like repeats. The smaller predicted protein did not contain the calpain catalytic domain and had only two zinc nger-like repeats. Calpains also have been found in the human brain. Their possible role in differentiation is illustrated by changes in calpain expression in the SH-SY5y neuroblastoma cells induced to differentiate by retinoic acid (RA) treatment. RA induces differentiation and neurite outgrowth in these cells. After 3 days of RA treatment, the levels of activated and precursor forms of calpain have been found to increase by >50% and 26%, respectively, in the particulate and cytosolic fractions of the cells. However, the levels of calpastatin, an endogenous inhibitor of calpain, were unchanged. Thus the process of neurite extension seems to be associated with a net activation of calpain (Grynspan et al. 1997). The calpaincalpastatin system seems to be regulated during processes of proliferation and differentiation of osteoprogenitor cells in response to the bone morphogenetic protein (Murray et al. 1997). Further evidence has been derived from the pattern in the expression of capn3 gene that is discernible during early foetal development. This could be interpreted as an indicator of its association with, if not participation in, morphogenesis. capn3 expression is detected only in skeletal muscle at this time. At an earlier stage, capn3
158
expression is found in the heart and it disappears subsequently. Variants of capn3 generated by alternative splicing are also detected in smooth muscle (Fougerousse et al. 1998). The requirement of both calpain and the proteasome proteolytic systems in the differentiation of myoblasts is exemplied by the work of Ueda et al. (1998). L8 myoblast cells grown in the absence of mitogen show enhanced levels of creatine kinase together with the differentiation of myotubes. However, when calpain inhibitor and the proteasome inhibitor lactacystin are added to these cultures, creatine kinase levels are markedly reduced together with inhibition of myotube differentiation. Although calpains have been implicated in neurodegenerative diseases, a diametrically opposite view is held by some that calpains may contribute to processes such as the remodelling of neuronal dendritic structures after neuronal injury. In murine cortical cultures subjected to dendritic injury, calpain did not seem to augment the injury. Quite the contrary, calpain appeared to assist dendritic recovery (Faddis et al. 1997). Similarly, the calpain inhibitor MDL28170 promotes neuronal recovery from injury by moderate hypoxia (Z.F. Chen et al. 1997). There are now clear indications that capn1 may be expressed in the early stages of the development of the skin (Michel et al. 1998, 1999). Michel et al. (1999) have provided denitive evidence for this. capn1 appears at day 54 of gestation of the human foetus, in the basal layer and in the periderm of the developing dermis. By day 125, capn1 is found in the granular layer. A possible link-up with differentiation is indicated by a signicant reduction of capn1 expression in biopsies obtained from harlequin ichthyosis, a condition known to result from abnormal terminal differentiation of the skin. The changes in capn1 expression were apparently specic for harlequin ichthyosis and were not encountered in other skin disorders. These ndings are compatible with the known ability and involvement of capn1 to inuence the proteolytic cleavage of prolaggrin to monomeric laggrin during terminal differentiation. It follows from the association between calpain and changes in cellular morphology that the cytoskeleton could be an important target of calpains. Emori and Saigo (1994) studied the expression of calpain in developing Drosophila embryos. In the early stages of cleavage of the fertilised eggs, calpain was found in the presumptive cleavage furrows and was co-localised with actin caps. As stated in an earlier section, calpains might regulate the levels of MAP components of the neuronal cytoskeleton (I. Fischer et al. 1991). Chakrabarti et al. (1993) studied the expression of calpains during brain development in rats, especially in relation to myelin formation. They found low capn2 levels during days 1 to 7 (postparturition), but these reached a peak between days 16 and 30. In rats that were more than 30 days old, roughly half of the calpain activity in the brain was found in the myelin component, whereas in 1 to 10-day old rats the majority of capn2 activity was cytosolic. Furthermore, this contrasted starkly with the pattern of expression of capn1, suggesting that capn2 is associated with the formation of the myelin sheath.
CALPAINS IN CELL PROLIFERATION AND APOPTOSIS

The involvement of calpain in the cell cycle traverse by proteolytic degradation of p53, which controls the cell cycle transition checkpoints, has been alluded to earlier. It is to be expected, therefore, that calpain will powerfully inuence cell proliferation
159
and growth. Calpeptin, an inhibitor of calpain has been found to inhibit the growth of the breast cancer cell lines MCF7, T47D, and ZR75 (E. Shiba et al. 1996). These cell lines are oestrogen receptor (ER) positive, but in a subsequent report it has been demonstrated that the calpeptin-mediated inhibition of growth is not due to a suppression of the hydrolysis of ER by calpain. This could be due to one or more of a variety of mechanisms that interfere with cell cycle control. There is a signicant inverse relationship between the expression of ER and EGFr in some of these cell lines. It would be interesting to see if the transduction of EGF signal via the cytoskeletal machinery is affected in any way by calpeptin. Aside from that, it is possible that calpeptin inuences p53 stability by inhibiting calpain. Apoptosis or programmed cell death is a natural physiological phenomenon. Cells undergoing apoptosis display distinctive morphological changes, such as condensation of nuclear chromatin and shrinkage of the cytoplasm. The cells break up into membrane-bound apoptotic bodies. The nuclear DNA is fragmented by Ca2+/Mg2+-dependent endonucleases. DNAse I and DNAse II have been regarded as putative candidates in DNA degradation, but recently Enari et al. (1998) have identied a caspase-activated DNA nuclease that is involved in the DNA degradation that occurs during apoptosis. The fragmentation of the DNA produces a characteristic ladder pattern of 180 to 200 bp of oligomers. Perturbations of the intracellular calcium levels are an important feature of apoptosis. High intracellular Ca2+ levels produced by calcium inux or its release from intracellular stores can lead to apoptosis (Orrenius et al. 1996). However, there is conicting evidence about the induction of apoptosis by calcium chelators and calcium ionophores. Nevertheless, the bcl2 family of genes, which is composed of inducers as well as inhibitors of apoptosis is known to regulate intracellular calcium (see Sherbet and Lakshmi, 1997b for references). Calcium signalling is known to activate a number of proteases. Among the notable ones are calpains and caspase. Both proteases have been implicated in apoptosis. Calpains often have been attributed with the ability to induce apoptotic cell death (Figure 21). They are said to be actively associated with T-cell activation and apoptosis (Squier et al. 1994; Sarin et al. 1995; S.J. Martin and Green, 1995). In mature T lymphocytes the induction of apoptosis by TCR is protease dependent (Sarin et al. 1995). The binding of the appropriate ligand of the TNF family to the Fas receptor initiates the activation of caspases and the down-stream protease cascade that leads ultimately to DNA degradation (see below). Dexamethasone is known to induce apoptosis of thymocytes, accompanied by Ca2+-dependent proteolytic activity. This is also accompanied by the autoproteolysis of the capn1 proenzyme, which suggests that calpain activation is taking place. Calpain inhibitors block apoptotic death (Squier et al. 1994). However, inhibitors of calpains have also been found to induce apoptosis. W. Zhu et al. (1995) found that two calpain inhibitors caused apoptosis of human prostate adenocarcinoma cells. However, in L1210 leukaemia cells, inhibition of calpain did not induce apoptosis (Wojcik et al. 1997). Apoptotic death can be induced in rat hippocampal pyramidal neurones in culture by exposing them to ABP and staurosporine. This can be inhibited by the calpain inhibitor MDL28170 (Jordan et al. 1997). There is a preliminary report that prevention of calpain activation produces resistance to necrosis in hepatocellular carcinoma cells
160
FIGURE 21 Involvement of the calcium-activated enzymes calpains and caspases in the induction of apoptosis. Raising intracellular calcium levels leads to activation of these enzymes and to apoptosis. In many instances it has been shown that when their activity is inhibited apoptosis is also suppressed. The Bcl-2 family has apoptosis-inducing genes as well as genes that inhibit apoptosis. These regulate intracellular calcium levels and inuence apoptosis through the caspase activation mechanism. The ced-9 gene of C. elegans encodes a protein that has apoptosis-inhibiting activity.
(A.S. Arora et al. 1995). However, necrosis and apoptosis are two distinct processes, and it would not be appropriate to discuss, in the same breath, how they are affected by calpain. A possible pathway by which calpain could inuence apoptosis has been suggested by Meredith et al. (1998) who showed that during apoptosis of human umbilical vein endothelial cells, the cytoplasmic tail of integrin 3 undergoes limited proteolysis. They showed not only that calpain is activated during apoptosis but also that calpain inhibitors prevent the proteolysis of the cytoplasmic domain of the integrin. Needless to say, more work needs to be done in order to establish the pathways of apoptosis that are inuenced by calpain.
CALPAINS IN CELL SPREADING AND MIGRATION

As stated often in these pages, the cytoskeletal dynamics are of overriding importance in signal transduction, cell morphology, and cell adhesion and locomotion. Calpain, by virtue of its proteolytic action, is bound to affect the integrity of the cytoskeleton and the adhesive and migratory properties of cells. Cell spreading is inhibited by the calpain inhibitors, calpeptin and MDL28170 (K.A. Potter et al. 1998). K.A. Potter et al. (1998) cloned a variant NIH3T3 cell line that overexpressed (two- to eight-fold) the calpain inhibitor, calpastatin. These cells markedly differed from the
161
parent cell line with respect to their morphology, spreading ability, and cytoskeletal characteristics. The variant cells failed to extend lamellipodia and showed abnormal lopodial extension and retraction. The calpain activity in these cells was found to be substantially reduced. Calpain inhibitors have been found to increase cell adhesiveness and reduce cell migration. A Chinese hamster ovary (CHO) cell line that expresses capn1 intrinsically at low levels has a low migratory ability (Huttenlocher et al. 1997). However, cell migration involves transient adhesion to and release from the substratum and these interactions require the linkage of the focal adhesion receptors to the cytoskeleton. The linkage of the cytoplasmic tail of integrin receptors is of crucial importance in the process of cell adhesion to the substratum. Calpain and similar proteins would affect it by cleaving the cytoplasmic domain of the receptor and may be regarded to be negatively regulating adhesion (Meredith et al. 1998). Palacek et al. (1998) have described migration as a function of cyclic membrane protrusion and adhesion at the leading edge and cytoskeletal contraction and detachment at the rear edge. They go on to demonstrate that calpain is involved in dissociation of the integrin receptor linkage with the cytoskeleton. These authors argue that an inhibition of integrin release by calpain decreases the speed of cell motility, and, inter alia, this may be due to the increased density of adhesive bonds or the strength of adhesion. Huttenlocher et al. (1997) have stated categorically that inhibitors of calpain increase cell adhesiveness and decrease the rate of detachment, whereas Meredith et al. (1998) regard calpains as negative regulators of adhesion. Because the adhesion of the leading edge and the retraction of the rear edge will necessitate a differential regulation of calpain, it would be interesting to see if the endogenous calpain inhibitor, calpastatin, might come into the adhesion dynamics. Calpastatin is itself subject to proteolysis by calpain (Nagao et al. 1994). For instance, very little is known about the intracellular localisation of calpastatin in relation to the direction or speed of cell locomotion. Furthermore, as indicated by the effects of calpain on integrin-mediated signal transduction, other components, such as talin, that are involved in the linkage of cytoplasmic domain of the integrin receptors with the cytoskeleton could conceivably form a part of the total picture.
CALPAINS IN INTEGRIN-MEDIATED CELL ADHESION AND SIGNAL TRANSDUCTION

The inhibition of integrin function is believed to be responsible for the downregulation of these adhesive and migratory abilities. Integrins are transmembrane proteins constituting focal adhesion plaques. Talin is a protein that links integrins to the cytoskeletal structures. Interestingly, talin has calpain susceptible sites. Therefore, calpain could affect the processes of cell adhesion and signal transduction, which are mediated by integrins. Using antibodies that recognise the calpain-susceptible sites of talin, Inomata et al. (1996) have demonstrated the involvement of calpain in integrin-mediated signal transduction. Calpain is able to cleave the cytoplasmic domain of 3 integrin (Du et al. 1995) and this could interrupt the signalling pathway. The focal adhesion kinase (pp125FAK) is a non-RTK associated with integrin-mediated signal transduction. Cooray et al. (1996) have argued that calpain
162
may have a role of terminating signal transduction in human platelets, by modifying pp125FAK by proteolysis and reducing its kinase activity as well as its subcellular location. Calpain indeed affects several events occurring after platelet aggregation, e.g., the attachment of the integrin tail to the cytoskeleton, as well as tyrosine phosphorylation of cytoskeletal protein and brin clot retraction (Schoenwaelder et al. 1997). Paxillin is another protein associated with focal adhesion plaques that is degraded by calpain (R. Yamaguchi et al. 1994).
CALPAINS IN CANCER GROWTH AND PROGRESSION

Calpain is signicantly involved, as discussed above, in a number of physiological processes that are integral features of the development and progression of cancer. We have seen that the suppressor phosphoprotein p53, which is closely linked with the control of cell cycle progression, is degraded by calpain. Mutations of p53 gene are a common event in tumorigenesis, and these lead to the abrogation of normal checkpoint control function exerted by wild-type p53. We have noted also that calpain is able to regulate the levels of cyclin D. Deregulation of the cell cycle using either target will lead to the deregulation of cell proliferation. Besides, one can also envisage a deregulation of the cell cycle under conditions where calpain is overexpressed per se or calpain activators are inappropriately expressed. Besides, there is some evidence that the calpains may inuence apoptosis in certain cell lines. This needs to be established beyond reasonable doubt, because there are reports to the effect that both calpain and its inhibitors are capable of inducing apoptosis. Tumour growth could be seen as a net outcome of these opposing factors of proliferation and apoptosis. A dynamic equilibrium between these two can result in growth stasis. Such a state of equilibrium also can apply to the growth of metastases and can lead to a state of dormancy of metastatic deposits. As shown in Figure 22, calpains and caspases may have positive or negative regulatory effects on tumour growth and metastatic spread. Although the expression of calpain in cancers has not been the subject of much investigation, there is some information on the association of calpain inhibitors with cancers. Squamous cell carcinomas of the lung have been reported to express a protein squamous cell lung carcinoma antigen (SCCA) that is able to inhibit capn1 (Kato, 1996). However, because SCCA inhibits other proteinases such as cathepsin L, it is difcult to assess the individual merit of calpain inhibition in squamous cell lung carcinoma. It should be borne in mind that the cathepsin family proteinases play an important role in tumour development and progression to the metastatic state, although, admittedly, cathepsin L has not been found to be as crucially important as other members of the family (see Sherbet and Lakshmi, 1997b). Braun et al. (1999a) have reported an enhanced expression of capn1 mRNA in clear cell renal carcinoma. The level of expression related closely with the presence of metastatic tumours in regional lymph nodes. Tumours with metastasis expressed capn1 mRNA at a higher level than tumours that had not spread to regional lymph nodes. Ceciliani et al. (1996a) isolated three low molecular weight proteins UK101, 114, and 150 from goat liver. These proteins are membrane associated. Of these,
163
Tumour growth Metastases
Apoptosis
Deregulation of cell proliferation
Endothelial cell apoptosis Antigenic response in host
Calpain/Caspase
Endogenous activators
FIGURE 22 Putative pathways of the involvement of calpain and caspase in the growth of the primary tumour and its metastases. The basic position is that these proteases can alter the steady state of population growth in both primary and secondary tumours. Caspase, but not calpain, has been credited with the ability to induce apoptosis of endothelial cells that can assist the entry of tumour cells into the vasculature. Endogenous activators of the enzymes have been suggested as a possible mode of immunising the host that can conceivably lead to loss of enzyme activation and potential loss of growth.
UK150 has been characterised to be a glycoprotein. UK114 has been suggested to be a marker for cancer (Ceciliani et al. 1996b). UK114 is a 14-kDa protein made up of 137 amino acid residues and shows sequence homology with the calpainactivating factor isolated from rat and bovine brain (Melloni et al. 1998a, 1998b). Furthermore, the goat liver UK114 also possesses calpain-activating properties (Melloni et al. 1998a). Bartorelli et al. (1996) demonstrated that administration of UK101 to patients with breast and colon carcinomas elicited signicant immune responses from the patients. Antibody titres against UK114 and 150 rose by 63 and 87% in breast and colon cancer patients, respectively. Although there has been very little background data on the levels of UK114 and 150 in patients with cancer, UK101 therapy appears to have been reasonably successful (Mor et al. 1997). It was administered to 217 patients with metastatic colon cancer. Twenty-ve patients in this trial showed reduction in metastatic mass, and 40% of the patients showed static disease. Patient survival correlated with both the baseline Karnofsky index score and anti-UK114 titre. Bussolati et al. (1997) have suggested that the antitumour effects are due to the cytolytic action of anti-UK114 antibodies. The latter were found to be cytolytic in vitro and were able to inhibit the growth of human tumours as xenografts in nude mice.
CALPAINS IN MYELODEGENERATIVE DISEASES

During early stages of brain development, calpains show a pattern of expression that is compatible with the view that they are mainly associated with the formation of the myelin sheath. Subsequent to this, they are found in the cytosol rather than in the myelin sheath. Myelin basic protein (MBP) and myelin-associated glycoprotein
164
are substrates of calpain. Banik et al. (1994) have shown that component I of human MBP is more susceptible to proteolysis by calpain than components II and III. They have identied two major and several minor cleavage sites in MBP. Because MBP is degraded in demyelinating diseases such as multiple sclerosis (MS), the potential role of calpains in the pathogenesis of MS has been studied in several laboratories. The presence of calpains 1 and 2 in myelin sheath was known for some time. High levels of capn1 and capn2 activity in myelin and premyelin were found to correlate with delayed myelination and high myelin turnover in the demyelinating paralytic tremor (PT)-mutant rabbit (Domanskajanik et al. 1992). Calpain activity has been reported to increase in MS tissue and cerebrospinal uid. Macrophages, lymphocytes, and reactive glial cells are the sources of the enhanced calpain activity. Calpains are found also in myelin-forming oligodendrocytes and Schwann cells (Banik et al. 1994). Both capn1 and capn2 occur in human lymphoid cell lines. Lymphocytic cell lines predominantly expressed capn1, but in monocytic cells the capn2 tended to be the major isoform (Deshpande et al. 1993). Activation of lymphoid cells with PMA and the calcium ionophore A23187 enhances the expression of both calpain protein and the corresponding mRNA (Deshpande et al. 1995a). Deshpande et al. (1995b) demonstrated further that activation of calpains was accompanied by the degradation of MBP and rat CNS myelin in vitro. A large proportion (60 to 80%) of the degradative activity was attributable to calpain, together with contributions from other proteases. It is conceivable that this degradation results in the exposure of antigenic epitopes. On the other hand, calpains occur in normal myelin and are associated with myelin formation during developmental stages. Therefore, calpains could be a normal constituent of myelin and may be involved in turnover of myelin proteins in the normal course of physiological events (Z.H. Li and Banik, 1995). Possibly, activated inltrating lymphoid cells generate a higher level of myelin breakdown and greater quantities of antigenic products. The status of the calpaincalpastatin system has been studied in experimental allergic encephalomyelitis (EAE), which is an animal model of MS, mainly in the laboratory of Shields and Banik. In Lewis rats with EAE, the optic nerves have been reported to show a marked increase in calpain content, although RT-PCR did not reveal any changes in calpain messenger RNA. Calpain-specic degradation of fodrin increased by 46% and the myelin-associated glycoprotein decreased by 25%. The endogenous inhibitor of calpain, i.e., calpastatin, was unaltered (Shields and Banik, 1998a,b). Human MS plaques reportedly show very similar alterations when compared with white matter from normal subjects (Shields et al. 1999a). Interestingly, alterations in calpain expression occur in peripheral lymphoid organs before the onset of symptomatic EAE (Shields et al. 1999b). MS plaques are characterised by reactive gliosis, inltration of inammatory cell, and a focal destruction of myelin and oligodendrocytes. An enhanced expression of calpain is said to occur in glial and inammatory cells (Shields et al. 1998a, 1998b). These observations putatively implicate calpain in the autoimmune-mediated demyelination process that occurs in EAE and MS. The regulation of the activity of this normal calpain component deserves serious study. Growth factors such as NGF, PDGF, and acidic as well as basic FGF exert different effects on capn1 and capn2 expression in transfected Schwann cells
165
(Neuberger et al. 1997). Neuberger et al. (1997) reported that cAMP and NGF inhibited both capn1 and capn2 activity. Both aFGF and bFGF enhanced capn1 activity by 37% and capn2 by 58%. In contrast, PDGF down-regulated both isoforms. Interestingly, the enhanced calpain activity following FGF treatment was associated with markedly reduced levels of calpastatin activity. In comparison, in cells exposed to cAMP and NGF, calpastatin levels were nearly comparable with those of control cells. These observations suggest that growth factors strongly affect the dynamic equilibrium between calpains and their endogenous inhibitor. The experiments of Neuberger et al. (1997) also indicate the possibility of their intracellular translocation. The expression of calpain isoforms as well as calpastatin isoforms has been studied in the EAE model (Shields and Banik, 1998). The transcription of neither the calpain isoforms nor calpastatin isoforms showed an up-regulation in EAE, but the expression of calpain protein increased more than fourfold. Calpastatin translation was also increased in EAE. This suggests another putative mechanism by which the calpaincalpastatin dynamics could be altered in myelodegenerative conditions.
CALPAINS IN MUSCULAR DYSTROPHY

Several forms of muscle myopathy are known. The major conditions are Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and limb girdle muscular dystrophy (LGMD). LGMD is recognised to be of two types: the autosomal-dominant type 1 (subtypes 1A1D) and the recessive type 2, with several subtypes. Calpain has been implicated in the pathogenesis of LGMD-2A.
ASSOCIATION OF CALPAINS WITH DUCHENNE MUSCULAR DYSTROPHY

Calpains are activated in many pathological and aberrant physiological conditions that result in muscle wastage. There is much evidence that calpains degrade a number of myobrillar proteins, and there is consensus that calpains participate actively in the early stages of myobril breakdown (F.C. Tan et al. 1988). Calpains as well as cathepsins have been associated with muscle bre degradation occurring in inammatory muscle myopathy (Kumamoto et al. 1997). The characteristic muscle weakness and the progressive degradation of striated muscle encountered in DMD and BMD appear to be attributable to calpain activity, besides the abnormalities in the DMD and BMD gene loci that alter the function of dystrophin and the dystrophinglycoprotein complex. DMD is generally associated with a loss of dystrophin, which links the sarcolemma with the actin cytoskeleton. Although the precise function of dystrophin is unclear, its loss is associated with increased calcium inux and high intracellular levels of calcium. Being calciumactivated enzymes, calpains were implicated in the pathogenesis of this chronic degenerative disease many years ago (Arahata and Sugita, 1989). Kumamoto et al. (1995) studied the expression of calpains and calpastatin in DMD and BMD. Calpains occurred at markedly elevated levels in muscle bres that had atrophied. In necrotic bres, both calpain and calpastatin were expressed at moderate levels, but hypertrophic and opaque bres manifested no activity at all. Ueyama et al. (1998) have recently conrmed the increase of capn1 and capn2 proteins and their mRNAs
166
in progressive muscular dystrophy as well as in ALS. However, capn3 appeared to be unaffected. These ndings suggest a possible deregulation of the calpaincalpastatin system as the cause of progressive muscle degeneration. Fasting has serious consequences in terms of calpain activation. A two-fold increase in the degradation of myobrillar protein of rabbit skeletal muscle occurred in response to fasting, together with an increase in mRNA levels encoding capn1 and capn2 as well as calpastatin mRNA levels (Ilian and Forsberg, 1992, 1994). Fasting raised the mRNA levels of cathepsin D and proteasome. These changes appear to be specic for skeletal muscle. Ilian and Forsberg (1994) did not nd any changes in the protease mRNA levels in other tissues such as liver, lung, or kidney. The transcription of the genes seems to be up-regulated to meet the need to mobilise muscle protein in response to the physiological insult. Therefore, Ilian and Forsberg (1992) suggest this explains why they did not encounter any increase in capn1 and capn2 protein levels. However, it is essential to know the stability of the messages, as well as the half-life of the enzymes, before relating the ndings to the continued requirement for protein mobilisation. It should be borne in mind also that other proteases would have contributed to the process of protein mobilisation as well. At any rate, Kumamoto et al. (1992) conrmed the earlier ndings of Ilian and Forsberg (1992) relating to the increase of calpains in response to fasting. They also demonstrated that capn1 and capn2 occurred in the Z-band, where the degradation of myobrils is initiated. Immunostaining studies showed that the Z-band contained twice as much calpain and calpastatin as the I-band or the A-band.
CALPAINS
AND
LIMB GIRDLE MUSCULAR DYSTROPHY
Three autosomal dominant and several autosomal-recessive loci have been identied. With the view of placing the following discussion in the proper perspective, but at the risk of a minor digression, details about the LGMD subtypes and the molecular features associated with them have been provided in Table 11. LGMD-2A is an autosomal-recessive form of muscular dystrophy. The skeletal muscle-specic homologue capn3 has been implicated in LGMD-2A. The capn3 gene is located in the region of chromosome 15q15.1q21.1 (Fougerousse et al. 1994). Five distinct genes have been identied with LGMDs, and capn3 as a putative LGMD-2A candidate gene (Chiannilkulchai et al. 1995; Beckmann et al. 1996). Recently, capn3 mutations have been found to co-segregate with the disease in families with LGMD-2A, and this has led to the suggestion that the disease is due to a defect in the enzyme rather than to abnormalities of any structural proteins (Richard et al. 1995). This has been conrmed by recent ndings that mutations of the gene result in the loss of proteolytic activity of capn3 (Y. Ono et al. 1998). Therefore, there seems to be a link between this loss of proteolytic activity of capn3 and the pathogenesis of LGMD2A. Spencer et al. (1997) did not nd capn3 in muscle biopsies from LGMD-2A patients, but did detect it in control subjects as well as in non-LGMD-2A patients. Capn3 is said to undergo rapid autolysis and it has a half-life of less than 1 hr, which could be one of the reasons for the failure of detection. Sorimachi et al. (1995) found that capn3 also interacts with connectin (titin), which spans the M- to Z-lines of muscle sarcomeres. They have, therefore, suggested that connectin may regulate
167
TABLE 11 Limb Girdle Muscular Dystrophy Types and Associated Molecular Features
LGMD Type/Subtype Associated Molecular Features Autosomal Dominant LGMD-1A LGMD-1B LGMD-1C Ref.
Caveolin-3 mutations, 3p25 Also in DMD and mdx mice
Minetti et al. (1998); McNally et al. (1998a) Vaghy et al. (1998)
LGMD-1D Autosomal Recessive Calpain, 15q15.1 Dysferlin mutations, 2p13 -Sarcoglycan missense, -sarcoglycan mutation and loss, 13q12 -Sarcoglycan mutation, 17q12-q21
LGMD-2A LGMD-2B (Myoshi myopathy) LGMD-2C
LGMD-2D
LGMD-2E LGMD-2F
-Sarcoglycan missense mutations, 4q12 -Sarcoglycan mutations, 17q11-q12
Chiannilkulchai et al. (1995); Moreira et al. (1997) J. Liu et al. (1998); Bashir et al. (1996, 1998) Van der Kooi et al. (1998); Jung et al. (1996); Moreira et al. (1997) Ettinger et al. (1997); McNally et al. (1998b); Moreira et al. (1997) Bonnemann et al. (1996); Moreira et al. (1997) Nigro et al. (1996); Duggan et al. (1997); Moreira et al. (1997)
Note: Jung et al. (1996) state that LGMD-2A2E all show abnormal expression of , , , and -sarcoglycans.
capn3. Obviously, there is a need for much further work to elucidate the role of capn3 in LGMD-2A, especially with a view to relating it to the signicance of the association of sarcoglycans with LGMD. As Spencer et al. (1997) have pointed out, capn3 was regarded as the agent responsible for the posttranslational generation of - and -sarcoglycans, but it may not be so involved.
14
Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia
CASPASES IN APOPTOTIC CELL DEATH

Calcium-mediated activation of caspases is also associated with apoptosis. The apoptotic pathway involves a cascade of proteolytic events mediated by caspases, their activators, and their repressors. Caspases are cysteine proteases of a family of interleukin-1-converting enzymes (ICE). A number of proteases ced-3, ced-4, and ced-9 have been identied in the nematode C. elegans. Of these, ced-3 is a caspase. A human homologue of the ced-4 protein, called apaf-1, has been described (Zou et al. 1997). Three new forms of apaf-1 have since been identied in mammalian cells (Hahn et al. 1999). The apaf-1 protein appears to promote cytochrome c-dependent activation of caspases (Zou et al. 1997; Kluck et al. 1997; P. Li et al. 1997). Cytochrome c has been found to be involved in caspase activation as an early event in many forms of apoptosis. However, it may not be involved in at least Fasinduced activation of caspase and apoptosis. (Vier et al. 1999). The chromatin condensation and nuclear fragmentation that occur in apoptosis involve, besides the caspases, caspase-activated DNAase (Enari et al. 1998; X.S. Liu et al. 1998). When apaf-1 is stably transfected into HL-60 cells, not only is apaf-1 overexpressed but there is also a marked induction of apoptosis (Perkins et al. 1998). According to two recent reports, apaf-1 (/) null mice died at day 15 of embryonic growth and showed reduced apoptotic cell loss and overgrowth of the brain. The null mutants also manifested marked craniofacial abnormalities. Markedly enhanced proliferation of neuronal cells was also encountered in these embryos. Apaf-1 (/) cells seemed to resist apoptosis, with an attendant impairment of caspase activation (Yoshida et al. 1998; Cecconi et al. 1998). In oncogene E1A-dependent induction of apoptosis, in which the caspase is activated by the oncogene product, the activated caspase-9 forms a complex with apaf-1 and cytochrome c (Fearnhead et al. 1998). As shown in Figure 21, some members of the bcl-2 family of genes can inhibit apoptosis. A general model has been proposed that the bcl-2 proteins inhibit apoptosis by binding to apaf-1, thereby preventing the activation of caspases. However, this model has been disputed by Morishi et al. (1999), who believe that the inhibition might not be a direct effect of the sequestration of apaf-1 by bcl-2 proteins.
169
170
Caspase-9 might initiate the programme of apoptosis or activate other caspases downstream and ultimately cleave downstream target proteins, which brings about apoptotic disintegration of the cell (Thornberry, 1997; Thornberry and Lazebnik, 1998). Cytochrome c-mediated activation of capase-9 seems to be followed by the activation of a string of other caspases. When cell extracts are depleted of caspase9, the activation of the downstream caspases does not occur. This indicates that activation of capsase-9 is obligatory for the activation of other caspases by cytochrome c (Slee et al. 1999). In Jurkat cells induced into apoptosis, caspase-3 occurs with caspase-6 in the activation complex. HSP60 is also found in this complex (Xanthoudakis et al. 1999); these authors have suggested that this HSP may participate in protein folding and aid in the proteolytic activation of caspase-3. In vitro, HSP60 aided the activation of the caspase by several caspase promoters. As many as ten caspases (caspases-1 to -10) have been identied. These form three natural groups based on their mode of activation from the corresponding procaspase form. Caspases-1, -2, -4, and -5 show autocatalytic activation from their pro-caspase form. The pro-caspases are believed to bind to activator molecules and lead to their oligomerisation and autoactivation. The second group composed of caspases-3, -6, -7, and -9 do not show autocatalytic activation. Lysosomal cysteine proteases may be involved in the activation of some caspases (Ishisaka et al. 1998). The overexpression of caspases-3 and -7 leads to apoptosis of the prostate cancer cell lines called LNCaP. In this system, caspase-7 seems to be activated by proteolysis (Marcelli et al. 1999). Caspases-8 and -9 undergo TNF-R1/Fas (CD95) mediated activation. The Fas ligand is a member of the TNF family. It is expressed in activated T cells. The apoptosis of target cells is induced by the binding of this ligand to its receptor (Nagata and Golstein, 1995; Nagata, 1997). The occupancy of Fas has been found to lead to the activation of caspases (Enari et al. 1995; Longthorne and Williams, 1997; Armstrong et al. 1996; Boldin et al. 1996) and of downstream proteases (Nagata, 1997; Fraser and Evan, 1996). The outcome of the function of this proteolytic cascade initiated by Fas binding is DNA fragmentation. The activation is believed to be due to the binding of caspase to the activated membrane receptor complex. Another line of evidence that strongly supports the role of caspases concerns the resistance of some cell systems to the induction of apoptosis by Fas receptor binding or other factors. Perara and Waldmann (1998) have used peripheral blood monocytes, which undergo spontaneous apoptosis unless the culture medium is supplemented with serum, growth factors, bacterial LPS, or cytokines. This induction of resistance to apoptosis is accompanied by a marked down-regulation of caspase8. Deciency of caspase-8 also makes the Jurkat cell line JB-6 resistant to Fasmediated apoptosis (Kawahara et al. 1998). T.S. Zheng et al. (1998) deny any role for caspases in the induction of Fas-mediated apoptosis of liver cells, but recognise that caspase-3 may be instrumental in bringing about morphological changes by cleaving substrate proteins. Equally, as suggested by Kawahara et al. (1998), cell killing triggered by Fas binding could take two pathways. One of these is via caspase8, and the other is more akin to necrosis that does not involve caspases. It has been suggested also that HSPs may interfere with apoptotic cell death. HSP70, for instance, seems to ensure cell survival by inhibiting caspase-3-dependent events of
171
apoptosis (Jaattela et al. 1998). However, the involvement of HSPs must be interpreted with some caution, especially in light of HSP60 being shown to be possibly associated with the activation complex of caspases. Caspases have several substrates such as poly (ADP-ribose) polymerase (PARP) (see below), lamin A, and several other cytoplasmic and nuclear proteins (see review by Zhivotovsky et al. 1997). The actin-binding proteins, -fodrin and gelsolin, are cleaved by caspase-3. Actin itself is cleaved by caspase. According to Mashima et al. (1999), this results in the generation of two actin fragments of 15 and 31 kDa. When introduced into cells, the 15-kDa actin fragment brings about morphological changes that are akin to apoptosis. The retinoblastoma susceptibility rb protein is also degraded by caspase. Caspase-3 cleaves CAMPK within its catalytic site (McGinnis et al. 1998). Nedd4, a ubiquitinprotein ligase, is cleaved by several caspases (Harvey et al. 1998). The cleavage of these proteins is associated with apoptosis, but the signicance of the process is not understood. The proteolytic cascade involves endonucleases downstream of caspases and calpain in the apoptotic pathway. Because caspase inhibitors also result in the inhibition of DNA fragmentation (X. Liu et al. 1997), it would appear that caspase may expose the DNA to endonuclease function by cleaving the chromatin-associated proteins such as lamin B, histone H1, and topoisomerase II (Neamati et al. 1995; Kaufmann, 1989). In the same vein, nuclear scaffold protease inhibitors block not only the cleavage of lamin but also DNA fragmentation. It seems then, that the cleavage of chromatin scaffold proteins by either pathway results in DNA fragmentation (Lazebnik et al. 1995). Not all caspases are inducers of apoptosis. Caspase-11 is a pro-inammatory caspase and is activated by the lysosomal protease cathepsin B (Schotte et al. 1998). Apoptotic changes triggered in nuclei by CaCl2 are inhibited by the inhibition of caspase-3 and to a lesser extent by caspase-1 (Juin et al. 1998). Hydrogen peroxide (at 50 M, but not at higher concentrations) induces apoptosis of Jurkat T lymphocytes and, in parallel, an enhancement of caspase activity is also detected. However, at H2O2 concentrations greater than 50 M, cells undergo necrosis (Hampton and Orrenius, 1997). Caspase seems to be involved in neuronal apoptosis following ischemic injury, and again caspase inhibitors appear to be able to protect neurones from undergoing apoptosis (Gorman et al. 1998). Overall, it seems reasonable to conclude that caspase plays a signicant role in the cell apoptosis occurring in response to a variety of extracellular signals. The evidence is based, as noted, mainly on the protective function of caspase inhibitors and also on the inhibition of the caspase substrate PARP. It ought to be stated, however, that in certain cell types, activation of caspase-3 does not automatically lead to apoptosis (Well et al. 1998). There could be other components in the pathway, e.g., the p36-MBP kinase (MBPK), the activation of which has been demonstrated recently by Kakeya et al. (1998). These authors showed that cytotrienin A, isolated from Streptomyces, induces apoptosis of HL-60 cells by activating MBPK. The broad-spectrum caspase inhibitor, Z-Asp-CH2-DCB, inhibited activation of MBPK as well as apoptosis. However, it did not inhibit the activation of other kinases such as c-jun N-terminal kinase/stressactivated protein kinase and p38 MAPK, which were also activated by cytotrienin A, albeit with a different kinetic pattern from MBPK activation. This suggests that
172
activation of MBPK is a component of the apoptosis-signalling pathway. The pathways of caspase and calpain involvement in apoptotic cell death are shown in Figure 21. It is fairly obvious from the above discussion that at present our knowledge of the mechanisms involved is rather limited.
POLY (ADP-RIBOSE) POLYMERASE AS A MARKER OF APOPTOSIS

The process of recovery from DNA damage involves the synthesis of poly (ADPribose) in response to strand breaks. The nuclear enzyme poly (ADP-ribose) polymerase (PARP) catalyses the cleavage of NAD+ into nicotinamide and adenosine 5diphosphoribose (ADP-ribose). PARP further catalyses the covalent linkage of poly (ADP-ribose) to the damaged sites in the DNA. PARP is activated upon binding to DNA strand breaks. It binds to DNA via its zinc nger domains (De Murcia and De Murcia, 1994; De Murcia et al. 1997). It links poly (ADP-ribose) to the nicks in the DNA and maintains its structural integrity until DNA excision repair is carried out by DNA polymerases (Lindahl et al. 1995). Caspases cleave this 113-kDa enzyme into an N-terminal DNA-binding fragment and a C-terminal catalytic fragment. Apoptosis is characterised by the cleavage of PARP and the appearance of the 89-kDa catalytic fragment is regarded as a marker event of apoptosis. There is a large body of evidence that PARP is involved in apoptosis. The ionophore A23187 has been reported to induce apoptosis in PC12 cells, when the ionophore is applied at low concentrations. This is accompanied by the activation of caspase-3. When caspase-3 is inhibited the process of apoptosis is also inhibited (Takadera and Ohyashiki, 1997). Dexamethasone and thapsigargin both induce apoptosis of WEHI mouse lymphoma cells, albeit by different pathways. Nevertheless, the activation of caspase-3 and cleavage of PARP accompanied the induction of apoptosis by both these agents. Both latter events were inhibited by overexpression of the bcl-2 gene (McColl et al. 1998). The protein encoded by the ced-9 gene of C. elegans is an inhibitor of apoptosis belonging to the bcl-2 gene family (Reed, 1997). A similar effect on apoptosis and PARP cleavage by caspase inhibitors had been demonstrated earlier in a different cell system by Bonfoco et al. (1996). The nature of PARP involvement is increasingly being appreciated. Caspase-3 activation and the triggering of PARP cleavage may be described as key events that occur in the induction of apoptosis. The poly (ADP-ribosylation) of nuclear proteins, followed by caspase-3-mediated PARP cleavage, is an early event of the apoptotic process, and it may indeed be an important requirement for apoptosis to proceed. The cleavage of PARP, internucleosomal DNA fragmentation, and morphological changes in nuclei do not occur if cells are depleted of PARP by using antisense strategy. These apoptosis-associated changes take place in cells with wild-type PARP (+/+) genotype, but are conspicuously absent in PARP (/) mutants (SimbulanRosenthal et al. 1998). Simbulan-Rosenthal et al. (1999) have since argued that PARP is also a component of the DNA replication complex, which includes several important proteins. The expression of these might be affected by PARP depletion, inhibit DNA replication, and commit the cells to the apoptotic pathway.
173
Notwithstanding these studies, the precise role played by PARP is not fully understood. The inhibition of PARP affects apoptosis. The activation of PARP by DNA damage results in NAD+ depletion and a depletion of ATP in the restoration of NAD+ levels. Therefore, cell death can be a result of the loss of ATP. In PARP / cells, ATP levels seem to be maintained and the cells are protected from necrotic cell death. However, in both PARP +/+ and PARP / mutants, cells can be equally susceptible to apoptosis (Ha and Snyder, 1999). In renal epithelial cells, hydrogen peroxide-induced PARP activation results in necrotic death due to NAD+/ATP depletion (Filipovic et al. 1999). Walisser and Thies (1999) found that endothelial cells exposed to hydrogen peroxide undergo cell death. Presumably, here too cell death may have been caused by NAD+ and consequent ATP depletion. However, when PARP is inhibited these cells switch to apoptosis. Other studies, however, suggest PARP inhibition may indeed protect cells from apoptosis (Richardson et al. 1999; Guo et al. 1998). A putative link-up between the function of caspases and PARP in apoptosis has emerged recently. Aoufouchi et al. (1999) transfected a PARP (/) cell line with the DNA-binding domain fragment of PARP (DBD), or mutants: DBDbd () which is defective in binding to DNA strand breaks, and DBDcl (), which resists cleavage by caspases. DBD transfected PARP (/) cells showed no changes in staurosporineinduced apoptosis. Cells that had been transfected by both mutants showed marked inhibition of apoptosis. Furthermore, the mutant DBDs inhibited the cleavage of the catalytic subunit of DNA-dependent protein kinase by caspase-3. These results suggest that PARP might be involved in the events leading to caspase activation. It seems, therefore, that it is possible to dissociate the DNA-binding properties of PARP from caspase activation. Aoufouchi et al. (1999) have suggested the possibility that PARP might interact with components involved in caspase activation.
CASPASE-MEDIATED APOPTOSIS AND CELL GROWTH INHIBITION IN TUMOUR EXPANSION

The relevance of caspase-mediated apoptosis in tumour development has been emphasised recently by the nding that enhanced expression of caspase-2 leads to a reversion of ras-induced transformation in NIH3T3 cells (Hiwasa and Nakagawara, 1998). Hiwasa and Nakagawara (1998) transfected caspase cDNAs into c-Ha-rastransformed cells. Enhanced expression of caspase-2 alone, but not caspases 1 and 2 together, resulted in a reduction in the ability of the transfected cell to grow in soft agar. This was accompanied by ras protein degradation, suggesting that the apparent reversion of the transformed phenotype was due to the degradation of the transforming gene product by caspase-2. However, Hiwasa and Nakagawara (1998) have not presented any evidence relating to the tumorigenicity of the ras-transformed cells, or any data on possible inhibition of tumorigenicity by the transfection of caspase-2 cDNA into these transformed cells. It is essential to recognise that cell transformation is a process quite distinct from tumorigenicity. Whether caspaseinduced apoptosis occurring in the developing tumour can control the growth of the tumour is still an open question. As stated elsewhere, apoptosis subserves an
174
important function in maintaining the kinetics of cell expansion in metastatic growth. By inference, there is no reason why a similar role may not be played by apoptosis in the control of primary tumour growth, or, to put it more precisely, primary tumour expansion. The apoptosis-mediated restriction of a cell population can be differentiated from control by inhibition of growth, although these might not represent totally independent mechanisms. This has been demonstrated in the case of TGF-, which inuences apoptosis as well as cell growth. However, in WEHI 231 cells, the caspase inhibitor BD-fmk can effectively inhibit TGF-induced apoptosis but not counteract TGF-mediated inhibition of cell growth (T.L. Brown et al. 1998). This apparent dichotomy of mechanisms is obvious also from the pathways of radiation-induced apoptosis. Ionising radiation induces the activation of caspase-3, which is followed by the induction of apoptosis. Ionising radiation induces rapid apoptosis in human lymphoblast cells expressing wild-type p53, but in cells where p53 is mutated or abrogated by viral oncoprotein, apoptosis is delayed as well as reduced (Yu and Little, 1998). In this instance apoptosis seems to occur in conjunction with growth control mediated by wild-type p53. On the other hand, caspases have been shown to cleave p53-induced cyclin-dependent kinase inhibitor p21waf1/cip1 and induce cells to undergo apoptosis (Y.K. Zhang et al. 1999). In the latter case, apoptosis seems to depend on proliferation. In contrast with p53-mediated apoptosis, VD3 has been reported to induce apoptosis of certain breast cancer cells independently of both p53 and caspases. This report is based on the observation that VD3 produces growth arrest and apoptosis of both MCF7 cells, which are p53 positive, and T47D cells that are p53-negative. Furthermore, this apoptotic induction is not inhibited by inhibitors of caspase, whereas TNF- or staurosporine-induced apoptosis is inhibited (Mathiasen et al. 1999). Whatever pathway apoptosis might take, it is inevitable that tumour growth and subsequent processes should be shaped by balancing forces of apoptosis and cell cycle control factors. Nonetheless, it is worthwhile to note here that ICE-like protease expression has been reported to correlate with progression and prognosis of neuroblastoma. The frequency of expression of ICE mRNA was markedly reduced in advanced-stage neuroblastomas as compared with early-stage tumours (Ikeda et al. 1997). The expression of caspase-3 in normal gastric mucosa, gastric adenomas and adenocarcinomas has been studied in some detail by Hoshi et al. (1998). They found that caspase-3 expression decreased from a high level (42% cells staining for caspase) in nonneoplastic gastric mucosa to a lower level (33%) in adenomas, and to a still lower level (17%) in adenocarcinomas. These differences were statistically signicant in spite of the large standard deviations of the mean. The caspase-3 positivity, of the three groups, correlated directly with apoptotic indices determined by the TUNEL method and inversely with proliferative indices provided by Ki67 labelling. These data are consistent with the view that a loss of apoptosis-mediated control over cell turnover is an important feature of tumour growth. Conversely, there is an implicit suggestion that ICE-like proteases may be involved in apoptosismediated regression of tumours, in the demonstration by Ikeda et al. (1997) that ICE protease staining could be co-localised in the nucleus with DNA fragmentation. In this context, one should also take note of the recent report that caspase-3 activity was found to increase in colonic carcinomas and adenomas as compared with normal
175
mucosa (Leonardos et al. 1999). Obviously, these observations may serve to conrm that the expression of the caspase is related more to the degree of apoptosis taking place in the tumour than to the degree of tumour progression. Donoghue et al. (1999) reported a difference in the pattern of caspase-3 distribution in B-cell diffuse large cell lymphoma. In immunohistochemistry, caspase-3 showed a diffuse distribution in the cytoplasm or a punctate or spotty pattern. The diffused pattern appeared to relate to poor prognosis, and the punctate pattern was associated with complete response to therapy. The authors have stated further that where the percentage of caspase-3-expressing cells was low, prognosis was poor. Not only are some of these ndings not compatible per se, but the study seems to raise more questions than it successfully answers. It should be conceded, however, that it would be unhelpful to attribute all apoptotic activity to caspases. Donoghue et al. (1999) found no correlation between apoptosis and caspase expression in the cells, but the degree of apoptosis was associated with poor prognosis. A nding of potential signicance is that the caspase distribution pattern might be signicant in terms of enzyme activity. Whether the punctate distribution might reects sequestration of the enzyme is worth further investigation. It should be recognised, nevertheless, that caspase expression may reect cancer progression and that caspases may actively promote metastatic deposition by a more direct route. Thus, a high proportion of in situ and invasive (58 and 90%, respectively) carcinomas of the breast stain strongly for caspase-3. Caspases-6 and -8 also are expressed at high levels more frequently in carcinomas than in hyperplasia. The enhanced expression correlated well with apoptotic indices in the samples. Furthermore, enhanced apoptosis was associated with poor prognosis (Vakkala et al. 1999a, 1999b). The formation of metastatic lesions depends on a cascade of events; prominent among them is the invasion of the vascular and endothelial systems by cells of the primary tumour. The entry into the circulatory system has been attributed to an active process of transmigration or diapedesis across the endothelial layer, as well as to the inherent structural defects often found in the endothelium. Recently, Kebers et al. (1998) found that several breast cancer cell lines, among them MCF7, MDA-MB231, T47D, and HT1080, induced a four-fold increase in apoptosis of human umbilical vein endothelial cells (HUVEC), with an attendant enhancement of caspase-3 activity. The interaction of MCF7 cells with HUVEC caused a transient increase in intracellular calcium levels (Lewalle et al. 1998), which, presumably, may have led to caspase activation. The induction of apoptosis required cellcell contact, because media conditioned by the growth of these cells were ineffective. Kebers et al. (1998) have further observed that lymphocytes do not induce apoptosis, suggesting that the apoptosis of endothelial cells might constitute a specic mechanism in the diapedesis of tumour cells. Whether caspase-mediated apoptosis of endothelial cells occurs in vivo is yet to be demonstrated. The outcome of tumour progression has often been assessed in relation to a single given variable as a prognostic factor. As noted above, in the context of caspases we have a paradoxical situation that in both caspase-positive and caspase-negative circumstances some relationship is noticed with tumour progression. As observed earlier, tumour growth and progression are a net outcome of the balancing forces of apoptosis and cell cycle control factors and cell proliferation. Perhaps it would
176
be more rewarding to reexamine the question of caspases in tumour progression in this light. For this viewpoint, the recent ndings of Volm and Koomagi (2000) are most encouraging. They have examined the relevance not only of caspase-3 but also of c-myc expression in the prognosis of non-SCLC. Volm and Koomagi (2000) have reported that caspase-3-negative patients had a median survival time of 41 weeks as compared with 79 weeks for caspase-3-positive patients. They then looked at cmyc expression and its inuence on prognosis. Patients who were c-myc negative had a median survival time of 89 weeks, whereas the median for c-myc-positive patients was 43 weeks. Not only did these two factors correlate inversely with survival, but, myc/caspase+ patients showed a median survival time of 102 weeks as compared with only 22 weeks for myc+/caspase patients. This clearly makes the point that more than one factor might inuence a given cellular feature and thereby determine the outcome of the disease. The study also serves to further emphasise that it would be unhelpful to try to evaluate a single prognostic factor, while other factors might be present that would impinge on the direction of cellular changes.
CASPASE-MEDIATED PROTEOLYSIS OF FODRIN: IMPLICATIONS FOR APOPTOSIS, CELL ADHESION, CELL MIGRATION, AND NEOPLASTIC TRANSFORMATION
Among the several substrates of caspases is the membrane protein called fodrin. The erythroid homologue of fodrin is known as spectrin, with which fodrin shares substantial amino acid sequence homology. Fodrin is attributed with the function of maintaining the structural integrity of the plasma membrane. Fodrin and spectrin form a major component of the skeletal network that underlies the plasma membrane (Levine and Willard, 1981; W.J. Nelson et al. 1990; Bennett and Lambert, 1991; Bennett and Gilligan, 1993). Fodrin (alias spectrin) isoforms are also found in the membranes of the Golgi apparatus (Devarajan et al. 1996, 1997; Beck et al. 1994, 1997; Godi et al. 1998; Fath et al. 1997; Stankewich et al. 1998), lysosomes (Hoock et al. 1997), and intracellular vesicles (Malchiodi-Albedi et al. 1993; Stankewich et al. 1998). Fodrin is an actin-binding protein; therefore, two further putative functions should also be considered. One of these is a presumptive involvement in the process of signal transduction, because fodrin has been found to be able to inhibit phospholipases A2 (PLA2), C, and D (Lukowski et al. 1996, 1998). In comparison, other cytoskeletal proteins such as actin and vimentin are far less efcient than fodrin (Lukowski et al. 1996). Phospholipases are closely associated with the generation of DAG and IP3 from PIP2. These are involved in the activation of downstream pathways of signal transduction that, in turn, involve the activation of appropriate protein kinases, such as PKC, and Ca2+ release from intracellular stores. Another potentially important function that can be attributed to fodrin is in inuencing cell adhesion and migration. Spectrin is required for neurite extension in neuroblastoma cells. Sihag et al. (1996) were able to inhibit neurite extension in NE2a/dl neuroblastoma cells with an antibody directed against the N-terminal domain of spectrin. The latter is known to interact with actin. The protein called
177
ankyrin mediates the link-up between membrane adhesion proteins and the spectrin cytoskeleton. The expression of some of these adhesion molecules has been shown to cause cell aggregation in which process both ankyrin and spectrin are recruited to the foci of adhesion (Dubreuil et al. 1996). In wound healing of corneal epithelium, fodrin becomes redistributed from its subplasma membrane location to a cytoplasmic location. This redistribution occurs soon after wounding of the epithelium. A similar redistribution occurs in response to PMA treatment and is inhibited by PKC inhibitors (Amino et al. 1995). Presumably these events are related to the cell migration that follows, although there is little direct evidence linking these. As stated above, fodrin (spectrin) is a substrate for caspases. Apoptosis induced by several different pathways has been shown to be accompanied by proteolysis of fodrin. Inhibition of apoptosis also results in the inhibition of fodrin proteolysis (Martin et al. 1995). This proteolysis seems to be produced by ICE/ced-3 proteases (Cryns et al. 1996; Vanags et al. 1996). Kouchi et al. (1997) believe that calpains are not associated with the cleavage of the 240-kDa subunit of fodrin in the apoptosis of rat thymocytes both in vivo and in vitro. However, Porn-Ares et al. (1998) do implicate calpains. Fodrin occurs in a variety of cell types including keratinocytes, chromafn cells, and renal epithelium, and in a variety of epithelial and broblast cell lines. Fodrin shows a homogeneous cytoplasmic and a discontinuous membrane distribution in benign melanocytic tumours, whereas normal melanocytes at the basal layer of the epidermis only faintly stain for fodrin at the plasma membrane. Overall, neoplastic cells show greater amounts of fodrin than their nonneoplastic counterparts (Tuominen et al. 1996). This observation has been conrmed in an immunohistochemical study of a variety of adenocarcinomas and squamous cell carcinomas by Sormunen et al. (1997). However, malignant melanomas contain subpopulations that do not express fodrin (Tuominen et al. 1996). This does not detract from any putative relationship between fodrin expression and malignancy, because malignant tumours are notoriously heterogeneous with respect to a large spectrum of cellular characteristics. Although much work needs to be done in this area, already there are clear indications that caspase-mediated alterations in fodrin expression and function might be involved in biological processes (e.g., apoptosis, cell adhesion, motility, and modulation of cell shape) that are inherent features of tumour development, dissemination, and metastasis.
CASPASES AND NEURONAL LOSS IN ALZHEIMERS DISEASE

Alzheimers disease is characterised by massive neuronal loss, which has been attributed, in recent years, to apoptotic cell death (Barinaga, 1998). The participation of caspases in inducing apoptosis has naturally led to the investigation of these proteinases, together with the bcl-2 family genes, in the pathogenesis of Alzheimers disease (Figure 23). Kitamura et al. (1998) showed that the expression of several bcl-2 family genes was up-regulated in Alzheimers disease. Caspases are involved in the induction of neuronal apoptosis (Bambrick and Krueger, 1999). Two genes known as PS (presenilin)-1 and PS2 have been associated in a mutated form with early onset of Alzheimers disease. The presenilins are integral proteins of the ER.
178
FIGURE 23 Possible pathways by which caspases may regulate neuronal apoptosis associated with Alzheimers disease. PS, presenilin proteins; PS-P, phosphorylated form of PS protein.
Their mutation could lead to a perturbation of calcium homeostasis in the cell with attendant aberrations of calcium-mediated processes of apoptosis as well as the processing of the amyloid precursor protein. Wild-type PS has been shown to promote neurite outgrowth in neuroblastoma cells grown in vitro (Dowjat et al. 1999). It is conceivable that mutations would affect this process as well. The involvement of PS proteins in apoptosis has been demonstrated by several experiments. For example, overexpression of the mutated PS protein can enhance apoptosis induced by several agents. PS2 antisense mRNA inhibits apoptosis. A new facet might be added to this story of PS2 involvement in neuronal apoptosis. A CBP has been suggested to play a part in this process. Stabler et al. (1999) have reported that a CBP, which they call calmyrin, interacts preferentially with PS2. Calmyrin consists of 191 amino acid residues and possesses two C-terminal EF-hands. It has been shown to interact with the cytoplasmic domain of the platelet integrin II b3, and is regarded as a putative regulator of the function of the platelet integrin (Naik et al. 1997). Stabler et al. (1999) found that calmyrin is myristoylated, and, in the modied form, it interacts with PS2. In HeLa cells, calmyrin is said to co-localise with PS2. The posttranslational modication of the protein could be required for targeting it to the membrane. This is reminiscent of myristoylation of recoverin and its function. Although, in the context of calmyrin, we can only draw inferences as to the signicance of complex formation between calmyrin and PS2, some preliminary observations made by Stabler et al. (1999) make interesting reading. They observed an enhancement apoptosis upon co-transfection of HeLa with PS2 and calmyrin. The PS proteins are substrates for caspases. Caspases cleave these at specic sites, e.g., aspartic acid 345/serine 346 and aspartic acid 329/serine 330. Caspase inhibitors as well as mutations at these sites inhibit cleavage of the proteins (Loetscher et al. 1997). Phosphorylation has been found to regulate the caspase sensitivity
179
of PS2. Two phosphorylation sites, at serine residues 326 and 329 have been identied in PS2. Both occur close to the site of cleavage by caspase-3. Cleavage of the protein is inhibited by phosphorylation of these residues, and the loss of this ability seems to inhibit apoptosis (Walter et al. 1999). However, there have been no attempts to date to determine if the phosphorylation of PS is related to disease activity. We do know that the sensitivity of the mutated PS1 and PS2 proteins to cleavage by caspases is not affected, which suggests that caspase-mediated modication of PS proteins might not be associated with the disease (Van de Craen et al. 1999). Also, PS proteins carrying mutations at the caspase cleavage site have been found to be functional (Brockhaus et al. 1998). Furthermore, the participation of PS genes themselves might have been overemphasised. Nonetheless, it has emerged from recent studies that caspases-2 and -3 show a marked increase in the brains of Alzheimers patients (Shimohama et al. 1999). In that study, Shimohama et al. (1999) also examined the expression of these caspases in developing rat brain. Their data have suggested some differential regulation of expression of these proteases. For instance, caspase-3 is highly expressed from 19-day-old embryos to 96-week-old embryos. However, high expression of caspase-2 begins only in approximately 4week old embryos. Although it does seem that there is a developmental regulation in rat embryos, the relation between the expression of these caspases and the process of ageing is not quite obvious. In any event, these data are at present insufcient to base any conclusions about whether the pathogenesis of Alzheimers disease is related to changes in the regulation of caspase expression. Another observation worthy of note is that caspase-3 can cleave PS2 at arginine 329 and generate a Cterminal peptide that might protect cells against apoptosis (Vito et al. 1997). Thus, caspase-mediated promotion of apoptosis by the modication of PS proteins could contain a self-regulatory mechanism. Vito et al. (1997) have suggested that the apoptosis-promoting function and the negative feedback signal provided by the Cterminal fragment generated by caspase activity could eventually determine the outcome. As discussed elsewhere Alzheimers disease is also characterised by abnormalities of the tau protein. This protein have been identied as a substrate for both caspases and calpain. The cleavage and dephosphorylation of tau has been shown to occur in neuronal apoptosis (Canu et al. 1998). Similarly, the amyloid precursor protein (APP), again a prominent feature of the disease, seems to be cleaved by caspase-3 (N.Y. Barnes et al. 1998). Not only does caspase-3 occur at high levels in Alzheimers brains, but the product of APP degradation co-localises with the caspase in degenerating neurones (Gervais et al. 1999). These observations represent another pathway involving caspase function in the pathogenesis of Alzheimers disease.
15
Parvalbumins in Neuronal Development, Differentiation, and Proliferation
The neuroepithelial ventricular zone of telencephalic ventricles gives rise to the majority of neurones and the glial component, which form the mammalian cerebral cortex. It has become increasingly obvious over the past few years that the various neuronal CBPs, such as calretinin, calbindin, neurocalcin, and parvalbumins, may each uniquely identify neuronal subpopulations across the cortical regions. Subpopulations with different CBPs may indeed represent metabolically distinctive cellular subtypes, and the presence of specic proteins may hold functional implications for the particular subtypes. These proteins may be playing different calcium-signalling roles in different cell types under different biological parameters. Parvalbumins are CBPs with three EF-hand domains. Three isoforms of PV may be distinguished: , , and CPV3. -PV, with its high afnity for Ca2+ is regarded as a calcium buffer. Furthermore, its distribution is much wider than that of the other isoforms. PVs occur in a neurone-specic fashion and are used as markers in the development and differentiation of neurones. A study of the localisation of PV in extraocular neurones has revealed moderate anti-PV immunoreactivity in motor neurones, but their axons show heavy staining, suggesting an intracellular segregation of PV (De La Cruz et al. 1998). De La Cruz et al. (1998) also state that PV is the only marker for extraocular motor neurones. The expression of PV seems to be related to differentiation. PV, among other calcium binding proteins such as calbindin D-28K and calretinin, shows age-related changes in the developing brain (Kishimoto et al. 1998; Majak et al. 1998). It has been reported that rhabdomyosarcoma cells that have been induced to differentiate, not only exhibit characteristic dendritic processes but also show a parallel increase in PV together with small increases in vimentin, desmin and neurone-specic enolase (Pappas et al. 1996). The regeneration of hair follicle cells, not involving mitosis, is another system in which the differentiation of supporting cells into hair cells has been shown to be accompanied by changes in the expression of CBPs, e.g., calbindin and parvalbumin (Steyger et al. 1997). Whether there are any tissue specic mechanisms regulating PV expression is unclear at present. Some preliminary work by
181
182
Castillo et al. (1995) does suggest this. These authors produced transgenic mice to express PV under the control of specic promoters. It is of some signicance that PV expression driven by a neurone-specic enolase promoter was at the highest level in the brain, but lower levels of expression were detectable in other tissues. It is also notable that ectopic PV expression in these transgenic mice did not produce developmental or behavioural abnormalities. -PV is referred to often as the onco-developmental parvalbumin or oncomodulin (OM). OM occurs in trophoblast cells, preimplantation embryos and neoplasms. Its distribution is far more restricted than that of -PV. The -PV isoform known as CBP-15, which has been reported to occur in the guinea pig organ of Corti (inner ear) (Thalmann et al. 1995), is identical to OM with respect to its 30 N-terminal amino acid residues. The identity between OM and CBP-15 has now been conrmed by the demonstration that isoform-specic anti-OM antibodies do cross-react with CBP-15 (Henzl et al. 1997). The OM gene has been mapped to the long arm of chromosome 5 of the mouse (Staubli et al. 1995). The calcium ion afnity of OM is lower when compared to that of -PV. OM may therefore have a calciumdependent regulatory function. Some investigators have addressed the question of a putative role for PV in cell proliferation. Blum and Berchtold (1994) have reported the expression of OM mRNA transcripts as well as the protein increases at the G1S boundary in two neoplastic cell lines (T14 and T10) that they tested. The increase was less marked in the T14 line when mitotically synchronised. However, because CaM shows a similar rise at the G1S interphase, the signicance of the rise in the levels of OM expression cannot be evaluated at present. It may be pointed out, however, that the expression of CaM and OM appeared to be differentially affected by the levels of extracellular calcium. When extracellular calcium levels were reduced, CaM expression increased by up to 60%, whereas OM levels decreased and the effects on both were reversed when the extracellular Ca2+ levels were increased (Klug et al. 1994). It is noteworthy that Klug et al. (1994) also state that the low extracellular calcium and the high CaM levels associated with it appeared to be best suited for cell growth. Andressen et al. (1995) transfected PV cDNA into a human ovarian carcinoma cell line but found a reduced mitotic rate in the cell lines carrying the exogenous PV cDNA. Furthermore, there were distinct alterations in cellular morphology and motility. Needless to say, further investigations into the inuences exerted by PV on these aspects of cellular behaviour seem worthwhile. CPV3 is an avian PV whose expression seems to be restricted to the thymus (Hapak et al. 1994a, 1994b). CPV3 has an isoelectric point (pI) of 4.6, which is somewhat lower than the pI of muscle PV. A full-length clone of CPV cDNA has 671 bp, and the protein product bears 68% sequence homology to OM. The CPV3 molecule contains cysteine residues at positions 18 and 72 and hence can form oligomers by means of disulphide bonds (Hapak et al. 1994b; Henzl et al. 1995).
16
Osteonectin in Cell Function and Behaviour
MOLECULAR STRUCTURE OF OSTEONECTIN

Osteonectin is a secreted glycoprotein associated with the extracellular matrix. It is also known by other epithets, such as SPARC (secreted protein acidic, rich in cysteine) and BM (basement membrane)-40. The protein is encoded by a single gene (Mason et al. 1986). Human osteonectin has a predicted molecular size of approximately 32 kDa (Swaroop et al. 1988). Human osteonectin has been cloned by PCR from an endothelial cell (EC) cDNA library, and this soluble biologically active monomer expressed in E. coli has 293 amino acid residues (Bassuk et al. 1996). Several homologues of osteonectin have been isolated, which share a high degree of amino acid sequence homology, with 61 to 65% of the 200-odd residues at the C-terminal region being identical. But they do show great deviation in the secondary structure of the Nterminal region (McKinnon et al. 1996; Soderling et al. 1997). According to McKinnon et al. (1996) the mouse homologues possess an exon structure similar to that of the osteonectin gene. Hafner et al. (1995) have reported the occurrence of a purine-rich stretch in the 5 end of the osteonectin gene. This region of bovine and murine genes shows marked sequence similarities, and it contains two GGA boxes with an intervening pyrimidine-rich spacer sequence. Hafner et al. (1995) also have shown that this region contains several regulatory domains. The GGA box 1 of the bovine promoter appears to be sufcient for maximal transcription of the gene, and the pyrimidine-rich spacer element seems, in contrast, to down-regulate gene expression. However, the GGA box does not seem to regulate gene expression, as in bovine cells. The transcriptional machinery obviously needs much further characterisation, especially to elucidate whether the regulatory domains described by Hafner et al. (1995) contribute to cell type-specic expression of osteonectin. Osteonectin has a C-terminal EF-hand domain (Yost and Sage, 1993; Maurer et al. 1995). Maurer et al. (1995) have identied two distinct domains (i.e., an acidic domain and a follistatin like [FC] domain), besides the EF-hand domain. Follistatin is a regulatory protein with multiple functions, notably inhibition of follicle-stimulating hormone. Follistatin has a characteristic and highly conserved amino acid sequence motif known as the follistatin domain. This domain has been shown to occur in a number of proteins, and these have been deemed as forming the follistatin family of proteins. Because osteonectin contains a follistatin domain, it has been regarded as a member of the follistatin family (Maurer et al. 1995; Phillips and
183
184
De Kretser, 1998). The EF-hand domain binds calcium with high afnity. Osteonectin from C. elegans, however, binds calcium with far less afnity as compared with the mammalian counterpart (Maurer et al. 1997). Calcium binding results in conformational changes in the molecule (Bassuk et al. 1996; Maurer et al. 1995; Takita and Kuboki, 1995). The EF-hand domain also mediates the Ca2+-dependent interaction of osteonectin with cells and the ECM. Other calcium-binding sites, which bind the cation with far less afnity, are also known to occur (see Table 13).
FUNCTIONS AND FUNCTIONAL DOMAINS OF OSTEONECTIN

Osteonectin is a highly conserved protein and is known to be expressed in a variety of organisms, from invertebrates to mammals (Damjanovski et al. 1997; Schwarzbauer and Spencer, 1993; Lane and Sage, 1994). It is not surprising therefore that, despite its restrictive nomenclature, it participates in a variety of cellular functions and cell behaviours (Table 12).
TABLE 12 Osteonectin in Cell Function and Behaviour

Embryonic differentiation and development Remodelling and rebuilding of the ECM Angiogenic and anti-angiogenic function Cell spreading Modulation of cell shape Intercellular and cellsubstratum adhesion Cell proliferation Tumour development and progression Source: Based on references discussed in the text.
Several individual molecular domains that participate in this physiological panoply have been identied with specic functions, by using peptide segments derived from different regions of the osteonectin molecule and antibodies raised against these peptides (Jendraschak and Sage, 1996). These domains and their putative functions are shown in Table 13.
REGULATION OF OSTEONECTIN EXPRESSION

The expression of osteonectin is regulated by several growth factors that can be closely identied with specic cellular properties such as cell proliferation, angiogenesis, etc. For instance, TGF induces the expression of osteonectin mRNA and protein in broblasts and other cell types (Wrana et al. 1991; M.J. Reed et al. 1994;
185
TABLE 13 Functional Domains of the Osteonectin Molecule

Amino Acid Stretch 351
Domain I
Subdomain 1.1
Sequence QTEVAEEIVEEETVVEETGV
Putative Function
II
52132
2.1
2.3
III IV
132227
3.2 4.2
Low-afnity Ca2+ binding; inhibition of cell adhesion, spreading; increase of PAI-1 expression QNHHCKHGKVCELDESNTP Ca binding; inhibition of EC proliferation; loss of focal adhesion; effect on cell cycle progression TLEGTKKGHKLHLDYIG Ca binding; follistatin homology; stimulation of EC proliferation and angiogenesis; plasmin sensitivity KNVLVTLYERDEGNNLLTEK Induction of MMP expression DLDNDKYIALEEWAGCFG EF-hand domain; inhibition of cell spreading, proliferation, and focal adhesion; binding to endothelial cells
Note: Domains III and IV might be involved in binding collagens (see text for references). PAI-1, plasminogen-activator inhibitor; EC, endothelial cell; MMP, matrix metalloproteinases. Source: Based on Jendraschak and Sage (1996).
Blazejewski et al. 1997). Also, PDGF, cytokines, and IGF-1 stimulate osteonectin synthesis and secretion (Chandrasekar et al. 1994). However, S. Nakamura et al. (1996) found that, in rabbit articular chondrocytes, IL-1 and -1 decreased osteonectin levels. In fact, IL-1 decreased osteonectin mRNA levels as well as glycosylation of the protein. S. Nakamura et al. (1996) also have shown that several other factors, among them TNF-, PMA, and bFGF, can down-regulate osteonectin expression. The down-regulation of its expression by bFGF might be related to a destabilisation of the osteonectin mRNA (Delany and Canalis, 1998; Delany et al. 1996). H. Shiba et al. (1995) found that bFGF reduced osteonectin synthesis as well as osteonectin mRNA in dental pulp cells. They suggested that the effects of the growth factor on odontoblast differentiation might occur at least in part at some pretranslational stage. In this context, it might be of interest to note that bFGF has been implicated in cell motility, proliferation, and angiogenesis, all features in which osteonectin is also putatively implicated. Furthermore, different growth factors and cytokines seem to affect the expression of ECM components, such as bronectin, laminin, and collagens, in a differential manner (Reed et al. 1994; H. Shiba et al. 1998). This provides additional support to the view that the effects on osteonectin
186
produced by the various biological response modiers might be identiable with their individual responses. It is small wonder, therefore, that osteonectin expression in cancers has been investigated with some vigour, albeit not with the degree that could be deemed appropriate in the context of the multifarious biological effects of osteonectin.
OSTEONECTIN IN THE REMODELLING OF THE EXTRACELLULAR MATRIX

The signicant role that is played by components of the ECM in cell motility and morphogenesis has been keenly appreciated by the scientic community. A secreted glycoprotein, osteonectin has been a prime candidate for participation in these processes. It was reported to be closely associated with the ECM in several normal as well as abnormal tissues (Dahlback et al. 1986). Several enzymes that are prominently associated with the membrane, refashion and remodel the ECM and in this way inuence cell behaviour. Among these are plasminogen activator (PA) and its inhibitor PAI, and a host of cathepsins MMPs (see Sherbet and Lakshmi, 1997b for review) (Figure 24). That osteonectin could be responsible for pericellular proteolysis and ECM remodelling is supported by several lines of evidence. Osteonectin is known to bind to a number of ECM components, such as collagen types IV and V and plasminogen (Mayer et al. 1991; Kelm et al. 1994; R.L. Xie and Long, 1996). Mayer et al. (1991) reported that osteonectin bound mainly collagen type IV, with other collagens (I, III, V, and VI) bound far less competitively. These binding interactions may involve specic regions of the molecule, as shown for collagens IV and V binding by osteonectin (Mayer et al. 1991; R.L. Xie and Long, 1996). Binding to ECM components could be a means of anchoring osteonectin to the ECM, so that it can participate in ECM remodelling. It also could serve as a cofactor in the formation of plasmin mediated by tissue plasminogen activator (tPA) (Kelm et al. 1994). On the other hand, osteonectin can induce the expression of PAI-1 as it has been shown to do in the case of bovine aortic endothelial cells (Hasselaar et al. 1991). A possible relationship between the expression of PAI-1 and osteonectin also has been encountered in senescent human diploid cells, where they appear to be up-regulated in parallel (S. Wang et al. 1996), although no causal linkup can be suggested on this basis. This would lead to a conservation of ECM components. The proteolytic feedback loop is completed by the demonstration that plasmin itself is capable of hydrolysing osteonectin in a limited way (Sage et al. 1984). Tremble et al. (1993) found that osteonectin up-regulated the expression of several MMPs, which included MMP1, MMP3, and MMP9, in rabbit synovial broblasts. Tremble et al. (1993) also identied domains of osteonectin that were involved with the regulation of MMP1 expression. It is unclear if up-regulation of MMPs alone is sufcient for ECM remodelling. In some systems, MMPs require activation. In human glioma U251.3 and brosarcoma HT1080, the MMP2 proenzyme is activated by MMP membrane type 1 (Deryugina et al. 1998). Whatever the downstream events may be, MMPs would be expected to refashion the expression and disposition of a number of ECM proteins. Furthermore, MMPs have been found
187
to produce a limited cleavage of osteonectin. Such cleavage is accompanied by a greatly enhanced binding of osteonectin to collagens I, IV, and V (T. Sasaki et al. 1997). The involvement of a specic site of cleavage (in helix C of the C-terminal calcium-binding module) has prompted T. Sasaki et al. (1997) to postulate that this enhanced afnity for collagens might be akin to a process of activation.
FIGURE 24 Feedback loop involving serine proteinases and matrix metalloproteinases (MMPs) in the remodelling of the extracellular matrix (ECM) by osteonectin. PAI, plasminogen-activator inhibitor; tPa, tissue plasminogen activator; uPa, urokinase-type plasminogen activator. (Based on references cited in the text.)
A remodelling of the ECM partly mediated by MMPs and their inhibitor TIMPs has recently been shown to be related to the expression of S100A4 in B16 murine melanoma and human astrocytoma-derived cell lines. The enhancement of invasive behaviour and metastatic spread, which occurs upon up-regulation of S100A4 expression, has been putatively linked with these events associated with the ECM (Merzak et al. 1994b; Lakshmi et al. 1997). It is not surprising therefore that, in light of the ECM changes produced by osteonectin, its role in embryonic development and differentiation, and other cell membrane-associated phenomena, such as cell adhesion, modulation of cellular morphology, cell proliferation, and wound healing, should have been investigated. It is also not surprising that such studies should be extended to neoplastic development, invasion, and dissemination.
OSTEONECTIN IN EMBRYONIC DEVELOPMENT AND DIFFERENTIATION

Osteonectin is secreted by a variety of tissues and its expression appears to be developmentally regulated. Osteonectin is highly expressed in the germ layers of the early mouse embryo, and the modulation of its expression follows a denite pattern in the course of embryogenesis (Holland et al. 1987; Nomura et al. 1988;
188
Sage et al. 1989a). Interference with this temporally controlled activation of osteonectin results in developmental abnormalities. Thus, when osteonectin antibodies are introduced into the blastocoel of Xenopus embryos, defective neurulation and organogenesis occur (Purcell et al. 1993). Microinjection of specic peptides derived from osteonectin induces abnormalities in the establishment of the embryonic axis in morphogenesis. The injection of osteonectin peptides containing FC domains and copper-binding sequences did not affect this process, but peptides containing the disulphide-bonded Ca2+-binding domain profoundly inhibited axial formation, leading to ventralisation of the embryos. The disulphide bonding seemed to be essential for axial inhibition, because peptides lacking cysteine residues were unable to induce axial abnormalities (Damjanovski et al. 1997). However, the recent work of Gilmour et al. (1998) suggests that osteonectin deciency may not damage developmental processes. They disrupted the osteonectin locus in murine embryonic stem cells and showed that the osteonectin-decient mice developed normally and were fertile. However, severe abnormalities of the eye, especially cataract formation and rupture of the lens capsule, developed around the age of 6 months. S.Y. Kim et al. (1997) have described the association of osteonectin in the sequence of events occurring during the development and maturation of chicken retina. Osteonectin is not only widely present in embryonic tissues, but it is also closely associated with organogenesis. In foetal rat lung, osteonectin is associated with epithelial cells of the airways during the pseudoglandular stage of lung morphogenesis. It is also found in the canalicular and saccular stages of branching morphogenesis of the lung (Strandjord et al. 1995). Using an in vitro model, Strandjord et al. (1995) were able to demonstrate that osteonectin antibodies inhibited the process of branching morphogenesis, resulted in the formation of dilated airways. The patterns of expression of bone matrix proteins, including osteonectin, suggest that these matrix proteins play different biological roles in the development and differentiation of mineralised tissues (Sommer et al. 1996). Distraction osteogenesis (bone lengthening) has afforded an excellent model that has indicated the involvement of osteonectin in various stages of differentiation (M. Sato et al. 1998). In cartilage differentiation, tissue transglutaminase catalyses the cross-linking of osteonectin into oligomers in situ, and this has been suggested as a major mechanism in the stabilisation of the cartilage matrix (Aeschlimann et al. 1995). Glycine residues 3 and 4 have been identied as the major amine acceptor sites, of which at least one is conserved in vertebrate osteonectin (Hohenadl et al. 1995).
MODULATION OF CELLULAR ADHESION, CELL SHAPE, AND MOTILITY BY OSTEONECTIN

It is to be expected that any modulation of the character of the ECM should be reected in changes in cellular features such as cellsubstratum and intercellular adhesion, as well as in changes in cell shape and motility. It was reported several years ago that osteonectin inhibited cell spreading and consequently altered cell shape, which indicated that cellsubstratum interaction was being inhibited (Sage et al. 1989b). Subsequently, Lane and Sage (1990) identied the osteonectin domains
189
responsible for this inhibition of spreading to be the N-terminal domain I and a sequence occurring in the C-terminal EF-hand domain. They raised antibodies against these peptides and demonstrated that the antibodies blocked the ability of osteonectin to inhibit cell spreading. Experimental overexpression of osteonectin, produced by transfection, in F9 embryonal carcinoma cells correlated with a rounded cell morphology, whereas transfection of antisense osteonectin cDNA constructs was found to restore cell spreading (Everitt and Sage, 1992). However, transfection of human osteonectin cDNA into human 293 and HT1080 cell lines has given somewhat contradictory results. Nischt et al. (1991) found no changes in cell spreading, proliferative rate, or adhesion behaviour of the transfected cell clones, even though they expressed osteonectin mRNA at high levels and secreted large quantities of the protein. Osteonectin can induce a loss of focal adhesion of bovine aortic endothelial (BAE) cells, and it appears to achieve this by a reorganisation of the submembranous cytoskeleton (Goldblum et al. 1994; Murphy-Ullrich et al. 1995). It would be a natural extension of the efforts described above to inquire into the mechanism by which osteonectin inhibits cell adhesion and spreading. As discussed earlier, osteonectin can alter the structural and functional characteristics of the ECM by inducing proteolytic degradation of its components, and thereby modulate cellsubstratum interaction. Some clues are provided for the presence of another mechanism by the observation that osteonectin brings about changes in the submembranous cytoskeleton. Extracellular signals may be transduced to the cytoskeleton via integrin receptors. Integrins are heterodimeric transmembrane glycoproteins. They are composed of two subunits: the and subunits. A large number of these subunits has been identied. They combine to form a vast array of integrins that function as receptors for ECM components such as bronectin, laminin, tenascin, osteopontin, thrombospondin, and vitronectin, as well as cell adhesion molecules such as VCAM and ICAM (Dedhar, 1990). These integrins form a link between the ligand and the cytoskeleton. There are at present no indications that the function of osteonectin is mediated by integrins, although we do know that it can bind to certain ECM components such as collagens and vitronectin, which do function through the agency of integrin receptors. Vitronectin is an adhesion-mediating protein. It is a secreted glycoprotein of predominantly hepatocyte origin. It occurs in the ECM of blood vessels and skin and has also been reported to occur in many tumours (Dahlback et al. 1986). It binds to v3 integrin. The expression of this integrin has been associated with the progression of cutaneous tumours and some neuroectodermal tumours (see Sherbet and Lakshmi, 1997b). In light of these observations, it is signicant that osteonectin can interact with vitronectin and modulate the adhesive properties of the latter (Rosenblatt et al. 1997). Interaction seems to occur between the heparin-binding region of vitronectin and the C-terminal calcium-binding domain of osteonectin, which is regarded as the region actively involved in the inhibition of cell adhesion and spreading. Rosenblatt et al. (1997) have demonstrated also that PAI-1 induces the binding of osteonectin with vitronectin. As stated previously, osteonectin is able to induce the expression of PAI. Thus the inhibitor seems to participate in the modulation of cell adhesion by two distinct pathways: by a direct route of inhibiting
190
PA-mediated excision and modication of ECM components, and by an indirect route of inducing the interaction between osteonectin and vitronectin. Whether other ECM components are similarly targeted by osteonectin largely remains to be investigated. Some ECM components such as bronectin and osteopontin can be eliminated. The binding of these to their integrin receptors is dependent on the presence of the RGD (arginineglycineaspartic acid) motif, which appears to function as a recognition signal for the binding of these ligands to their respective receptors. Sage (1992) has shown that inhibition of cell spreading by osteonectin is not affected by the RGD peptide. It may be suggested on the basis of this nding that neither bronectin nor osteopontin enters into any interaction with osteonectin. However, one cannot yet exclude this possibility, because the adhesion of some cell types, such as osteocytes and osteoblasts, to osteonectin and osteopontin is strongly inhibited by the RGD peptide (Arden et al. 1996).
MODULATION OF CELL PROLIFERATION BY OSTEONECTIN

Osteonectin can inhibit or stimulate cell proliferation. This faculty appears to depend on the cell type and possibly also on the physiological environment. Osteonectin expression has been found in some instances to closely correlate with the processes of cell proliferation and differentiation, upon exposure to hormonal or growth factor milieus. The modulation of endothelial cell proliferation by osteonectin, pursued rather relentlessly by the research group of Sage, is a prime example of its function. Some of their work is discussed below. Osteonectin can inhibit proliferation of microvascular endothelial cells induced by VEGF (Kupprion et al. 1998). In sharp contrast with effects on ECs, in dental pulp mesenchymal cells, bFGF enhances osteonectin expression and also acts as a potent mitogen (H. Shiba et al. 1995). In osteoblasts, oestrogen-induced differentiation and inhibition of cell proliferation is totally unrelated to the levels of osteonectin (Robinson et al. 1997). However, wound healing, where cell proliferation and migration are important processes, does involve osteonectin. Osteonectin is a component of the alpha granules of platelets, and along with thrombospondin it participates in platelet aggregation (Kelm and Mann, 1990; Clezardin et al. 1991). A high level of osteonectin immunoreactivity has been observed in the healing epithelium of the cornea, during a 6-day period following wounding, and then osteonectin reactivity falls off (Latvala et al. 1996). As shown in Table 13, two domains have been identied that correspond with these putative functions. Osteonectin has been shown transiently to inhibit the progression of vascular endothelial cells at the mid-G1 phase of the cell cycle. A similar inhibition is produced also by the peptide 2.1 (Funk and Sage, 1991). This inhibition was not accompanied by a rounding-up of cells. This is not surprising, because rounding-up of cells is a prelude to cell division, and cessation of division is usually followed by a phase of cell spreading. Peptides from domain IV containing the EF-hand motifs produced strong growth inhibition, e.g., peptide 4.2, which seemed to cooperate with peptide 2.1 (Sage et al. 1995). Funk and Sage (1991) noticed further that the inhibition of cell spreading was produced by peptide 1.1,
191
which did not inhibit cell cycle progression. However, peptide 2.1 stimulated the proliferation of broblasts derived from human foreskin and foetal bovine ligament. This stimulation was strictly concentration dependent and occurred only in the range of 0.1 to 0.4 mM of the peptide. Higher levels of the peptide resulted in growth inhibition (Funk and Sage, 1993). Another peptide; i.e., peptide 2.3, stimulated the proliferation of broblasts. Overall, osteonectin appears to be able to control the process of proliferation in a cell type-specic fashion, in which different domains of the molecules participate. Although the function of different peptides in vitro cannot directly form the basis for inferring the function of osteonectin in vivo, it is possible that differential effects on cell proliferation, adhesion, and spreading could be seen as an attribute of different congurations of the molecule assumed upon calcium binding. There will be signicant changes in the -helical content of the molecule in relation to calcium binding. As pointed out by Maurer et al. (1996) conformational changes may occur within a domain. Furthermore, different distant domains may be linked and thereby alter their orientation relative to one another. One also needs to take into account the posttranslation changes, such as glycosylation and phosphorylation, which profoundly affect biological activity. Glycosylation, for instance, has a regulatory effect on the binding of osteonectin to collagen V (R.L. Xie and Long, 1995, 1996).
EFFECTS OF OSTEONECTIN ON ANGIOGENESIS

Neovascularisation is a complex process consisting of several events, such as the endothelial cell proliferation and their migration toward the source of the angiogenic factor. The endothelial cells align themselves end to end to form a sprout, which then develops a lumen (reviewed by Sherbet and Lakshmi, 1997b). The currently available evidence for the induction or inhibition of angiogenesis by osteonectin is still somewhat indirect and rather scanty. This is based on the observation that osteonectin inuences the proliferation of endothelial cells. Further, certain growth factors that are known to be angiogenic also enhance osteonectin expression, and factors that have been shown to be able to inhibit angiogenesis also inhibit osteonectin expression. Thus, TGF induces both angiogenesis and osteonectin expression, whereas IL-1 inhibits both. Some factors such as PA, PAI, and TIMPs might indirectly affect osteonectin expression and also inuence angiogenesis. The uncertainty arises when one considers the effects of factors such as bFGF and TNF. bFGF is an inducer of angiogenesis. However, it down-regulates osteonectin expression in endothelial cells, although its effect is manifestly the opposite in mesenchymal cells. As discussed above, there is evidence that VEGF, which is demonstrably angiogenic (Zhang H.T. et al. 1995), inhibits osteonectin expression. Similarly, TNF induces angiogenesis but down-regulates osteonectin. The presence of osteonectin in the ECM of vascular structures and its proven ability to induce loss of focal adhesion of endothelial cells, and to reorganise the submembranous cytoskeleton, are clear indications of a positive effect on endothelial cell motility (see Table 14). Unfortunately, there is very little direct evidence that osteonectin induces angiogenesis. Some ndings could be construed as direct evidence. For instance, the presence of osteonectin in enhanced quantities has been found to accompany the
192
TABLE 14 Correlation between Angiogenesis and Modulation of Osteonectin Expression by Growth Factors and ECM Components
Effect on Osteonectin Status/Effect of Osteonectin on Effector Agent + +2b b b d
Effector Agent TGF PA IL-1 TIMPs Thrombospondin PAI VEGF bFGF TNF
Effect on Angiogenesisa + + c + +
Note: The table provides a summary of the evidence that links, directly or indirectly, angiogenesis with osteonectin expression. Although osteonectin contains domains that putatively possess both angiogenic and anti-angiogenic properties, the table, constructed on the basis of work discussed in the text, underlines the need for more denitive experiments in order to establish whether osteonectin is involved in the regulation of angiogenesis.
a
+, up-regulation; , down-regulation of expression. Osteonectin inuences the expression of the effector agent. c TIMPs 14 are known to inhibit angiogenesis (Sherbet and Lakshmi, 1997b; Blavier et al. 1999). d bFGF is believed to up-regulate osteonectin in mesenchymal cells.
b
formation of capillary-like structures by endothelial cells both in vitro and in vivo (Iruela-Arispe et al. 1991a, 1991b; Lane et al. 1994). Mendis et al. (1998) noticed an enhanced expression of osteonectin mRNA in the early stages of the formation of blood vessels following injury to adult rat cerebral cortex. However, one does not know whether changes in osteonectin levels occur as a consequence of induction of angiogenesis by some other factor, and whether osteonectin itself is a causative agent or merely an epi-phenomenon. One could concede that, prima facie, osteonectin seems to be involved in the regulation of angiogenic processes, but there is an acute need for more substantive data, such as, for instance, by transfecting osteonectin into appropriate recipient cancer cells and determining if the expression alters microvascular density. Technically this could be demanding, notably because osteonectin contains domains with both angiogenic and anti-angiogenic properties, but it is certainly an achievable goal.
193
OSTEONECTIN EXPRESSION IN CANCER DEVELOPMENT AND PROGRESSION

Osteonectin seems to be able to inuence most of the biological properties of the cell that are highly relevant in the context of cancer development, growth, and dissemination. The most obvious reasons for this are that osteonectin is able to initiate changes in the ECM, cell adhesion and spreading, and also in the cytoskeleton. It has been found to inuence cell proliferation, and apparently, it can regulate vascularisation. The elucidation of these properties has inevitably led to the study of its expression in cancer development and progression. Osteonectin reportedly occurs in a wide spectrum of cancerous tissues from human subjects. Porter et al. (1995) found high osteonectin immunoreactivity in invasive tumours of the GI tract, breast, lung, kidney, ovary, brain, and adrenal cortex. They state that normal tissues show low levels of reactivity. It is somewhat paradoxical that trophoblast cells, which are a highly invasive cell component of the placenta, show low levels of reactivity. Nevertheless, bone extracts and osteonectin itself have been found to enhance the motility in vitro of prostate epithelial cells as well as prostate cancer cells (Jacob et al. 1999). The presence of osteonectin has also been demonstrated in normal as well as adenoma of human thyroid (BurgiSaville et al. 1997). However, there is a reasonable body of evidence that suggests a close association of osteonectin expression with the progression of human melanomas. Osteonectin is not expressed by normal melanocytes and it is weakly expressed in a small proportion (4/25) of nevocellular nevi. The level of osteonectin expression is moderate in a majority (13/14) of dysplastic nevi. However, the expression occurs invariably and at a very high level in both primary (7/7) and metastatic (29/29) melanomas (Ledda et al. 1997). A few studies have also been carried out on the relevance of osteonectin as a marker for the progression of breast cancer. According to Bellahcene and Castronovo (1995), osteonectin is only weakly expressed in benign breast disease, but the expression is very strong in both in situ and invasive carcinomas. The presence of oestrogen receptors in breast cancer is regarded as an indicator of differentiation and good prognosis, and their absence is suggestive of clinically aggressive disease. Apparently, there is an inverse relationship between ER status and osteonectin expression. Tumour samples that were low in ER content tended to contain fourfold higher levels of osteonectin mRNA as compared with tumours with high ER levels (Graham et al. 1997). It would be of much interest, in this context, to examine whether osteonectin expression relates directly to the presence of EGFr, because ER-negative breast cancers often tend to be EGFr positive. There is thus an apparent relationship of osteonectin levels to disease progression. This has been conrmed in another study, in which Podhajcer et al. (1996) found that the osteonectin gene is expressed at a high level in invasive human breast carcinoma and also in metastatic lymph nodes. Osteonectin transcripts were found in the broblast stroma. Furthermore, high levels of expression of stromelysin-3 also accompanied high osteonectin expression. Osteonectin seems to be able to activate MMP2 in the invasive breast cancer cell lines MDA-MB231 and BT549, but not in the noninvasive MCF7 cells.
194
This ability was associated with the osteonectin peptide 1.1 (Gilles et al. 1998). Although Gilles et al. (1998) also found some attenuation of TIMP-2, they have suggested that the invasive propensity of the breast cancer cell lines is most likely attributable to osteonectin-mediated activation of MMP2. There can be little doubt that metalloproteinase expression and the expression of their inhibitors corresponds closely with invasive potential of cancers (see Sherbet and Lakshmi, 1997b). The apparent relationship between osteonectin and MMP does not necessarily reect a causal connection. There is the distinct possibility that a common effector, such as bFGF, may regulate both. Nonetheless, the outcome could be an alteration in the invasive nature of the tumour. Bellahcene and Castronovo (1995) have stated also that high expression of osteonectin was associated with calcication of the lesions. However, contrary to the ndings of Bellahcene and Castronovo (1995), Hirota et al. (1995) reported no correlation between osteonectin expression and the development of foci of calcication. It ought to be stated, nonetheless, that in other cellular systems a relationship does seem to subsist between osteonectin levels and calcication. Human dental pulp cells maintained in tissue culture express osteonectin, and they also contain calcied nodules. The level of osteonectin closely correlates with the level of calcication. When these cells are treated with bFGF, osteonectin expression is reduced and calcication of the ECM is abolished (H. Shiba et al. 1995). M. Sato et al. (1997) used a human salivary cancer cell line and have reached similar conclusions. These cells produce tumours when implanted into nude mice. When treated with VD3, tumour growth rate was reduced and calcied foci appeared in the tumours. In parallel, M. Sato et al. (1997) also found the expression of osteonectin mRNA in these treated tumours. Whether the expression of osteonectin, together with other bone matrix components such as osteopontin and bone sialoprotein, could have some bearing on the propensity of breast tumours to metastasise to the bone, is currently being debated. Osteopontin has been implicated in tumour cell motility (Xuan et al. 1995; Sung et al. 1998). Oates et al. (1996, 1997) transfected Rama-37, a rat mammary epithelial cell line, with genomic DNA fragments from a human mammary carcinoma cell line. The transfectants were found to produce tumours with metastasising ability. They then isolated a cell line from a metastatic tumour and compared its mRNA prole with a control cell line, by subtractive hybridisation. One of the mRNAs strongly expressed, (nine-fold greater in the metastatic cell line as compared with the nonmetastatic parent line), was that for osteopontin. An increase in the level of expression does not constitute irrefutable evidence of a relationship to metastatic ability. Oates et al. (1996) did show that similar transfection of Rama-37 cells with DNA from benign tumours did not result in elevated expression of osteopontin. Some of these early studies have been conrmed recently. The levels of osteopontin have been found to be low in nontumorigenic cells and tumour cells with low metastatic ability. The osteopontin-transfectant cells as well as cells exposed to exogenous osteopontin have been reported to make marked gains in invasive ability (Tuck et al. 1999). What is even more interesting is the observation by these authors that, under both experimental conditions, the gain in invasive ability was accompanied by increases in the expression of uPA mRNA as well as the uPA protein. This
195
has marked similarities with the effects of osteonectin on cell invasion. One should recognise, nonetheless, that VD3 can act independently of osteopontin, because although it can enhance the expression of both osteonectin and osteopontin, VD3 indeed inhibits cell proliferation and invasion. Its ability to inhibit invasion can be directly linked with the down-regulation of PAs and MMPs together with an upregulation of the endogenous MMP inhibitors. These arguments simply emphasise the need to include osteopontin in studies of osteonectin involvement in cancer cell invasion and metastasis. It would be relevant to cite here the observations of Jacob et al. (1999), who found that bone extracts as well as osteonectin enhanced the motility of tumour cells that normally metastasised to the bone, e.g., breast and prostate cancer. However, cell lines derived from tumours that do not normally metastasise to the bone did not respond in this way. Jacob et al. (1999) postulate, in consonance with the discussion above, that this apparent differential effect of osteonectin on cell motility could be due to the ability of osteonectin to induce tumour-associated metalloproteinase activity. An activation of uPA together with enhanced proliferative potential has been reported in MCF7 cells exposed to soluble factors secreted by the osteogenic cell line SaOS-2. MCF7 cells are normally only weakly invasive, but they appeared to become more invasive when cultured on an ECM produced by SaOS-2 cells. Furthermore, this acquisition of invasive potential seemed to be related to the ability of the ECM to induce uPA expression in MCF7 cells (Martinez et al. 1999). Although these ndings are of considerable signicance in the context of cancer invasion, it is necessary to take into account a number of related facts and factors, lest one be led into an alley of overinterpretation of the data. It should be recognised that both MCF7 and MDA cells do synthesise uPA, although MCF7 cells do so at a far lower level than do MDA-MB231 cells. It should be recognised also that ability to synthesise uPA is not itself directly related to the invasive ability of cancer cells. Urokinase receptors as well as PAIs enter into the equation. Undoubtedly there is a large body of correlative evidence derived from the study of plasminogen activator expression in a host of human tumours. Nonetheless, all potential interacting factors need to be checked before one can be certain that one is, indeed, dealing with uPA as the major instigator of invasive potential. Martinez et al. (1999) state that the enhancement of invasive behaviour was found only in the ER-positive MCF7 cells, but not in the ER-negative MDA-MB231 cells. This is consistent with the experiments described by Hachiya et al. (1995), who found that oestrogen enhanced uPA as well as tPA expression and also enhanced the invasive ability of breast cancer cells. They also demonstrated that PA expression is regulated by oestrogen, because tamoxifen blocked the production of PA and also inhibited the invasive ability. Among other factors that come into the reckoning is the hepatocyte growth factor (HGF). HGF bears sequence homology to PA. PA is known to activate HGF (Mars et al. 1993). Furthermore, HGF is a potent mitogen and can induce angiogenesis as well as vascular invasion (Hildebrand et al. 1995). It would be worth recalling here that the ER status correlates inversely with EGFr in these cell lines. EGF is another modulator of the invasive behaviour of cancer cells. Whether the changes in the invasive behaviour might have been mediated by EGF receptors is a moot point.
196
As stated previously in this section, osteonectin expression is said to be inversely related to ER status, with high expression of osteonectin being found in ER-negative cells. Furthermore, it also has been claimed that osteonectin stimulates MMP expression in MDA cells, but not in MCF7 cells. It may be that uPA is more relevant in the context of breast cancer cell invasion than are MMPs. Overall, there is a reasonable body of evidence to suggest the ectopic expression of these bone matrix proteins might have serious implications for the osteotropic metastasis of breast cancer. However, the mechanisms involved remain to be elucidated. Although the above discussion suggests a direct relationship between malignancy and levels of osteonectin expression, in ovarian epithelial cells an inverse relationship has been reported. Mok et al. (1996) found high osteonectin expression in normal ovarian epithelial cells, but this was markedly reduced in ovarian carcinoma cells. They also transfected osteonectin cDNA into SKOV3 cell, which is an ovarian carcinoma cell line. This resulted in reduced growth rate in vitro, and furthermore, these cells were less tumorigenic when implanted into nude mice. These results suggest and impute a tumour suppressor property to osteonectin. This is supported by recent work by Vial and Castellazzi (2000). The expression of osteonectin is down-regulated when cells are transformed by oncogenes. Such a downregulation is noticed in chick embryo broblasts transformed by v-src or v-jun oncoproteins. When the protein is reexpressed, these cells retain the transformed phenotype but lose their ability to form brosarcomas in vivo (Vial and Castellazzi, 2000). In other words, in the experimental model osteonectin does seem to behave like a tumour suppressor. Nonetheless, it would be reasonable to expect further conrmation of the possible tumour suppressor function. Some of these uncertainties are compounded by the view expressed in some quarters that osteonectin might not be a reliable marker for osteosarcoma (Grundmann et al. 1995; Park et al. 1996). An overall view of the present status of osteonectin in relation to cancer progression ought to be ambivalent. There is a need for far more extensive investigation of human tumour types. Above all, much more experimental work is needed to establish the various postulates that bring together the putative functions of osteonectin with the altered biological properties of the cancer cell. In a sense, therefore, it would be premature to dive into investigations of clinical material without appropriate groundwork. This is especially important with respect to osteonectin function, because the protein contains domains that reputedly possess antagonistic functions. The scientic community has not even begun to unravel the mechanisms by which these antagonistic functions become expressed in the physiological setting. Equally, it would be unreasonable to delay the investigation of the relevance of osteonectin in tumour classication, or its potential in clinical management of patients, if indeed it has even the semblance of predictive value.
OSTEONECTIN INVOLVEMENT IN OTHER DISEASE STATES

A number of nonneoplastic diseases have been linked with osteonectin. Among these are rheumatoid arthritis (RA) and osteoarthritis (OA). Immunostaining studies of
197
cartilage and synovial specimens from patients with RA and OA have been reported (S. Nakamura et al. 1996; Nanba et al. 1997). Osteonectin immunoreactivity is found in chondrocytes in the supercial and middle zones of the cartilage from RA and OA joints, but these zones of normal cartilage are either devoid of any activity or stain only too weakly. Osteonectin expression is greatly increased in synovial cells from these patients. S. Nakamura et al. (1996) have also reported that, on average, the osteonectin content of synovial uid from RA patients was ten-fold greater than that of synovial uid from OA patients. Nanba et al. (1997) have made the interesting and valuable observation that osteonectin is detected in the early stages of OA, and they point out that this could be signicant in terms of disease progression, because cartilage calcication is closely related to the stage of the disease. Liver brosis appears to be associated with enhanced levels of osteonectin. According Inagaki et al. (1996), osteonectin is virtually undetectable in normal liver, but osteonectin gene transcripts were abundantly expressed, mainly in the lipocytes, which markedly increase in number in brosis. Similar increases in osteonectin levels have been described by Blazejewski et al. (1997). These authors reported that cultured myobroblasts from liver synthesised osteonectin. They further observed that in normal liver myobroblasts occured in small numbers, but that they proliferated markedly in brosis and synthesise ECM proteins. Consistent with this, osteonectin mRNA expression was low in normal liver but was markedly enhanced in brotic liver. Osteonectin expression is reported to decrease at the early stages of diabetesrelated renal enlargement in experimental animals (Gilbert et al. 1995). As noted by Gilbert et al. (1995), renal enlargement is a mixture of several events occurring in the ECM along with cell hypertrophy and hyperplasia, and one can see how osteonectin might be implicated in the pathogenesis of the disease.
OSTEONECTIN HOMOLOGUES AND THEIR PUTATIVE TUMOUR SUPPRESSOR PROPERTIES

Osteonectin is probably a typical member of a large family of proteins whose members share several important structural features and perform similar functions. In recent years several cDNAs have been isolated that show a high degree of structural homology to osteonectin. Hevin (MAST9, SC1), QR1, testin, and tsc36/FRP are members of the osteonectin family. Hevin was rst identied in endothelial venules of lymphoid tissue (Girard and Springer, 1995, 1996). The hevin gene is located on chromosome 1 (Claeskens et al. 2000). The hevin cDNA encodes a protein that shows a substantial sequence homology to osteonectin, SC1 (of murine origin), and QR1 isolated from quail (Girard and Springer, 1995). Monomeric hevin is 75 kDa in size and possibly forms dimers in vitro. Bendik et al. (1998) have characterised the full-length cDNA, which has 2808 nucleotides and an ORF of 1992 nucleotides corresponding to a 75-kDa protein. Overall, all osteonectin homologues identied thus far show a molecular organisation similar to that of osteonectin, and, in particular, possess the characteristic FC domain and EF-hand motifs in the calcium-binding domain.
198
Hevin is expressed ubiquitously in most normal tissues and in diseased tissues of nonneoplastic origin. There may be differences in the patterns of their distribution in normal tissues, as demonstrated for SC1 (Soderling et al. 1997). The expression of hevin has been reported to be down-regulated in many neoplasms, e.g., in nonsmall cell lung carcinome (NSCLC) (Bendik et al. 1998) and adenocarcinoma of the prostate (P.S. Nelson et al. 1998). Claeskens et al. (2000) transfected hevin cDNA into HeLa-35 cells, which do not express indigenous hevin. The transfected hevin cDNA negatively regulated proliferation and seemed to block G1S transition of cells. Therefore, Claeskens et al. (2000) suggest that hevin may be a putative suppressor gene. However, it might be premature to label a gene as a suppressor gene merely on the basis of possible inhibition of cell proliferation. It ought to be stated, in defence of the postulate, that hevin does possess anti-adhesion properties, can inhibit focal adhesion of cells and confers a rounded morphology on cells (Girard and Springer, 1996). Needless to say, that this area is worthy of pursuit and should provide interesting results relating to the relevance of these molecules in cell migration, diapedesis, and cancer dissemination.
17
S100 Proteins: Their Biological Function and Role in Pathogenesis
There has been an unremitting effort by the scientic community in the past two decades aimed at identifying the genetic determinants of the invasive and metastasising properties of cancer cells. This has been based on the premise that the acquisition of these seemingly aberrant abilities ows from the expression of a candidate gene or a group of genes that might be connected with the process of metastasis. However, one can muster very little direct evidence for the metastasis gene concept. Indeed, a review of recent research would suggest that alterations of the proliferative status, motility, and cellcell and cellsubstratum adhesion of cells, may be seen as the basic requirement for cancer growth, invasion, and metastasis, as well as other nearly analogous processes such as cell differentiation, growth, and morphogenesis. Quite obviously, therefore, a vast array of genes can be identied whose expression profoundly affects cancer cell behaviour as well as cell differentiation, growth, and morphogenesis (Sherbet and Lakshmi, 1997b). It can be argued that neoplastic transformation of cells, accompanied by the acquisition of enhanced proliferative potential, motility, and heterotypic adhesion properties, can be attributed to an overexpression or inappropriate expression of genes that are associated with extracellular signal transduction, and those required in normal physiological function. The S100 proteins, which form the main focus of this section, have been shown to be capable of modulating enzyme function and altering cytoskeletal dynamics. They can bind to a variety of cellular target proteins, and possibly by this means control cell cycle progression. Furthermore, much evidence has accumulated that shows these proteins are associated with terminal cell differentiation; they can promote remodelling of the ECM, alter cell shape, motility, and enhance the invasive behaviour, and metastatic spread of cancer. Here we attempt to link the physical and physiological alterations occurring in a cell consequent to overexpression of S100 proteins with the salient phenotypic aspects of biological behaviour of cells. The S100 family of proteins comprises a large number of CBPs. As can be seen in Table 15, a majority of them are found in a 2.05-Mbp segment of human genomic DNA of chromosome 1q21 region (Marenholz et al. 1996; Mischke et al. 1996; Schafer et al. 1995). The S100 family of genes, including those coding for prolaggrin (FLG)
199
200
TABLE 15 S100 Family of Calcium-Binding Proteins: Their Chromosomal Location and Putative Physiological Function
S100 Nomenclature S100A1 (S1001) Chromosomal Location (Human) Putative Physiological Function and in Pathogenesis
S100A2/S100L S100A3/S100E
S100A4 (18A2/mts1, CAPL, p9Ka, metastasin, calvasculin, FSP1) S100A5/S100D S100A6 (calcyclin, 2A9, CACY, caltropin) S100A7 (psoriasin)
1q21 (Dorin et al. Associated, together with S100B, with 1990; Engelkamp cardiomyopathy; differentially modulated in tissues et al. 1993; Schafer in experimental diabetes (Zimmer et al. 1997); see et al. 1995) text 1q21 (Engelkamp et Putatively associated with tumour suppression; see al. 1993) text 1q21 Specically expressed in the skin; participation in the differentiation of hair follicles; found in glial tumours 1q21 See text
1q21 1q21 1q21 (Hardas et al. 1996) 1q21
S100A8 (MRP8)a S100A9 (MRP14)a
Associated with acute myeloid leukaemia (Calabretta et al. 1986a,b) and melanomas; see text Psoriatic skin and other skin diseases; regarded as a potential marker for squamous cell carcinoma of the bladder; see text Together with MRP-14, associated with cystic brosis, rheumatoid arthritis (Odink et al. 1987; Fanjul et al. 1995); inammatory bowel disease, allograft rejection, recruitment of neutrophils and monocytes to delayed-type hypersensitivity inammatory sites (Dunn et al. 1996); might be involved in the regulation of the inammatory process, transendothelial migration of monocytes (Kerkhoff et al 1991a, 1999b)
S100A10 (p11, calpactin light chain, 42C) S100A11 (S100C)
S100A12 (calgranulin C, P6, CGRP, CAAF1)
1q21 (Mooglutz et al. 1995; Wicki et al. 1996a) 1q21 (Wicki et al. 1996a; Yamamura et al. 1996)
Differentially expressed in uveal melanomas and cell lines derived from them (Van Ginkel et al. 1998). Gene with 3 coding exons; exons 2 and 3 encode peptide of 92 amino acid residues (Wicki et al. 1996a, Yamamura et al. 1996a); 2 EF-hand domains; isolated from neutrophils, bovine amniotic uid (Guignard et al. 1995; Hitomi et al. 1996, 1998); also found in foetal epidermal keratinocytes, squamous epithelia; oesophageal epithelium; differentiation-related expression, but no known association with cell proliferation; associated with Moorens ulcer
201
TABLE 15 (CONTINUED) S100 Family of Calcium-Binding Proteins: Their Chromosomal Location and Putative Physiological Function
S100 Nomenclature CAAF2 Chromosomal Location (Human) Putative Physiological Function and in Pathogenesis 2 EF-hand domains; ca. 30% sequence homology with CAAF1; 63% homology with S100A7 (Hitomi et al. 1996) ca. 50% homology to S100A5 and ca. 60% to S100A12; mouse A13 shows ca. 87% homology to human A13; mRNAs expressed at high levels in skeletal muscle, heart, kidney, ovary, pancreas (Wicki et al. 1996b) Associated with Alzheimers disease; Downs syndrome, neurite extension factor; occurs in glial and Schwann cells, cytoskeletal disruption; possibly inuences cell cycle progression via activation of Ndr protein kinase Isolated from human placenta (Becker et al. 1992; Emoto et al. 1992); cutaneous sensory signal transduction; progression of prostate cancer; see text Filaggrin IF monomers, aggregation of laments in keratinocyte differentiation; reduced expression in and possible aberrant interaction with keratin laments in ichthyosis; rheumatoid arthritis
S100A13
1q21 (Wicki et al. 1996b)
S100B/S100
21q22 (Duncan et al. 1989)
S100P
4p16 (Schafer et al. 1995) 1q21 (Marenholz et al. 1996; Mischke et al. 1996)
FLG (prolaggrin)
Trichohyalin (THH) Repetin (rptn) (mouse)
Calbindin D-9K (intestinal calcium-binding protein)

a
1q21 (Fietz et al. 1992) Mouse chromosome Overexpression in mouse skin tumours (Krief, P., 3 (Krief et al. personal communication) 1997) Xp22.2
MRP8 and 14 are regarded as myeloid-specic proteins. Shapiro et al. (1999) have identied a B-cellspecic antigen, which appears to possess N-terminal sequence homology with human MRP8. The antigen also shows homology to other S100 proteins too, albeit at a far lower level. Shapiro et al. (1999) have argued, on rather tenuous grounds, that because MRP8 is not expressed in lymphocytes, this antigen might represent a new member of the S100 family. Source: Data collated from Heizmann (1996), Sherbet and Lakshmi (1997b), and references cited in the table and the text.
and trichohyalin (THH), which are associated with terminal differentiation of keratinocytes, are arranged in the following order: 1 cenS100A10S100A11THH FLGIVL (involucrin)LOR (loricrin)S100A9S100A12S100A8S 100A7S100A6S100A5S100A4S100A3S100A2S100A13S100A11qtel. S100B is an exception in that it occurs on chromosome 21q22 (Duncan et al. 1989).
202
A similar clustering and synteny of S100 genes is found in mouse chromosome 3, and the organisation shows a great resemblance to that of the human S100 gene cluster. Eight mouse S100 genes are organised in the same way as the human genes. The linkage between S100A8 and S100A9 and the linkage of the array S100A3S100A4S100A5S100A6 are conserved. However, S100A1 and S100A13 are separated, unlike as in the human gene cluster. Therefore, much of the organisation seems to be conserved in evolution, with a degree of rearrangement of some of the genes (Ridinger et al. 1998). S100 proteins are in general of a small molecular size. Although some of them, e.g., S100B() and S100A(), have been known for a long time and much information has accumulated over the years, the function of the majority is still largely unknown. There have been signicant advances in recent years in our understanding of the involvement of some S100 proteins in normal and aberrant physiology. Especially noteworthy are the close association some of these proteins show in the abnormal behaviour of cancer cells, and the effects of their elevated expression in tumours and the consequent modulation of cytoskeletal dynamics, cell mobility, and cell proliferation.
S100 PROTEINS IN CELL DIFFERENTIATION, MOTILITY, AND CANCER INVASION PROFILAGGRIN (FLG)
IN
KERATINOCYTE DIFFERENTIATION
The consortium of genes at the 1q21 region, whose physical mapping has been referred to above, includes a number of genes associated with the terminal differentiation of human epidermis. This collection of genes has been called epidermal differentiation complex (EDC) (Mischke et al. 1996). This complex has been most lucidly described by Marenholz et al. (1996) as being composed of three groups of genes: (1) structural genes such as IVL, LOR, and others; (2) genes coding for the IF-associated proteins FLG and THH; and (3) S100A1S100A10. FLG and THH are synthesised in the granular layer of the epidermis and participate in the aggregation and aligning of keratin IF during the terminal differentiation of keratinocytes. Both proteins contain EF-hand calcium-binding domains and are functionally associated with Ca2+-dependent processing (see below). Krieg et al. (1997) have cloned a novel gene, the repetin gene, from the mouse. This gene has marked similarities to FLG and THH with respect to genomic organisation and EFhand calcium-binding domain characteristics. Repetin, together with FLG and THH, could represent a subfamily of S100 genes (Table 16). The Molecular Characteristics of Prolaggrin Human FLG is a 400-kDa phosphoprotein that is found in the granular cells of the epidermis. Its N-terminal region contains two calcium-binding motifs similar to the EF-hand calcium-binding domains found in the S100 family of proteins. The two calcium-binding sites differ markedly with respect to their calcium-binding afnity (Presland et al. 1995). The FLG molecule consists of several units of laggrin linked
203
TABLE 16 Genomic and Molecular Features of Prolaggrin, Trichohyalin and Repetin

Feature Exons/introns EF-hands Repeat elements FLG 3 exons, 2 introns Two N-terminal exons I and II Filaggrin units THH rptn 3 exons, 2 introns Two N-terminal exons I and II Involucrin-like
Two N-terminal Involucrin-like
Source: Based on Markova et al. (1993); S.C. Lee et al. (1993); Presland et al. (1995); Krieg et al. (1997).
together. The molecule undergoes proteolytic cleavage at the sites of linkage during terminal differentiation, generating monomeric laggrin units with an average molecular size of 42 kDa. The laggrin units also acquire specic C- and N-terminal sequences as a result of this proteolytic processing (Resing et al. 1993a; Thulin and Walsh, 1995). The processing of FLG is a two-stage process that generates intermediate products with laggrin repeats. An endopeptidase, FLG endopeptidase 1 (PEP1), involved at this rst stage of the cleavage of the linkers has been identied. The functional specicity of this enzyme has been suggested to be due to phosphorylation of FLG (Resing et al. 1995). PEP1 is a serine protease. Also involved in this proteolytic processing are leupeptin-sensitive cysteine protease and protein phosphatases. The former has features resembling calpain (Resing et al. 1993b, 1995). The calpains are Ca2+-activated cysteine proteases. Yamazaki et al. (1997) have shown that -calpain is capable of processing FLG (Figure 25).
FIGURE 25 The generation of laggrin monomers from prolaggrin, with enzymatic cleavage of linking peptides occurring between laggrin repeats. One of the enzymes implicated in this process is -calpain. This enzyme is made up of a large 80-kDa subunit and a 30-kDa subunit. Calpain is activated by the binding of calcium to CaM-like domains that occur in both subunits. (Based on Resing et al. 1993a, 1993b, 1995; Saido et al. 1993; Yamazaki et al. 1997; Kam et al. 1997).
204
The expression of FLG is markedly reduced in ichthyosis. This has been attributed to its regulation at the posttranscriptional level. A decimation of FLG levels in keratinocytes from ichthyosis-affected patients has been reported, but the decrease in FLG mRNA expression amounted to only 30 to 60% of controls (Nirunsuksiri et al. 1995). Another reason for this could be the stability of the gene transcript. The FLG transcripts in keratinocytes of ichthyosis subjects were found to be far more unstable and possessed a shorter half-life than the transcripts in normal keratinocytes (Nirunsuksiri et al. 1998). The normal function of FLG might also depend on the state of its phosphorylation. It has been proposed that phosphorylation may prevent FLG from causing premature aggregation of keratin laments and their packaging into storage granules. It has been suggested also that phosphorylation might be involved in the proteolytic processing of FLG into laggrin monomers (see Figure 25). The conversion of the highly phosphorylated FLG into nonphosphorylated laggrin appears to depend on protein phosphatases PP-2A and PP-1. Keratinocytes show a high level of PP-2A and PP-1 activity, but in harlequin ichthyosis, the activity of these phosphatases is greatly reduced, with a resultant disruption of laggrin generation (Kam et al. 1997). It has been reported that PKC- can mediate FLG phosphorylation. This, together with the solubilisation of FLG, indicates that phosphorylation may be involved in the generation of laggrin monomers (Old et al. 1997). Filaggrin monomers are formed during terminal differentiation of keratinocytes. A major process occurring at this stage of differentiation is the aggregation of keratin laments. This occurs in association with FLG monomers. It is believed that prolaggrin itself is unable to accomplish this. Haydock et al. (1993) transfected antisense FLG constructs into rat epidermal cell lines. In transfectants expressing the antisense mRNA, the processing of FLG was suppressed, and the differentiation in vitro of keratinocytes was markedly affected as a consequence. The generation of laggrin also produces marked changes in cell morphology and affects nuclear integrity. Cell adhesion to substratum also appears to be affected (Dale et al. 1997). FLG occurs in keratohyalin granules, and a recent study by Dale et al. (1997) suggests that the insoluble native form of FLG is required in their formation, which indicates that the linker peptides have a role to play in this process. Prolaggrin and laggrin might be involved in the pathobiology of rheumatoid arthritis (RA). They show a distribution pattern similar to the antigens recognised by antibodies that are used as markers for RA. These so-called anti-keratin antibodies have been shown to recognise epidermal laggrin and are demonstrably anti-laggrin antibodies (Simon et al. 1995). Antibodies against laggrin have been employed as a diagnostic aid, and their use might complement that of anti-keratin antibodies in diagnosing RA (Vincent et al. 1998). The possibility has been envisaged that the pathogenesis of RA could involve a loss of immunological tolerance to laggrin (Berthelot et al. 1995). Abnormal interaction between keratin laments and laggrin may be involved in some cases of epidermolytic ichthyosis. Here, although prolaggrin/laggrin are expressed at an enhanced level, no abnormalities of this protein have been encountered (Ishida-Yamamoto et al. 1994). Abnormal expression of FLG as well as K6 and K16 laments is found in keratinocytes from the congenital condition of
205
harlequin ichthyosis. FLG expression is either severely down-regulated or totally undetectable in ichthyosis vulgaris. The possible reasons for this have been discussed above.
TRICHOHYALIN (THH)
Trichohyalin is another protein that is associated with IF. THH is a 248-kDa protein that is found in trichohyalin granules and possesses two EF-hand-like calciumbinding domains at its N-terminal end. The protein could occur as a exible rod approximately 215 nm in length (S.C. Lee et al. 1993). It associates with and forms regular and precise arrays of keratin IF in the medulla and inner root sheath cells of hair follicles (Oguin et al. 1992). THH appears to undergo specic sequential posttranslational modications to produce highly cross-linked rigid laments. Tarcsa et al. (1997) have proposed that the insoluble cytoplasmic THH is rst modied by peptidyl arginase to convert it into a less organised and soluble molecule. These THH molecules are then cross-linked by the agency of Ca2+-dependent transglutaminases to generate rigid laments that interact with keratin laments. THH is also found in nonfollicular epithelia, e.g., liform papillae of tongue epithelium (E.J. OKeefe et al. 1993). It occurs, together with FLG, in epithelia that possess IF containing K6/K16 keratins, which has led to the suggestion that THH may specifically mediate aggregation of IF containing these keratins (Manabe and Oguin, 1994). The association of THH with a possible differentiation pathway has prompted studies into THH expression in skin tumours (Manabe et al. 1996). Preliminary ndings also are available with regard to the possible involvement of repetin (rptn) in the progression of skin tumours in the mouse (P. Krieg, personal communication, 1998). Apparently, rptn mRNA is expressed at low levels in normal mouse epidermis. Treatment with TPA, which is a tumour promoter, results in a transient overexpression of the mRNA. In benign tumours this overexpression becomes constitutive. Benign papillomas show far higher levels of rptn mRNA than normal epidermis. During progression into carcinomas, the steady-state levels of rptn mRNA are reduced. Compatible with this, in a few squamous cell carcinomas investigated, rptn mRNA has been found to occurs at lower levels as compared with papillomas. There is a hint of the possibility that rptn gene expression may be involved as an early event in the progression of skin tumours. These studies are too preliminary in nature to merit serious discussion at present. However, the recognition of the potential of these genes in the context of cancer development does merit citation. Needless to say, they could lend themselves as possible candidates for markers of differentiation in the development and progression of skin tumours.
EFFECTS OF S100 PROTEINS ON CELL DEFORMABILITY AND CELLULAR MORPHOLOGY

The ability to alter the malleability of the membrane and cell shape and regulation of intercellular adhesion are two essential requirements for facilitating cell motility, diapedesis, and invasiveness. Changes in cellular morphology also occur in terminal
206
differentiation and when differentiation is experimentally induced. For instance, in neuroblastoma cells exposed to retinoic acid, changes occur in cell shape with attendant changes in S100 family gene calcyclin (Tonini et al. 1991). Calcyclin appears to be associated with the tangential migration of proliferating cells of the subventricular zone of the lateral ventricle into the olfactory lobe and their differentiation into interneurones. A subpopulation of astrocytes in the tangential migration pathway seems to express calcyclin (Yamashita et al. 1997). The migration of subventricular zone (SVZ) cells is believed to be organised by the polysialylated neural cell adhesion molecule (PSA-NCAM). Neural cell adhesion molecules (NCAM) and cadherins have been studied extensively in the context of neurite regeneration and neuronal adhesion. Whereas cadherins are Ca2+ dependent for their function, NCAMs are not. It is of considerable interest, therefore, that the migration of the SVZ cells mediated by an NCAM is associated with calcyclin expression. Intracellular calcium levels regulate cellular morphology. Cells appear to alter their shape in response to subtle changes in intracellular Ca2+. Thus calcium transients produced by the mobilisation of calcium from intracellular stores can have effects that differ profoundly from those induced by inuxed calcium. Caffeine induces transient increases in calcium in the dendrites and spines of hippocampal neurones. An increase in the size of existing dendrites and possibly also the formation of new dendrites occurs in association with these calcium transients (Korkotian and Segal, 1999). In contrast, glutamate receptor activation by NMDA and glutamate itself produces a collapse of dendritic spines, together with an infux of calcium. This leads to comparatively large changes in the levels of intracellular calcium. The loss of dendritic spines corresponds with the loss of lamentous actin. Actin-stabilising agents counteract the effects of NMDA (Segal, 1995; Halpain et al. 1998). Inuxed calcium and calcium mobilised from intracellular stores might have independent roles (Haverstick et al. 1991). It also has been recognised that calcium released from intracellular stores might activate specic components of the signal transduction pathway (Parker and Sherbet, 1992). One can then hardly overemphasise the potential inuence CBPs can exert on cellular morphology. S100 proteins alter cell shape and motility apparently by virtue of their ability to alter cytoskeletal dynamics. Inhibition of S100B, which is predominantly expressed in glial and Schwann cells, has been shown to result in changes in cellular morphology as well as in the organisation of the cytoskeleton of glial cells in culture (Selinfreund et al. 1990). Selinfreund et al. (1990) introduced antisense S100B oligonucleotides, which were placed under the control of a dexamethasone-inducible promoter, into rat C6 glioma cells. When the antisense constructs were induced to express, the levels of S100B decreased with concomitant changes in cell morphology. They assumed a attened morphology and displayed an organised network of microlaments. The neurite extension factor, which bears sequence homology to S100B, is a homodimer of S100B-like subunits. This factor actively promotes neurite extension (Kligman and Marshak, 1985). S100 proteins are expressed in the dendritic cells in human transitional cell carcinoma of the bladder, and the invasive potential of these tumours has been found to correlate with the presence of cells expressing S100 protein (Inoue et al. 1993). The occurrence of S100 proteins together with
207
smooth muscle actin and vimentin has been reported in a metaplastic carcinoma of the breast invading the chest wall as well as in recurrent tumour (Harb et al. 1995). Reeves et al. (1994) generated transgenic S100B mice in which S100B mRNA expression was two- to seven-fold greater than that in control mice. They found enhanced expression of glial brillary acidic protein (GFAP) and also enhanced neurite proliferation in the transgenic mice. Similarly, the expression of S100A4 appears to correlate with the in vitro invasive potential of glioma cells in culture (Merzak et al. 1994b). The ability of S100A4 to interact with nonmuscle myosin supports the view that it takes part in cellular motility (Takenaga et al. 1994a, 1994b). S100A4 is highly expressed during embryonic development in the highly invasive mesenchymal elements (Klingelhofer et al. 1997). These effects appear to be due to an interaction of the S100 proteins with the cytoskeletal machinery of the cell. NGF markedly enhances calcium uptake (see Kozak et al. 1992). Many of the effects of NGF on PC12 cells, for instance, are accompanied by alterations in the expression of S100 proteins. An S100 protein called p11 (S100A10), identied some years ago, which shows a high degree of sequence homology to p42C. The latter is induced in PC12 cells by treatment with NGF (Masiakowski and Shooter, 1988). p11 binds to and inhibits the phosphorylation of a tyrosine residue of annexin (p36). It has been found that a complex composed of p11 dimer and two subunits of annexin p36 is involved with the process of actin bundling and in the linkage of the actin component to the cell membrane (Kligman and Hilt, 1988). Zimmer et al. (1998) transfected S100A1 cDNA in antisense orientation into PC12 cells. These transfectants showed a marked reduction in S100A1 expression. When these transfectant cells were exposed to NGF, there was a marked increase in neurite formation. In parallel, Zimmer et al. (1998) also found increased levels of -tubulin. The antisense S100A1 clones also showed reduced anchorage-dependent growth. S100B has been reported to form a complex with the cytoskeletal tau protein, and this complex appears to inhibit microtubule polymerisation (Baudier et al. 1982; Baudier and Cole, 1988). S100 proteins show a substantial expression in brain tissues; they are found in astrocytes and oligodendrocytes, and their expression seems to be functionally linked with their morphology and differentiation. These functions may be associated with changes in the cytoskeletal elements, their organisation, and disposition. S100 proteins have several putative target cellular proteins; interactions with these might inuence a panoply of cellular features (Table 17). S100B has been shown to inhibit the polymerisation of GFAP (Bianchi et al. 1993). It appears to interfere with the early stages of GFAP polymerisation, reducing the rate of assembly of GFAP subunits, besides actively promoting depolymerisation of GFAP polymers (Bianchi et al. 1994). A specic domain in GFAP interacts with S100 proteins, and a peptide known as TRTK-12, which bears homology to this domain of GFAP, has been shown to be able to counteract the inhibition by S100 of the polymerisation of GFAP monomers (Bianchi et al. 1996). Bianchi et al. (1996) have suggested further that S100B achieves this by sequestering GFAP monomers. This view is in sharp contrast with a recent observation that S100B promotes the extension of MAP-2, and might indeed promote the reassembly and stabilisation of the cytoskeleton (Nishi et al. 1997). S100A4 also has been shown to be associated with cytoskeletal elements
208
TABLE 17 S100 Proteins and Their Intracellular Targets

S100 Protein S100A1 S100A4 Target Molecule Phosphoglucomutase Myosin II Nonmuscle tropomyosin F-actin Tubulin monomers p53 S100A8/A9 (MRP8/14) S100A10 (p11) S100A11 (S100C) Membrane arachidonic acid Annexin II Binds to actin laments and inhibits Mg2+ATPase; annexins Phosphoglucomutase GFAP CapZ actin-capping protein Ndr nuclear serine/threonine kinase Calponin Functional Association Stimulation Cell locomotion Cell locomotion Cell locomotion Cell proliferation; locomotion Cell proliferation Translocation to cell membrane Cytoskeletal reorganisation Reorganisation of the cytoskeleton; putative participation in growth and differentiation Stimulation Inhibition of GFAP polymerisation Actin polymerisation Inuence on cell cycle progression by the activation of Ndr kinase Interaction with membrane phospholipids Ref. Zimmer et al. (1995); Landa et al. (1996) Ford et al. (1997) Takenaga et al. (1994b) Davies et al. (1993); Gibbs et al. (1994) Lakshmi et al. (1993) Parker et al. (1994a, 1994b) Klempt et al. (1997) Kaczanbourgois et al. (1996) Zhao et al. (2000); Naka et al. (1994); Mailliard et al. (1996); see text Landa et al. (1996) Bianchi et al. (1993) Kilby et al. (1997) Millward et al. (1998)
S100B
Fujii et al. (1995)
(Davies et al. 1993; Gibbs et al. 1994; Takenaga et al. 1994a). In S100A4 transfected Rama-29 cells, the S100A4 staining pattern markedly resembles that of phalloidin, which binds to F-actin laments (B.R. Davies et al. 1993; Gibbs et al. 1994). Such an association seems to result in cytoskeletal disorganisation (Watanabe et al. 1992, 1993), possibly related to the ability of the protein to promote microtubule depolymerisation (Lakshmi et al. 1993). Takenaga et al. (1994c) demonstrated that the binding of S100A4 to nonmuscle tropomyosin was calcium dependent and identied amino acid residues 39107 of S100A4 as the binding site. They showed further by immunoblotting that part of S100A4 associated with Triton-insoluble cytoskeletal components. Indeed, in situ, S100A4 could be localised together with tropomyosin in microlament bundles. In NIH3T3 and 3Y1 tissue culture cells S100A4 is found diffusely distributed in the cytoplasm, but some of the protein is also found in association with actin stress
209
bres (Takenaga et al. 1994b). S100A4 also has been shown to interact specically with myosin II at the rate of 3 moles of S100A4 per mole of MHC. However, it does not interact with myosin I or II. Further, this interaction might destabilise myosin laments (Ford et al. 1997). The S100A4-binding site has been identied. It occurs in the rod region and is mapped to a sequence, between residues 1909 and 1937, at the C-terminal end of MHC (Kriajevska et al. 1998). These authors have shown that S100A4 inhibits PKC-mediated phosphorylation of MHC at serine residue 1917. It seems possible that S100A4 might inuence tropomyosin-assisted myosinactin interactions. The distribution of A and B isoforms of nonmuscle myosin II, as discussed earlier, shows a marked relationship to processes associated with cell locomotion. This might afford a mechanism by which S100A4 could regulate the cytoskeletal machinery and inuence locomotion. S100 protein has been reported to show calcium-dependent binding to calponin, a protein that can bind to CaM, actin, and tropomyosin. An N-terminal 22-kDa domain of calponin was involved in the interaction with S100 as well as with actin, tubulin, CaM and tropomyosin (Fujii et al. 1994, 1997). Calponin also interacts with membrane phospholipids, and these interactions are inhibited by S100 (Fujii et al. 1995). Most of these observations come from in vitro studies and thus no inferences can be drawn at present as to their relevance to in vivo situations, especially in light of the nding that calponin does not affect tubulin polymerisation (Fujii et al.1997). Nevertheless, Kilby et al. (1997) showed that certain C-terminal amino acid residues and N-terminal residues valine 8 to aspartic acid 12 of S100B may be the domains that are involved in its interaction with the actin-capping protein CapZ. This lends itself strongly in favour of S100 interactions with their target proteins as being a consistent feature in determining cell behaviour. There is, therefore, a complex series of interactions between S100 proteins and cytoskeletal structures, which in turn may be related to membrane uidity and exibility, and this could well inuence the shape and motility of cells. These interactions may also be indicative of terminal differentiation, as shown experimentally by Tonini et al. (1991). These authors found an enhancement of S100 expression in neuroblastoma cells that were induced to differentiate using retinoic acid. Zimmer and Landar (1995) studied the expression of S100A and B in the differentiation of PC12 cells and L6S4 skeletal muscle cells. Both proteins are expressed in the undifferentiated as well as the differentiated state. S100A1 protein expression was higher in differentiated cells, but the expression of its mRNA showed no change in differentiated L6 cells, and in fact mRNA levels decreased in PC12 with differentiation. This would suggest that S100 expression is regulated at the posttranscriptional level. The S100 protein MRP14 (S100A9) is phosphorylated during monocytic differentiation. Two phosphorylated isoforms of MRP14 have been detected. MRP14 seems to respond to enhanced Ca2+ levels by undergoing a selective translocation from the cytoplasm to the cell membrane (Van den Bos et al. 1996). In fact, heterodimers of MRP8 and 14 are capable of binding specically to membrane arachidonic acid (Klempt et al. 1997). The association of S100 proteins with syncytioblast maturation and differentiation in the placenta has been reported recently. S100A10, which was formerly known as p11, forms a heteromeric association with
210
annexin II that is linked to cytoskeletal structures, and both annexin II and p11 show a progressive increase in association with placental differentiation (Kaczanbourgois et al. 1996). Apart from the obvious implications for membrane-related activities of the cell, there may also be other functions subserved by the association of S100 proteins with cytoskeletal components. MRP8 and MRP14 are both activated by PKC phosphorylation before they are secreted, and the activated proteins associate themselves with tubulin laments before release, which seems to suggest that such an association could provide a route for the release of MRPs (Rammes et al. 1997).
CELL ADHESION
AND INVASIVE
POTENTIAL
OF
CANCER CELLS
Adhesive interaction between cells as well as between cell and substratum is an important ingredient of cellular motility. This cellular faculty is dependent on not only the cell membrane-associated adhesive macromolecules but also their temporal and spatial distribution in the cell membrane. It follows, therefore, that alterations in the overall expression of adhesion-mediating components of the cell membrane as well as their topographical distribution should entail alterations in the adhesive property. As a converse of this, one would expect to see changes either in the nature or in the pattern of distribution of cell membrane components in association with the acquisition of invasive potential. In inquiries into the question of whether S100 proteins inuence the adhesive abilities of cells, a testable postulate would be to see if they regulated the expression on cell membrane components that participate in cell adhesion. Historically there is much evidence that calcium signalling is an important event in the induction of cell motility. Calcium signalling in processes such as the penetration of the oocyte by the sperm cell involves G-protein-mediated increases in intracellular calcium levels (Florman et al. 1989). The migration of neutrophils appears to depend on the enhancement of intracellular calcium levels (Marks and Maxeld, 1990). Chemoattractants have been known to induce calcium inux (Milne and Coukell, 1991). They produce rapid changes in the intracellular Ca2+ levels together with cell spreading, formation of pseudopodia, motility and its direction, and phagocytosis. However, blocking of the intracellular Ca2+ transients seems to have little effect on these biological features, indicating the possible involvement of other factors (Hendey and Maxeld, 1993). Nonetheless, Hendey and Maxeld (1993) report that cell motility on physiological substrates such as bronectin and vitronectin are dependent on intracellular calcium levels. Cell surface integrin receptors possess calcium-binding domains similar to EF-hands (Tuckwell et al. 1992). The calcium-binding motifs of the subunit of M2 integrin of leukocytes show similarities to as well as differences from EF-hand motifs (Oxvig and Springer, 1998). Savarese et al. (1992) found that type IV collagen, which induces cell motility, also brings about increases in intracellular calcium levels, presumably by the release of Ca2+ from intracellular stores. Therefore, it would seem that intracellular calcium transients closely relate to the cellular interaction with substratum mediated by ECM components such as bronectin and collagen. These interactions could be important events in the initiation of calcium signalling. The S100 proteins MRP8 (S100A8) and MRP14 (S100A9) are expressed abundantly in the cytoplasm of neutrophils.
211
These proteins associate with vascular endothelia and presumably aid in the diapedesis of cells across the endothelium. Newton and Hogg (1998) have found that MRP14 regulates the function of 2-integrin, an adhesion-mediating glycoprotein. The function of MRP14 appears to be restricted to just this regulatory process. The function of MRP14 itself may be regulated by MRP8. Apparently, the formation of a heterodimer between MRP8 and MRP14 can inhibit the function of MRP14. However, the nature of 2-integrin regulation by MRP14 remains to be elucidated, although Newton and Hogg (1998) do state that integrin does not function as a receptor for MRP14. Many components of the ECM are associated with and known to participate actively in cell motility and cancer invasion (see Sherbet and Lakshmi, 1997b). More pertinent to the topic under discussion here is the observation by Savarese et al. (1992) that type IV collagen, which stimulates motile behaviour in melanoma cells, also increases intracellular calcium levels. The effect seems to be mediated by the release of Ca2+ from intracellular stores rather than by an inux of extracellular calcium. Another ECM component, the transmembrane glycoprotein CD44, has been the subject of much debate with regard to its putative role in cancer invasion and metastasis (see Sherbet and Lakshmi, 1997b). CD44 is a membrane-associated adhesive glycoprotein that mediates intercellular adhesion by virtue of its function as a hyaluronate receptor. It has been attributed with enhancing the invasive ability of cells (Radotra et al. 1994; Merzak et al. 1994a). In this context may be cited recent studies of CD44 expression in B16 melanoma cells in which the expression of S100A4 was experimentally altered (Lakshmi et al. 1997). CD44 expression did not increase when S100A4 expression was up-regulated. However, an enhanced expression of S100A4 produced a redistribution of CD44 into a patchy focal pattern. Because S100A4 is able to depolymerise cytoskeletal elements, it has been suggested that the redistribution of the glycoprotein could be a result of cytoskeletal depolymerisation and the resultant enhanced lateral mobility of CD44 molecules leading to the aggregation and patching of CD44 receptors (Lakshmi et al. 1997). Fully compatible with this line of argument are the recent ndings that CD44 molecules localise in cholesterol-rich domains of the plasma membrane. These molecules enjoy restricted lateral mobility because of the cholesterol-rich nature of the membrane domain in which they are localised and also the integrity of the actin cytoskeleton. A disruption of the integrity of the actin cytoskeleton results in an increase in the partitioning of CD44 to the cholesterol-rich domains (Oliferenko et al. 1999). Hyaluronic acid, which is a ligand for CD44, induces such patching, and these CD44 patches are associated with a preferential accumulation of and binding to plaques of the cytoskeletal protein called ankyrin (Bourguignon et al. 1993; Welsch et al. 1995). The interaction of CD44 with ankyrin occurs when CD44 is phosphorylated by Rho kinase, which, in turn, is activated by the GTPase activity of Rho A bound to CD44v. The phosphorylation of CD44 enhances its interaction with ankyrin. Bourguignon et al. (1999) have shown that microinjection of the catalytic domain of Rho kinase produces membrane rufing. This can be inhibited by CD44 antibodies and by the microlament inhibitor cytochalasin D.
212
It is obvious from these observations that the induction of cell motility by CD44 is a complex process. The redistribution of CD44 into patches occurring under conditions of high S100A4 could provide the tumour cells with discrete and strongly adhesive foci that may promote their invasive behaviour by increasing and strengthening anchorage and intercellular interaction (Lakshmi et al. 1993). Overall, these data indeed suggest that S100A4 may be linked with the cytoskeleton. Further evidence, albeit circumstantial, that S100A4 might associate with the cytoskeleton, has come from recent work by Keirsebilck et al. (1998a). They investigated the status of E-cadherin and S100A4 in two murine tumour cell lines and found tha E-cadherin expression correlated inversely with S100A4 expression in these cell lines. E-cadherin is a transmembrane CBP, which is linked to the actin cytoskeleton via a number of linking elements (see Figure 13). E-cadherin is a putative suppressor of invasive and metastatic abilities (see Sherbet and Lakshmi, 1997b). Low levels of E-cadherin expression have been reported to correlate with poor prognosis of a variety of human cancers. Therefore, the inverse relationship between it and S100A4 is compatible with the invasion- and metastasis-promoting ability of the latter. The exact mechanics of this relationship have yet to be elucidated. Keirsebilck et al. (1998a) found an alteration in the intracellular distribution of - and -catenins, which link the cytoplasmic tails of Ecadherin to the actin cytoskeleton. Some cellular proteins, such as the adherence junction protein, are known to bind to this cytoplasmic domain of E-cadherin. APC protein, which is believed to function as a tumour suppressor protein, is known to compete with E-cadherin for binding to -catenin (Hulsken et al. 1994). Arguably, the suppressor effect of cadherin does not depend on merely the presence of cadherin per se, but also on its being present in a functional state in its entirety as a complex with the linking proteins. Three forms of cadherincatenin complexes may occur in cells. The form with one cadherin molecule linked to a -catenin/catenin or plakoglobin/-catenin is the conventional from depicted in Figure 13. In the second type of complex, the extracellular domains of cadherin dimerise in a parallel fashion. A proposed third type is one in which the extracellular domains of two conventional complexes belonging to two neighbouring cells associate in an antiparallel fashion (Chitaev and Troyanovsky, 1998). Further, Chitaev and Troyanovsky (1998) have suggested that an antiparallel association of dimerised complexes of neighbouring cells is responsible for intercellular adhesion, and that this is dependent on not only extracellular calcium but also the presence of the catenin components. It is apparent from this discussion that suppression of invasion by cadherin is a complex process, and at present it is difcult to envisage how S100A4 and cadherin antagonise each other. If the mechanism proposed by Chitaev and Troyanovsky (1998) were valid, one would not expect an inverse relationship between cadherin and S100A4 expression, for the latter would be expected to promote the dimerisation of cadherin complexes by enhancing the lateral mobility of the complexes in the cell membrane. Nonetheless, it would be of considerable interest to see if S100A4 affects in any way the interaction of E-cadherin with the catenins.
213
S100 PROTEINS
IN
REMODELLING
OF THE
EXTRACELLULAR MATRIX
ECM undergoes constant changes in terms of remodelling and renewal in a host of biological processes, such as morphogenesis, cell differentiation, wound healing, angiogenesis, cell motility, and also in the invasive and metastatic behaviour of cancers. A wide variety of proteinases are involved in the remodelling of the ECM. These enzymes and their inhibitors are known to be genetically regulated (see Sherbet and Lakshmi, 1997b). That ECM remodelling might involve S100A4 was demonstrated some time ago by Merzak et al. (1994). In glioma cell cultures, S100A4 expression appears to be inversely related to the expression of TIMP-2 gene, which encodes an inhibitor of tumour-associated metalloproteinases. Interestingly, S100A4 is highly expressed in cell lines derived from invasive gliomas, as compared with cell lines obtained from noninvasive localised brain tumours or foetal brain cells. On the basis of these observations, it has been suggested that the invasive property of glioma cells could be a result of the uninhibited function of tumourassociated proteinases and that this lack of regulation of the proteinases concerned might be due to inhibition of TIMP-2. In a recent report, Bjornland et al. (1999) claimed that TIMP-2 up-regulation can occur independently of the state of S100A4 expression. They isolated several clones from a highly metastatic osteosarcoma cell line, which expressed S100A4 at different levels on account of being transfected with an anti-S100A4 ribozyme. It is somewhat intriguing that in the clones with low or intermediate S100A4 expression, both MMPs and TIMP-1, were down-regulated. With a complex experimental system such as this, it is difcult to visualise how MMP homeostasis could be altered by parallel modulation, in the same direction, also of the MMP inhibitor TIMP-1 (also see below). Several other studies also have suggested that the expression of S100A4 might correlate with that of MMPs. De Vouge and Mukherjee (1992) found that both S100A4 and transin-2 are up-regulated in parallel in rat kidney cells transformed by Ki-ras. K. Andersen et al. (1998) found that IL-1 down-regulated the expression of TIMP-1 in osteosarcoma cells that express S100A4 at high levels. In contrast, in cells with low level of S100A4 expression, IL-1 did not change TIMP expression. K. Andersen et al. (1998) have suggested that the effects of bFGF, which also affects TIMP, might not be related to the levels of S100A4 expression. They suggest that IL-1 functions synergistically with S100A4. It should be borne in mind, however, that both bFGF and IL-1 effects on TIMP could have been mediated by osteonectin, because bFGF is known to regulate the expression of osteonectin. In mesenchymal cells, bFGF is known to up-regulate the expression of osteonectin (Shiba et al. 1995). IL-1, on the other hand, down-regulates the expression of osteonectin. Osteonectin has been shown to modulate MMP expression, and possibly also that of its inhibitor. There is no evidence at present for the latter. However, in many systems studied to date, the MMP/TIMP system is regulated as a unit, with MMP up-regulation more often than not being a result of down-regulation of TIMP or vice versa. None of the experiments described here provide any indication of how S100A4 achieves TIMP2 inhibition. This remains to be elucidated. It should be recognised that the remodelling of the ECM and the downstream effects of such changes on cell adhesion and motility might be directly linked with
214
the reorganisation of the cytoskeleton. We know from the work of Irigoyen et al. (1997) that cytoskeletal reorganisation leads to, probably among other things, an up-regulation of uPA. Irigoyen et al. (1999) have further shown that this up-regulation occurs via the ras/erk-signalling pathway. The ability of S100A4 to alter the organisation of the cytoskeleton has been satisfactorily demonstrated. Besides, De Vouge and Mukherjee (1992) have shown that cell transformation by the ras oncogene results in the up-regulation of S100A4 as well as an MMP. Furthermore, heat shock can down regulate not only S100A4 expression (Albertazzi et al. 1998a) but also the expression of membrane type I MMP together with an inhibition of in vitro invasive ability (Sato et al. 1999). Under these circumstances one would be amply justied in considering this to be a putative link between S100A4 expression and ECM remodelling and the associated changes in membrane properties and cell behaviour. S100A4 is generally believed to be a cytosolic protein. However, its putative participation in ECM remodelling suggests that it might be secreted into the ECM. The ndings of Duarte et al. (1999) seem to conrm this view. They reported that S100A4 is secreted under both in vivo and in vitro conditions. They further reported that S100A4 inhibits the process of mineralisation and bone nodule formation in bone marrow cell cultures. It is possible, therefore, that the apparent modulation of TIMP activity and any consequential effects on the character of the ECM described by Merzak et al. (1994) could have resulted from the action of the S100A4 secreted into the ECM. The ECM-modulating effects of S100A4 are also implicit in a set of observations relating to the epithelialmesenchymal transformation that occurs in vertebrates. An important feature of this transformation is that it is associated with the acquisition of the ability to invade and migrate into the ECM (Hay, 1995). Several genes are switched on during this transformation, among them S100A4, which was called the broblast-specic protein 1 (FSP1). Although the broblast specicity is debatable, it has been shown that antisense S100A4 suppressed the expression of the gene and at the same time suppressed epithelialmesenchymal transformation (Okada et al. 1997). Okada et al. (1997) also have gone on to demonstrate that EGF and TGF 1, both capable of inducing cellular motility, also induce S100A4 expression in epithelial cells. A somewhat simplied conclusion that ows from the above discussion is that S100 proteins play a very signicant role in cell motility, cell adhesion, and the invasive behaviour of cancer cells. It is also obvious that many questions remain unanswered, especially those regarding the interaction of these proteins with the cytoskeletal elements, i.e., the mode of their function as agents that promote cytoskeletal disassembly or inhibit the polymerisation of cytoskeletal monomeric elements. We do, however, have some signicant leads as to how S100 proteins might be involved in the apparent regulation of tumour-associated proteinases.
S100 PROTEINS
IN
CELL PROLIFERATION
There is a general acceptance of the concept that cancers possess a high proliferative potential and that this apparent potential is more a consequence of the loss of
215
homeostatic growth control than of an increase in the rate of proliferation. Inevitably, S100 genes, whose expression correlates remarkably well with invasive and metastatic potential of cancers, have also been investigated for their relationship to growth deregulation in cancers. It is signicant, therefore, that some of the major S100 proteins are expressed in a cell cycle-specic manner. Indeed, some members of the S100 family such as S100A4 were originally cloned from highly proliferative tissues and were described as proliferation-related proteins. Two questions can be posed in order to elucidate the relationship between growth regulation and the expression of S100 proteins. One question is whether the presence of S100 proteins correlates with the growth fraction and proliferative behaviour of tumours, or with tumour cells or normal cells in culture. Recently, S100B has been attributed with a role in the regulation of the cell cycle. The evidence takes the form of the demonstration of the ability of S100B to bind to and activate in vitro the nuclear serine/threonine protein kinase called Ndr, in response to changes in the intracellular calcium levels. An N-terminal regulatory region of Ndr seems to be involved in the Ca2+-dependent binding of S100B, and this region contains sequences that are characteristic of CaM/S100 binding (Millward et al. 1998). S100B also seems to be able to regulate the intracellular activity of Ndr. Millward et al. (1998) have proposed a biological role for this interaction between S100B and Ndr and the regulation of the activity of the latter. In melanomas that overexpress S100B, the CaM inhibitor W7 inhibits the activation of Ndr, but not in those that lack S100B expression. The Ndr kinase was identied by Millward et al. (1995). It seems to belong to a subfamily of protein kinases that shows 40 to 60% sequence similarities among the members of the family. These kinases have been implicated strongly in the progression of the cell cycle as well as in the regulation of cell morphology. Therefore, Millward et al. (1998) have argued that an overactivation of Ndr by S100B might be related in some way to the development and progression of melanomas. However, as Millward et al. (1998) have pointed out, some melanomas do overexpress S100B without also showing an overactivation of Ndr. Therefore, they suggest that a mechanism of negative control of Ndr might exist. This postulate requires that this negative regulation is inactivated by loss-of-function mutations of the Ndr regulator. S100B has been reported to stimulate sciatic nerve regeneration and may function as a growth factor for peripheral nerve axons (Haglid et al. 1997). Growth may be regulated by a homeostasis of cell proliferation and apoptotic loss of cells. S100B has been reported to rescue motor neurones from apoptotic death (Iwasaki et al. 1997). The ability of S100B to inuence actin dynamics might be involved in this effect. The status of actin polymerisation has been found to regulate apoptosis. Induction of polymerisation leads to apoptosis, whereas inhibitors of polymerisation block apoptosis (J.Y. Rao et al. 1999). As alluded to above, S100B can inhibit actin polymerisation. Therefore the ndings of Iwasaki et al. (1997) are compatible with those of J.Y. Rao et al. (1999). On the other hand, it has been found that S100B may lead to apoptotic death by releasing nitric oxide (Hu et al. 1997). The calcyclin gene is expressed more frequently in epithelial-type or Schwann cells than in neuroblastic cells. This is compatible with the difference in their growth potential. A consequence of the induction of differentiation of neuroblastoma cells
216
by retinoic acid is an enhancement of calcyclin expression together with arrest of cell growth (Tonini et al. 1991). It is yet uncertain if calcyclin indeed negatively regulates growth, because, in total contrast, a higher proportion of melanoma cells shows the presence of calcyclin in the vertical growth phase (Weterman et al. 1993). It may be pointed out, however, that S100B expression has been reported to increase in neuroblastoma differentiation (Tsunamoto et al. 1988). Tonini et al. (1991) found an inhibition of growth rate in cells where S100B levels had been experimentally reduced. This is not compatible with the ndings of Tsunamoto et al. (1988) that reduction of growth rates invariably accompany the induction of differentiation. Nonetheless, S100A4 expression is up-regulated by positive proliferative responses such as those imparted by growth factors and down-regulated by negative responses elicited by hyperthermia, with parallel changes in the size of the S phase fraction. For instance, human tumour cells that are high expressors of EGFr also tend to overexpress S100A4 (Sherbet et al. 1995). In murine BL6 melanoma cells and human HUT cells, which are a heat-resistant variant derived from HepG2 cells, hyperthermia down-regulates S100A4 gene expression. In parallel, there is a decrease in the size of the S phase fraction and an increase in the doubling time of cells (Sherbet et al. 1996; Albertazzi et al. 1998a). Hyperthermia, which is known to increase intracellular calcium levels (Furukawa et al. 1997), induces the synthesis of several heat shock (cognate) proteins. Among the most prominent in terms of their relevance to cell proliferation are HSP70 and HSP28. The microtubule-interacting protein MIP-90 has been reported to show extensive sequence homology to HSP90 (Cambiazo et al. 1999). HSP70 has been known for some time to show a cell cycle-related expression. This HSP seems to be essential for mitotic division in the early developmental stages of the sea urchin Paracentrotus lividus. HSP70 has been reported to accumulate in the mitotic apparatus, and further, antibodies raised against HSP70 have been shown to inhibit mitosis (Sconzo et al. 1999). It would seem that HSP70 is an important ingredient of cell division and could play a role in the cell division machinery. In sharp contrast, HSP28 seems to exert an inhibitory effect on cell proliferation, and furthermore, HSP28 function might have a genetic basis. This is obvious from the experimental work described by Albertazzi et al.(1998a), where there was a remarkable differential increase in HSP28 as compared with the non-HSP28 component of total cellular HSP. Thus, in the BL6-derived HTG murine melanoma cells exposed to hyperthermia, HSP28 expression increased by 3.5-fold as compared with control BL6 cells. The corresponding increase in the non-HSP component of the HTG cells was only 22%. The enhancement of HSP28 together with a down-regulation of S100A4 (mts1) is compatible with previous observations that HSP28 is a growth-inhibitory protein. HSP28 is induced by hyperthermia in normal lymphocytes and macrophages as well as in leukaemia cells. Its induction has been reported to coincide with peak proliferative activity and the onset of growth arrest (Spector et al. 1992, 1993, 1994). HSP28 is highly expressed in quiescent keratinocytes as compared with their proliferating counterparts (Honore et al. 1994). A low molecular weight heat shock protein, HSP27, has been reported to be expressed abundantly in normal squamous epithelia of the oesophagus, but is markedly down-regulated in Barretts metaplasia and adenocarcinoma of the oesophagus
217
(Soldes et al. 1999). The HSP27 gene has been transfected into A375 melanoma cells and A431 cells. In transfectant cells obtained from both cell lines, overexpression of the HSP was associated with reduced proliferative rates (Kinder-Mugge et al. 1996). Presumably, HSP27 and 28 are the same molecular species. Antibodies against HSP27 have been detected in sera of breast cancer patients but not of normal subjects. Furthermore, their presence in sera correlated with improved patient survival (Conroy et al. 1998). It may be suggested that this reects less aggressive disease on account of the growth-inhibitory properties of the HSP. It is interesting to note also that cells over-expressing HSP27 show a delay in tumorigenicity (Kinder-Mugge et al. 1996). This delay is compatible with inhibition of cell proliferation. However, after this initial delay tumours did appear, but these were devoid of HSP27 expression and they showed growth properties similar to control tumours. Again, this supports the close link between HSP27 expression and proliferative potential. It would be interesting to study how HSP28 functions as an intermediary in the inhibition of proliferation accompanying the S100A4 down-regulation. There are two pointers to the possible mode of action of this low molecular weight HSP. HSP27 binds both - and -tubulin and is also found in association with microtubules (Hino et al. 2000). We know that S100A4 markedly inuences cytoskeletal dynamics. Therefore, Albertazzi et al. (1998a) have postulated HSP28 might be involved in direct interaction with S100A4. There are several reports that HSP can bind to p53. Normal human cell lines exposed to hyperthermia show G1 arrest together with an increase in the expression of p53, but not in rb protein phosphorylation (Miyakoda et al. 1999). In light of these ndings, the suggestion by Albertazzi et al. (1998a) that there might exist a complete regulatory loop involving S100A4, HSP, and p53 in the control of the G1S checkpoint of the cell cycle, seems highly credible (Figure 26).
CELL CYCLE-RELATED EXPRESSION
OF
S100 PROTEINS
The progression of the cell cycle involves a sequence of events that includes calcium signalling. Calcium signalling involves both an inux of extracellular calcium and an elevation of intracellular calcium levels by the release of Ca2+ from intracellular stores. Mitogenic agents generate calcium signals via the IP3-signalling pathway. These calcium signals might control the entry of resting cells into the cell cycle, G1S and G2M transitions as well as the exit of the cells from mitosis. Mitogens activate calcium inux into the cells, which does seem to control the progression of the cell cycle (Barbiero et al. 1995). In the mitotic phase, transient increases of Ca2+ occur between metaphase and anaphase. An increase in cytosolic calcium is associated also with the process of cell division, i.e., cytokinesis. Calcium channel blockers can inhibit cell proliferation by arresting cells in the G0G1 phase (Zeitler et al. 1997). Fertilisation of the ovum results in a rapid rise in Ca2+. This is required for the cleavage of the fertilised egg (Whitaker and Patel, 1990), which can be blocked by chelating Ca2+ (Zucker and Steinhardt, 1978). Unexpectedly, mobilisation of cell calcium and calcium channel activation also have been found to inhibit the passage of cells at the G1S and G2M checkpoints (Vanolah and Ramsdell, 1996). It may be that calcium sequestration by CBPs controls intracellular levels of calcium
218
FIGURE 26 Hypothetical view of interaction between S100A4, HSP28, and p53 in the regulatory loop that might control G1S transition of cells. (From Albertazzi et al. 1998a.) Reprinted by permission of the publisher Mary Ann Liebert Inc.
and thereby regulates the role of calcium signalling in cell cycle progression. Therefore, much attention has been focused on the potential role of S100 proteins and indeed other calcium-sequestering proteins in cell proliferation. Furthermore, much information is currently available identifying individual CBPs with intervention and function at specied stages of the cell cycle. Ca2+ has been regarded also as playing an important part in the cycle of meiotic cell division. The question of whether S100 proteins regulate growth can be answered in two parts. The rst part of the answer is the unequivocal association between the expression of certain S100 proteins and specic stages of the cell cycle. It was noticed some years ago that S100B and calcyclin genes were expressed predominantly in the G1 phase of the cell cycle (Hirschhorn et al. 1984). Selinfreund et al. (1990) showed that intracellular levels of S100B can be reduced by introducing antisense S100B oligonucleotides into cells. This resulted in a decrease in the growth rate of the cells. The induction of differentiation of neuroblastoma cells by retinoic acid enhances the expression of calcyclin mRNA. This is accompanied by arrest of cell proliferation, and cells appear to accumulate in the G1 phase of the cell cycle (Tonini et al. 1991). The mechanism by which calcyclin might be regulating the cell cycle is not fully understood at present. Calcyclin could indirectly affect cell cycle progression. It has been found to interact with great specicity with the annexin CAP50 (Minami et al. 1992; Hidaka and Mizutani, 1993). As discussed elsewhere, the expression of annexins is closely associated with cell cycle progression and,
219
furthermore, CAP-50 shows a predominant nuclear location (Hidaka and Mizutani, 1993). Minami et al. (1992) have suggested, in light of the apparent specicity of calcyclin binding to CAP-50, that this annexin could be regulated by calcyclin. Therefore, one cannot exclude the possibility that calcyclin might inuence and regulate the progression of the cell cycle and cell growth by an indirect route. S100A4 (mts1) was cloned by Jackson-Grusby et al. (1987), who showed that it could be induced to express by serum stimulation of growth-arrested cells and that its expression increased during the S phase of the cell cycle. The NGF induces the synthesis of two proteins in rat PC12 cells, which were originally referred to as 42C and 42A. 42C and 42A are regarded as the rat homologues S100A10 and S100A4, respectively. Not only does NGF-induced neuronal differentiation induce 42C and 42A, but the cells concomitantly exit from mitosis and enter the G0 phase (Masiakowski and Shooter, 1988). From this it seems not only that some S100 proteins may be specically expressed in relation to the stage of progression of the cell division cycle, but they could conceivably also regulate cell cycle progression itself. Some denitive evidence is now available that shows that S100A4 may regulate cell cycle progression. As stated previously, in B16 and human HUT cells exposed to hyperthermia, the S100A4 is down-regulated, with a reduction in the size of the S phase fraction (Albertazzi et al. 1998b). Parker et al. (1994b) transfected an S100A4 gene, which was placed under the control of a dexamethasone-inducible MMTV (murine mammary tumour virus) promoter, into B16 murine melanomas. They showed that the transfected cells, in which the gene was switched on by exposure to dexamethasone, contained substantially larger S phase fractions as compared with the control cells. These observations suggest that the S100A4 gene could be driving the cells into the S phase. Another line of evidence is provided by the work of Parker and Sherbet (1992), who found that verapamil, a specic blocker of L-type calcium channels, down regulated the expression of S100A4. Subsequently, several investigators have demonstrated that verapamil can inhibit not only cell proliferation (Brocchieri et al. 1996; Zeitler et al. 1997; Hoffman et al. 1998) but also invasion and metastasis (Farias et al. 1998). Interestingly, verapamil reduces the expression of certain membrane-associated metalloproteinases and urokinase-type PA. An enhanced expression of S100A4, on the other hand, is associated with a remodelling of the ECM in a way that is conducive to enhanced cell motility and invasiveness.
POSTULATED MECHANISM
OF
CELL CYCLE CONTROL
BY
S100A4
The mechanisms by which S100A4 exerts control over the progression of the cell cycle are yet unclear. However, much circumstantial evidence has been adduced to support the concept that this protein might form complexes with certain cellular target proteins, such as the suppressor p53 phosphoprotein that has been regarded as the guardian of the genome. When cellular DNA is damaged p53 protein is expressed, and this appears to block the transition of the damaged cells from G1 into the S phase, until DNA repair takes place. In B16 melanoma cells, up-regulation of S100A4 (18A2/mts1) expression is associated with an enhanced level of p53, as detected by immunohistochemical methods. This has been attributed to the formation
220
of a complex of S100A4 with p53 (Parker et al. 1994a, 1994b). Such a complex formation would have the effect of stabilising and enhancing the half-life of p53, and as a consequence p53 becomes detectable by immunohistochemical staining procedures. The formation of a complex between S100A4 and p53 could effectively sequester p53 and abrogate its G1S checkpoint control, resulting in an increase in the size of the S phase fraction. One might then ask why the cells do not continue to accumulate in the S phase, but successfully transit from G2 into mitosis. It has been shown recently that p53 is also involved in the control of the G2M transition of cells (Michalowitz et al. 1990; Stewart et al. 1995; Pellegata et al. 1996). One of the proposed mechanisms of this involves the participation of a protein called stathmin. Stathmin is a 19-kDa cytosolic phosphoprotein. It is up-regulated in many neoplasms as well as in highly proliferating normal tissues (Luo et al. 1994; Rowlands et al. 1995; Friedrich et al. 1995; Bieche et al. 1998). Even immortalisation, an event regarded as a prelude to neoplastic transformation, of primary embryonic broblasts, results in a four-fold enhancement of stathmin expression and a substantial increase in the rate of proliferation. However, in this experimental system there were no changes in stathmin expression upon oncogenic transformation (Mistry and Atweh, 1999). It would seem, therefore, that modulation of stathmin expression is a feature of cell proliferation rather than of neoplastic transformation. This statement is amply substantiated below. The expression of stathmin is down-regulated when wild-type p53 is expressed. Using inducible p53 constructs, it has been demonstrated that when p53 is switched on the stathmin gene is down-regulated together with the arrest of cells in the G2 phase. Indeed, p53 may be capable of down-regulating the stathmin promoter. Ahn et al. (1999) have shown that a decrease in the expression of stathmin occurs in immortalised human and murine cells in parallel with induction of wild-type p53 by DNA-damaging agents. Of interest in the context of the function of S100A4 is the observation that stathmin destabilises the cytoskeleton (Marklund et al. 1996; DiPaolo et al. 1997). It sequesters free tubulin and inhibits tubulin polymerisation (Andersen et al. 1997; Curmi et al. 1997, 1999). Thus S100A4 and stathmin seem to share several properties, of which the most prominent are the inuence they exert on cell proliferation and their postulated interaction with p53. A conundrum, in the observed association between S100A4 and cell cycle progression, is why cells that are induced by S100A4 to enter the S phase do not accumulate in the S phase but successfully negotiate the G2M checkpoint into mitosis. Some recent work has shown that stathmin expression closely parallels that of S100A4. Indeed, changes in S100A4 expression seem to be tightly coupled with corresponding changes in the expression of stathmin gene. Thus, stathmin expression changes when cell proliferation is inhibited by hyperthermia. This happens also when cells are growing exponentially, i.e., with increasing growth rates, or when full serum supplement is restored to serum starved cells (Sherbet and Cajone, 2000; Cajone and Sherbet, 2000) (Table 18). There are two schools of thought about how stathmin might be involved with cell proliferation. One school believes that the G2M transition of cells is associated
221
TABLE 18 Coupling of S100A4 and Stathmin Gene Expression in Relation to State of Proliferation
Experimental Condition Hyperthermia (HUT cells) (decreased growth rate) HeLa cells, 24-, 48-, and 72-hr cultures S100A4 Stathmin Growth Rate
Source: Based on Sherbet and Cajone (2000) and Cajone and Sherbet (2000). Reprinted by permission of the publisher Kluwer Academic Publishers.
with enhanced stathmin expression (Eustace et al. 1995; Jones et al. 1995). The other proposes that phosphorylation of stathmin is the critical event that allows the cells to make the transition (Marklund et al. 1994; Larsson et al. 1995; Duraj et al. 1995; Beretta et al. 1995; Lawler et al. 1998). In the studies cited above, Sherbet and Cajone (2000) and Cajone and Sherbet (2000) have not investigated the state of stathmin phosphorylation, for the correlation between stathmin gene expression and cell proliferation was highly signicant. S100 proteins have been shown to be able to modulate PKC-mediated phosphorylation of proteins (Kriajevska et al. 1998). S100A4 might function by inhibiting the phosphorylation of stathmin, which is regarded by some as essential for stathmin function. Therefore, the potential significance of posttranslational modication of stathmin cannot be excluded. As stated earlier, previous work has demonstrated that the interaction of S100A4 and p53 might sequester the latter and abrogate its checkpoint control at the G1S checkpoint (Parker et al. 1994a, 1994b). However, after this successful transition into the S phase, one would expect them to accumulate in the S phase. This does not seem to happen. Although the size of the S phase fraction does increase upon induction of S100A4 expression, cells do not appear to be held back at the G2M checkpoint. It is in this context that the parallel modulation of the expression of S100A4 and stathmin genes assumes some signicance. Cajone and Sherbet (2000) have postulated that S100A4 might directly induce an up-regulation of stathmin expression, enabling the cell to enter into mitosis. Because wild-type p53 has been known to down-regulate stathmin expression, it has been suggested that S100A4 might sequester p53 and abrogate its control over stathmin function, and thereby enable cells to make the G2M transition (Figure 27). There is simplicity about this concept, which emphasises the possibility of S100A4 being involved at both G1S and G2M transition checkpoints. This postulate needs to be tested rigorously, for this is the rst CBP that has been shown not only to have close links with cell cycle progression but also to be intricately involved in the two main control checkpoints of the cell cycle.
222
M + p53 Stathmin G2
-
G1
S100A4
P + + p53
p53
S100A4
FIGURE 27 The putative involvement of S100A4 in G1S and G2M transition in the cell cycle. As discussed in the text, S100A4 may drive the cells into the S phase by sequestration of p53. Stathmin is up-regulated as well as phosphorylated during the G2M transition. Wildtype p53 is said to down-regulate stathmin and might block the G2M transition by this method. Because stathmin expression parallels that of S100A4, the gure represents the postulate that S100A4 might itself up-regulate stathmin and promote G2M transition, or this effect is routed through p53 sequestration.
S100A ISOFORMS
S100A protein occurs as two isoforms made up of homo- or heterodimers of the two subunits called the and subunits. These two subunits comprise 93 and 91 amino acid residues respectively, and share 58% amino acid sequence homology (Isobe and Okuyama, 1978, 1981). S100A0 is a homodimer of two subunits, and S100A is a heterodimer of an and subunit. S100B is made up of two subunits. The S100A isoforms as well as S100B occur predominantly in the brain. S100A0 forms a minor component of S100 proteins in the neurones and peripheral nerves (Isobe et al. 1984). S100A0 is also associated with the sarcolemma and the SR (Haimato and Kato, 1987; Donato et al. 1989). Because the SR holds the intracellular stores of calcium, S100A0 could be involved in the mobilisation of calcium from these intracellular stores. Fano et al. (1989) have shown that S100A0 does induce calcium mobilisation from SR-terminal cisternae, which were isolated from rat skeletal muscle. The levels of S100A0 in the bloodstream of patients with acute myocardial infarction have been investigated on the premise that the protein may be released into the bloodstream from damaged heart muscle. In a group of patients investigated, Usul et al. (1990) found a nearly fourfold increase of S100A0 in the serum that eventually increased to roughly 20-fold higher than the levels of the protein found in control subjects. However, there was considerable variation between patients. Nevertheless, such increases were not encountered in patients with angina pectoris. Usul et al. (1990) have, therefore, suggested that this might provide a method for differentiating between myocardial infarction and angina pectoris.
223
S100A2 AS A PUTATIVE TUMOUR SUPPRESSOR

It is evident from the foregoing discussion that a majority of S100 family proteins appear to possess the ability to promote tumour progression by enhancing the proliferative and invasive potential of tumours. One member of the S100 protein family, namely S100A2, is believed to function as a tumour suppressor. S100A2 was isolated more than a decade ago by Glenney et al. (1989). It is found in certain cell types in the kidney, lung, and breast epithelium. It is moderately expressed in the liver, and cardiac and skeletal muscle, but not encountered in the adrenal gland, the intestine, or the brain. Lee et al. (1991) identied several cDNA clones that were highly expressed in normal tissues but suppressed in corresponding tumour-derived cells. Among these clones was a transcript of an S100 family gene. Subsequently, its expression was reported to be down-regulated in breast tumour cells. Exposure of these cells to 5-aza-2-deoxycytidine produced a reexpression of this gene, which suggested that its expression was normally suppressed in the tumour cells by hypermethylation (Lee et al. 1992). That hypermethylation of this gene is responsible for the loss of its expression in breast cancer cell lines has been conrmed recently (Wicki et al. 1997). A loss of S100A2 expression has been reported in several breast cancer cell lines (Pedrocchi et al. 1994). A marked loss of expression has been found in human sarcomas (E. Horvig, personal communication, 1998). S100A2 expression has been found in only 7% (of 107) of human sarcomas. In contrast, S100A4 and A6 expression was detected in 38 and 48%, respectively, of the specimens. This suggests a preponderant loss of S100A2 expression. However, there was no obvious correlation with clinical features or patient survival. In human astrocytomas also there is a conspicuous loss of S100A2 expression, whereas, in contrast, several other S100 proteins, notably S100A1, A4, and A6, are markedly expressed (Camby et al. 1999). The suppressor function of S100A2, however, is not so clear-cut either in normal melanocytes or in melanomas. Thus, in normal melanocytes S100A2 is expressed at very low levels or is virtually undetectable. Neither is its expression up-regulated in malignant melanoma (L.B. Andersen et al. 1996). S100A2 staining has been reported in the basal layer of the epidermis and in hair follicles, but none has been found in naevi. Also, only a small proportion (4/39) of primary cutaneous melanomas and none of 14 metastatic lesions stained for S100A2 (Boni et al. 1997). A further report has appeared on S100A2 expression in epidermal cell types and epithelial tumours of the skin. Again the basal cells, epithelial cells of the sebaceous glands, and epithelial cells of hair follicles stained positive for S100A2. Also immunoreactive were basal cell as well as squamous cell carcinomas (Shrestha et al. 1998). Overall, the evidence available to date does not lend itself to a rm interpretation that S100A2 has a suppressor function or that its expression is associated with advanced stages of tumour progression. This view is also supported by the data published by Maelandsmo et al. (1997). The differences in the levels of S100A2 expression between naevi and cell lines derived from primary tumours seem to be more marked than those between the primary and metastatic lesions. This suggests that a down-regulation of the gene could occur in the early stages of development of these tumours. Xia et al. (1997) have cast further doubts about the suppressor
224
function of S100A2. They found this gene to be highly expressed in basal and squamous cell carcinomas of the skin and oral cavity, although in situ hybridisation studies have indicated that the amounts of S100A2 occurring in the tumour cells themselves were limited. The majority of the protein was found in the hyperplastic epidermis around the tumour. Xia et al. (1997) found no differences in the expression of the protein in primary tumours and metastatic lesions. Ilg et al. (1996) studied the binding of antibodies against several S100 proteins, including S100A2, and noticed a marked difference in their intracellular localisation. S100A2 was located predominantly in the nucleus, but S100A6 occurred mainly around the nucleus. They also found that the binding pattern of S100A2 antibodies differed from that of antibodies against S100A4, the expression of which has been widely reported to promote tumour-progression (see below). Although there is some evidence supporting the putative tumour suppressor function of S100A2, most of the research deals with its expression in tumour-derived cell lines, and there are no signicant studies on the status of its expression in human tumours themselves. The observation made by Xia et al. (1997) relating to the presence of S100A2 in hyperplastic epidermis incidentally serves to emphasise a putative relationship between the expression of this protein and the proliferative state of cells. This relationship now will seem more secure with the nding that EGF up-regulated S100A2 expression in organ cultures of human skin. EGF also markedly up-regulated the expression of S100A2 mRNA in immortalised human keratinocytes in culture. In both cases, the EGF effects could be blocked by using PD153035, which is a specic inhibitor of EGFr tyrosine kinase (Stoll et al. 1998). These experiments demonstrate the requirement of EGFr activation for the up-regulation of S100A2 expression, and therefore, suggest the presence of a direct relationship between S100A2 and mitogenic stimulation. With this background of great ambiguity regarding the suppressor function of S100A2, one should look to some recent evidence that S100A2 function might be mediated by wild-type p53. Tan et al. (1999) have identied putative p53-binding sites in the promoter of S100A2. In vitro, wild-type p53 seems to transactivate S100A2. This transactivation is blocked by dominant negative p53 mutants. This can be deemed as evidence that S100A2 might inuence cell proliferation with the mediation of p53. Thus p53 mediation may yet prove to be an underlying mechanism in the regulation of cell proliferation by S100 proteins.
S100A3 EXPRESSION IN CELL DIFFERENTIATION AND NEOPLASIA MOLECULAR FEATURES

OF
S100A3
S100A3 is a cysteine-rich CBP that binds to Ca2+ with low afnity but binds with high afnity to Zn2+ (Engelkamp et al. 1993; Fohr et al. 1995). As many as 10 out of 101 amino acids of the protein are cysteine residues. A subfamily of S100 proteins that binds Zn2+ with high afnity can be identied. These proteins bind four Zn2+ ions per protein monomer (Fohr et al. 1995). S100A3 may bind two Zn2+ ions per monomer. One zinc atom binds to four cysteine residues and the second atom binds
225
to one histidine residue. Therefore, a commonality of the zinc-binding domain and its organisation and stabilisation seems to be evident, and this has obvious similarities to the GAL4 zinc nger transcription factor from yeast. However, S100A3 binds to Zn2+ with far less afnity than that displayed by zinc nger transcription factors. The binding of zinc by S100A3 seems to be a well-coordinated process (Fritz et al. 1998), and might have effects on its conformational and functional states. The S100A3 gene occurs in the S100 gene cluster on the human chromosome 1q21 and on mouse chromosome 3.
S100A3 EXPRESSION GLIOMAS
IN
CELL DIFFERENTIATION
AND
HUMAN
S100A3 seems to be expressed specically in skin and derivative tissues (Kizawa et al. 1996, 1998; Boni et al. 1997) and shows a specic pattern of localisation in the subcompartments in human and murine follicles. High expression of S100A3 has been found in the cuticle sheath and cortical cells undergoing terminal differentiation. This has led to the suggestion that it might participate in the differentiation of the cuticle cell and the formation of the hair shaft (Kizawa et al. 1998; Takizawa et al. 1999). The expression of S100A3 has been studied in a series of human astrocytomas of various grades of malignancy (Camby et al. 1999). These authors have reported that they were able to differentiate between grade I (WHO) pilocytic astrocytomas and grades IIIV tumours on the basis of their expression of S100A3. Further, on this basis they suggest grade IIIV astrocytomas form a group of astrocytomas that are biologically distinct from WHO grade I tumours. On the other hand, the expression of S100A3 could be merely an indicator of the degree of anaplasia.
S100A4 IN CANCER DEVELOPMENT AND PROGRESSION S100A4 EXPRESSION

AND
METASTATIC POTENTIAL
OF
CANCERS
The considerable inuence that S100A4 exerts over cell adhesion, motility, and cell proliferation has prompted the investigation of the possible relevance of its expression in tumour development and their progression to the metastatic state. S100A4 is found in a variety of normal tissues of both murine and human origin (Grigorian et al. 1994; Gibbs et al. 1995). It occurs in normal adult rat tissues such as smooth muscle, brown adipose tissue, and the liver. Other tissues, e.g., normal breast tissue, endothelia, absorptive and keratinised epithelia, neuronal, as well as some cells of the haematopoietic system, also contain S100A4. S100A4 is predominantly intracellular in distribution, but Gibbs et al. (1995) believe that in breast tissue, it may occur as an extracellular secreted protein. There could be some speciesspecic differences in tissue distribution of the protein (M. Davies et al. 1995). In spite of this apparently generalised expression of S100A4, it ought to be recalled here that there are reports that S100A4 (FSP1) is expressed in a broblastspecic manner. Not only this, S100A4 (FSP1) is described as one of the genes
226
whose expression is associated with the epithelialmesenchymal transformation encountered in chordates (Hay, 1995). The broblast-specic expression has been attributed to the presence of a cis-acting element in the 5-anking region of the rst intron of the gene. This sequence is believed to be active in broblasts but not in epithelial cells. Okada et al. (1998) have shown further that the 5-bp sequence TTGAT (177 to 173) interacts specically with nuclear extracts obtained from broblasts. In addition to its wide distribution in normal tissues, high levels of S100A4 have also been recorded in human as well as in murine tumours with high metastatic potential (Ebralidze et al. 1990). A clear correlation seems to exist between the level of S100A4 mRNA and protein and the metastatic potential of Dunning rat prostate carcinoma cell lines (Ke et al. 1997). Indeed, some of this evidence might be construed as suggesting that S100A4 (FSP1) is a developmentally regulated gene. The aberrant expression of S100A4 in neoplasia could be viewed as reecting abnormalities in the process of de-differentiation associated with cancer development and progression. In addition to the empirical correlation, substantial experimental work in recent years has allowed a strong link to be established between the levels of S100A4 expression and metastatic potential. In the B16 murine melanoma, modulation of its expression levels alters metastatic behaviour (Parker et al. 1991). Upon transfection with the S100A4 gene, benign rat mammary epithelial cells have been found to become more tumorigenic and acquire a higher potential for metastasis (B.R. Davies et al. 1993). Although these results were unambiguous, the process of transfection of exogenous genes has been subjected often to the criticism that it could lead to genomic perturbation as a consequence of a stable integration of an exogenous gene. Kerbel et al. (1987) noted that calcium phosphate-mediated DNA transfer also changed cellular behaviour. They reported that when CBA/J mouse adenocarcinomas were transfected with a vector carrying only a marker gene but not transforming genes, 17% of transfectant cells showed lung colonisation. Equally, there are reports that transfection of extraneous DNA does not alter biological behaviour in any way (Jamieson et al. 1990a, 1990b). Nonetheless, it is imperative that the expression of S100A4 is linked with changes in the biological behaviour of cells. Therefore, to obviate this potential criticism, B16 melanoma cell lines have been transfected with S100A4 (18A2/mts1) and placed under the control of the dexamethasone-inducible MMTV promoter. In these transfectants, switching on the exogenous gene markedly enhances localisation of the transfectant cells in the lung (Parker et al. 1994b). In the same investigation, Parker et al. (1994b) also transfected the inducible construct into a dexamethasone receptor negative B16 cell line and demonstrated that the exogenous gene is not switched on. These experiments have established conclusively that S100A4 enhances lung colonisation by B16 tumour cells. It is worthy of note, however, that Parker et al. (1994b) did not determine whether S100A4 transfectants were capable of spontaneous metastasis. As is often pointed out, much of this work is still open ended. There are one or two points of criticism that need to be taken into account. One of these is that dexamethasone is known to up regulate the expression of other EF-hand proteins such as calcineurin. Because the S100A4 transfectants were exposed to dexamethasone to switch on
227
S100A4 expression, these cells should have been tested for calcineurin expression. Furthermore, tail vein injection of cells often allows them to bypass the initial hurdles that cells released from a primary tumour have to face, and they have direct access to the lung. Thus, although one can accept that S100A4 enhances the localisation of cells in the lung, there are no data on the effects of S100A4 on the release of cells from the primary tumour, their ability to gain access to the blood vessels, and their ability to withstand or escape the immunological surveillance by the host. The recent studies of Lloyd et al. (1998) have answered some of these questions. They transfected the human S100A4 gene into the rat benign mammary tumour cell line, Rama-37. Transfectant cells that expressed S100A4 mRNA at high levels were capable of forming primary tumours in syngeneic rats and were also able to metastasise spontaneously. In contrast, those transfectant cells that expressed S100A4 at low levels were unable to form secondary tumours. Furthermore, they have shown for the rst time that metastatic deposits that expressed S100A4 mRNA were able to form primary tumours that were also capable of metastasising spontaneously. In other words, a constitutive expression of the gene does confer metastasising properties. Another piece of documentation, which supports the metastasis-promoting ability of S100A4, is the generation of S100A4 transgenic lines, e.g., Tg463 and Tg507, of GRS/A mice by Ambartsumian et al. (1996). The GRS/A primary strain rather characteristically shows a high incidence of mammary tumours that do not appear to possess metastatic ability. In contrast, the S100A4 transgenic strains developed metastatic tumours in approximately 40% of the animals. Ambartsumian et al. (1996) also demonstrated the presence of S100A4 in both primary and metastatic tumour. Simultaneously, M.P.A. Davies et al. (1996) carried out similar experiments on transgenic S100A4 mice. In the S100A4-expressing animals, mammary tumours were palpable earlier than in corresponding control animals, and macroscopic deposits of tumours were detectable in the lungs. There is thus adequate proof that high expression of S100A4 is conducive to metastatic dissemination. Conversely, the transfection of antisense constructs into malignant cell lines has resulted in the reduction of metastatic potential (Grigorian et al. 1994). The ability of antisense S100A4 mRNA to suppress metastatic potential has been conrmed in further experiments with highly metastatic lines of the Lewis lung carcinoma. Several clones with transfected antisense S100A4 mRNA have been isolated, and all of them proved to have very low metastatic potential (Takenaga et al. 1997a). In a novel experimental approach, Maelandsmo et al. (1996) transfected human osteosarcoma cells that expressed S100A4 at a high level with a ribozyme directed against the gene transcript. With the destruction of the S100A4 mRNA by the ribozyme, the osteosarcoma cells showed no skeletal metastases when injected intracardially into nude mice. They also found that the degradation of S100A4 mRNA had no effect on the proliferation of the cells either in vitro or in vivo. On the basis of the latter results, Maelandsmo et al. (1996) seem to be suggesting that metastatic ability may be dissociated from proliferative ability. This is an interesting postulate, but, so far as S100A4 is concerned there is little doubt that its high level of expression leads to high growth rates. Compatible with the association of high S100A4 expression with malignancy is the recent demonstration that the S100A4 gene may be down-regulated in benign
228
mammary epithelial cells. It would appear that S100A4 transcription is inhibited by a cis-acting sequence, related to the consensus recognising GC factor, occurring 1.3 kbp upstream of the transcription start site. An inverse relationship has been noted between the GC-factor and S100A4 mRNA levels (Chen et al. 1997). In conclusion, one may justiably regard the currently available body of evidence, relating to the conferment of invasive and metastatic abilities by S100A4, as virtually irrefutable. It ought to be stated, nonetheless, that there has appeared a solitary report to the effect that S100A4 shows no relationship to cell proliferation, invasion, or metastasis. This report described the transfer of S100A4 sense constructs into MCF7 cells, with no apparent effects on in vitro invasion or proliferation. The transfectants produced no metastasis in mice, but there was marked tumour necrosis and abnormalities in tumour vasculature (Onischenko et al. 1996). There are a number of points to be taken into account with regard to this work. Grigorian et al. (1996) also used MCF7 cells for transfection of S100A4 and found that the transfected cells acquired marked metastatic properties. This is in sharp contrast with the ndings of Onischenko et al. (1996). MCF7 cells are known to express the putative metastasis suppressor gene nm23 at a high level (Sherbet et al. 1995). S100A4 and nm23 genes might be coordinately regulated (Parker et al. 1991; Hsu et al. 1997). Admittedly, the signicance of nm23 expression in breast cancer cells may not be rated high. However, Albertazzi et al. (1998b) have reported recently that the expression status of both S100A4 and nm23 taken together correlated far more powerfully with the clinical aggressiveness of breast cancer than when the status of S100A4 alone was considered. In other words, it would have been advisable for Onischenko et al. (1996) to look also at nm23 expression in their experiments. There have been no other reports so far that S100A4 has any effects on neovascularisation or cell death by necrosis or apoptosis. Without prejudice to their ndings, however, it would be worth remarking that these latter effects could be collateral changes associated with the integration of the exogenous gene into MCF7 cells. The chances of this happening have been obviated by Grigorian et al. (1996) by adopting a different experimental strategy of transfecting the gene under the control of an inducible promoter. Some ambivalent views have been expressed also by Chiaramonte et al. (1998). These authors used a murine mammary adenocarcinoma cell line, TS/A, and two murine melanoma cell lines, B16-A and B78H1. All three cell lines responded to IFN- treatment with an increase in metastatic ability. However, when they tested these IFN--treated cells for S100A4 expression, no changes were noticed in TS/A cells. Further, of the two melanomas only B16-A showed an enhancement of S100A4 expression. The effects of IFN- seem to be somewhat variable. It has been found to enhance transcription in human macrophages (Grigorian et al. 1994). On the other hand, IFN- down-regulates S100A4 expression in human colon adenocarcinoma cell lines. This effect is dependent on membrane-associated IFN- receptors and is not shown by IFN- or - (Takenaga, 1999). Chiaramonte et al. (1998) have, therefore, quite legitimately argued that different genes may be associated with the metastatic behaviour of different tumour types. It would be useful to pursue these observations, even in the face of the virtually overwhelming evidence for a major role for S100A4 in tumour progression.
229
Whether S100A4 promotes the metastatic spread of tumours by inuencing cellular properties at a specic stage of the metastatic cascade is not clear at present. In light of the alleged inhibition of the microvasculature by S100A4, Cajone and Sherbet (2000) have employed heme oxygenase-1 (HO-1) gene as a marker in a series of experiments. Heme oxygenase (HO-1) is a stressresponse protein. The induction of HO-1 gene is believed to play a protective role against both heme- and nonheme-mediated oxidant injury. Many agents such as hyperoxia, bacterial LPS, hydrogen peroxide, interleukin, prostaglandins, TPA, and hyperthermia, among others, are known to induce HO-1 (Choi and Alam, 1996). The HO-1 gene expression is regulated by the presence of responsive elements that can bind these agents. Another agent called 4-hydroxynonenal (4-HNE) is also a potent inducer of HO-1 expression. 4-HNE is a product of the peroxidation of membrane lipids and it has been shown to be able to induce the synthesis of HSPs (Cajone et al. 1989; Cajone and Bernelli-Zazzera, 1989). One of the HSPs induced by 4-HNE is a 32-kDa protein (Allevi et al. (1995). This HSP32, apparently associated with oxidative stress, seems to be none other than HO-1. Although total identity between these two proteins has not been fully established, HO-1 has been referred to, not infrequently, as HSP32 (Choi and Alam, 1996). Cajone and Sherbet (2000) modulated the levels of S100A4 expression by using agents that are known to up-regulate HO-1 expression. They found that the HO-1 expression was up-regulated irrespective of the effects of the agent on S1004 expression (Table 19). Grigorian et al. (1994) found that LPS has a variable effect on S100A4 expression in inammatory macrophages. In these cells it reduced S100A4 expression in the rst 3hr but then up-regulated it over the next four hours.
TABLE 19 The Lack of Relationship between Expression of S100A4 and Heme Oxygenase (HO-1) Genes
Cells/Treatment S100A4 HO-1
HUT 37,a 42C 3T3 normal serum + 4-HNE 3T3 serum-starved cells + 10% FCS 3T3 + TPA 3T3 + 4-HNE a HUT is a heat-resistant clone of cells derived from HepG2 cells. Source: Based on data from Cajone and Sherbet (2000). (Reprinted by permission of the publisher Kluwer Academic Publishers.
In other words, there was a total dissociation of the expression of the two genes. This might throw some light on the possible function of this protein in cancer metastasis. HO-1 has been reported to have a marked effect on tumour angiogenesis.
230
Transfection of human HO-1 gene has been found to produce a two-fold increase in neovascularisation (Deramaudt et al. 1998). Indeed, some agents that induce HO1 gene expression are also inducers of angiogenesis. For instance, the HO-1 gene contains response elements for LPS, IL-6, and prostaglandins (Camhi et al. 1995, 1996; Kozumi et al. 1995), all of them known inducers of angiogenesis. These are powerful indicators that that S100A4 might not be associated with induction of neovascularisation. However, the effects of S100A4 expression on the cellular properties that are highly relevant in other compartments of the metastatic cascade, such as the phase of expansive growth of the primary tumour, phase of invasion, and possibly also the growth of overt metastases, are virtually indisputable (see also Figure 28 and Table 21).
CLINICAL POTENTIAL PROGNOSIS
OF
S100A4
AS A
MARKER
FOR
CANCER
S100A4 has attracted a great deal of attention in recent years because of the wide variety of physiological events and parameters of biological function that it appears to inuence. However, in contrast with several other putative markers of malignancy, investigations into the possible clinical value of this gene are of recent origin. This is because much of the proposed work has been dampened by the somewhat illinformed criticism and reservations expressed about it. Fortunately, a great deal of research into the basic aspects of S100A4 function has been carried out, which has greatly emphasised the importance of S100A4 in the clinical assessment of the disease. Some of the work, such as that of Pedrocchi et al. (1994), Grigorian et al. (1996), and Maelandsmo et al. (1996), used tumour cell lines derived from human tumours, and these authors have shown that the levels of S100A4 expression were related to invasive and metastatic ability. Sustained studies of human tumours aimed at determining the relevance of S100A4 in predicting progression of the disease have followed. These have been carried out mainly in melanomas, breast cancer, and astrocytic tumours.
S100A4
IN
HUMAN BREAST CANCER
Pedrocchi et al. (1994) rst described the importance of S100A4 in breast cancer cell lines. Recently, Albertazzi et al. (1998b) have published a detailed study of S100A4 expression, not only in relation to the degree of aggressiveness of the disease, but also as to how it relates to other clinical markers such as the status of oestrogen and progesterone receptors and the degree of tumour differentiation. Albertazzi et al. (1998b) also employed another marker, nm23-H1. nm23 is a putative metastasis suppressor gene identied in murine melanomas (Steeg et al. 1988a, 1988b), of which two human homologues have been cloned (Rosengard et al. 1989; Stahl et al. 1991). Several reports in the literature suggest an inverse relationship between the level of expression of nm23 mRNA or the gene product, nucleoside diphosphate (NDP) kinase, and metastatic potential. Such an inverse relationship has been described in some forms of human cancer, e.g., melanoma (Florenes et al. 1992; Caligo et al. 1994). In colorectal and gastric carcinomas, nm23
231
expression has been reported to be down-regulated substantially in metastatic tumour as compared with the primary carcinoma (Ayhan et al. 1993; H. Nakayama et al. 1993). An inverse relationship between nm23/NDP kinase expression and metastatic potential has been reported to occur in hepatocellular carcinoma (T. Nakayama et al. 1992). Low levels of NDP kinase expression have been correlated with reduced survival of patients with inltrating ductal carcinoma of the breast (Barnes et al. 1991; Royds et al. 1993). However, several studies repudiate this inverse relationship (Haut et al. 1991; Hailat et al. 1991; Keim et al. 1992; Zhou et al. 1993; Sawan et al. 1994; Saitoh et al. 1996; Srivasta et al. 1996; Aldersio et al. 1998). A study of a large series of breast carcinomas has shown that NDP kinase expression is neither related to disease relapse or patient survival, nor does it correlate with other prognostic factors such as tumour grade, oestrogen and progesterone receptor status and p53 expression (Sawan et al. 1994). Stephenson et al. (1998) examined 412 cases of lung carcinoma for nm23/NDP kinase expression. They found no relationship of its expression to metastatic disease or prognosis. Similarly, overexpression of nm23H1 in epithelial ovarian carcinomas is associated with lower survival and signicantly poor prognosis in early stage and well-differentiated carcinomas (Schneider et al. 2000). Some of the problems associated with the observed lack of inverse correlation between nm23/NDP kinase expression and metastatic progression might be due to the tacit assumption that NDP kinase function and the putative metastasis-suppressor properties are interrelated and inseparable. Contrary to this, it has been argued that these properties can indeed be dissociated. H.Y. Lee and Lee (1999) transfected a cDNA coding for a mutant form of nm23-H1 that lacked NDP kinase activity into human prostate carcinoma cells. Nonetheless, the transfectant cells showed reduced invasive ability, in the same way as cells that had been transfected with wild-type cDNA. Although these observations do suggest that the two properties of nm23 are dissociable, conceptually the putative metastasis-suppressor function of nm23 becomes even harder to appreciate. This somewhat equivocal correlation, between nm23/NDP kinase expression and tumour progression, has led to the suggestion and actual demonstration that this gene might be coordinately regulated with the metastasis-promoting S100A4 gene in the B16 murine melanoma (Parker et al. 1991). These authors used MSH and RA to modulate the levels of S100A4 expression and found that the expression of nm23 levels showed corresponding changes. As a result, the ratio of their expression remained virtually constant. Similar results have been described more recently by Hsu et al. (1997) in CH27 human lung cancer cells, which essentially conrm the ndings of Parker et al. (1991). Indeed, there is evidence that S100A4 expression promotes the depolymerisation of tubulin, whereas nm23 expression has the opposite effect (Nickerson and Wells, 1988; Lakshmi et al. 1993). Whether there is a direct regulatory link between S100A4 and nm23 remains to be established. It is unclear whether S100A4 is itself involved in some way with the negative regulation of nm23. The 1q21 locus, which harbours the S100A4 gene, also contains the human homologue of the PRUNE gene of Drosophila melanogaster. The prune eye colour phenotype of Drosophila is attributed to null mutations of the PRUNE gene. The occurrence of one mutant copy of awd (abnormal wing disc) (the
232
Drosophila homologue of nm23-H1) with the prune genotype is lethal. This suggests that nm23-H1 and the prune protein might interact. The prune protein has indeed been found to interact with nm23-H1 and to negatively regulate the latter. In further conrmation, it has been noticed that the interaction is impaired when nm23-H1 is mutated (Reymond et al. 1999). The emergence of this negative regulation of nm23H1 by the prune protein does not explain the inverse relationship shown to subsist between S100A4 and nm23-H1. Perhaps it would be fruitful in future studies relating to the expression of these genes also to measure PRUNE expression as well. With this background, Albertazzi et al. (1998b) focused on the expression of S100A4 (h-mts1) as well as that of nm23 in human breast cancer. This was with a view to determining their individual abilities to serve as markers, and further to check whether a combination of the status of their expressions might enable one to obtain a more accurate assessment of the metastatic potential of the tumours. The data presented by Albertazzi et al. (1998b) show that high S100A4 expression is associated with metastatic spread to the regional lymph nodes. The expression of nm23 on its own did not show a statistically signicant inverse correlation with nodal spread. However, the expression status of the two genes, taken together, strongly correlated with nodal metastasis. Furthermore, the correlation was more signicant when S100A4 and nm23 were considered in combination,as compared with S100A4 on its own (Table 20). This suggests that, although nm23 did not seem relevant to nodal metastasis, a more accurate assessment of nodal status could be derived by looking at the expression of both S100A4 and nm23 genes. A further indication that S100A4 expression status might be related to the clinical aggressiveness of the tumour is provided by another line of evidence. Albertazzi et al. (1998b) have reported that breast cancers with no detectable expression of S100A4 were ER and PgR positive. With increased expression of S100A4, the tumours could possibly progress toward a hormone-independent state. Such progression has been demonstrated in vitro. MCF7 breast cancer cell lines that were transfected with and producing S100A4 have been reported to acquire hormone-independent growth (Grigorian et al. 1996). This is compatible with the generally held view that ER/PgRnegative breast cancers are clinically more aggressive and also tend to be EGFr positive. The ER/PgR status of breast cancers generally correlates inversely with EGFr status, which has been regarded by some as an indicator of poor prognosis (Sainsbury et al. 1987a, 1987b; Bolla et al. 1990; Hainsworth et al. 1991). This ts with the ndings of Sherbet et al. (1995) that breast cancer cell lines that were high expressors of S100A4 tended also to be high expressors of EGFr. Furthermore, the recent nding that EGF is able to induce the expression of S100A4 in epithelial cells (Okada et al. 1997) may be deemed as supporting the relationship between EGFr expression and cancer prognosis. A correlation between the expression of type 1 growth factor receptor family and prognosis has also emerged from the recent work. Rudland et al. (2000) found that S100A4 positivity of breast cancer correlated signicantly with the expression of c-erbB2 and c-erbB3. The prognostic signicance of c-erbB2 has been well established and overexpression of this receptor is associated with poor prognosis. However, there are no data relating to the prognostic value of c-erbB3 nor c-erbB4 (Angus et al. 2000). Albertazzi et al. (1998b) also looked at tumour differentiation, but this did not correlate with S100A4 expression.
233
TABLE 20 Relationship Between S100A4, S100A4v, and nm23 Expression to Nodal Spread of Breast Cancer
Gene Expressed S100A4 nm23 S100A4+nm23 S100A4v+b S100A4/S100A4vb
a
Relationship to Nodal Spreada + ++ +
+, statistically signicant relationship (p < .05); ++, strong relationship (p < .01). b The expression of S100A4v (the splice variant form) alone did not relate to nodal spread; however, tumours that express neither S100A4 nor S100A4v do not tend to spread to the regional lymph nodes. Only 27% of carcinomas expressing neither h-mts1 nor h-mts1v showed metastatic spread to the lymph nodes; 57% (4/7) of carcinomas in which only the variant isoform was detectable showed nodal metastasis. This apparent difference did not reach statistical signicance. Source: Based on Albertazzi et al. (1998a, 1998b). Reprinted by permission of the publisher Mary Ann Liebert Inc.
The clinical data, together with the state of expression of steroid receptors and the expression levels of S100A4 and nm23 genes, were analysed using articial neural networks (ANNs) for accuracy of prediction of nodal spread of the carcinomas. Naguib et al. (1997) previously had demonstrated that ANNs could be used to predict nodal involvement in breast cancer. In that study several established as well as experimental cancer markers had been analysed, and ANN techniques were found to be capable of dissecting and identifying the most powerful predictors of nodal metastasis. The ANN analyses, provided by Albertazzi et al. (1998b) as well as by Naguib et al. (1998), have also supported the conclusion that, overall, S100A4 expression is associated with and indicative of more aggressive forms of the disease. The investigation of Albertazzi et al. (1998b) does emphasise the view that when complemented with nm23, S100A4 could provide a powerful marker for predicting breast cancer prognosis. On the other hand, S100A4 might be a signicant and independent marker for prognosis. An investigation of a large series of breast cancer over a period of 16 years has led Rudland et al. (2000) to conclude that S100A4 expression correlates strongly with patient survival. The Rudland group found S100A4 mRNA in both epithelial and stromal components of breast cancers and, further, that the levels of S100A4 are higher in carcinomas as compared with benign breast tumours (Nikitenko et al. 2000). They have also reported that 80% of patients who were S100A4 negative were alive after 19 years of follow-up, whereas only
234
11% of S100A4-positive patients were alive at the end of this period. The correlation between S100A4 negativity and survival was highly statistically signicant. Interestingly, Rudland et al. (2000) found that S100A4 expression correlated only marginally with axillary lymph nodal involvement. Nevertheless, S100A4positive patients who were also node positive had poorer survival than S100A4 patients with no nodal involvement. The involvement of regional lymph nodes is regarded as the most consistent predictor of prognosis (Angus et al. 2000). The poorer prognosis of the S100A4+/node+ group may be related to whether or not S100A4 is expressed by tumour cells in the regional lymph nodes. Overall, the work reviewed here leaves little room for doubt that S100A4 is associated with tumour malignancy and prognosis. With the large body of evidence of the active participation of S100A4 in a wide spectrum of cellular functions, a demonstration of its association with metastatic cells of human breast cancer would be the coup de grace that is long awaited and that might satisfy the scientic cognocenti. As stated previously, two splice variants of S100A4 have been reported so far (Ambartsumian et al. 1995; Albertazzi et al. 1998c). The larger variant, described by Ambartsumian et al. (1995), occurs in many tissues, although with differences in the levels of expression. Albertazzi et al. (1998c) found only the shorter splice variant (h-mts1v) in the series of breast cancers that they had investigated. They did not detect the expression of either variant in a number of tumour cell lines. The apparently highly specic nature of its expression is somewhat inexplicable. Nonetheless, Albertazzi et al. (1998c) found that h-mtsv expression did tend to correlate with nodal spread of breast cancers, albeit the correlation was not as persuasive as in the case of the unspliced S100A4 transcript. Notwithstanding the positive nature of these ndings, it ought to be stated here that assessing the state of expression of the S100A4 protein, not merely that of its mRNA, is equally important for providing a total picture of the relevance of the gene to progression of cancer. It is the occurrence of the functional protein that would determine the nature of the downstream events that dene the degree of aggressiveness of the disease. As Ambartsumian et al. (1999) observed recently, S100A4 mRNA is expressed in all organs of S100A4 transgenic mice that they had developed. However, the protein is not expressed in organs that do not normally express the S100A4 gene in the wild-type animals. Therefore, it is imperative that information concerning the expression of the protein is collated at the same time as the mRNA levels are measured. This would take into account the fact that there may exist mechanisms that regulate the translation of the mRNA transcripts and possibly also decay of the protein in the normal course of cellular events.
S100A4
IN
OTHER FORMS
OF
HUMAN CANCER
Among other forms of human cancer that have been studied for S100A4 expression is human melanoma. Maelandsmo et al. (1997) detected high S100A4 levels only in approximately 50% of melanomas, but benign nevi also showed roughly similar levels of S100A4. Quite obviously, S100A4 bears no relationship to the clinical state of disease in this case. A criticism that can be made of this study is the method of assessment of gene expression as undetectable, low, moderate, and high. It would
235
have been helpful if the authors had given the actual values of signal densities and provided the range of values of each group. The ndings of Shrestha et al. (1998) generally support those of Maelandsmo et al. (1997). Shrestha et al. (1998) found little or no S100A4 in several cell types derived from normal skin. Furthermore, neither did basal cell carcinomas or squamous cell carcinomas show any S100A4 immunoreactivity. The apparent lack of correlation of S100A4 with melanoma progression is in sharp contrast with the high degree of correlation found in B16 melanoma cell lines. One should state, notwithstanding, that a direct comparison of expression of the gene in tissue culture cell lines and tumour material, which is heterogeneous in cell composition, is probably not helpful, nor can it lead to meaningful conclusions. In human gliomas, the expression of S100A4 was found to correlate with the degree of malignancy as indicated by the World Health Organization (WHO) histological grading. However, the changes in S100A4 were far less striking than those occurring in S100A1, which signicantly correlated with malignancy (Camby et al. 1999). It would be worthwhile to bear in mind that the histological grading of these tumours is a differentiation-related feature, and often tumour behaviour may not strictly conform to characteristics dened for a particular histological group. Nonetheless, intrinsically the degree of differentiation would be inversely related to malignancy, and therefore the correlation reported between S100A4 and gliomas of different grades may be seen as generally supporting the involvement of S100A4. With the added knowledge that S100A4 expression is higher in cell lines derived from higher grade gliomas as compared with lower grade tumours, it would not be premature to conclude that S100A4 may play an important role in the malignancy of gliomas. The expression of S100A4 in colorectal tumours seems to show a clear correlation with disease progression. The expression of the gene is low in colonic broblasts. Normal mucosal tissue and adenomas have been described as showing comparable levels of expression, but expression level is markedly higher in adenocarcinomas. Cells lines derived from adenocarcinomas also show high levels of S100A4 expression. Immunohistochemical studies have generally conrmed these Northern analyses of S100A4 expression. Of much greater interest is the observation that although adenomas are negative, carcinomatous foci within the adenomas have been found to stain for S100A4 (Takenaga et al. 1997b). Quite obviously, the expression of S100A4 correlates well with the progression of colonic tumours.
S100A6 (CALCYCLIN) IN CANCER

The pattern of expression of calcyclin, now carrying the new nomenclature of S100A6, has been studied in several forms of human cancer, e.g., melanomas (Weterman et al. 1992, 1993), salivary gland tumours (J.W. Huang et al. 1996), chondro-osseous tumours (Muramatsu et al. 1997), and squamous cell carcinomas of the oral mucosa (Berta et al. 1997). Muramatsu et al. (1997) have reported that calcyclin, together with S100A1 and A4, was particularly strongly associated with the development of chondro-osseous tumours. Enhanced calcyclin expression has been reported in squamous cell carcinoma of the oral mucosa but not in benign
236
mucosal lesions. This was accompanied also by an increased expression of the Hras oncogene (Berta et al. 1997). Several genes are differentially expressed in rat mammary tumours, which are induced by the carcinogen N-methyl-N-nitrosourea (NMU). L.H. Young et al. (1996) detected several differentially expressed cDNA clones. Of these, one clone showed strong homology to calcyclin. The expression of this clone was ten-fold greater in tumour tissue than in normal tissue. Pedrocchi et al. (1994) investigated several breast cancer cell lines and noticed marked differences among S100 proteins in their relationship to aggressive behaviour. Calcyclin did not appear to bear any relationship to tumour aggressiveness. However, its expression was higher in the vertical phase of growth of melanomas and correlated with metastatic potential of melanomas (Weterman et al. 1992, 1993). Van Ginkel et al. (1998) found S100A6 to be differentially expressed between normal uveal melanocytes and uveal melanomas and cell lines derived from them. Also differentially expressed were annexins V and VI, CaM, S100A11, and S100B. Although they have suggested that S100A6 expression may relate to the malignancy of uveal melanomas, it would be difcult to dissociate the effects of S100A6 from that of other CBPs. As Sudo and Hidaka (1999) have shown, the CBPs may interact with one another. The correlation of calcyclin expression with metastatic potential has been conrmed by two recent studies (Boni et al. 1997; Maelandsmo et al. 1997). In the rst study, all of 39 primary cutaneous melanomas tested showed intense cytoplasmic staining for calcyclin. Also 9 of 14 metastatic tumours were found to be calcyclin positive. In the second study, S100A6 expression was reported to correlate with patient survival times (Maelandsmo et al. 1997). In a series of human astrocytomas, Camby et al. (1999) found the expression of S100A6 to be clearly related to their WHO grading. Thus, grades I and II tumours could be distinguished, on the basis of S100A6 expression, from the more malignant grade III and IV astrocytomas. However, as discussed previously, there is some ambiguity about the relationship between calcyclin expression and tumour growth. Some investigators regard calcyclin as a negative regulator of growth on the basis that induction of differentiation in tumours can enhance calcyclin expression. It may be that the involvement of this S100 protein has other facets in its relationship with tumour aggressiveness than are currently appreciated and investigated. For example, it has been shown to bind to annexin XI-A (Sudo and Hidaka, 1999). This interaction seems to be of a highly specic nature. This may have some functional implication for S100A6, because annexin expression shows a marked relationship to cell cycle progression and, furthermore, the expression of annexins is linked with deregulation of growth of cancers.
THE BIOLOGICAL PROPERTIES OF S100A7 (PSORIASIN) STRUCTURE

AND
MOLECULAR PROPERTIES
OF
S100A7
Psoriasin (S100A7) has been traditionally associated with psoriatic skin lesions. The S100A7 gene coding for this protein has been described as the psoriasis-susceptibility gene. It spans 2.7 kb of the genome and consists of three exons and two introns. The gene has been mapped to the S100 gene family cluster at chromosome
237
1q21 (Hardas et al. 1996). The S100A7 protein has been isolated from psoriatic skin. It is a ca. 11-kDa cytoplasmic protein with PI of 6.2. It is also secreted into the extracellular compartment. S100A7 is structurally similar to other S100 proteins. However, there are some important differences, especially with regard to the Nterminally located EF-hand calcium-binding domain. The latter contains a distorted loop, and notably, in S100A7 this EF-hand lacks an important calcium-binding residue. Consequently, it binds no more than one calcium ion per monomer (Brodersen et al. 1998). S100 family proteins are also known to bind other divalent cations besides Ca2+, and their binding to the proteins can often be functionally more signicant than their binding of Ca2+. Recombinant S100A7 indeed binds Ca2+, Zn2+, and Mg2+. It can bind seven Ca2+ ions in the presence of KCl and several in the presence of NaCl. Therefore, in the secreted state, S100A7 could bind Ca2+ and, in the cellular compartments, possibly binds higher amounts of calcium. It can bind eight Zn2+ ions in the presence of KCl and four in the presence of NaCl (Vorum et al. 1996). The binding of these cations produces conformational changes in the protein. A subsequent picture of calcium-binding described by Brodersen et al. (1998) suggests a greatly reduced calcium-binding ability, and moreover, their ndings are not compatible with the view that calcium-binding brings about conformational changes in S100A7. It may be that insofar as S100A7 is concerned, calciumbinding may be functionally less signicant than Zn2+. Recently, Brodersen et al. (1999) have identied a Zn2+-binding site in S100A7 that contains three histidine and one aspartate residues. It is of much interest to note that these residues present the Zn2+ in a way characteristic of certain metalloproteinases, which are important components of the ECM. It also seems from the work of Brodersen et al. (1999), that the absence of Zn2+ may have collateral effects on the organisation of the distorted EF-hand loop. This might result in reduced calcium-binding by S100A7. Although the zinc-binding site occurs in other S100 proteins as well, the reason why calcium-binding is functionally the most signicant event in a majority of these proteins could be the absence of collateral Zn2+-induced changes.
S100A7
IN
SKIN PATHOLOGY
S100A7 (psoriasin) was originally isolated from psoriatic skin and seen to be associated clearly with inammatory disease of the skin. Retinoic acid treatment has been shown to induce S100A7 expression in skin but not in other tissues. This suggests a tissue-specic regulation of its expression. Expression occurs at a low level in untreated epidermal keratinocytes but not in broblasts or melanocytes (Tavakkol et al. 1994). S100A7 has been shown to function as a chemotactic factor for CD4+ T lymphocytes and to mediate inammation. However, it is structurally distinct from conventional lymphokines (J.Q. Tan et al. 1996). S100A7 has been shown to form complexes with the epidermal-type fatty acid-binding protein (EFABP). EFABP, which shows an increased expression in psoriatic skin, is believed to be involved in the transport of cytosolic fatty acids. S100A7EFABP complexes occur in the cytoplasm of differentiating keratinocytes derived from psoriatic skin (Hagens et al. 1999). The functional signicance of the complex is not understood
238
at present. Two possibilities have been suggested: keratinocyte differentiation and/or the transport and targeting of cytosolic fatty acids. Semprini et al. (1999) have suggested that the S100A7 gene may not be a candidate gene for familial psoriasis susceptibility. Several other genes occurring in the 1q21 cluster are said to be overexpressed in psoriasis (Hardas et al. 1996). This may support the contention that S100A7 is not a candidate gene for psoriasis. Furthermore, the 1q21 gene cluster contains a collection of genes associated with the terminal differentiation of epidermis. As discussed in an earlier section, genes in this cluster are also actively involved in the control of cell proliferation. Primary human keratinocytes exposed in vitro to growth factors show an up-regulation of not only S100A7 but also MRPs 8 and 14, S100A11. Also expressed in these cells are the actin-binding non-EF-hand proteins, gelsolin and annexin (Olsen et al. 1995). Because of the wide-spectrum genetic modulations involved, the relative signicance of the overexpression of individual genes of this cluster, is very difcult to assess in relation to pathogenesis of psoriasis. It is inevitable that the overexpression of other CBP genes at the locus will have repercussions on the expression of S100A7, because not only does calcium bind S100A7, but calcium is also able to up-regulate the synthesis of S100A7 (Hoffmann et al. 1994). It follows, therefore, that a significant sequestration of intracellular calcium by other CBPs would affect S100A7 expression.
S100A7
IN
NEOPLASTIC DISEASE
The possibility that S100A7 expression in keratinocytes might reect the state of their proliferation and differentiation has made it easier to appreciate why A7 is expressed in both normal and malignant keratinocytes in culture (P.H. Watson et al. 1998). Early studies indicated that S100A7 is expressed in breast cancers as well as cell lines derived from breast cancers, but it was not detectable in control tissues (Moog-Lutz et al. 1995). Leygue et al. (1996) compared its expression in ductal carcinoma in situ and invasive ductal carcinoma. They detected S100A7 mRNA only in the in situ carcinomas. It would be inappropriate to extrapolate from these limited studies on the possible relationship between expression of the gene and invasive behaviour of the carcinoma. In this context it might be interesting to note that S100A7 has been found to enhance the adhesion of neutrophils to epidermal cells (Von den Driesch et al. 1998). Unhappily, most of these studies are very preliminary in nature. Nonetheless, the results are signicant enough to warrant further investigation and conrmation. There have been tentative investigations of the possibility that S100A7, as a secreted protein, could be a potential marker in certain tumour types. In squamous cell carcinoma of the bladder, urinary S100A7 has been suggested to be a useful noninvasive marker for follow-up of patients (Ostergaard et al. 1999). S100A7 is expressed, together with markers for keratinised stratied squamous epithelia, in squamous cell carcinoma of the bladder. S100A7 expression is localised to the socalled squamous pearls (Celis et al. 1996). As expected, S100A7 is also detectable in the urine of patients. There is a lack of persuasive data on the relationship between S100A7 expression and the state of malignancy of bladder carcinoma. However, the
239
expression of several other important genes of the 1q21 cluster is often up-regulated with S100A7 as a consortium. This seems to be ample justication for evaluating these genes as a potential noninvasive prole of markers for bladder cancer.
S100A8 AND S100A9 PROTEINS IN INFLAMMATORY DISEASES

S100A8 and S100A9 proteins, previously known by the designations MRP8 and MRP14, respectively, are CBPs of the S100 protein family (Kligman and Hilt, 1988; Kerkhoff et al. 1998). They contain two EF-hand domains, one at either end of the molecule, and they are separated by a central hinge region. The EF-hands themselves are anked by hydrophobic regions. Besides Ca2+, they can also bind Zn2+ ions. The latter might regulate some of the functional properties of these proteins. S100A8 and A9 are regarded as myeloid-specic proteins and show an enhanced presence in activated phagocytes. Increased plasma levels of these proteins have been reported in patients with inammatory conditions, such as rheumatoid arthritis, cystic brosis, and chronic bronchitis. Although localised predominantly in the cytosol, they may be secreted by activated monocytes (Rammes et al. 1997). It would seem that they are translocated from the cytosol to the tubulin cytoskeleton and the plasma membrane. The targeting of these molecules to this cellular compartment could be a result of phosphorylation. Guignard et al. (1996) found that translocation occurred after the proteins were phosphorylated. As an extracellular protein, several functions have been envisaged and attributed to S100A8/A9. Kerkhoff et al. (1999a) have suggested they might be involved in the transendothelial migration of monocytes. Among other suggested roles are growth inhibition and cytostasis. There is some interesting evidence that they may regulate the inammatory process by virtue of being able to form complexes with arachidonic acid and aid its transport and metabolism (Kerkhoff et al. 1999b). The binding of arachidonic acid by the S100A8/A9 heterodimeric complex has been found to be dependent on Zn2+ binding. Whereas Ca2+ binding seems to be conducive to the binding of arachidonic acid, Zn2+ binding appears to reverse this process. Many of these studies are still in an incipient stage and much further work is required in order to elucidate the mechanisms by which S100A8 and A9 putatively carry out this range of function. Until then, most of the suggested mechanisms will remain in the realm of speculation.
S100A11 (S100C) AND POSSIBLE MODES OF ITS FUNCTION

S100A11 was isolated and characterised by Ohta et al. (1991). It is an 11-kDa protein with two EF-hand domains and shows 41 and 37% sequence homology with S100A and S100B proteins. The S100A11 gene has been localised to the S100 gene cluster at chromosome 1q21 (Moog-Lutz et al. 1995; Wicki et al. 1996a,b). The protein has been shown to bind to actin and inhibit actin-activated Mg2+-ATPase
240
FIGURE 28 The biological processes inuenced by S100 proteins. This is especially true of S100A4, as discussed here. In the context of cancer, the major inuences are on cell motility, adhesive properties, and proliferation. The effects exerted by S100 proteins on cytoskeletal dynamics seem to be important in maintaining or modulating cell morphology and also highly pertinent in cell proliferation and signal transduction. Overall, this illustration focuses on the breadth of biological effects brought about by S100 proteins and the need to regard S100 genes, such as S100A4, as normal genes, whose inappropriate expression could lead to aberrant biological behaviour, rather than as oncogenes or metastasis genes. (From Sherbet and Lakshmi, 1997b, 1998.) Reprinted by permission of the publisher Academic Press.
activity of smooth muscle cells (Zhao et al. 2000). Zhao et al. (2000) have, in fact, shown that the inhibition of the myosin-ATPase enzyme is not due to the depolymerisation of actin laments but it is a direct effect of actin binding by S100A11. The cytoskeletal binding action is probably not unique to this protein. The association of S100A4 with the cytoskeleton has now been amply demonstrated (Figure 28 and Table 17). Moreover, S100A4 also binds to the rod region of myosin and inhibits actin-activated ATPase (Ford et al. 1997). The function subserved by S100A11 is yet unclear. Apart from the obvious consequences of Mg2+-ATPase inhibition to the functioning of the smooth muscle, it is of interest to note that S100A11 interacts also with annexins (Naka et al. 1994; Mailliard et al. 1996; Seemann et al. 1996), a property it shares with S100A10 (Kaczanbourgois et al. 1996). These interactions of S100A11 and S100A10 could be important indicators of their cellular function. PKC-mediated regulation of annexin function involves certain amino acid residues of the S100 proteins that have been implicated also in the interaction between annexins and S100A11 and S100A10. The interaction with cytoskeletal elements quite obviously implicates them in the regulation of the cytoskeletal machinery. Furthermore, there is a potential, albeit indirect link between these S100 proteins and cell proliferation and differentiation, and, by analogy with S100A4 function, it might be involved in cancer invasion too. This aspect of potential inuences of S100A10 and S100A11 has received almost no attention to date.
241
S100P IN CANCER PROGRESSION

S100P was isolated from human placenta and characterised some years ago (T. Becker et al. 1992; Emoto et al. 1992). It has received much attention in recent years. S100P consists of 95 amino acid residues and possesses two EF-hands that bind calcium with different afnity. The high-afnity EF-hand occurs at the Cterminal and the one with low afnity occurs at the N-terminal region of the protein. The protein also binds other divalent ions such as Zn2+ at the C-terminal region and Mg2+ at the N-terminal end. The binding of Ca2+ and Mg2+ has been shown to alter the conformational state of S100P, and it has been postulated that the biological properties of the protein may be altered by a Ca2+//Mg2+ switch (Gribenko and Makhatadze, 1998; Gribenko et al. 1998).
S100P
AND ITS
PUTATIVE FUNCTIONS
The presence of S100P in skin sensory corpuscles has been described by some investigators (Delvalle et al. 1995; Albuerne et al. 1998). Intense immunostaining for S100P has been encountered in the lamellar cells of Meissner corpuscles and the inner core cells of Pacinian corpuscles (Albuerne et al. 1998). In the avian Herbst corpuscles also S100P is known to be associated with the inner core cells. These studies have found no evidence of S100P in the central axons of the sensory corpuscles. The protein may be putatively involved in sensory signal transduction. Both these studies also refer to the occurrence of other S100 proteins. That S100P might contribute, together with S100A, to the structural integrity of the sensory apparatus is suggested by the nding that the pattern of expression of S100 proteins, including S100P, was not altered in skin samples obtained from patients with spinal cord injury. However, there was a reduction in the number of sensory corpuscles that expressed these proteins (Albuerne et al. 1998). Frank and Wolburg (1996) studied the events associated with wound healing and tissue repair following injury to the optical nerve in the rat. They have reported the appearance of reactive astrocytes staining for S100P about 6 days after the nerve injury, and these astrocytes might be involved in the restructuring of the nerve bres. Some preliminary observations have been reported about the expression of S100P in relation to the progression of carcinoma of the prostate. Androgens are actively involved in the growth and function of the prostate. Carcinomas of the prostate have notably been described as being androgen dependent at early stages of their development, but becoming independent of this hormone during progression of the disease. Averboukh et al. (1996) found that the androgen-responsive cell line LNCaP-FGC, derived from prostate cancer, expresses S100P gene, but this is downregulated within 30 hr after androgen deprivation. On the basis of these observations, they suggest that S100P might be involved in the aetiology of prostate cancer. One would concede that the expression of the gene might be regulated by androgen, but this is a far cry from demonstrating its implication in the pathogenesis of prostate cancer. One could even tentatively suggest that S100P might conceivably function as a suppressor gene. A number of true metastasis-suppressor genes, as contrasted with tumour-suppressor genes, have been cloned.
242
In evaluating the potential role of S100P one should also take account of the fact that androgens regulate the expression of PSA. They regulate PSA gene transcription by means of the androgen-responsive elements that occur in the promoter region of the PSA gene (Riegman et al. 1991). Probably cis-acting elements are involved also in the AR-mediated transcription of PSA (J.Y. Zhang et al. 1997). The PSA-related protein, human glandular kallikrein-1, is also regulated by androgens. The gene that encodes it contains an androgen-responsive element (Murtha et al. 1993). A proportion of PSA occurs in the form of a complex with other proteins such as 1-antichymotrypsin. The ratio of bound to free PSA is greater in carcinomas than in BPH. Therefore, the proportion of free PSA found in the serum has been regarded as a reasonable tumour marker (Becker and Lilja, 1997; Stenman et al. 1999). Sartor et al. (1997) studied the rate of increase of PSA and have concluded that a rapid increase in the level of PSA indicated metastatic disease, in contrast with a moderate rise in PSA, which was associated with local recurrence. However, Bangma et al. (1995) did not nd any relationship between PSA expression and progression of the disease. Chopra et al. (1996) found PSA expression in normal prostate, BPH, and carcinomas. Some recent experimental work has shown that PSA can inhibit endothelial cell proliferation and also inhibit the angiogenic effects of FGF and VEGF. In the mouse model, PSA has been found to reduce metastatic deposition of tumour cells in the lungs (Fortier et al. 1999). The apparent antiangiogenic and anti-metastatic effects of PSA have raised further doubts about its reliability as a marker of tumour progression. However, because PSA forms complexes with proteases other than antichymotrypsin, there is room for renements of its utility as a diagnostic agent. PSA is a kallikrein-like serine protease and can conceivably bring about changes in the ECM that might be conducive to tumour cell invasion. Indirect evidence for this comes from the observation that the in vitro invasive behaviour of LNCaP cells is inhibited when the proteolytic activity of PSA is experimentally suppressed (Webber et al. 1995). In vivo, the invasive behaviour of prostate carcinoma correlates with PSA concentration in serum (Bostwick et al. 1996). The PSA-related prostatespecic kallikrein is also regulated by androgen (Murtha et al. 1993), and its expression has been reported to increase from benign epithelium through intraepithelial neoplasia to carcinomas (Darson et al. 1997). Therefore, PSA alone could be viewed as being responsible for the invasive behaviour and metastatic spread. However, one must reconcile with the increasing androgen independence of the process of progression of this cancer. In the midst of contradictory ndings, there seems to be a consensus view that PSA expression might in fact be maintained or even increase with progression. However, the uncertainty about the relationship between PSA and tumour progression might yet allow one to dissociate the potential inuence on tumour progression of S100P from that of PSA. Therefore, in spite of these various complications arising from androgen-mediated regulation, S100P deserves to be studied further. For example, it would be of much interest to know the state of its expression in BPH, and whether there are any discernible changes in expression in the progression of disease from the state of intraepithelial neoplasia to invasive adenocarcinoma.
243
Of considerable practical signicance is the recent report by Bertram et al. (1998) of the strong association between S100P, and to a lesser extent S100A4, expression and drug resistance. S100P was highly expressed in doxorubicin-resistant cells as compared with those sensitive to the drug. The reasons for this association can only be speculated. At any rate, the need to exploit the practical value of this nding should far outweigh the desire to understand the underlying mechanisms.
POTENTIAL VALUE OF S100 PROTEINS AS MARKERS OF CANCER PROGRESSION AND PROGNOSIS

The establishment of a marker for monitoring disease progression and possibly as an aid in predicting prognosis should contain two basic elements. One of these is that the expression of the putative marker should show clear and unequivocal empirical correlation with disease state. The second, more important element is that the putative marker should manifestly function via a mechanism that involves one or more fundamental processes of the life of the cell. Whether measuring the expression of S100 genes has any role to play in the clinical management of human cancer is an area that now clearly warrants investigation. Early indications are that this could be useful. Serum levels of S100 proteins have been measured in melanomas. Elevated levels of generic S100 proteins may be related to metastatic outcome (Buer et al. 1997; Drummer et al. 1997). S100 proteins were detectable in the serum of 79.3% patients with metastatic disease as compared with 4% of stage I/II patients and 21.4% of stage III patients. Furthermore, there was a sharp decline in S100 levels in two patients in remission (Drummer et al. 1997). The detection of higher serum levels also has been reported in all stages of cutaneous melanomas, as compared with levels found in normal subjects (Abraham et al. 1997). Immunohistochemical staining of cutaneous tumours has revealed intense staining for S100A6 in all primary lesions and in 64% (9/14) of metastatic lesions studied; but staining for S100A4 was found to be weak in these neoplasms (Boni et al. 1997). S100A4 expression has been studied in human colorectal neoplasms. Normal colonic mucosa and adenomas have been found to contain comparable levels of S100A4, but adenocarcinomas express considerably larger amounts of S100A4 mRNA. Furthermore, immunohistochemical analyses have revealed metastatic tumours to be strongly S100A4 positive (Takenaga et al. 1997b). It has been reported recently that, in human inltrating ductal carcinomas of the breast, S100A4 (h-mts1) expression correlates strongly with the potential to metastasise to axillary lymph nodes. S100A4 expression also inversely correlates with oestrogen and progesterone receptor status. These observations suggest that S100A4 (h-mts1) expression might reect the aggressiveness of breast cancers (Albertazzi et al. 1998b). Albertazzi et al. (1998b) also reported the occurrence of a variant hmts1v, a shorter transcript of S100A4 (h-msts1) in which exon 1 (1a, 1b) is spliced out (Albertazzi et al. 1998c). Their data have suggested the possibility that the expression of this variant might also be indicative of aggressive disease, albeit not as powerful a marker as S100A4 (h-mts1) itself. Further work needs to be done on
244
the expression of both S100A4 (h-mts1) and h-mts1v in breast cancer as well as in other forms of cancer in order to consolidate these ndings before pronouncing on the clinical value of S100A4 expression in tumours. Nevertheless, there are rm data concerning the ability of S100A4 to enhance the invasive and metastatic propensities of experimental tumour models as well as in certain human neoplasms. Of the S100 family proteins, S100A4 seems most satisfactorily to fulll both the criteria stated above. It appears to function via the modulation of the cytoskeletal machinery of the cell and by controlling the progression of the cell cycle. These observations provide considerable support for the thesis that S100A4 expression might be a powerful marker, from the S100 family of proteins, of cancer progression and prognosis.
Epilogue
The range of biological parameters inuenced by calcium-binding proteins in general and S100 proteins in particular is indeed most impressive (see Figure 28). CBPs are highly versatile proteins, and highly signicant is their mediation of several critical events closely aligned to and identiable with specic compartments of the metastatic cascade (Table 21). Six compartments can be identied in tumour development, invasion, metastatic dissemination, and the development of overt metastatic lesions. In all these, CBPs and S100 proteins, in particular, will inuence the cellular properties that are highly associated with the behavioural or phenotypic event relevant to that specic metastatic compartment. This seems to begin at the beginning, in the initiation of neoplastic transformation and the expansive growth of the tumour. The transduction of signals originating from aetiological agents or down-stream signals of TGFs or mitogenic stimuli often takes the calcium-signalling pathway. The ow of information is mediated by CBPs. The CBPs may be regarded as the translators and the harbingers of the calcium messages. They undergo conformational changes upon binding to Ca2+. A consequence of this seems to be the acquisition of the ability to recognise target proteins and translate the calcium signal into a biochemical function reecting a phenotypic property. Also often involved in the pathway is a coordinated functioning of kinases and phosphatases, as, for instance, in the transduction of the TCR binding by its antigen, in which calcineurin phosphorylation plays an important part in information ow.
TABLE 21 Metastatic Compartments and Relevant Cellular Properties Inuenced by Calcium-Binding Proteins
Metastatic Compartment A. B. C. D. E. F. Aetiology Cell proliferation, apoptosis ECM remodelling, modulation of cell adhesion, cell shape, motility, cytoskeletal dynamics Cytoskeletal dynamics, cell membrane malleability, cell motility, angiogenesis Heterotypic adhesion Cell proliferation, apoptosis Relevant Events of the Metastatic Cascade Neoplastic transformation Tumour development, expansive growth Invasion Extravasation from vascular system Arrest of cells at metastatic targets Development of overt metastases
245
246
The expansive growth of tumours is a reection of a net increase of the cell population size, which is a function of total cell proliferation and apoptotic cell loss. The deregulation of the cell cycle seems to be the prime cause of this expansive phase of tumour development, and it is very appropriate that North (1991) described cancer as a disease of the cell cycle. As we have seen, S100 proteins not only show cell cycle-related expression, but individual proteins such as S100A4 might be closely linked with the regulation of cell cycle progression in conjunction with other regulatory proteins such as p53, rb, and stathmin. The activity of these proteins is exquisitely coordinated to achieve effective regulation. Calcium signalling is an integral part of the process of apoptosis, and the apoptotic pathway contains several target enzymes activated by calcium. This is equally important in the development of metastatic deposits, but the kinetics of cell proliferation and apoptotic loss in primary tumours and metastatic deposits are not strictly comparable. We have discussed the potential effects of Ca2+-mediated activation of caspases and calpains in the deregulation of cell proliferation and induction of apoptosis. The inuence of VD3 on cell differentiation with attendant inhibition of proliferation is a good example of the interplay of VDRE-mediated activation of gene transcription and the control over this exerted by osteocalcin. The latter in turn is regulated by TGF. The invasive phase of the metastatic cascade is replete with examples of CBP activity. There is much empirical evidence that high levels of S100 family proteins occur in association with enhanced invasive propensity. Besides, some of these proteins appear to take part in the remodelling of the extracellular matrix, which, as we have noted, gures prominently in cell adhesion and invasive behaviour. S100A4 and osteonectin are prime examples of ECM-modulating CBPs. Both seem to participate in its remodelling by having recourse to regulating the proteolytic activity associated with the ECM. The regulation of the contractile machinery of the cell is obviously of considerable importance in cell motility. The regulatory component of the actomyosin assembly, i.e., MLC, binds calcium in its function of myosin-ATPase regulation and generation of contractile forces. MLCs themselves undergo reversible phosphorylation through the agency of Ca2+/CaM-dependent kinases and phosphatases. The phosphorylation of MLCs seems to be involved in the invasion of vascular endothelium by activated PMN. As we have noted already, not only does calcium signalling via the activation of calpains and caspases severely affect tumour development, but we have also established the involvement of these enzymes on the adhesive and invasive behaviour of cells. The transmembrane glycoprotein cadherin forms a complex with -catenin and -catenin and links-up with the actin cytoskeleton. This complex is a complete example of calcium signalling mediated by cadherin, which has calcium-binding domains in the extracellular part of the molecule, and the catenin serving an adhesion function as well as providing a signal transduction machinery. Furthermore, there might be a feedback mechanism present, with cadherin negatively regulating the signalling function of -catenin. The cadherincatenin complex, at the same time, is also essential for the physical process of formation of adherens junctions. The cellular properties of heterotypic adhesion and deregulation of cell proliferation kinetics would apply equally also to the development of overt metastatic deposits. Nonetheless, one should recognise that the adhesive properties required of a tumour
Epilogue
247
cell trying to gain entry into the vascular system are probably totally different in character from those required for extravasation of the tumour cell in the target organ. On the other hand, one might speculate that the seeding of tumour cells at the target organ might be a reection of the induction of apoptosis of endothelial cells rather than by a conventional form of diapedesis across the epithelium. Notwithstanding the downstream pathway of calcium signalling, a retardation of the initial event of calcium inux into cells seems to affect profoundly the invasive behaviour of cells as well as prolong survival times of mice bearing human tumour xenografts. The vascularisation of the tumour and the decimation of tumour cells that have gained access to the vascular compartment are the major gaps in our understanding of CBP activity. Osteonectin has been attributed with both angiogenic and antiangiogenic abilities. There is very little direct evidence that one can cite for either attribute. Some indirect evidence is available that suggests that S100A4 may not be involved with tumour vascularisation. However, there is also a report that claims that S100A4 can reduce the size and density of tumour-associated microvasculature. It might be recalled here that activation of calcium inux brings about major changes in the shape of endothelial cells, which can lead to enhanced permeability and promote the diapedesis of leukocytes. Activation of calcium inux also induces angiogenesis. Furthermore, certain agents that inhibit calcium signal transduction also seem to be capable of inhibiting angiogenesis. Whether CBPs intervene in this aspect of signal transduction is uncertain at present, but this is a distinct possibility. We know that calcium-dependent NOS is involved in the remodelling of vascular endothelia and in angiogenesis. Furthermore, the expression of endothelial NOS in carcinomas correlates with tumour grade. Hence, one of the strategies attempted to control metastatic spread was aimed at inhibiting angiogenesis by blocking the calcium-signalling pathway mediated by calcium-dependent NOS. That calcium signalling might be implicated in the regulation of angiogenesis is, therefore, not altogether in the realm of scientic speculation. There is much scope for intensive investigation of this aspect of the function of CBPs in the metastatic spread of cancer. It is now safe to say that the study of CBPs in general and of S100 family proteins, such as S100A4, in particular have much to offer in the understanding of cell behaviour in a normal cellular environment as well as their behaviour upon neoplastic transformation. This conclusion is based on the extensive evidence that links CBPs, beyond reasonable doubt, with cellular properties that are essential features of tumour growth, differentiation, invasion, and metastatic dissemination. There is much to learn and much to be gained.
References
Abe H, Obinata T, Minamide LS, Bamburg JR. (1996). Xenopus laevis actin depolymerising factor colin. A phosphorylation-regulated protein essential for development. J Cell Biol 132, 871885. Abraham HD, Fuller LC, Du Vivier AWP, Higgins EM, Sherwood RA. (1997). Serum S100 protein: A potentially useful prognostic marker in cutaneous melanoma. Br J Dermatol 137, 381385. Adam T, Arpin M, Prevost MC, Gounon P, Sansonetti PJ. (1995). Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella exneri into HeLa cells. J Cell Biol 129, 367381. Adams AEM, Shen WY, Lin CS, Leavitt J, Matsudaira P. (1995). Isoform-specic complementation of the yeast sac6 null mutation by human mbrin. Mol Cell Biol 15, 6975. Adamus G, Amundson D. (1996). Epitope recognition of recoverin in cancer associated retinopathy. Evidence for calcium dependent conformational epitopes. J Neurosci Res 45, 863872. Adamus G, Ortega H, Witkowska D, Polans A. (1994). Recoverin. A potent uveitogen for the induction of photoreceptor degeneration in Lewis rats. Exp Eye Res 59, 447455. Aeschlimann D, Kaupp O, Paulsson M. (1995). Transglutaminase-catalysed matrix crosslinking in differentiating cartilage. Identication of osteonectin as a major glutaminyl substrate. J Cell Biol 129, 881892. Ahern-Djamali SM, Conner AR, Bachmann C, Kastenmeier AS, Reddy SK, Beckerie MC, Walter U, Hoffmann FM. (1998). Mutations in Drosophila enable and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional role for Ena/VASP homology domain-1 (EVH1) and EVH2 domains. Mol Biol Cell 9, 21572171. Ahmed A, Dunk C, Kniss D, Wilkes M. (1997). Role of BEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. Lab Invest 76, 779791. Ahn J, Murphy M, Kratowicz S, Wang A, Levine AJ, George DL. (1999). Down-regulation of the stathmin/Op18 and FKBP25 genes following p53 induction. Oncogene 18, 59545958. Ahn SH, Sawada H, Ro JY, Nicolson GL. (1997). Differential expression of annexin I in human mammary ductal epithelial cells in normal and benign and malignant breast tissue. Clin Exp Metastasis 15, 151156. Ai ZW, Misra S, Susa M, Verticovski L, Cohen CM. (1995). Phosphatidylinositol 3-kinase activity in murine erythroleukaemia cells during DMSO-induced differentiation. Exp Cell Res 219, 454460. Aikawa M, Kim HS, Kuroo M, Manabe I, Watanabe M, Yamaguchi H, Yazaki Y, Nagai R. (1995). Phenotypic modulation of smooth muscle cells during progression of human atherosclerosis as determined by altered expression of myosin heavy chain isoforms. Ann NY Acad Sci 748, 578585. Airaksinen M, Eilers J, Garaschuk O, Thoenen H, Konnerth A, Meyer M. (1997). Ataxia and altered dendritic calcium signalling in mice carrying a targeted null mutation of the calbindin D28K gene. Proc Natl Acad Sci USA 94, 14881493.
249
250
Alaiya AA, Franzen B, Fujioka K, Moberger B, Schedvins K, Silversvard C, Linder S, Auer G. (1997). Phenotypic analysis of ovarian carcinoma. Polypeptide expression in benign, borderline and malignant tumours. Int J Cancer 73, 678683. Albertazzi E, Cajone F, Lakshmi MS, Sherbet GV. (1998a). Heat shock modulates the expression of the metastasis related mts1 gene and cell proliferation of murine and human cancer cells. DNA Cell Biol 17, 17. Albertazzi E, Cajone F, Leone BE, Naguib RNG, Lakshmi MS Sherbet GV. (1998b). The expression of metastasis associated genes h-mts1 and nm23 in carcinoma of the breast is related to disease progression. DNA Cell Biol 17, 335342. Albertazzi E, Cajone F, Sherbet GV. (1998c). Characterisation of a splice variant of the metastasis associated h-mts1 (S100A4) gene expressed in human inltrating carcinomas of the breast. DNA Cell Biol 17, 10031008. Albuerne M, Lopez S, Naves FJ, Martinez-Almagro A, Represa J, Vega JA. (1998). S100 alpha and S100 beta proteins in human cutaneous sensory corpuscles. Anat Rec 251, 351-359. Aldersio M, Cenci M, Valli C, Russo A, Bazan V, Dadanoni G, Cucciarre S, Carreca I, Maculuso MP, Momasino RM, Vecchlone A. (1998). nm23-H1 protein, DNA ploidy and S-phase fraction in relation to overall survival and disease free survival in transitional cell carcinoma of the bladder. Anticancer Res 18, 4225-4230. Alekseev AM, Shulga-Morskoy SV, Zinchenko DV, Shulga-Morskaya SA, Suchkov DV, Vaganova SA, Senin II, Zargarov AA, Lipkin VM, Akhtar M, Philippov PP. (1998). Obtaining and characterisation of EF-hand mutants of recoverin. FEBS Lett 440, 116118. Ali IU, Schriml LM, Dean M. (1999). Mutational spectra of PTEN/MMAC1 gene; a tumour suppressor with lipid phosphatase activity. J Natl Cancer Inst 91, 19221932. Allbritton NL, Meyer T, Stryer L. (1992). Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258, 18121815. Allevi P, Anastasia M, Cajone F, Ciuffreda P, Sanvito AM. (1995). Structural requirements of aldehydes produced in LPO for the activation of heat shock genes in HeLa cells. Free Radical Biol Med 18, 107116. Allore RJ, Friend WC, OHanlon DR, Neilson KM, Baumal R, Dunn RJ, Marks A. (1990). Cloning and expression of the human S100beta gene. J Biol Chem 265, 15537-15543. Ambartsumian N, Klinegelhofer J, Grigorian M, Karlstrom O, Sidenius N, Georgiev G, Lukanidin E. (1999). Tissue-specic post-transcriptional down-regulation of expression of the S100A4 (mts1) gene in transgenic mice. Invasion Metastasis 18, 96104. Ambartsumian NS, Grigorian MS, Larsen IF, Karlstrom O, Sidenius N, Rygaard J, Georgiev G, Lukanidin E. (1996). Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene. Oncogene 13, 16211630. Ambartsumian N, Tarabykina S, Grigorian M, Tulchinsky E, Hilgaard E, Georgiev G, Lukanidin E. (1995). Characterisation of two splice variants of metastasis associated gene mts1. Gene 159, 125130. Ambs S, Bennettu WP, Merrian WG, Ogunfusika MO, Oser SM, Khan MA, Jones RT, Harris CC. (1998). Vascular endothelial growth factor and nitric oxide synthase expression in human lung cancer and relation to p53. Br J Cancer 78, 233239. Ames JB, Porumb T, Tanaka T, Ikura M, Stryer L. (1995). Amino terminal myristoylation induces co-operative calcium binding to recoverin. J Biol Chem 270, 45264533. Ames JB, Tanaka T, Stryer L, Ikura M. (1994). Secondary structure of myristoylated recoverin determined by 3-dimensional heteronuclear NMR. Implications for the calcium myristoyl switch. Biochemistry 33, 1074310753.
References
251
Amino K, Takahashi M, Honda Y, Fujimoto T. (1995). Redistribution of fodrin in an in vitro wound healing model of the corneal epithelium. Exp Eye Res 61, 501508. Andersen K, Maelandsmo GM, Hovig E, Fodstad O, Loennechen T, Winberg JO. (1998). Interleukin-1 alpha and basic broblast growth factor induction of matrix metalloproteinases and their inhibitors in osteosarcoma cells is modulated by the metastasis associated protein CAPL. Anticancer Res 18, 32993303. Andersen LB, Xia LQ, Stoll S, Zhao XP, Elder JT. (1996). Lineage-specic CaN19 expression in human skin. Lack of expression in normal melanocytes. J Dermatol Sci 12, 6972. Andersen SSL, Ashford J, Tournebize R, Gavet O, Sobel A, Hyman AA, Kersenti E. (1997). Mitotic chromatin regulates phosphorylation of stathmin/Op-18. Nature 389, 640643. Anderson RGW. (1998). The caveolae membrane system. Annu Rev Biochem 67, 199225. Andre E, Brink M, Gerisch G, Isenberg G, Noegel A, Schleicher M, Segall JE, Wallraff E. (1989). A Dictyostelium mutant decient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis. J Cell Biol 108, 985995. Andresen K, Tom TD, Strand M. (1991). Characterisation of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni. J Biol Chem 266, 1508515090. Andressen C, Blumcke I, Celio MR. (1993). Calcium binding proteins. Selective markers of nerve cells. Cell Tissue Res 271, 181208. Andressen C, Gotzos V, Berchtold MW, Pauls TL, Schwaller B, Fellay B, Celio MR. (1995). Changes in shape and motility of cells transfected with parvalbumin cDNA. Exp Cell Res 219, 420426. Angle JM, Joyal JL, Black ED, Alli H, Sacks DB, Madara JL, Turner JR. (1998). Protein kinase C-mediated phosphorylation of myosin light chain kinase (MLCK) inhibits myosin light chain (MLC) phosphorylation and results in increased transepithelial resistance (TER). Gastroenterology 114, G1421. Angus B, Lennard TWJ, Naguib RNG, Sherbet GV. (2000). Analysis of molecular prognostic factors in breast cancer by articial neural networks. In: Articial neural networks in cancer diagnosis, prognosis and patient management, eds. Naguib RNG, Sherbet GV, CRC Press, Boca Raton, FL. Ankarcrona M, Dypbukt JM, Orrenius S, Nicotera P. (1996). Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Lett 394, 321324. Aoufouchi S, Yelamos J, Milstein C. (1999). Inhibition of apoptosis of a PARP/ cell line transfected with PARP DNA-binding domain mutants. J Mol Biol 290, 943949. Aprikian AG, Han K, Chevalier S, Bazinet M, Viallet J. (1996). Bombesin specically induces intracellular calcium mobilisation via gastrin-releasing peptide receptors in human prostate cancer cells. J Mol Endocrinol 16, 297306. Arahata K, Sugita H. (1989). Dystrophin and the membrane hypothesis of muscular dystrophy. Trends Pharmacol Sci 10, 437439. Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, Caroni P. (1998). Regulation of actin dynamics through phosphorylation of colin by LIM-kinase. Nature 393, 805809. Arden EM, Nijweide PJ, Vander-Plas A, Albas MJ, Mackie EJ, Horton MA, Helfrich MH. (1996). Adhesive properties of isolated chick osteocytes in vitro. Bone 18, 305313. Armstrong RC, Aja T, Xiang JL, Gaur S, Krebs JF, Hoang K, Bai X, Korsmeyer J, Karanewsky DS, Fritz LC, Tomaselli KJ. (1996). Fas-induced activation of the cell death protease CPP32 is inhibited by bcl-2 and ICE family protease inhibitors. J Biol Chem 271, 1685016855. Arnold DB, Heintz N. (1997). A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells. Proc Natl Acad Sci USA 94, 88428847.
252
Arola J, Heikkila P, Voutilainen R, Kahri AI. (1993). Role of adenylate cyclase-cyclic AMP dependent signal transduction in the ACTH induced biphasic growth effect of rat adrencortical cells in primary culture. J Endocrinol 13, 451. Arora AS, Degroen PC, Croall DE, Gores GJ. (1995). Hepatocellular carcinoma cells resist necrosis during anoxia by preventing activation of calpains. FASEB J 9, 143. Arora PD, McCulloch CAG. (1996). Dependence of broblast migration on actin severing activity of gelsolin. J Biol Chem 271, 2051620523. Arpin M, Friederich E, Algrain M, Vernel F, Louvard D. (1994). Functional differences between L-plastin and T-plastin informs. J Cell Biol 127, 19952008. Asch HL, Head K, Dong Y, Natoli F, Winston JS, Connolly JL, Asch BB. (1996). Widespread loss of gelsolin in breast cancers of humans, mice and rats. Cancer Res 56, 48414845. Atlas D, Adler M. (1981). -Adrenergic antagonists as possible calcium channel inhibitors. Proc Natl Acad Sci USA 78, 12371241. Attawia MA, Herbert KM, Laurencin CT. (1995). Osteoblast-like cell adherence and migration through 3-dimensional porous polymer matrices. Biochem Biophys Res Commun 213, 639644. Averboukh L, Liang P, Kantoff PW, Pardee AB. (1996). Regulation of S100P expression by androgen. Prostate 29, 350355. Avrova SV, Borovikov YS, Emova NN, Chacko S. (1998). Calcium modulates conformational changes in F-actin induced by smooth muscle meromyosin. FEBS Lett 430, 266268. Ayhan A, Yasui W, Yukozaki H, Kitadai Y, Tahara E. (1993). Reduced expression of nm23 protein is associated with advanced tumour stage and distant metastasis in human colorectal carcinomas. Virchows Arch B 63, 213218. Babb SG, Matsudaira P, Sato M, Correia I, Lim SS. (1997). Fimbrin in podosomes of monocyte-derived osteoclasts. Cell Motility Cytoskeleton 37, 308325. Baehrecke H. (1996). Ecdysone signal cascade and regulation of Drosophila metamorphosis. Arch Insect Biochem Physiol 33, 231244. Baimbridge KG, Celio MR, Roger JH. (1992). Calcium binding proteins in the nervous system. Trends Neurosci 15, 303308. Baimbridge KG, Miller JJ, Parkes CO. (1982). Calcium binding protein distribution in the rat brain. Brain Res 239, 519525. Balciuniene J, Hohansson K, Sandgren O, Wachtmeister L, Holmgren G, Forsman K. (1995). A gene for autosomal dominant progressive cone dystrophy (CORDS5) maps to chromosome 17p12-p13. Genomics 30, 281286. Baldan B, Navazio L, Friso A, Mariani P, Meggio F. (1996). Plant calreticulin specically and efciently phosphorylated by protein kinase CK2. Biochem Biophys Res Commun 221, 498502. Ballweber E, Giehl K, Hannapel E, Huff T, Jockusch BM, Mannherz HG. (1998). Plant prolin induces actin polymerisation from actin beta-thymosin complexes and competes directly with beta thymosins and with negative co-operativity with DNAse 1 for binding to actin. FEBS Lett 425, 251255. Ballweber E, Hannappel E, Niggemeyer B, Mannherz HG. (1994). Induction of polymerisation of actin from the actin-thymosin beta-4 complex by phalloidin, skeletal subfragment 1, chicken intestinal myosin-1 and free ends of lamentous actin. Eur J Biochem 223, 419426.
References
253
Balzar M, Bakker HAM, Briare de Bruijn IH, Fleuren CJ, Warnaar SO, Litvinov SV. (1998). Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule. Mol Cell Biol 18, 48334843. Bambrick LL, Krueger BK. (1999). Neuronal apoptosis in mouse trisomy 16. Mediation by caspases. J Neurochem 72, 17691772. Banerjee C, Stein JL, Van Wijnen AJ, Frenkel B, Lian JB, Stein GS. (1996). Transforming growth factor beta-1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1 binding site. Endocrinology 137, 19912000. Bangma CH, Hop WCJ, Schroder FH. (1995). Serial prostate specic antigen measurements and progression in untreated conned (stage-T0 to stage 3NXMO, grade 1 to grade 3) carcinoma of the prostate. J Urol 154, 14031406. Banik NL, Chou CH, Deibler GE, Krutzch HC, Hogan EL. (1994). Peptide bond specicity of calpain. Proteolysis of human myelin basic protein. J Neurosci Res 37, 489496. Banik NL. (1992). Pathogenesis of myelin breakdown in demyelinating diseases. Role of proteolytic enzymes. Crit Rev Neurobiol 6, 257271. Bao L, Loda M, Zetter BR. (1998). Thymosin beta 15 expression in tumour cell lines with varying metastatic potential. Clin Exp Metastasis 16, 227233. Bao LR, Loda M, Janmey PA, Stewart R, Anandapte B, Zetter BR. (1996). Thymosin beta15. A novel regulator of tumour cell motility up-regulated in metastatic prostate cancer cells. Nature Med 2, 13221328. Barbiero G, Munaron L, Antoniotti S, Baccino FM, Bonelli G, Lovisolo D. (1995). Role of mitogen-induced calcium inux in the control of the cell cycle in Balb-C3T3 broblasts. Cell Calcium 18, 542556. Barford D. (1996). Molecular mechanisms of the protein serine threonine phosphatases. Trends Biochem Sci 21, 407412. Barille S, Pellat-Deceunynck C, Bataille R, Amiot M. (1996). Ectopic secretion of osteocalcin, the major non-collagenous bone protein by the myeloma cell line NCI-H929. J Bone Mineral Res 11, 466471. Barinaga M. (1998). Is apoptosis key in Alzheimers disease? Science 281, 1303-1304. Barnes NY, Li L, Yoshikawa K, Schwartz LM, Oppenheim RW, Milligan CE. (1998). Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurones. J Neurosci 18, 58695880. Barnes R, Masood S, Barker E, Rosengard AM, Coggin DL, Crowell T, King CR, PorterJordan K, Wargotz ES, Liotta LA, Steeg PS. (1991). Low nm23 protein expression in inltrating ductal breast carcinomas correlates with reduced patient survival. Am J Pathol 139, 245250. Barnes TM, Hodgkin J. (1996). The tra-3 sex determination gene of Caenorhabditis elegans encodes a member of the calpain regulatory protease family. EMBO J 15, 44774484. Baron MD, Davison MD, Jones P, Patel B, Critchely DR. (1987). Isolation and characterisation of a cDNA encoding a chick alpha-actinin. J Biol Chem 262, 25582561. Barstead RJ, Kleiman L, Waterston RH. (1991). Cloning, sequencing, and mapping of an alpha-actinin gene from the nematode Caenorhabditis elegans. Cell Motility Cytoskeleton 20, 6978. Bartorelli A, Biancardi C, Cavalca V, Clemente C, Ferrara R, Arzani C, Bailo M, Botta M. (1996). Enzyme-lined immunosorbent assays (ELISA) for the detection of antibodies to UK114 and UK150. J Tumour Marker Oncol 11, 6780.
254
Barwise JL, Walker JH. (1996). Annexin II, annexin IV, annexin V and annexin VI relocate in response to rises in intracellular calcium in human foreskin broblasts. J Cell Sci 109, 247255. Bashir R, Britton S, Strachan T, Keers S, Vaadaki E, Lako M, Richard I, Marchand S, Bourg N, Argov Z, Sadeh M, Mahjneh I, Marconi G, Passos-Bueno MR, Moreira ED, Zatz M, Beckmann JS, Bushby K. (1998). A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb girdle muscular dystrophy type 2B. Nature Genet 20, 3742. Bashir R, Keers S, Strachan T, Passos-Bueno R, Zatz M, Weissenbach J, Le Pasler D, Meisler M, Bushby K. (1996). Genetic and physical mapping at the limb girdle muscular dystrophy locus (LGMD-2B) on chromosome 2p. Genomics 33, 4652. Bass J, Chiu G, Argon Y, Steiner DF. (1998). Folding of insulin receptor monomers is facilitated by the molecular chaperones, calnexin and calreticulin and impaired by rapid dimerisation. J Cell Biol 141, 637646. Bassuk JA, Baneyx F, Vernon RB, Funk SE, Sage EH. (1996). Expression of biologically active human SPARC in Escherichia coli. Arch Biochem Biophys 325, 819. Bastianelli E, Pochet R. (1994). Calbindin D28K, calretinin, and recoverin immuno-reactivities in developing chick pineal gland. J Pineal Res 17, 103111. Batiuk TD, Kung LN, Halloran PF. (1997). Evidence that calcineurin is rate-limiting for primary human lymphocyte activation. J Clin Invest 100, 18941901. Baudier J, Briving C, Deinum J, Haglid K, Sorskog L, Wallin M. (1982). Effects of S100 protein and calmodulin on Ca2+ induced disassembly of brain microtubule proteins in vitro. FEBS Lett 147, 167. Baudier J, Cole RD. (1988). Interaction between microtubule associated protein and S100b regulate phosphorylation by Ca2+/calmodulin dependent protein kinase II. J Biol Chem 263, 58765883. Bayle JH, Elenbaas B and Levine AJ. (1995). The carboxyl terminal domain of the p53 protein regulates sequence-specic DNA binding through its non-specic nucleic acid binding activity. Proc Natl Acad Sci USA 92, 57295733. Beck KA, Buchanan JA, Malhotra V, Nelson WJ. (1994). Golgi spectrin. Identication of an erythroid beta-spectrin homologue associated with the Golgi complex. J Cell Biol 127, 707723. Beck KA, Buchanan JA, Nelson WJ. (1997). Golgi membrane skeleton. Identication and localisation and oligomerisation of a 195 kDa ankyrin isoform associated with the Golgi complex. J Cell Sci 110, 12391249. Becker C, Lilja H. (1997). Individual prostate specic antigen (PSA) forms as prostate tumour markers. Clin Chim Acta 257, 117132. Becker H, Stengl G, Stein M, Federlin K. (1995). Analysis of proteins that interact with the IL-2 regulatory region in patients with rheumatic diseases. Clin Exp Immunol 99, 325330. Becker T, Gerke V, Kube E, Weber K. (1992). S100P, a novel Ca2+-binding protein from human placenta. Eur J Biochem 207, 541547. Beckmann JS, Richard I, Broux O, Fougerousse F, Allamand V, Chiannikulchai N, Lim LE, Duclos F, Bourg N, Brenguier L, Pasturaud P, Quertier F, Roudaut C, Sunada Y, Meyer J, Dincer P, Lefranc G, Merlini L, Topaloglu H, Tome FMS, Cohen D, Jackson CE, Campbell KP, Fardeau M. (1996). Identication of muscle-specic calpain and betasarcoglycan genes in progressive autosomal recessive muscular dystrophies. Neuromuscular Disorders 6, 455462.
References
255
Beggs AH, Arahata K, Watkins SC, Knoll JHM, Kunkel LM. (1994). Cloning of a novel human non-muscle alpha-actinin and characterisation of alpha-actinin complex in nemaline rod myopathy (NRM). Am J Human Genet 55, 732S. Beggs AH, Byers TJ, Knoll JHM, Boyce FM, Bruns GAP. (1992). Cloning and characterisation of two human skeletal muscle alpha-actinin genes on chromosome 1 and chromosome 11. J Biol Chem 267, 92819288. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W. (1998). Functional interaction of an axin homologue, conduction, with beta catenin, APC, and GSK3 beta. Science 280, 596599. Behrens J, Von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W. (1996). Functional interaction of beta catenin with the transcription factor LEF-1. Nature 382, 638643. Bellagamba C, Hubaishy I, Bjorge JD, Fitzpatrick SL, Fujita DJ, Waisman DM. (1997). Tyrosine phosphorylation of annexin II tetramer is stimulated by membrane binding. J Biol Chem 272, 31953199. Bellahcene A, Castronovo V. (1995). Increased expression of osteonectin and osteopontin, two bone matrix proteins in human breast cancer. Am J Pathol 146, 95100. Benbernou N, Esnault S, Shin HCK, Fekkar H, Gueroumou M. (1997). Differential regulation of IFN-gamma, IL-10 and inducible nitric oxide synthase in human T cells by cyclic AMP dependent signal transduction. Immunology 91, 361368. Bendik I, Schrami P, Ludwig CU. (1998). Characterisation of MAST9/hevin, a SPARC-like protein, that is down-regulated in non-small cell lung cancer. Cancer Res 58, 626629. Bennett JP, Zaner KS, Stossel TP. (1984). Isolation and some properties of macrophage alphaactinin. Evidence that it is not an actin gelling protein. Biochemistry 23, 50815086. Bennett V, Gilligan DM. (1993). The spectrin-based membrane skeleton and micro scale organisation of the plasma membrane. Annu Rev Cell Biol 9, 2766. Bennett V, Lambert S. (1991). The spectrin skeleton: From red cells to brain. J Clin Invest 87, 14831489. Beretta L, Dubois MF, Sobel A, Bensaude O. (1995). Stathmin is a major substrate for mitogen-activated protein kinase during heat shock and chemical stress in HeLa cells. Eur J Biochem 227, 388395. Berg JM. (1990). Zinc ngers and other metal binding domains. Elements for interactions between macromolecules. J Biol Chem 265, 65136516. Berman DM, Gilman AG. (1998). Mammalian RGS proteins. Barbarians at the gate. J Biol Chem 273, 12691272. Berman DM, Wilkie TM, Gilman AG. (1996). GAIP and RGS4 are GTPase-activating proteins for the G (i) subfamily of G protein alpha subunits. Cell 86, 445452. Berridge MJ. (1993). Inositol trisphosphate and calcium signalling. Nature 361, 315325. Berta GN, Ghezzo F, DAvolio A, Zulian P, Carbone V, Racca S, Vercellino V, Di Carlo F. (1997). Enhancement of calcyclin gene RNA expression in squamous cell carcinoma of the oral mucosa but not in benign lesions. J Oral Pathol Med 26, 206210. Berthelot JM, Maugars Y, Frost A, Youinou P. (1995). Rheumatoid arthritis and laggrin. A review. Rev Rheumatisme 62, 127138. Bertram J, Palfner K, Kiddemann W, Kneba M. (1998). Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin-resistant cell lines. Comparison of mRNA differential display reverse transcription polymerase chain reaction and subtractive suppressive hybridisation for the analysis of differential gene expression. Anticancer Drugs 9, 311317.
256
Bhattacharya D, Steinkotter J, Melkonian M. (1993). Molecular cloning and evolutionary analysis of the calcium modulated contractile protein, centrin, in green algae and land plants. Plant Mol Biol 23, 12431254. Bianchi R, Garbuglia M, Verzini M, Giambanco I, Ivanenkov VV, Dimlich RVW, Jamieson GA, Donato R. (1996). S-100 (alpha and beta) binding peptide (TRTK-12) blocks S100/GFAP interaction. Identication of a putative S-100 target epitope within the head domain of GFAP. Biochim Biophys Acta 1313, 258267. Bianchi R, Gianbanco I, Donato R. (1993). S100 protein, but not calmodulin, binds to the glial brillary acidic protein and inhibits its polymerisation in a Ca2+ dependent manner. J Biol Chem 268, 1266912674. Bianchi R, Verzini M, Garbuglia M, Gianbanco I, Donato R. (1994). Mechanisms of S100 protein dependent inhibition of glial brillary acidic protein (GFAP) polymerisation. Biochim Biophys Acta 1223, 354360. Bieche I, Lackhar S, Becette V, Cifuentes-Diaz C, Sobel A, Liderau R, Curmi PA. (1998). Over-expression of the stathmin gene in a subset of human breast cancer. Br J Cancer 78, 701709. Bingham PM, Chou TB, Mims I, Zachar Z. (1988). On/off regulation of gene expression at the level of splicing. Trends Genet 4, 134138. Bini L, Magi B, Marzocchi B, Arcuri F, Tripodi S, Cintorino M, Sanchez JC, Frutiger S, Hughes G, Pallini V, Hochstrasser DF, Tosi P. (1997). Protein expression proles in human breast ductal carcinoma and histologically normal tissue. Electrophoresis 18, 28322841. Birkenbeil H. (1996). Involvement of calcium in prothoracicotropic stimulation of ecdysone synthesis in Galleria mellonella. Arch Insect Biochem Physiol 33, 3952. Bjornland K, Winberg JO, Odegaard OT, Hovig E, Loennechen T. (1999). S100A4 involvement in metastasis. Deregulation of matrix metalloproteinases and tissues of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme. Cancer Res 59, 47024708. Blaheta RA, Hailer NP, Brude N, Wittig B, Oppermann E, Leckel K, Harder S, Scholz M, Weber S, Encke A, Markus BH. (1998). Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967). Potent immunosuppression by inhibition of T-cell inltration through allogeneic endothelium. Immunology 94, 213220. Blaheta RA, Hailer NP, Brude N, Wittig B, Leckel K, Oppermann E, Bachmann M, Harder S, Cinatl J, Scholz M, Bereiter-Hahn J, Weber S, Encke A, Markus BH. (2000). In vitro analysis of verapamil-induced immunosuppression. Potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells. Transplantation 69, 588597. Blanchard A, Ohanian V, Critchley D. (1989). The structure and function of alpha-actinin. J Muscle Res Cell Motility 10, 280289. Blanchard S, Barwise JL, Gerke V, Goodall A, Vaughan PFT, Walker JH. (1996). Annexins in the human neuroblastoma SH-SY5Y. Demonstration of relocation of annexin II and annexin V to membranes in response to elevation of intracellular calcium by membrane depolarisation and by the calcium ionophore A23187. J Neurochem 67, 805813. Blavier L, Henriet P, Imren S, Declerck YA. (1999). Tissue inhibitors of matrix metalloproteinases in cancer. Ann NY Acad Sci 678, 108119. Blazejewski S, Le Bail B, Boussarie L, Blanc JF, Malaval L, Okubo K, Saric J, Bioulac Sage P, Rosenbaum J. (1997). Osteonectin (SPARC) expression in human liver and in cultured human liver myobroblasts. Am J Pathol 151, 651657.
References
257
Bloomqvist C, Risteli L, Risteli J, Virkkunen P, Sarna S, Elomaa I. (1996). Markers of type I collagen degradation and synthesis in the monitoring of treatment response in bone metastases from breast cancer. Br J Cancer 73, 10741079. Blum JK, Berchtold MW. (1994). Calmodulin-like effect of oncomodulin on cell proliferation. J Cell Physiol 160, 455462. Bogatcheva NV, Gusev NB. (1995). Computer-assisted prediction of phospholipid binding sites of caldesmon and calponin. FEBS Lett 363, 269272. Boldin MP, Goncharov TM, Golstev YV, Wallach D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1 and TNF receptor-induced cell death. Cell 85, 803815. Bolla M, Chedin M, Souvignet C, Marron J, Arnould C, Chambaz E. (1990). Estimation of growth factor receptor in 177 breast cancers: Correlation with prognostic factors. Breast Cancer Res Treat 16, 97102. Bombardieri E, Martineti A, Miceli R, Mariani L, Castellani MR, Seregni E. (1997). Can bone metabolism markers be adopted as an alternative to scintigraphic imaging in monitoring bone metastases from breast cancer? Eur J Nuclear Med 24, 13491355. Bonfoco E, Zhivotovsky B, Rossi AD, Aguilar-Santalises M, Orrenius S, Lipton SA, Nicotera P. (1996). Bcl-2 delay apoptosis and PARP cleavage induced by NO donors in GT1-7 cells. Neuroreport 8, 273276. Boni R, Burg G, Doguoglu A, Ilg EC, Schafer BW, Muller B, Heizmann CW. (1997). Immunohistochemical localisation of Ca2+ binding S100 proteins in normal skin and melanocytic lesions. Br J Dermatol 137, 3943. Bonnemann CG, Passos-Bueno MR, McNally EM, Vainzof M, Moreira ED, Marie SK, Pavenello RCM, Noguchi S, Ozawa E, Zatz M, Kunkel LM. (1996). Genomic screening for -sarcoglycan gene mutations: Mis-sense mutations may cause severe limb girdle muscular dystrophy type 2E (LGMD-2E). Hum Mol Genet 5, 19531961. Bonnet D, Lemoine FM, Frobert Y, Bonner ML, Baillou C, Najman A, Guigon M. (1996). Thymosin beta-4, inhibitor for normal haematopoietic progenitor cells. Exp Haematol 24, 776782. Borgatti P, Mazzoni M, Carini C, Neri LM, Marchisio M, Bertolaso L, Previati M, Zauli G, Capitani S. (1996). Changes in nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation. Exp Cell Res 224, 7278. Bostwick DG, Qian JQ, Bergstralh E, Dundore P, Dugan J, Myers RP, Oesterling JE. (1996). Prediction of capsular perforation and seminal vesicle invasion in prostate cancer. J Urol 155, 13611367. Bottini F, Maxxocco K, Abhondi T, Tonini GP. (1994). Identication of an AP-1-like sequence in the promoter region of calcyclin, an S100-like gene. Enhancement of binding during retinoic acid-induced neuroblastoma cell differentiation. Neurosci Lett 181, 3538. Bouloumie A, Schini-Kerth VB, Busse R. (1999). Vascular endothelial growth factor upregulates nitric oxide synthase expression in endothelial cells. Cardiovasc Res 41, 773780. Bourguignon LYW, Jin HT. (1995). Identication of the ankyrin binding domain of the mouse T lymphoma cell inositol 1,4,5-trisphosphate (IP3) receptor and its role in the regulation of IP3-mediated internal Ca2+ release. J Biol Chem 270, 72577260. Bourguignon LYW, Lokeshwar VB, Chen X, Kerrick WGL. (1993). Hyaluronic acid induced lymphocyte signal transduction and HA receptor (GP85/CD44)-cytoskeleton interaction. J Immunol 151, 66346644.
258
Bourguignon LYW, Zhu HB, Shao LJ, Zhu D, Chen YW. (1999). Rho kinase (ROK) promotes CD44v (3,8-10)-ankyrin interaction and tumour cell migration I metastatic breast cancer cells. Cell Motility Cytoskeleton 43, 269287. Braga VMM, Del Maschio A, Machesky L, Dejana E. (1999). Regulation of cadherin function by Rho and Rac. Modulation by junction maturation and cellular contact. Mol Biol Cell 10, 922. Braun C, Engel M, Seifert M, Theisinger B, Seitz G, Zang KD, Walter C. (1999a). Expression of calpain 1 messenger RNA in human renal cell carcinoma. Correlation with lymph node metastasis. Int J Cancer 84, 69. Braun C, Engel M, Theisinger B, Welter C, Seifert M. (1999b). Capn-8. Isolation of a new mouse calpain isoenzyme. Biochem Biophys Res Commun 260, 671675. Braunewell KH, Spilker C, Behnisch T, Gundelnger ED. (1997). The neuronal calcium sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells. Amino terminal myristoylation determines functional activity. J Neurochem 68, 21292139. Bresnick AR, Maupin P, Pollard TD. (1996). Localisation of myosin light chain kinase in dividing cells. Mol Biol Cell 7, 1156. Brewer JM, Wunderlich JK, Ragland WL. (1990). The aminoacid sequence of avian thymic hormone, a parvalbumin. Biochemie 72, 653660. Brindle NPJ, Holt MR, Davies JE, Price CJ, Critchley DR. (1996). The focal adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domains in vinculin. Biochem J 318, 753757. Brocchieri A, Saporiti A, Moroni M, Porta C, Tua A, Grignani G. (1996). Verapamil inhibits to different extents agonist-induced Ca2+ transients in human tumour cells and in vitro tumour cell growth. Invasion Metastasis 16, 5664. Brockhaus M, Grunberg J, Rohrig S, Loetscher H, Wittenburg N, Baumeister R, Jacobsen H, Haass C. (1998). Caspase-mediated cleavage is not required for the activity of presenilins in amyloidogeneis and NOTCH signalling. Neuroreport 9, 14811486. Brodersen DE, Etzerodt M, Madsen P, Celis JE, Thorgersen HC, Nyborg J, Kjeldgaard M. (1998). EF-hands at atomic resolution. The structure of human psoriasin (S100A7) solved by MAD phasing. Structure 6, 477489. Brodersen DE, Nyborg J, Kjeldgaard M. (1999). Zinc binding site of an S100 protein revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in the Zn2+-loaded and Zn2+-free states. Biochemistry 38, 16951704. Brodie C, Bogi K, Acs P, Lazarovici P, Petrovics G, Anderson WB, Blumberg PM. (1999). Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells. Cell Growth Differentiation 10, 183191. Bronner F, Pansu D, Stein WD. (1986). An analysis of intestinal calcium transport across the rat intestine. Am J Physiol 250, G561G569. Brown JP, Delmas PD, Malaval L, Meunier PJ. (1984). Serum bone gla- protein. A specic marker for bone formation in post-menopausal women. Lancet 1, 10911093. Brown TL, Patil S, Basnett RK, Howe PH. (1998). Caspase inhibitor BD-fmk distinguishes transforming growth factor beta-induced apoptosis from growth inhibition. Cell Growth Differentiation 9, 869875. Bruchovsky N, Snoek R, Rennie PS, Akakura K, Goldenberg SL, Gleasve M. (1996). Control of tumour progression by maintenance of apoptosis. Prostate S6, 1321. Buer J, Probst M, Franzke A, Duensing S, Haindl J, Volkenandt M, Wittke F, Hoffmann R, Ganser A, Atzpodien J. (1997). Elevated serum levels of S100 and survival in metastatic melanoma. Br J Cancer 75, 13731376.
References
259
Bunting M, Tang W, Zimmerman GA, McIntyre TM, Prescott SM. (1996). Molecular cloning and characterisation of a novel human diacylglycerol kinase zeta. J Biol Chem 271, 1023010236. Burgi-Saville ME, Gerber H, Peter HJ, Paulsson M, Aeschlimann D, Glaser C, Kaempf J, Ruchti C, Sidiropoulos I, Burgi U. (1997). Expression pattern of extracellular matrix components in native and cultured normal human thyroid tissue and in human toxic adenoma tissue. Thyroid 7, 347356. Burridge K, Feramisco JR. (1981). The non-muscle alpha-actinins are calcium-sensitive actin binding proteins. Nature 294, 565567. Burridge K, Nuckolls G, Otey C, Pavalko F, Simon K, Turner C. (1990). Actin membrane interaction in focal adhesions. Cell Differentiation Dev 32, 337342. Bussolati G, Guena M, Bussolati B, Millesimo M, Botta M, Bartorelli A. (1997). Cytolytic and tumour inhibitory antibodies against UK114 protein in the sera of cancer patients. Int J Oncol 10, 779785. Cai WZ, Hu L, Foulkes JG. (1996). Transcription modulating drugs. Mechanism and selectivity. Curr Opin Biotechnol 7, 608615. Cairns P, Okami K, Halachmi S, Halalchmi N, Esteller M, Herman JG, Jen J, Isaacs WB, Bova GS, Sidransky D. (1997). Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res 57, 49975000. Cajone F, Bernelli-Zazzera A. (1989). The action of 4-Hydroxynonenal on heat shock gene expression in cultured hepatoma cells. Free Radical Res Commun 7, 189194. Cajone F, Salina M, Bernelli-Zazzera A. (1989). 4-hydroxynonenal induces a DNA-binding protein similar to the heat shock factor. Biochem J 162, 977979. Cajone F, Sherbet GV. (2000). Stathmin is involved in S100A4-mediated regulation of the cell cycle. Clin Exp Metastasis (in press). Cala SE. (1999). Determination of a putative phosphate-containing peptide in calreticulin. Biochem Biophys Res Commun 259, 233238. Calabretta B, Battini R, Kaczmarck L, De Riel JK, Baserga R. (1986). Molecular cloning of a cDNA for a growth factor inducible gene with strong homology to S100 gene, a calcium binding protein. J Biol Chem 261, 1262812632. Calabretta B, Venurelli D, Kaczmarek L, Narni F, Talpaz M, Anderson B, Beran M, Baserga R. (1986b). Altered expression of G1 specic genes in human malignant myeloid cells. Proc Natl Acad Sci USA 83, 14951498. Califano D, Monaco C, Sanelli G, Guiliano A, Veronese ML, Berlingieri MT, De Franciscis V, Berger N, Trapasso F, Santoro M, Viglietto G, Fusco A. (1998). Thymosin beta 10 gene over-expression correlated with the highly malignant neoplastic phenotype of transformed thyroid cells in vivo and in vitro. Cancer Res 58, 823828. Caligo MA, Grammatico P, Cipolline G, Varesco L, Delporto G, Bevilacqua G. (1994). A low nm23-H1 gene expression identifying high malignancy human melanomas. Melanoma Res 4, 179184. Calvert PD, Klenchin VA, Bownds MD. (1995). Rhodopsin kinase inhibition of recoverin. Function of recoverin myristoylation. J Biol Chem 270, 2412724129. Cambiazo V, Gonzalez M, Isamit C, Maccioni RB. (1999). The beta isoform of heat shock protein hsp-90 is structurally related to human microtubule interacting protein MIP-90. FEBS Lett 457, 343347. Camby I, Nagy N, Lopes MB, Schafer BW, Maurage CA, Ruchoux MM, Murmann P, Pochet R, Heizmann CW, Brotchi J, Salmon I, Kiss R, Decaestecker C. (1999). Supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas are characterised by a differential expression of S100 proteins. Brain Pathol 9, 119.
260
Cameron AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV, Snyder SH. (1995). Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ ux. Cell 83, 463472. Camhi SL, Alam J, Choi AMK. (1996). Transcriptional activation of the mouse heme oxygenase gene (HO-1) by interleukin-6 requires co-operation between 5 distal enhancer and the proximal promoter. Am J Respir Crit Care Med 153, A29. Camhi SL, Alam J, Otterbein I, Sylvester SL, Choi AMK. (1995). Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. Am J Respir Cell Mol Biol 13, 387398. Campbell HD, Fountain S, Young IG, Claudiaos C, Hoheisel JD, Chen KS, Lupski JR. (1997). Genomic structure, evolution and expression of human FLII, a gelsolin and leucine-rich repeat family member. Overlap with LLGL. Genomics 42, 4654. Campbell KP, Leung AT, Sharp AH. (1988). The biochemistry and molecular biology of the dihydropyridine-sensitive calcium channel. Trends Neurosci 11, 425430. Campbell MJ, Elstner E, Holden S, Uksodovic M, Koefer HP. (1997). Inhibition of proliferation of prostate cancer cells by a 19-nor-hexauoride vitamin D-2 analogue involves the induction of p21 (waf1), p27 (kip1) and E-cadherin. J Mol Endocrinol 19, 1527. Canu N, Dus L, Barbato C, Ciotti MT, Brancolini C, Rinaldi AW, Novak M, Cattaneo A, Bradbury A, Calissano P. (1998). Tau cleavage and dephosphorylation in cerebellar granule neurones undergoing apoptosis. J Neurosci 18, 70617074. Cao Y, Motomura K, Ohtsuru A, Matsumoto T, Yamashita S, Kosaka M. (1997). Prolin gene expression and regulation in a temperature-sensitive breast cancer cell line, TsFT101. Pugers Arch Eur J Physiol 434, 341345. Carafoli E, Garcia-Martin E, Guerini D. (1996). The plasma membrane calcium pump. Recent developments and future perspectives. Experientia 52, 10911100. Carafoli E, Genazzani A, Guerini D. (1999). Calcium controls the transcription of its own transporters and channels in developing neurones. Biochem Biophys Res Commun 266, 624632. Carafoli E. (1997). Plasma membrane calcium pump. Structure, function and relationships. Basic Res Cardiol 92, 5961. Cargnello R, Celio MR, Schwaller B, Gotzos V. (1996). Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after differentiation. Biochim Biophys Acta 1313, 201208. Carillo S, Pariat M, Steff AM Jariel-Encontre I, Poulat F, Berta P, Peichaczyk M. (1996). PEST motifs are not required for rapid calpain mediated proteolysis of c-fos protein. Biochem J 313, 245251. Carlier MF, Didry D, Erk I, Lepault J, Van Troys ML, Van de Kerckhove J, Perelroizen I, Yin H, Doi YK, Pantaloni D. (1996). T-beta 4 is not a simple G-actin sequestering protein and interacts with F-actin at high concentrations. J Biol Chem 271, 92319258. Carlsberg C, Polly P. (1998). Gene regulation by vitamin D3. Crit Rev Eukaryotic Gene Expression 8, 1942. Carpintero P, Delamo FF, Anadon R, Gomez-Marquez J. (1996). Thymosin beta (10) messenger RNA expression during early post-implantation mouse development. FEBS Lett 394, 103106. Castellano F, Montcourrier P, Guillemot JC, Gouin E, Machesky L, Cossart P, Chavrier P. (1999). The inducible recruitment of Cdc42 or WASP to a cell surface receptor triggers actin polymerisation and lopodium formation. Curr Biol 9, 351360. Castets F, Grifn WST, Marks A, Van Eldik IJ. (1997). Transcriptional regulation of the human S100 beta gene. Mol Brain Res 46, 208216.
References
261
Castillo MB, Celio MR, Andressen C, Gotzos V, Rulicke T, Berger MC, Weber J, Berchtold MW. (1995). Production and analysis of transgenic mice with ectopic expression of parvalbumin. Arch Biochem Biophys 317, 292298. Castresana J, Saraste M. (1995). Does Vav bind to F-actin through a CH domain? FEBS Lett 374, 149151. Castro CY, Stephenson MA, Gondo MM, Cagle PT. (1998). Prognostic implications of calbindin-D28K in lung cancer. Modern Pathol 11, A172. Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA, Gruss P. (1998). Apaf1 (Ced-4 homologue) regulates programmed cell death in mammalian development. Cell 94, 727737. Ceciliani F, Biancardi C, Cavalca V, Ferrara R, Botta M, Bailo M, Arzani C, Berra B, Ronchi S, Bartorelli A. (1996a). Structural characterisation of the small molecular weight proteins present in UK101. J Tumour Marker Oncol 11, 6366. Ceciliani F, Faotto L, Negri A, Colombo I, Berra B, Bartorelli A, Ronchi S. (1996b). The primary structure of UK114 antigen. FEBS Lett 393, 147150. Celio MR. (1990). Calbindin D28K and parvalbumin in the rat nervous system. Neurosci 35, 375475. Celis JE, Rasmussen HH, Vorum H, Madsen P, Honore B, Wolf H, Orntoft TF. (1996). Bladder squamous cell carcinomas express psoriasin and externalise it to the urine. J Urol 155, 21052112. Chafel MM, Shen WY, Matsudaira P. (1995). Sequential expression and differential localisation of L-mbrin, and T-mbrin during differentiation of the mouse intestine and yolk sac. Dev Dynamics 203, 141151. Chakrabarti AK, Banik NL, Lobo DC, Terry EC, Hogan EL. (1993). Calcium-activated neutral proteinase (calpain) in rat brain during development. Compartmentation and role in myelination. Dev Brain Res 71, 107113. Chan DC, Wynshaw-Boris A, Leder P. (1995). Formin isoforms are differentially expressed in the mouse embryo and are required for normal expression of FGF-4 and SHH in the limb bud. Development 121, 31513162. Chan YM, Tong HQ, Beggs AH, Kunkel LM. (1998). Human skeletal muscle-specic alphaactinin-2 and -3 isoforms form homodimers and heterodimers in vitro and in vivo. Biochem Biophys Res Commun 246, 134139. Chandrasekar S, Harvey AK, Johnson MG, Becker GW. (1994). Osteonectin/SPARC is a product of articular chondrocytes/cartilage and is regulated by cytokines and growth factors. Biochim Biophys Acta 1221, 714. Chang CD, Mukai H, Kuno T, Tanaka C. (1994). cDNA cloning of an alternatively spliced isoform of the regulatory subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin B-alpha2). Biochim Biophys Acta 1217, 174180. Chang HY, Takei K, Sydor AM, Born T, Rusnak F, Hay DG. (1995). Asymmetric retraction of growth cone lopodia following focal inactivation of calcineurin. Nature 376, 686690. Chard PS, Bleakman D, Christakos S, Fullmer CS, Miller RJ. (1993). Calcium buffering properties of calbindin D (28K) and parvalbumin in rat sensory neurones. J Physiol 472, 341357. Chard PS, Jordan J, Marcuccilli CJ, Miller RJ, Leiden JM, Roos RP, Ghadge GD. (1995). Regulation of excitatory transmission at hippocampal synapses by calbindin D28K. Proc Natl Acad Sci USA 92, 51445148. Chatterjee A, Tanaka T, Parrish JE, Herman GE. (1995). Rened mapping of caltractin in human Xq28 and in the homologous region of the mouse X-chromosome places the gene within the bare patches (BPA) and striated (STR) critical regions. Mammalian Genome 6, 802804.
262
Chen CK, Inglese J, Lefkowitz RJ, Hurley JB. (1995). Ca2+-dependent interaction of recoverin with rhodopsin kinase. J Biol Chem 270, 1806018066. Chen DS, Davies MPA, Rudland PS, Barraclough R. (1997). Transcriptional down-regulation of the metastasis inducing S100A4 (p9Ka) in benign but not in malignant rat mammary epithelial cells by GC-factor. J Biol Chem 272, 2028320290. Chen DS, Rudland PS, Chen HL, Barraclough R. (1999). Differential reactivity of the rat S100A4 (p9Ka) gene to sodium bisulte is associated with differential levels of the S100A4 mRNA in rat mammary epithelial cells. J Biol Chem 274, 24832491. Chen KS, Gunaratne PH, Hoheisel JD, Young IG, Miklos GLG, Greenberg F, Shaffer LG, Campbell HD, Lupski JR. (1995). The human homologue of the Drosophila melanogaster ightless-I gene (FLII) maps within the Smith-Magenis microdeletion critical region in 17p11.2. Am J Hum Genet 56, 175182. Chen LH, Marechal V, Moreau J, Levine AJ, Chen JD. (1997). Proteolytic cleavage of the mdm2 oncoprotein during apoptosis. J Biol Chem 272, 2296622973. Chen NQ, Davis AT, Canbulat EC, Liu YX, Goueli S, McKenzie BA, Eccleston ED, Ahmed K, Holtzman JL. (1996). Evidence that casein kinase 2 phosphorylates hepatic microsomal calcium binding proteins 1 and 2 but not 3. Biochemistry 35, 82998306. Chen P, Murphy-Ullrich JE, Wells A. (1996). Role for gelsolin in actuating epidermal growth factor receptor mediated cell motility. J Cell Biol 134, 689698. Chen ZF, Schottler F, Lee KS. (1997). Neuronal recovery after moderate hypoxia is improved by the calpain inhibitor MDL28170. Brain Res 769. 188192. Cheon J, Ko SC, Gardner TA, Shirakawa T, Gotoh A, Kao C, Chung LWK. (1997). Chemogene therapy. Osteocalcin promoter-based suicide gene therapy in combination with methotrexate in a murine osteosarcoma model. Cancer Gene Ther 4, 359365. Cheung WT, Richards DE, Rogers JH. (1993). Calcium binding by chick calretinin and rat calbindin D28K synthesised in bacteria. Eur J Biochem 215, 401410. Chiannilkulchai N, Pasturaud P, Richard I, Auffray C, Beckmann JS. (1995). A primary expression map of the chromosome 15q15 region containing the recessive of the limb girdle muscular dystrophy (LGMD2A) gene. Human Mol Genet 4, 717725. Chiaramonte R, Fasola S, Lollini PL, Giovanni CD, Comi P. (1998). Is mts1 (S100A4) gene involved in the metastatic process modulated by gamma interferon? Pathobiology 66, 3840. Chiavegato A, Bochatonpiallat ML, Damore E, Sartore S, Gabbiani G. (1995). Expression of myosin heavy chain isoforms in mammary epithelial cells and in myobroblasts from different brotic settings during neoplasia. Virchows Arch Pathol 426, 7786. Chiavegato A, Pauletto P, Sartore S. (1996). Smooth muscle type myosin heavy chain isoforms in bovine smooth muscle and non-muscle tissues. Biol Cell 86, 2738. Childs TJ, Watson MH, Novy RE, Lin JJC, Mak AS. (1992). Calponin and tropomyosin interactions. Biochim Biophys Acta 1121, 4146. Chin ER, Olson EN, Richardson JA, Yano Q, Humphries C, Shelton JM, Wu H, Zhu WG, Bassel-Duby R, Williams RS. (1998). A calcineurin-dependent transcriptional pathway controls skeletal muscle bre type. Genes Dev 12, 24992509. Chitaev NA, Troyanovsky SM. (1998). Adhesive but not lateral E-cadherin complexes require calcium and catenins for their formation. J Cell Biol 142, 837846. Choi AMK, Alam J. (1996). Heme oxygenase-1. Function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15, 919. Choi YH, Lee SJ, Nguyen P, Jang JS, Lee J, Wu ML, Takano E, Maki M, Henkart PA, Trepel JB. (1997). Regulation of cyclin D1 by calpain protease. J Biol Chem 272, 2847928484.
References
263
Chopra DP, Grignon DJ, Joiakin A, Mathieu PA, Mohamed A, Sakr WA, Powell IJ, Sarkar FH. (1996). Differential growth factor responses of epithelial cell cultures derived from normal human prostate, benign prostatic hyperplasia, and primary prostate carcinoma. J Cell Physiol 169, 269280. Christakos S, Gabrielides C, Rhoten WB. (1989). Vitamin D dependent calcium binding proteins. Chemistry, distribution, functional considerations and molecular biology. Endocrinol Rev 10, 326. Christensen EI, Birn H, Verroust P, Moestrup SK. (1998). Megalin-mediated endocytosis in renal proximal tubule. Renal Failure 20, 191199. Christensen EI, Moskaug JO, Vorum H, Jacobsen C, Gundersen TE, Nykjaer A, Blomhoff R, Willnow TE, Moestrup SK. (1999). Evidence for an essential role of megalin in transepithelial transport of retinol. J Am Soc Nephrol 10, 685695. Christenson MA, Means AR. (1993). Co-ordinate regulation of messenger RNAs from multiple calmodulin genes during myoblast differentiation in vitro. J Cell Physiol 154, 343349. Christenson RH. (1997). Biochemical markers of bone metabolism. An overview. Clin Biochem 30, 573593. Claeskens A, Ongenae N, Neefs JM, Cheyns P, Kaijen P, Cools R, Kutoh E. (2000). Hevin is down regulated in many cancers and is a negative regulator of cell growth and proliferation. Br J Cancer 82, 11231130. Clarke BL, Moore DR, Blalock JE. (1994). Adrenocorticotropic hormone stimulates a transient calcium uptake in rat lymphocytes. Endocrinology 135, 17801786. Clayton L, Hall A, Johnson MH. (1999). A role for Rho-like GTPases in the polarisation of mouse eight cell blastomeres. Dev Biol 205, 322331. Clemens TL, Tang H, Maeda S, Kesterson RA, Demayo F, Pike JW, Gundberg CM. (1997). Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes. J Bone Mineral Res 12, 15701576. Clezardin P, Malaval L, Moarel MC, Gulchard J, Lecompte T, Trzeciak MC, Dechavanne M, Breton-Gorlus J, Delmas PD, Kaplan C. (1991). Osteonectin is an alpha granule component involved with thrombospondin in platelet aggregation. J Bone Mineral Res 6, 10591070. Clipstone NA, Crabtree GR. (1992). Identication of calcineurin as a key signalling enzyme in lymphocyte activation. Nature 357, 695697. Cohen BE, Lee G, Arispe N, Pollard HB. (1995). The cyclic 3-5-adenosine monophosphate binds to annexin I and regulates calcium dependent membrane aggregation and ion channel activity. FEBS Lett 377, 444450. Coleman RE, Purohit OP, Black C, Vinholes JJF, Schlosser K, Huss H, Quinn KJ, Kanis J. (1999). Double blind, randomised, placebo controlled, dose-nding study of oral ibandronate in patients with metastatic bone disease. Ann Oncol 10, 311316. Collinson SK, Liu SL, Clouthier SC, Benser PA, Doras JL, Sanderson KE, Kay WW. (1996). The location of four mbrin-encoding genes, agfA, mA, sefA, and sefD, on the Salmonella enteritidis and/or S. typhimurium. Gene 169, 7580. Colucci S, Gianelli G, Grano M, Faccio R, Quaranta V, Zallone AZ. (1996). Human osteoblastlike cells selectively recognise laminin isoforms, an event that induces migration and activates Ca2+-mediated signals. J Cell Sci 109, 15271535. Conroy SE, Faulds GB, Williams W, Latchman DS, Isenberg DA. (1994). Detection of autoantibodies to the 90-kDa heat shock protein in systemic lupus erythematosus and other autoimmune diseases. Br J Rheumatol 33, 923926.
264
Conroy SE, Sasieni PD, Amin V, Wang DY, Smith P, Fentiman IS, Latchman DS. (1998). Antibodies to heat shock protein 27 are associated with improved survival in patients with breast cancer. Br J Cancer 77, 18751879. Constantin B, Meerschaert K, Van de Kerckhove J, Gettemans J. (1998). Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions. J Cell Sci 111, 16951706. Cooper DMF. (1991). Inhibition of adenylate cyclase by Ca2+. A counterpart to stimulation by Ca2+/calmodulin. Biochem J 278, 903904. Cooper HL, Feuerstein N, Noda M, Bassin RH. (1985). Suppression of tropomyosin synthesis. A common biochemical feature of oncogenesis by structurally diverse retroviral oncogenes. Mol Cell Biol 5, 972983. Cooper NR. (1985). The classical complement pathway. Activation and regulation of the rst complement component. Adv Immunol 37, 151216. Cooray P, Yuan YP, Schoenwaelder SM, Mitchell CA, Salem HH, Jackson SP. (1996). Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain. Biochem J 318, 4147. Coppolino MG, Dedhar S. (1998). Calreticulin. Int J Biochem Cell Biol 30, 553558. Coppolino MG, Demaurex N, Grinstein S, St Arnaud R, Dedhar S. (1996). Calreticulinknockout embryonic stem cells are decient in integrin-mediated adhesion but not calcium stores. Mol Biol Cell 7, 1419. Coppolino MG, Woodside MJ, Demaurex N, Grinstein S, St Arnaud R, Dedhar S. (1997). Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 386, 843847. Copray JCVM, Liem RSB, Kernell D. (1996). Calreticulin expression in spinal motoneurones of the rat. J Chem Neuroanat 11, 5765. Corneliussen B, Holm M, Walterson Y, Onions J, Hallberg B, Thornell A, Grundsrom T. (1994). Calcium/calmodulin inhibition of basic helix-loop-helix transcription factor domains. Nature 368, 760764. Couet J, Sargiacomo M, Lisanti MP. (1997). Interaction of a receptor tyrosine kinase, EGFr, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities. J Biol Chem 272, 3042930438. Counts JL, McClain RM, Goodman JI. (1997). Comparison of effect of tumour promoter treatments on DNA methylation status and gene expression in B6C3F1 and C57BL/6 mouse liver and in B6C3F1 mouse liver tumours. Mol Carcinogenesis 18, 97106. Crawford C, Brown NR, Willis AC. (1993). Studies of the active site of m-calpain and the interaction with calpastatin. Biochem J 296, 135142. Crawford HC, Fingleton BM, Rudolph-Own LA, Goss KJH, Rubinfeld B, Polakis P, Matrisian LM. (1999). The metalloproteinase matrilysin is a target of beta catenin transactivation in intestinal tumours. Oncogene 18, 28832891. Creutz CE, Kambouris NG, Snyder SL, Hamman HC, Nelson MR, Liu W, Rock P. (1992). Effects of the expression of mammalian annexins in yeast secretory mutants. J Cell Sci 103, 11771192. Creutz CE, Liou A, Snyder SL, Brownawell A, Willison K. (1994). Identication of the major chromafn granule-binding protein. J Biol Chem 269, 3203532038. Criddle RA, Zheng MH, Dick IM, Callus B, Prince RL. (1997). Oestrogen responsiveness of renal calbindin-D 28K gene expression in rat kidney. J Cell Biochem 65, 340348. Crivici A, Ikura M. (1995). Molecular and structural basis of target recognition by calmodulin. Annu Rev Biophys Biomol Struct 24, 85116. Croall DE, De Martino GN. (1991). Calcium-activated neutral protease (calpain) system. Structure, function and regulation. Physiol Rev 71, 813847.
References
265
Cruz-Ortega R, Cushman JC, Ownby JD. (1997). cDNA clones encoding 1,3-beta-glucanase and a mbrin-like cytoskeletal protein are induced by Al toxicity in wheat roots. Plant Physiol 114, 14531460. Cryns VL, Bergeron L, Zhu H, Li HL, Yuan JY. (1996). Specic cleavage of alpha-fodrin during Fas- and tumour necrosis factor-induced apoptosis is mediated by an interleukin1 beta-converting enzyme ced-3 protease distinct from the poly (ADP-ribose) polymerase protease. J Biol Chem 271, 3127731282. Cunningham EB. (1995). The human erythrocyte membrane contains a novel 12-kDa inositol phosphate binding protein that is an immunophilin. Biochem Biophys Res Commun 215, 212218. Curmi PA, Anderson SSL, Lachkar S, Gavet O, Kersenti E. (1997). The stathmin/tubulin interaction in vitro. J Biol Chem 272, 2502925036. Curmi PA, Gavet O, Charbaut E, Ozon S, Lochkar-Colmerauer S, Manceau V, Siavoshian S, Maucuer A, Sobel A. (1999). Stathmin and its phosphoprotein family. General properties, biochemical and functional interaction with tubulin. Cell Struct Func 24, 345357. Curnow KM, Pascoe L, Davies E, White PC, Corvol P, and Clauser E. (1995). Alternatively spliced human type-1 angiotensin II receptor messenger RNAs are translated at different efciencies and encode two receptor isoforms. Mol Endocrinol 9, 12501262. Cuthbertson KSR. (1983). Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol. J Exp Zool 226, 311314. Czisch M, Scheicher M, Horger S, Voelta W, Holak TA. (1993). Conformation of thymosin beta (4) in water determined by NMR spectroscopy. Eur J Biochem 218, 335344. Czurylo EA, Kulikova N, Dabrowska R. (1997). Does calponin interact with caldesmon? J Biol Chem 272, 3206732070. Da Silva ACR, Reinach FC. (1991). Calcium binding induces conformational changes in muscle regulatory proteins. Trends Biochem Sci 16, 5357. Dahlback K, Lofberg H, Dahlback B. (1986). Localisation of vitronectin (S-protein of complement) in normal human skin. Acta Dermatol Venereol 66, 461467. Dai E, Stewart M, Ritchie B, Mesaeli N, Raha S, Kolodziejczyk D, Hobman ML, Liu LY, Etches W, Nation N, Michalak M, Lucas A. (1997). Calreticulin, a potential vascular regulatory protein, reduces intimal hyperplasia after arterial injury. Arterioscler Thromb Vasc Biol 17, 23592368. Dale BA, Presland RB, Lewis SP, Underwood RA, Fleckman P. (1997). Transient expression of epidermal laggrin in cultured cells causes collapse of intermediate lament networks with alteration of cell shape and nuclear integrity. J Invest Dermatol 108, 179187. Dalil-Thiney N, Bastianelli E, Pochet R, Reperant J, Versaux-Botteri C. (1998). Recoverin and hippocalcin distribution in the lamprey (Lampreta uviatilis) retina. Neurosci Lett 247, 163166. Dallas SL, Garrett IR, Oyajobi BO, Dallas MR, Boyce BF, Bauss F, Radl J, Mundy GR. (1999). Ibandronate reduces osteolytic lesions but not tumour burden in a murine model of myeloma bone disease. Blood 93, 16971706. Damajanovski S, Karp X, Funk S, Sage EH, Ringuete MJ. (1997). Ectopic expression of SPARC in Xenopus embryos interferes with tissue morphogenesis. Identication of a bioactive sequence in the C-terminal hand. J Histochem Cytochem 45, 643655. Darnell JE. (1997). STATs and gene regulation. Science 277, 16301635. Darson MF, Pacelli A, Roche P, Rittenhouse HG, Wolfert RL, Young CY, Klee GG, Tindall DJ, Bostwick DG. (1997). Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma. A novel prostate cancer marker. Urology 49, 857862.
266
Davies BR, Davies MPA, Gibbs FEM, Barraclough R, Rudland PS. (1993). Induction of metastatic phenotype by transfection of a benign mammary epithelial cell line with the gene for p9Ka, a rat calcium binding protein, but not with the oncogene EJ-ras-1. Oncogene 8 9991008. Davies M, Harris S, Rudland P, Barraclough R. (1995). Expression of the rat, S100-related, calcium binding protein gene, p9Ka, in transgenic mice demonstrates different patterns of expression between these two species. DNA Cell Biol 14, 825832. Davies MPA, Rudland PS, Robertson L, Parry EW, Jolicoeur P, Barraclough R. (1996). Expression of the calcium-binding protein S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of mammary tumours. Oncogene 13, 16311637. Davis HW, Crimmins DL, Thoma R, Garcia JGN. (1996). Phosphorylation of calmodulin in the rst calcium binding pocket by myosin light chain kinase. Arch Biochem Biophys 332, 101109. De la Cruz RR, Pastor AM, Martinez-Guijarro FJ, Lopez-Garcia C, Delgado-Garcia JM. (1998). Localisation of parvalbumin, calretinin, and calbindin D28K in identied extraocular motoneurones and internuclear neurones of the cat. J Comp Neurol 390, 377391. De Luca CI, Davies PL, Samis JA, Elce JS. (1993). Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit. Biochim Biophys Acta 1216, 8193. De Marzo AM, Marchi VL, Yang ES, Veeraswamy R, Lin XH, Nelson WG. (1999). Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis. Cancer Res 59, 38553860. De Mattos-Dutra A, DeFreitas M, Pessoa-Pureur R. (1998). Calcineurin dephosphorylates neurolament subunit NF-M and beta tubulin of the cytoskeletal fraction of rat cerebellum. Med Sci Res 26, 111113. De Murcia G, De Murcia JM. (1994). Poly (ADP-ribose) polymerase. A molecular nick sensor. Trends Biochem Sci 19, 172176. De Murcia JM, Niedergang C, Trucco C, Ricoul M, Dutrillaux B, Mark M, Oliver FJ, Masson M, Dierich A, Le Meur M, Walztinger C, Chambon P, De Murcia G. (1997). Requirement of poly (ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proc Natl Acad Sci USA 94, 73037307. De Raad S, Comte S, Nef P, Lenz SE, Gundelnger ED, Cox JA. (1995). Distribution pattern of 3 neural calcium binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retinal. Histochem J 27, 524535. De Vouge MW, Mukherjee BB. (1992). Transformation of normal rat kidney cells by v-Kras enhances expression of transin 2 and an S-100-related calcium-binding protein. Oncogene 7, 109119. De Waard M, Pragnall M, Campbell KP. (1994). Ca2+ channel regulation by a conserved subunit domain. Neuron 13, 495503. Dear TN, Matena K, Vingren M, Boehm T. (1997). A new subfamily of vertebrate calpains lacking a calmodulin-like domain. Implications for calpain regulation and evolution. Genomics 45, 175184. Decorti G, Malusa N, Furlan G, Candussio L, Klugmann FB. (1999). Endocytosis of gentamicin in a proximal tubular renal cell line. Life Sci 65, 11151124. Dedhar S. (1990). Integrins and tumour invasion. BioEssays 12, 583590. Dedhar S. (1994). Novel functions for calreticulin. Interaction with integrins and modulation of gene expression. Trend Biol Sci 19, 269271. Degen E, Williams DB. (1991). Participation of a novel 88 kDa protein in the biogenesis of murine class I histocompatibility molecules. J Cell Biol 112, 10991115.
References
267
Delaney SJ, Hayward DC, Barleben F, Fischbach KF, Mikios GL. (1991). Molecular cloning and analysis of small optic lobes. A structural brain gene of Drosophila melanogaster. Proc Natl Acad Sci USA 88, 72147218. Delany AM, Canalis E. (1998). Basic broblast growth factor destabilises osteonectin mRNA in osteoblasts. Am J Physiol 43, C734C740. Delany AM, Young MF, Canalis E. (1996). Basic broblast growth factor (bFGF) downregulates osteonectin expression in osteoblasts by transcriptional mechanisms. J Bone Mineral Res 11, T413. Delmas PD, Demiaux B, Malaval L, Chapuy MC, Eduard C, Meunier PJ. (1986). Serum bone gamma carboxyglutamic acid containing protein in primary hyperthyroidism and in malignant hypercalcemia. J Clin Invest 77, 985991. Delmas PD, Malaval L, Arlot M, Meunier PJ. (1985). Serum bone gla-protein compared to bone histomorphometry in endocrine diseases. Bone 6, 329341. Delmas PD, Wahner HW, Mann KG, Riggs BL. (1983). Assessment of bone turnover in postmenopausal osteoporosis by measurement of serum bone gla-protein. J Lab Clin Med 102, 470476. Delvalle ME, Ciriaco E, Bronzeti E, Albuerne M, Naves FJ, Germana G, Vega JA. (1995). Calcium binding proteins in avian Herbst and Grandry sensory corpuscles. Anat Rec 243, 272281. Denissenko MF, Chen JX, Tang MS, Pfeifer GP. (1997). Cytosine methylation determines hot spots of DNA damage in the human p53 gene. Proc Natl Acad Sci USA 94, 38933898. Denning GM, Leidal KG, Hoist VA, Iyer SS, Pearson DW, Clark JR, Nauseef WM, Clark RA. (1997). Calreticulin biosynthesis and processing in human myeloid cells. Demonstration of signal peptide cleavage and N-glycosylation. Blood 90, 372381. Denton KJ, Stretch JR, Gatter KC, Harris AL. (1992). A study of adhesion molecules as markers of progression of melanomas. J Pathol 167, 187191. Deramaudt BMJM, Braunstein S, Remy P, Abraham NG. (1998). Gene transfer of human heme oxygenase into coronary endothelial cells potentially promotes angiogenesis. J Cell Biochem 68, 121127. Deryugina EL, Bourdon MA, Reisfeld RA, Strongin A. (1998). Remodelling of collagen matrix by human tumour cells requires activation and cell surface association of matrix metalloproteinase-2. Cancer Res 58, 37433750. Desdouits F, Buxbaum JD, Desdouits-Magnen J, Nairn AC, Greengard P. (1996). Amyloid beta peptide formation in cell free preparations. Regulation by protein kinase C, calmodulin, and calcineurin. J Biol Chem 271, 2467024674. Deshpande RV, Goust JM, Banik NL. (1993). Differential distribution of calpain in human lymphoid cells. Neurochem Res 18, 767773. Deshpande RV, Goust JM, Chakrabarti AK, Barbosa E, Hogan EL, Banik NL. (1995a). Calpain expression in lymphoid cells. Increased messenger RNA and protein levels after activation. J Biol Chem 270, 24972505. Deshpande RV, Goust JM, Hogan EL, Banik NL. (1995b). Calpain secreted by activated human lymphoid cells degrades myelin. J Biol Chem 42, 259262. Devarajan P, Stabach PR, De Matteis MA, Morrow JS. (1997). Na, K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin ankyrin G119 skeleton in Madin Darby canine kidney cells. Proc Natl Acad Sci USA 94, 1071110716. Devarajan P, Stabach PR, Mann AS, Ardito T, Kashgarian M, Morrow JS. (1996). Identication of a small cytoplasmic ankyrin (Ank[G119]) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus. J Cell Biol 133, 819830.
268
Dewhurst LO, Gee JW, Rennie IG, MacNeil S. (1997). Tamoxifen, 17-beta oestradiol and the calmodulin antagonist J8 inhibit human melanoma cell invasion through bronectin. Br J Cancer 75, 860868. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandol PP. (1998). PTEN is essential for embryonic development and tumour suppression. Nature Genet 19, 348355. Di Nublile MJ, Huang S. (1997). Capping of the barbed ends of actin laments by a high afnity prolin-actin complex. Cell Motility Cytoskeleton 37, 211225. DiPaolo G, Antosson B, Kassel D, Riederer BM, Grenningloh G. (1997). Phosphorylation regulates the microtubule-destabilising activity of stathmin and its interaction with tubulin. FEBS Lett 416, 149152. Doglioni C, Tos APD, Laurino L, Iuzzoliono P, Chiarelli C, Celio MR, Viale G. (1996). Calretinin, a novel immunocytochemical marker for mesothelioma. Am J Surg Pathol 20, 10371046. Dohlman GH, Thorner J. (1997). RGS proteins and signalling by heterotrimeric G proteins. J Biol Chem 272, 38713874. Dolphin AC, Page KM, Berrow NS, Stephens GJ, Canti C. (1999). Dissection of the calcium channel domains responsible for modulation of neuronal voltage-dependent calcium channels by G proteins. Ann NY Acad Sci 868, 160164. Domanskajanik K, Denechaud B, Inomata M, Kawashima S, Zalewska T. (1992). Calciumactivated neutral protease (CANP) in normal and dysmyelinating mutant paralytic tremor rabbit myelin. Mol Chem Neuropathol 16, 273288. Dominguez L, Yunes RMF, Fornes MW, Burgos M, Mayorga LS. (1999). Calcium and phospholipase A2 are both required for the acrosome reaction mediated by G-proteins stimulation in human spermatozoa. Mol Reprod Dev 52, 297302. Donato R, Giambanco I, Aisa MC, Di Geronimo C, Ceccarelli P, Rambotti MG, Spreca A. (1989). Cardiac S-100A0 protein. Purication by a simple procedure and related immunocytochemical and immunochemical studies. Cell Calcium 10, 8192. Donoghue S, Baden HS, Lauder I, Sobolewski S, Pringle JH. (1999). Immunohistochemical localisation of caspase-3 correlates with clinical outcome in B-cell diffuse large cell lymphoma. Cancer Res 59, 53865391. Dorin JR, Emslie E, Van Heiningen V. (1990). Related calcium binding proteins map to the same region of chromosome 1 and to an extended region of synteny on mouse chromosome 3. Genomics 8, 420426. Dowjat WK, Wisniewski T, Efthimiopoulos S, Wisniewski HM. (1999). Inhibition of neurite outgrowth by familial Alzheimers disease-linked presenilin-1 mutations. Neurosci Lett 267, 141144. Downs AMR, Ward KA, Peachey RDG. (1997). Subungual squamous cell carcinoma in Dariers disease. Clin Exp Dermatol 22, 277279. Drechsel DN, Hyman AA, Cobb MH, Kirschner MW. (1992). Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol Biol Cell 3, 11411154. Dresselhaus T, Hagel C, Lorz H, Kranz E. (1996). Isolation of full-length cDNA encoding calreticulin from a PC library of in vitro zygotes of maize. Plant Mol Biol 31, 2334. Droillard MJ, Guclu J, Le Caer JP, Mathieu Y, Guern J, Lauriere C. (1997). Identication of calreticulin-like protein as one of the phosphoproteins modulated in response to oligogalacturonides in tobacco cells. Planta 202, 341348. Drummer R, Joller-Jemelka HI, Boni R, Burg G. (1997). Serum S100. A marker for disease monitoring in metastatic melanoma. Dermatology 194, 208212.
References
269
Du XP, Saido TC, Tsubuki S, Indig FE, Williams MJ, Ginsberg MH. (1995). Calpain cleavage of the cytoplasmic domain of the integrin beta (3) subunit. J Biol Chem 270, 2614626151. Duarte WR, Iimura T, Takenaga K, Ohya K, Ishikawa I. (1999). Extracellular role of S100A4 calcium binding protein in the periodontal ligament. Biochem Biophys Res Commun 255, 416420. Dubreuil RR, MacVicar G, Dissanayake S, Liu CH, Homer D, Hortsch M. (1996). Neuroglianmediated cell adhesion induces assembly of the membrane skeleton at cell contact sites. J Cell Biol 133, 647655. Ducibella T, Anderson E, Albertini DF, Aalberg J, Rangarajan S. (1988). Qualitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation. Dev Biol 130, 184197. Ducibella T, Kuraswa S, Duffy P, Kopf GS, Schultz RM. (1993). Regulation of the polyspermy block in the mouse egg. Maturation-dependent differences in cortical granule exocytosis and zona pellucida modications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C. Biol Reprod 48, 12511257. Duggan DJ, Manchester D, Stears KP, Mathews DH, Hart C, Hoffman EP. (1997). Mutations in the -sarcoglycan gene (LGMD-2F) are a rare cause of autosomal recessive limb girdle muscular dystrophy. Neurology 48, 49001. Duhaiman AS, Bamburg JR. (1984). Isolation of brain alpha-actinin. Its characterisation and comparison of its properties with those of muscle alpha-actinins. Biochemistry 23, 16001608. Dumonteil E, Barry H, Meissner G. (1994). Effects of palmitoyl carnitine and related metabolites on the avian Ca2+-ATPase and Ca2+ release channel. J Physiol 479, 2939. Duncan LM, Bouffard D, Howard C, Mihm MC, Byers HR. (1996). In situ distribution of integrin alpha2 beta1 and alpha-actinin in melanocytic proliferations. Modern Pathol 9, 938943. Duncan RJ, Higgins J, Dunn RJ, Allore R, Marks A. (1989). Rened localisation of the human genome encoding the subunit of the S100 protein (S100B) and conrmation of a subtle t(9,21) translocation using in situ hybridisation. Cytogenet Cell Genet 50, 234235. Dunlap K, Luebke JI, Turner TJ. (1995). Exocytotic Ca2+ channels in mammalian neurone. Trends Neurosci 18, 8998. Dunn CJ, Chosay JG, Essani NA, Cruz R, Manning AM, Galinet LA, Eversole RR, Yem AW, Deibel MR. (1966). Increased expression of neutrophil MRP8 and MRP14 is associated with vascular adhesion molecule activation and differential leukocyte inltration in delayed-type hypersensitivity suggesting a pro-inammatory role for S100 calcium binding proteins. Int J Immunopathol Pharmacol 9, 7994. Duraj J, Kovcikova M, Sedlak J, Koppel J, Sobel A, Chorvath B. (1995). The protein kinase C inhibitor blocks phosphorylation of stathmin during TPA-induced growth inhibition of human pre-B leukaemia REH6 cells. Leukaemia Res 19, 457461. Dutartre H, Davoust J, Gorvel JP, Chavrier P. (1996). Cytokinesis arrest and redistribution of actin cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42HS. J Cell Sci 109, 367377. Eastell R, Robins SP, Colwell T, Assiri AMA, Riggs BL, Russell RGG. (1993). Evaluation of bone turnover in type-1osteoporosis using biochemical markers specic for both bone formation and bone resorption. Osteoporosis Int 3, 255260. Ebralidze A, Florene V, Lukanidin E, Fostad O. (1990). The murine mts1 gene is highly expressed in metastatic but not in non-metastatic human tumour cells. Clin Exp Metastasis 8 (suppl. 1), 35.
270
Edelman CF, George SE, Means AR, Schulman H, Yaswen P. (1994). Selective activation and inhibition of calmodulin-dependent enzymes by a calmodulin-like protein found in human epithelial cells. Eur Biochem 226, 725730. Edward M, Gold JA, Mackie RM. (1989). Modulation of melanoma cell adhesion to basement membrane components by retinoic acid. J Cell Sci 93, 155161. Edward M, Gold JA, Mackie RM. (1992). Retinoic acid-induced inhibition of metastatic melanoma cell lung colonisation and adhesion to endothelial and sub-endothelial extracellular matrix. Clin Exp Metastasis 10, 6167. Edward M, Mackie RM. (1990). Retinoic acid-induced inhibition of lung colonisation and changes in the synthesis properties of glycosaminoglycans of metastatic B16 melanoma cells. J Cell Sci 94, 537543. Efstathiou JA, Liu D, Wheeler JMD, Kim HC, Beck NE, Ilyas M, Karayiannakis AJ, Mortensen NJM, Kmiot W, Playford RJ, Pignatelli M, Bodmer WF. (1999). Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells. Proc Natl Acad Sci USA 96, 23162321. Egea J, Espinet C, Comella JX. (1998). Calmodulin modulates mitogen-activated protein kinase activation in response to membrane depolarisation in PC12 cells. J Neurochem 70, 25542564. Eggleton P, Reid KBM, Kishore U, Sontheimer RD. (1997). Clinical relevance of calreticulin in systemic lupus erythematosus. Lupus 6, 564571. Ehlers MD, Zhang S, Bernhardt IP, Huganir RL. (1996). Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84, 745755. Eichinger L, Bahler M, Dietz M, Eckerskorn C, Shleicher M. (1998). Characterisation and cloning of a Dictyostelium Ste20-like protein kinase that phosphorylates the actin binding protein severin. J Biol Chem 273, 1295212959. Eichinger L, Noegel AA, Schleicher M. (1991). Domain structure in actin binding proteins. Expression and functional characterisation of truncated severin. J Cell Biol 112, 665676. El-Hayek RC, Valdivia C, Valdivia HH, Hogan K, Coronado R. (1993). Activation of the Ca2+-release channel of skeletal muscle sarcoplasmic reticulum by palmitoyl carnitine. Biophys J 65, 779789. Elwess NL, Filoteo AG, Enyedi A, Penniston JT. (1997). Plasma membrane Ca2+ pump isoforms 2a and 2b are unusually responsive to calmodulin and Ca2+. J Biol Chem 272, 1798117986. Elwess NL, Filoteo AG, Enyedi A, Penniston JT. (1998). Plasma membrane calcium pump isoform 2 has an unusually high afnity for calmodulin and calcium. Biophys J 74, A42. Emori Y, Saigo K. (1994). Calpain localisation changes in co-ordination with actin-related changes during early embryonic development of Drosophila. J Biol Chem 269, 2513725142. Emoto Y, Kobayashi R, Akatsuka H, Hidaka H. (1992). Purication and characterisation of a new member of the S100 protein family from human placenta. Biochem Biophys Res Commun 182, 12461253. Enari M, Hug H, Nagata S. (1995). Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature 375, 7881. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. (1998). A caspaseactivated DNase that degrades DNA during apoptosis and its inhibitor ICAD. Nature 391, 4350. Enderlin S, Norman AW, Celio MR. (1987). Ontogeny of the calcium binding protein calbindin D28K in the rat nervous system. Anat Embryol 177, 1528.
References
271
Endo T, Masaki T. (1984). Differential expression and distribution of chicken skeletal muscletype and smooth muscle-type alpha-actinins during myogenesis in culture. J Cell Biol 99, 23222332. Engelkamp E, Schafer BW, Mattei MG, Erne P, Heizmann CW. (1993). Six S100 genes are clustered on human chromosome 1q21. Identication of two genes coding for two previously unreported calcium-binding proteins S100D and S100E. Proc Natl Acad Sci USA 90, 65476551. Engelman JA, Lee RJ, Karnezis A, Bearss DJ, Webster M, Siegel P, Muller WJ, Windle JJ, Pesell RG, Lisanti MP. (1998). Reciprocal regulation of neu tyrosine kinase activity and caveolin-l protein expression in vitro and in vivo. Implications for breast cancer. J Biol Chem 273, 2044820455. Enyedi A, Elwess NL, Filoteo AG, Verma AK, Paszty K, Penniston JT. (1997). Protein kinase C phosphorylates the a forms of plasma membrane Ca2+ pump isoforms 2 and 3 and prevents binding of calmodulin. J Biol Chem 272, 2752527528. Errabolu R, Sanders MA, Salisbury JL. (1994). Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles. J Cell Sci 107, 916. Ettinger AJ, Feng GP, Sanes JR. (1997). -sarcoglycan, a broadly expressed homologue of the gene mutated in limb girdle muscular dystrophy 2D. J Biol Chem 272, 3253432538. Eustace W, Johnson B, Jones NA, Rowlands DC, Williams A, Guest SS, Brown G. (1995). Down regulation but not phosphorylation of stathmin is associated with induction of HL-60 cell growth arrest and differentiation by physiological agents. FEBS Lett 364, 309313. Everitt EA, Sage EH. (1992). Expression of SPARC is correlated with altered morphologies in transfected F9 embryonal carcinoma cells. Exp Cell Res 199, 134146. Faddis BT, Hasbani MJ, Goldberg MP. (1997). Calpain activation contributes to dendritic remodelling after brief exitotoxic injury in vitro. J Neurosci 17, 951959. Fagotto F, Funayama N, Gluck U, Gumbiner BM. (1996). Binding to cadherins antagonises the signalling activity of beta catenin during axis formation in Xenopus. J Cell Biol 132, 11051114. Famulski KS, Paterson MC. (1999). Defective regulation of Ca2+/calmodulin-dependent protein kinase II in gamma irradiated ataxia telangiectasia broblasts. FEBS Lett 453, 183186. Fanjul M, Renaud W, Merten M, Guy-Crotte O, Hollande E, Figarelle C. (1995). Presence of MRP8 and MRP14 in pancreatic cell lines. Differential expression and localisation in CFPAC-1 cells. Am J Physiol 268, C1241C1251. Fano G, Marsili V, Angelella P, Aisa MC, Giambanco I, Donato R. (1989). S100A0 protein stimulates Ca2+-induced Ca2+ release from isolated sarcoplasmic reticulum vesicles. FEBS Lett 255, 381384. Farias EF, Ghiso JAA, Ladeda V, Joffe EBD. (1998). Verapamil inhibits tumour protease production, local invasion and metastasis development in murine carcinoma cells. Int J Cancer 78, 727734. Farkas LM, Suter-Crazzolara C, Unsicker K. (1997). GDNF induces the calretinin phenotype in cultures of embryonic striatal neurones. J Neurosci Res 50, 361372. Fath KR, Trimbur GM, Burgess DR. (1997). Molecular motors and a spectrin matrix associate with Golgi membranes in vitro. J Cell Biol 139, 11691181. Fattoum A. (1997). Contractile events in smooth muscle cells. M S Med Sci 13, 777789. Fazely F, Ledninko N. (1990). Retinoid acid and epidermal growth factor binding in retinoidmediated invasion suppressed lung carcinoma cells. Anticancer Res 10, 667670.
272
Fearnhead HO, Rodriguez J, Govek EE, Guo W, Kobayashi R, Hannon G, Lazebnik YA. (1998). Oncogene-dependent apoptosis is mediated by caspase-3. Proc Natl Acad Sci USA 95, 1366413669. Feinberg AP, Vogelstein B. (1983). Hypomethylation of ras oncogenes in primary human cancer. Biochem Biophys Res Commun 111, 4754. Feinberg J, Heitz F, Benyamin Y, Roustan C. (1996a). The N-terminal sequence (5-20) of thymosin 4 binds to monomeric actin in an -helical conformation. Biochem Biophys Res Commun 222, 127132. Feinberg J, Lebart MC, Benyamin Y, Roustan C. (1997b). Localisation of calcium sensitive binding site for gelsolin on actin sub-domain I. Implications for severing process. Biochem Biophys Res Commun 233, 6165. Feinberg J, Mery J, Heitz F, Benyamin Y, Roustan C. (1997a). Conformational and functional studies of three gelsolin sub-domain I synthetic peptides and their implication in actin polymerisation. Biopolymers 41, 645655. Feinberg J, Mery J, Heitz F, Beyamin Y, Roustan C. (1996b). Correlations between biological activity and structural properties for two short homologous sequences in thymosin beta4 and gelsolin. Int J Peptide Protein Res 47, 6269. Ferrary E, Cohen-Tannoudji M, Pehau-Arnaudet G, Lapillonne A, Athman R, Ruiz T, Boulouha L, El Marjour F, Doye A, Fontaine JJ, Antony C, Babinet C, Louvard D, Jaisser F, Robine S. (1999). In vivo, villin is required for Ca2+-dependent F-actin disruption in intestinal brush borders. J Cell Biol 146, 819829. Ferreira A, Kincaid R, Kosik KS. (1993). Calcineurin is associated with the cytoskeleton of cultured neurones and has a role in the acquisition of polarity. Mol Biol Cell 12, 12251238. Fietz MJ, Rogers GE, Eyre HJ, Baker E, Callen DF, Sutherland GR. (1992). Mapping of the trichohyalin gene co-localisation with the prolaggrin, involucrin, and loricrin genes. J Invest Dermatol 99, 542544. Filipovic DM, Meng XM, Reeves WB. (1999). Inhibition of PARP prevents oxidant-induced necrosis but not apoptosis in LLC-PK1 cells. Am J Physiol Renal Physiol 46, F428F436. Finlay CA, Hinds PW, Tan TH, Eiyahu D, Oren M, Levine AJ. (1988). Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with altered half-life. Mol Cell Biol 8, 531539. Fischer I, Romano-Clarke G, Grynspan F. (1991). Calpain-mediated proteolysis of microtubule associated proteins MPA1B and MAP2 in developing brain. Neurochem Res 16, 891898. Fischer R, Julsgart J, Berchtold MW. (1998). High afnity calmodulin target sequence in the signalling molecule PI 3-kinase. FEBS Lett 425, 175177. Flaherty KM, Zozulya S, Stryer L, DB. (1993). 3-dimensional structure of recoverin, a calcium sensor in vision. Cell 75, 709716. Fleet A, Ashworth R, Kubista H, Edwards H, Bolsover S, Mobbs P, Moss SE. (1999). Inhibition of EGF-dependent calcium inux by annexin VI is splice form-specic. Biochem Biophys Res Commun 260, 540546. Flood G, Kahana E, Gilmore AP, Rowe AJ, Gratzer WB, Critchley DR. (1995). Association of structural repeats in the alpha-actinin rod domain. Alignment of inter-subunit interactions. J Mol Biol 252, 227234. Florenes VA, Aamdal S, Myklebost O, Maelandsmo GM, Bruland OS, Fostad O. (1992). Levels of nm23 messenger RNA in metastatic malignant melanomas: Inverse correlation to disease progression. Cancer Res 52, 60886091.
References
273
Florman HM, Tombes RM, First NL, Babcock DF. (1989). An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis. Dev Biol 135, 133146. Fohr UG, Heizmann CW, Engelkamp D, Schafer BW, Cox JA. (1995). Purication and cation binding properties of the recombinant human S100 calcium binding protein A3, an EFhand motif with high afnity for zinc. J Biol Chem 270, 2105621061. Folger PA, Berg WJ, De Jesus Z, Fong Y, Pardee J. (1999). A mammalian severin replaced gelsolin in transformed epithelial cells. Cancer Res 59, 53495355. Folpe AL, Gown AM. (1997). Calretinin, a mesothelioma associated protein, is a sensitive marker of ovarian serous carcinoma. Lab Invest 76, 575. Ford HL, Silver DL, Kachar B, Sellers JR, Zain SB. (1997). Effect of mts1 on the structure and activity of non-muscle myosin II. Biochemistry 36, 1632116327. Forrest ARR, McCormack AK, De Souza CPC, Sinnamon JM, Tonks ID, Hayward NK, Ellem KAO, Gabrielli BG. (1999). Multiple splicing of cdc25B regulate G2/M progression. Biochem Biophys Res Commun 260, 510515. Fortier AH, Nelson BJ, Grella DK, Holaday JW. (1999). Anti-angiogenic activity of prostate specic antigen. J Natl Cancer Inst 91, 16351640. Fougerousse F, Durand M, Suel L, Pourquie O, Delezoide AL, Romero NB, Abitbol M, Beckmann JS. (1998). Expression of genes (capn3, SGCA, SGCB and TTN) involved in progressive muscular dystrophies during early human development. Genomics 48, 145156. Fougerousse F, Broux O, Richard I, Allaand V, De Souza AP, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Chiannilkulchai N, Hillaire D, Bui H, Chumakoov I, Weissenbach J, Cherif D, Cohen D, Beckmann JS. (1994). Mapping of a chromosome 15 region involved in limb girdle muscular dystrophy. Human Mol Genet 3, 285293. Fox SB, Turner GDH, Gatter KC, Harris AL. (1995). The increased expression of adhesion molecules ICAM-3, E-selectin and P-selectin on breast cancer endothelium. J Pathol 177, 369376. Fra AM, Mastroiani N, Mancini M, Pasqualetto E, Sitia R. (1999). Human caveolin-1 and caveolin-2 are closely linked genes co-localised with WI-5336 in a region of 7q31 frequently deleted in tumours. Genomics 56, 355356. Francia G, Mitchell SD, Moss SE, Hanby AM, Marshall JF, Hart IR. (1996). Identication by differential display of annexin VI, a gene differentially expressed during melanoma progression. Cancer Res 56, 38553858. Frank M, Wolburg H. (1996). Cellular reactions at the lesion site after crushing of the rat optic nerve. Glia 16, 227240. Frantz GD, Tobin AJ. (1994). Cellular distribution of calbindin D28K messenger RNAs in the adult mouse brain. J Neurosci Res 37, 287302. Franz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, Tocci MJ, OKeefe SJ, ONeill EA. (1994). Calcineurin acts in synergy with PMA to inactivate IB/MAD3, an inhibitor of NF-B. EMBO J 13, 861870. Franzen B, Linder S, Alaiya AA, Eriksson E, Fujioka K, Bergman AC, Jornvall H, Auer G. (1997). Analysis of polypeptide expression in benign and malignant human breast lesions. Electrophoresis 18, 582587. Franzen B, Linder S, Uryu K, Alaiya AA, Hirano T, Kato H, Auer G. (1996). Expression of tropomyosin isoforms in benign and malignant human breast lesions. Br J Cancer 73, 909913. Fraser A, Evan G. (1996) A licence to kill. Cell 85, 781784.
274
Fraser IDC, Copeland O, Wu B, Marston SB. (1997). The inhibitory complex of smooth muscle caldesmon with actin and tropomyosin involves three interacting segments of the C-terminal domain 4. Biochemistry 36, 54835492. Frederick MJ, Rodriguez LV, Johnston DA, Darnay BG, Grimm EA. (1996). Characterisation of the Mr 65,000 lymphokine-activated killer proteins phosphorylated after tumour target binding. Evidence that pp65a and 65b are phosphorylated forms of L-plastin. Cancer Res 56, 138144. Friederich E, Kreis TE, Louvard D. (1993). Villin-induced growth of microvilli is reversibly inhibited by cytochalasin-D. J Cell Sci 105, 765775. Friederich E, Van Compernolle K, Louvard D, Van de Kerckhove J. (1999). Villin function in the organisation of the actin cytoskeleton. Correlation of in vivo effects to its biochemical activities in vitro. J Biol Chem 274, 2675126760. Friedman PA. (1999). Calcium transport in the kidney. Curr Opin Nephrol Hypertension 8, 589595. Friedrich B, Gronberg H, Landstrom M, Gullberg M, Bergh A. (1995). Differentiation-stage specic expression of oncoprotein-18 in human and rat prostatic adenocarcinoma. Prostate 27, 102109. Fritz G, Heizmann CW, Kroneck PMH. (1998). Probing the structure of the human Ca2+- and Zn2+-binding protein S100A3. Spectroscopic investigations of its transition metal complexes, and three-dimensional structure model. Biochim Biophys Acta 1448, 264276. Fujii T, Hiromori T, Hamamoto M, Suzuki T. (1997). Interaction of chicken gizzard smooth muscle calponin with brain microtubules. J Biochem 122, 344351. Fujii T, Oomatsuzawa A, Kuzumaki N, Kondo Y. (1994). Calcium dependent regulation of smooth muscle calponin by S100. J Biochem 116, 121127. Fujii T, Yamana K, Ogoma Y, Kondo Y. (1995). Interaction of calponin with phospholipids. J Biol Chem 117, 9991003. Fujimoto T, Miyawaki A, Mikoshiba K. (1995). Inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to the actin lament. J Cell Sci 108, 715. Fujimoto T. (1993). Calcium pump of the plasma membrane is localised in caveolae. J Cell Biol 120, 11471157. Fujino I, Yamada N, Miyawaki A, Hasegawa M, Furuichi T, Mikoshiba K. (1995). Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor messenger RNAs in various mouse tissues. In situ hybridisation study. Cell Tissue Res 280, 201210. Fujita T. (1999). Senescence marker protein-30 (SMP30). Structure and biological function. Biochem Biophys Res Commun 254, 14. Fukada T, Ohtani T, Yoshida Y, Shirogane T, Nishida K, Nakajima K, Hibi M, Hirano T. (1998). STAT3 orchestrates contradictory signals in cytokine-induced G(1)-S cell cycle transition. EMBO J 17, 66706677. Fukata M, Nakagawa M, Kuroda S, Kaibuchi K. (1999). Cell adhesion and Rho small GTPases. J Cell Sci 112, 44914500. Fukui Y, Masuda H, Takagi M, Takahashi K, Kiyokane K. (1997). The presence of h2-calponin in human keratinocyte. J Dermatol Sci 14, 2936. Fukushima A, Motohashi Y, Sakuma K. (1998). Transcriptional regulation of rat calbindin expression during development determined by bacterially expressed protein. J Nut Sci Vitaminol 44, 137149. Fulceri R, Knudsen J, Giunti R, Volpe P, Nori A, Benedetti A. (1997). Fatty acyl-CoA acylCoA-binding protein complexes activate the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum. Biochem J 325, 423428.
References
275
Fulceri R, Nori A, Gamerucci A, Volpe P, Giunti R, Benedetti A. (1994). Fatty acyl-CoA esters induce calcium release from terminal cisternae of skeletal muscle. Cell Calcium 15, 109116. Fullmer CS. (1997). Lead-calcium interactions. Involvement of 1,25-dihydroxy vitamin D. Environmental Res 72, 4555. Fulton D, Gratton JP McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulous A, Sessa WC. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597601. Funk SE, Sage EH. (1991). The Ca2+-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells. Proc Natl Acad Sci USA 88, 26482652. Funk SE, Sage EH. (1993). Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and broblasts. J Cell Physiol 154, 5363. Furnari FB, Lin H, Huang HS, Cavenee WK. (1997). Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc Natl Acad Sci USA 94, 1247912484. Furukawa F, Fujii K, Horiguchi Y, Matsuyoshi N, Fujita M, Toda K, Imamura S, Wakita H, Shirahama S, Takigawa M. (1997). Roles of E- and P-cadherin in the human skin. Microsc Res Technique 38, 343352. Fyrberg E, Kelly M, Ball E, Fyrberg C, Reedy MC. (1990). Molecular genetics of Drosophila alpha-actinin. Mutant alleles disrupt Z-disk integrity and muscle insertions. J Cell Biol 110, 19992011. Gagnon AM, Welsh JE. (1997). Modulation and phosphorylation of calbindin-D 28K correlates with protein kinase C activation. Biochem Cell Biol 75, 3340. Gallagher PJ, Stull JT. (1997). Localisation of an actin binding domain in smooth muscle myosin light chain. Mol Cell Biochem 173, 5157. Galler S, Hilber K, Gohlsch B, Pette D. (1997). Two functionally distinct myosin heavy chain isoforms in slow skeletal muscle bres. FEBS Lett 410, 150152. Gallo O, Masini E, Morbidelli L, Franchi A, Fini-Strochi I, Vergari WA, Ziche M. (1998). Role of nitric oxide in angiogenesis and tumour progression in head and neck cancer. J Natl Cancer Inst 90, 587596. Gamosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, Ehrlich M. (1983). The 5-methylcytosine content of DNA from human tumours. Nucleic Acis Res 11, 68836894. Gander JC, Bustos-Castillo M, Stuber D, Hunziker W, Celio M, Schwaller B. (1996). The calcium binding protein calretinin-22K, an alternative splicing product of the calretinin gene is expressed in several colon adenocarcinoma cell lines. Cell Calcium 20, 6372. Garbers DL, Kopf GS. (1980). The regulation of spermatozoa by calcium and cyclic nucleotides. Adv Cyclic Nucleotides Res 13, 251306. Garbers DL, Tubb DJ, Hyne RV. (1982). A requirement of bicarbonate for Ca2+-induced elevations of cyclic AMP in guinea pig spermatozoa. J Biol Chem 257, 89808984. Garcia JGN, Verin AD, Herenyiova M, English D. (1998). Adherent neutrophils activate endothelial myosin light chain kinase: Role in transendothelial migration. J Appl Physiol 84, 18171821. Garcia JGN, Verin AD, Schaphorst K, Siddiqui R, Patterson CE, Csortos C, Natarajan V. (1999). Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60 (src). Am J Physiol 20, L989L998. Garcia-Rostan G, Tallini G, Herrero A, DAquila TG, Carcangiu ML, Rimm DL. (1999). Frequent mutation and nuclear localisation of beta catenin in anaplastic thyroid carcinoma. Cancer Res 59, 18111815.
276
Gaub HE, Fernandez JM. (1998). The molecular elasticity of individual proteins studied by AFM-related techniques. Alexander von Humboldt Magazine No. 71. 1118. Geier BM, Wiech H, Schiebel E. (1996). Binding of centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components KAR1p and SPC110p. J Biol Chem 271, 2833628374. Geiger B, Ginsberg D, Salomon D, Volberg T. (1990). The molecular basis for the assembly and modulation of adherens-type junctions. Cell Differentiation Dev 32, 343353. Gervais FG, Xu DG, Robertson GS, Vaillancourt JP, Zhu YX, Huang JQ, Le Blanc A, Smith D, Rigby M, Shearman MS, Clarke FE, Zheng H, Van der Ploeg LHT, Ruffolo SC, Thornberry NA, Xanthoudakis S, Zamboni RJ, Roy S, Nicholson DW. (1999). Involvement of caspases in proteolytic cleavage of Alzheimers beta precursor protein and amyloidogenic A beta peptide formation. Cell 97, 395406. Gianini G, Celnenti E, Cici R, Marziali G, Sorrentino V. (1992). Expression of a ryanodine receptor-Ca2+ channel that is regulated by TGF-beta. Science 257, 9194. Giarre M, Semenov MV, Brown AMC. (1998). Wnt signalling stabilises the dual function protein -catenin in diverse cell types. Ann NY Acad Sci 857, 4355. Gibbs FEM, Barraclough R, Platt-Higgins A, Rudland PS, Winkinson MC, Parry EW. (1995). Immunocytochemical distribution of the calcium binding protein p9Ka in normal rat tissues. Variation in the cellular location in different tissues. J Histochem Cytochem 43, 169180. Gibbs FEM, Wilkinson MC, Rudland PS, Barraclough R. (1994). Interaction in vitro of p9Ka, the rat S-100 related metastasis-inducing calcium binding protein. J Biol Chem 268, 1899218999. Gilbert RE, McNally PG, Cox A, Dziadek M, Rumble J, Cooper ME, Jerums G. (1995). SPARC gene expression is reduced in early diabetes-related kidney growth. Kidney Int 48, 12161225. Gill RK, Christakos S. (1995). Regulation by oestrogen through the 5-anking region of the mouse calbindin D 28K gene. Mol Endocrinol 9, 319326. Gilles C, Bassuk JA, Pulyaeva H, Sage EH, Foidart JM, Thompson EW. (1998). SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines. Cancer Res 58, 55295536. Gillespie GY, Soroceanu L, Manning TJ, Gladson CL, Rosenfeld SS. (1999). Glioma migration can be blocked by non-toxic inhibitors of myosin II. Cancer Res 59, 20762082. Gilman AG. (1989). G proteins and regulation of adenylyl cyclase. JAMA 262, 18191825. Gilmore AP, Parr T, Patel B, Gratzer WB, Critchley DR. (1994). Analysis of the phasing of four spectrin-like repeats in alpha-actinin. Eur J Biochem 225, 235242. Gilmour DT, Lyon GJ, Carlton MBL, Sanes JR, Cunningham JM, Anderson JR, Hogan BLM, Evans MJ, Colledge WH. (1998). Mice decient for the secreted glycoprotein SPARC/osteonectin/BM-40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J 17, 18601870. Gimona M, Small JV. (1996). Calponin. In: Biochemistry of smooth muscle contraction, ed. Barany M, Academic Press, New York, pp. 91103. Girard JP, Springer TA. (1995). Cloning from puried high endothelial venule cells of hevin, a close relative of the anti-adhesive extracellular matrix protein SPARC. Immunity 2, 113123. Girard JP, Springer TA. (1996). Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules. J Biol Chem 271, 45114517. Glass-Marmor L, Beitner R. (1997). Detachment of glycolytic enzymes from cytoskeleton of melanoma cells induced by calmodulin antagonists. Eur J Pharmacol 328, 241248.
References
277
Glass-Marmor L, Morgenstern H, Beitner R. (1996). Calmodulin antagonists decrease glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP and viability of melanoma cells. Eur J Pharmacol 313, 265271. Glenney JR, Kindy MS, Zokas L. (1989). Isolation of a new member of the S100 protein family. Aminoacid sequence, tissue, and subcellular distribution. J Cell Biol 108, 569578. Godi A, Sanone I, Pertile P, Devarajan P, Stabach PR, Morrow JS, Di Tullio G, Polishuck R, Petrucci TC, Luinin A. et al. (1998). ADP ribosylation factor regulates spectrin binding to the Golgi complex. Proc Natl Acad Sci USA 95, 86078612. Goedert M. (1996). Tau protein and the neurobrillary pathology of Alzheimers disease. Ann NY Acad Sci 777, 121131. Gold JS, Bao L, Ghoussoub RAD, Zetter BR, Rimm DL. (1997). Localisation and quantitation of expression of the cell motility related protein thymosin beta 15 in human breast tissue. Mod Pathol 10, 11061112. Goldberg D, Polly P, Eisman JA, Morrison NA. (1996). Identication of an osteocalcin gene promoter sequence that binds AP1. J Cell Biochem 60, 447457. Goldblum SE, Ding X, Funk SE, Sage EH. (1994). SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function. Proc Natl Acad Sci USA 91, 34483452. Goldsmith SC, Pokala M, Shen WY, Fedorov AA, Matsudaira P, Almo SC. (1997). The structure of an actin cross-linking domain from human mbrin. Nature Struct Biol 4, 708712. Goligorsky MS, Abedi H, Noiri E, Takhtajan A, Lense S, Romanov V, Zachary I. (1999). Nitric oxide modulation of focal adhesions in endothelial cells. Am J Physiol 276, C1271C1281. Golla R, Philp N, Safer D, Chintapalli J, Hoffman R, Collins L, Nachmias VT. (1997). Coordinate regulation of the cytoskeleton in 3T3 cells over-expressing thymosin beta (4). Cell Motility Cytoskeleton 38, 187200. Gomez-Marquez J, Delamo FF, Carpintero P, Anadon R. (1996). High levels of mouse thymosin beta (4) messenger RNA in differentiating P19 embryonic cells and during development of cardiovascular tissues. Biochim Biophys Acta 1306, 187193. Goode BL, Drubin DG, Lappalainen P. (1998). Regulation of the cortical actin cytoskeleton in budding yeast by twinlin, a ubiquitous actin monomer-sequestering protein. J Cell Biol 142, 723733. Gorczyca WA, Polans AS, Surgucheva IG, Subbaraya I, Baehr W, Palczewski K. (1995). Guanylyl cyclase activating protein. A calcium sensitive regulator of phototransduction. J Biol Chem 270, 2202922036. Gorczynska E, Spaliviero J, Handelsman DJ. (1994). The relationship between 3,5-cyclic adenosine monophosphate and calcium in mediating follicle stimulating hormone signal transduction in Sertoli cells. Endocrinology 134, 293300. Gormon AM, Orrenius S, Cecctelli S. (1998). Apoptosis in neuronal cells. Role of caspases. Neuroreport 9, R49R45. Gorodovikova EN, Gimelbrant AA, Senin II, Philippov PP. (1994b). Recoverin mediates the calcium effect upon rhodopsin phosphorylation and cGMP hydrolysis in bovine retina rod cells. FEBS Lett 349, 187190. Gorodovikova EN, Senin II, Philippov PP. (1994a). Calcium sensitive control of rhodopsin phosphorylation in the reconstituted system consisting of photoreceptor membranes, rhodopsin kinase and recoverin. FEBS Lett 353, 171172.
278
Goto K, Heymont JL, Klein-Nulend J, Kronenberg HM, Demay MD. (1996). Identication of an osteoblastic silencer element in the rst intron of the rat osteocalcin gene. Biochemistry 35, 1100511011. Gotzos V, Schwaller B, Gander JC, Bustos-Castillo M, Celio MR. (1996a). Heterogeneity of expression of the calcium binding protein calretinin in human colonic cancer cell lines. Anticancer Res 16, 34913498. Gotzos V, Vogt P, Celio MR. (1996b). The calcium binding protein calretinin is a selective marker for malignant pleural mesotheliomas of the epithelial type. Pathol Res Practice 192, 137147. Gould MN. (1997). Cancer chemoprevention and therapy by monoterpenes. Environ Health Perspect 105, 977979. Graceffa P, Adam LP, Morgan KG. (1996). Strong interaction between caldesmon and calponin. J Biol Chem 271, 3033630339. Graceffa P. (1995). Cross linking and uorescence study of the COOH-terminal and NH2terminal domains of intact caldesmon bound to actin. J Biol Chem 270, 3018730193. Graceffa P. (1997). Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin. Biochemistry 36, 37923801. Graether SP, Heinonen TYK, Raharjo WH, Jin JP, Mak AS. (1997). Tryptophan residues in caldesmon are major determinants for calmodulin binding. Biochemistry 36, 364369. Graham JD, Balleine RL, Milliken JS, Bilous AM, Clarke CL. (1997). Expression of osteonectin mRNA in human breast tumours is inversely correlated with oestrogen receptor content. Eur J Cancer 33, 16541660. Graves CB, Gale RC, Lurino JP, McDonald JM. (1986). The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin-stimulated phosphorylation of calmodulin. J Biol Chem 261, 1042910438. Gribenko A, Lopez MM, Richardson JM, Makhatadze GI. (1998). Cloning, over-expression, purication, and spectroscopic characterisation of human S100P. Protein Sci 7, 211215. Gribenko AV, Makhatadze GI. (1998). Oligomerisation and divalent ion binding properties of the S100P protein. A Ca2+/Mg2+ switch model. J Mol Biol 283, 679694. Grigorian M, Ambartsumian N, Lykkesfeldt AE, Bastholm L, Elling F, Georgiev G, Lukanidin E. (1996). Effect of mts1 (S100A4) expression on the progression of human breast cancer cells. Int J Cancer 67, 831841. Grigorian MS, Tulchinsky E, Burrone O, Tarabykina S, Georgiev G, Lukanidin E. (1994). Modulation of mts1 expression in mouse and human tumour cells. Electrophoresis 15, 463468. Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, Zaidi MS, Wisniewski HM. (1986a). Microtubule-associated protein tau. A component of Alzheimers paired helical laments. J Biol Chem 261, 60846089. Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wisniewski HM, Binder LI. (1986b). Abnormal phosphorylation of the microtubule-associated protein tau in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA 83, 49134917. Grundmann E, Ueda Y, Schneiderstock R, Roessner A. (1995). New aspects of cell biology in osteosarcoma. Pathol Res Pract 191, 563570. Gruver CL, Demayo F, Goldstein MA, Means AR. (1993). Targeted developmental overexpression of calmodulin induces proliferative and hypertrophic growth of cardiomyocytes in transgenic mice. Endocrinology 133, 376388. Grynspan F, Grifn WB, Mohan PS, Shea TB, Nixon RA. (1997). Calpains and calpastatin in SH-SY5Y neuroblastoma cells during retinoic acid-induced differentiation and neurite outgrowth. Comparison with the human brain calpain system. J Neurosci Res 48, 181191.
References
279
Gu JG, Tamura M, Pankov R, Danen EHJ, Takino T, Matsumoto K, Yamada KM. (1999). Shc and FAK differentially regulate cell motility and directionality modulated by PTEN. J Cell Biol 146, 389403. Gualberto A, Marquez G, Carballo M, Youngblood GL, Hunt SW, Baldwin AS, Sobrino F. (1998). P53 transactivation of the HIV-1 long terminal repeat is blocked by PD 144795, a calcineurin inhibitor with anti-HIV properties. J Biol Chem 273, 70887093. Guerini D, Garcia-Martin E, Gerber A, Volbracht C, Leist M, Merino CG, Carafoli E. (1999). The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurones is modulated by Ca2+. J Biol Chem 274, 16671676. Guerini D, Garcia-Martin E, Zecca A, Guidi F, Carafoli E. (1998). The calcium pump of the plasma membrane. Membrane targeting calcium binding sites, tissue specic isoform expression. Acta Physiol Scand 163, 265273. Guignard F, Mauel J, Markert M. (1995). Identication and characterisation of a novel human neutrophil protein related to the S100 family. Biochem J 309, 395401. Guignard F, Mauel J, Markert M. (1996). Phosphorylation of myeloid-related proteins MRP14 and MRP8 during neutrophil activation. Eur J Biochem 241, 265271. Guldberg P, Straten PT, Birck A, Ahrenkiel V, Kirkin AF, Zeuthen J. (1997). Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma. Cancer Res 57, 36603663. Gumbiner BM. (1996). Cell adhesion. The molecular basis of tissue architecture and morphogenesis. Cell 84, 345357. Gunji K, Skolnick C, Bednarczuk T, Benes S, Ackrell BAC, Cochran B, Kennerdell JS, Wall JR. (1998). Eye muscle antibodies in patients with ocular myesthenia gravis. Possible mechanism for eye muscle inammation in acetylcholine-receptor antibody-negative patients. Clin Immunol Immunopathol 87, 276281. Gunning P, Weinberger R, Jeffrey P. (1997). Actin and tropomyosin isoforms in morphogenesis. Anat Embryol 195, 311315. Gunthert U, Hofmann M, Rudy W, Reber S, Zoller M, Haussmann I, Matzku S, Wenzel A, Ponta H, Herrlich P. (1991). A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65, 1324. Guo TL, Miller MA, Datar S, Shapiro IM, Shenker BJ. (1998). Inhibition of poly (ADPribose) polymerase rescues human T lymphocytes from methylmercury-induced apoptosis. Toxicol Appl Pharmacol 152, 397405. Guo W, Campbell KP. (1995). Association of triadin with the ryanodine receptor and calsequestrin in the lumen of the sarcoplasmic reticulum. J Biol Chem 270, 90279030. Ha HC, Snyder SH. (1999). Poly (ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci USA 96, 1397813982. Hachiya T, Nakano H, Wake N, Sueishi R. (1995). In vitro invasiveness of human breast cancer cells through plasminogen activator activity independently regulated by hormones and transforming growth factor -1. Cancer J 8, 1320. Hafner M, Zimmermann K, Pottgieser J, Krieg T, Nischt R. (1995). A purine-rich sequence in the human BM-40 gene promoter region is a prerequisite for maximum transcription. Matrix Biol 14, 733741. Hagens G, Roulin K, Holz R, Saurat JH, Hellman U, Siegenthaler G. (1999). Probable interaction between S100A7 and EFABP in the cytosol of human keratinocytes from psoriatic scales. Mol Cell Biochem 192, 123128. Haglid KG, Yang Q, Hamberger A, Bergman S, Wilderberg A, Danielsen N. (1997). S-100 stimulates neurite outgrowth in the rat sciatic nerve grafted with acellular muscle transplants. Brain Res 753, 196201.
280
Hahn C, Hirsch B, Jahnke D, Durkop H, Stein H. (1999). Three new types of apaf-1 in mammalian cells. Biochem Biophys Res Commun 261, 746749. Hailat N, Keim DR, Melhem RF, Zhu X, Eckerskorn C, Bordeur GM, Reynolds CP, Seeger RC, Lottspeich F, Strahler JR, Hanash SM. (1991). High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplication. Clin Invest 88, 341345. Hailer NP, Blaheta RA, Harder S, Scholz M, Encke A, Markus BH. (1994). In vitro investigations on immunosuppression with verapamil. Zentrallblatt Chir 119, 347354. Haimoto H, Kato K. (1987). S100A0 (alpha-alpha) protein, a calcium-binding protein, is localised in the slow twitch muscle bre. J Neurochem 48, 917923. Hainsworth PJ, Henderson MS, Stillwell RG, Bennett RC. (1991). Comparison of EGFr, cerbB2 product and ras p21 immunohistochemistry as prognostic markers in primary breast cancer. Eur J Surg Oncol 17, 915. Hait WN, Gesmonde J, Cheng E. (1995). Effects of KS-501, KS-502 and their enentiomers on calmodulin-sensitive enzyme activity and cellular proliferation. Biochem Pharmacol 50, 6974. Hait WN, Gesmonde JF, Lazo JS. (1994). Effect of anti-calmodulin drugs on the growth and sensitivity of C6 glioma cells to bleomycin. Anticancer Res 14, 17111721. Hajkova L, Sjogren CB, Korenbaum E, Nordberg P, Karlsson R. (1997). Characterisation of a mutant prolin with reduced actin-binding capacity. Effects in vitro and in vivo. Exp Cell Res 234, 6677. Hakii H (1986). Thapsigargin. A histamine secretagogue, is a non-12-O- tetradecanoylphorbol-13-acetate (TPA) type tumour promoter in two stage mouse skin carcinogenesis. J Cancer Res Clin Oncol 111, 177181. Hakkarainen M, Wahlfors J, Myohanen S, Hiltunen MO, Eskelinen M, Johansson R, Janne J. (1996). Hypermethylation of calcitonin gene regulatory sequences in human breast cancer as revealed by genomic sequencing. Int J Cancer 69, 471474. Halpain S, Hipolito A, Saffer L. (1998). Regulation of F-actin stability in dendritic spines by glutamate receptors and calcineurin. J Neurosci 18, 98359844. Hamman HC, Gaffey LC, Lynch KR, Creutz CE. (1988). Cloning and characterisation of a cDNA encoding bovine endonexin (chromobindin-4). Biochem Biophys Res Commun 156, 660667. Hammond C, Helenius A. (1995). Quality control in the secretory pathway. Curr Opin Cell Biol 7, 523529. Hampton M, Orrenius S. (1997). Dual regulation of caspase activity by hydrogen peroxide. Implications for apoptosis. FEBS Lett 414, 552556. Han X, Li G, Li GH, Lin KC. (1997). Interaction between smooth muscle alpha-actinin and lipid bilayers. Biochemistry 36, 1036410371. Hanahisa Y, Yamaguchi M. (1998). Stimulatory effect of calcium binding protein regucalcin on phosphatase activity in the brain cytosol of rats with different ages. Brain Res Bull 46, 347351. Handley HH, Yu J, Yu DTY, Singh B, Gupta RS, Vaughan JH. (1996). Autoantibodies to human heat shock protein (hsp) 60 may be induced by Escherichia coli GroEL. Clin Exp Immunol 103, 429435. Hanein D, Matsudaira P, De Rosier DJ. (1997a). Evidence for a conformational change in actin induced by mbrin (N375) binding. J Cell Biol 139, 387396. Hanein D, Rost LE, Matsudaira P, De Rosier DJ. (1997b). 3D structures of F-actin decorated with two different actin-binding domains. Biophys J 72, A240.
References
281
Hanski C, Riede E, Gratchev A, Foss HD, Bohn C, Klussmann E. (1997). MUC2 gene suppression in human colorectal carcinomas and their metastases. In vitro evidence of the modulatory role of DNA methylation. Lab Invest 77, 685695. Hapak RC, Zhao HM, Boschi JM, Henzl MT. (1994a). Novel avian thymic parvalbumin displays high degree of sequence homology to oncomodulin. J Biol Chem 269, 52885296. Hapak RC, Zhao HM, Henzl MT. (1994b). Oligomerisation of an avian thymic parvalbumin. Chemical evidence for a Ca2+-specic conformation. FEBS Lett 349, 295300. Harb JM, Komorowski RA, Vitali CM. (1995). Metaplastic breast carcinoma invading chest wall. Ultrastruct Pathol 19, 439443. Hard T, Kellenbach E, Boelens R, Maler BA, Dahlman K, Freedman LP, Carlstedt-Duke J, Yamamoto KR, Gustaffson JA, Kaptein R. (1990). Solution structure of the glucocorticoid receptor DNA-binding domain. Science 249, 157160. Hardas BD, Zhao XP, Zhang J, Xia LQ, Stoll S, Elder JT. (1996). Assignment of psoriasin to human chromosomal band 1q21. Co-ordinate overexpression of clustered genes in psoriasis. J Invest Dermatol 106, 753758. Harris MR, Yu YYL, Kindle CS, Hansen TH, Solheim JC. (1998). Calreticulin and calnexin interact with different protein and glycan determinants during the assembly of MHC class I. J Immunol 160, 54045409. Harrison JK, Mickevicius CK, Gnegy ME. (1988). Differential regulation by calmodulin of basal, GTP- and dopamine-stimulated adenylate cyclase activities in bovine striatum. J Neurochem 51, 345352. Hart MJR, De Los Santos R, Albert IN, Rubinfeld B, Polakis P. (1998). Down regulation of beta catenin by human axin and its association with the APC tumour suppressor, beta catenin and GSK3 beta. Curr Biol 8, 573581. Harvey KF, Harvey NL, Michael JM, Parasivam G, Waterhouse N, Alnemri ES, Watters D, Kumar S. (1998). Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis. J Biol Chem 273, 1352413530. Hasegawa M, Smith MJ, Goedert M. (1998). Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly. FEBS Lett 437, 207210. Hashimoto Y, Shindookada N, Tani M, Takeuchi K, Toma H, Yokota J. (1996). Identication of genes differentially expressed in association with metastatic potential of K-1735 murine melanoma by messenger RNA differential display. Cancer Res 56, 52665271. Hasselaar P, Loskutoff DJ, Sawdey M, Sage EH. (1991). SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells. J Biol Chem 266, 1317813184. Hasson T, Gillespie PG, Garcia JA, MacDonald RB, Zhao YD, Yee AG, Mooseker MS, Corey DP. (1997). Unconventional myosins in inner ear sensory epithelia. J Cell Biol 137, 12871307. Hasson T, Heintzelman MB, Santossacchi J, Corey DP, Mooseker MS. (1995). Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type IB. Proc Natl Acad Sci USA 92, 98159819. Hasson T, Mooseker MS. (1995). Molecular motors, membrane movements and physiology. Emerging roles for myosins. Curr Opin Cell Biol 7, 587594. Hauschka PV, Lian JB, Cole DE, Gundberg CM. (1989). Osteocalcin and matrix gla protein. Vitamin K-dependent proteins in bone. Physiol Rev 69, 9901047. Haussler MR, Haussler CA, Jurutka PW, Thompson PD, Hsieh JC, Remus LS, Selznick SH, Whiteld GK. (1997). The vitamin D hormone and its nuclear receptor. Molecular actions and disease states. J Endocrinol 154, S57S73.
282
Haut M, Steeg PS, Willson JKV, Markowitz SD. (1991). Induction of nm23 expression in human colonic neoplasms and equal expression in colon tumours of a high and low metastatic potential. J Natl Cancer Inst 83, 712716 Haverstick DM, Engelhard DH, Gray LS. (1991). Three intracellular signals for cytotoxic Tlymphocyte-mediated cell killing. Independent roles for protein kinase C, Ca2+ inux and Ca2+ release from internal stores. J Immunol 146, 33033313. Hay ED. (1995). An overview of epithelio-mesenchymal transformation. Acta Anat 154, 820. Hayakawa K, Okagaki T, Higashi-Fujime S, Kohama K. (1994). Bundling of actin laments by myosin light chain kinase from smooth muscle. Biochem Biophys Res Commun 199, 786791. Hayakawa K, Okagaki T, Ye LH, Samizo K, Higashi-Fujime S, Takagi T, Kohama K. (1999). Characterisation of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin laments in vitro. Biochim Biophys Acta 1450, 1224. Hayashi S, Rubinfeld B, Souza B, Polakis P, Wieschaus E, Levine A. (1997). A Drosophila homologue of the tumour suppressor gene adenomatous polyposis coli down regulates beta catenin but its zygotic expression is not essential for the regulation of Armadillo. Proc Natl Acad Sci USA 94, 242247. Haydock PV, Blomquist C, Brumbaugh S, Dale BA, Holbrook KA, Fleckman P. (1993). Antisense prolaggrin RNA delays and decreases prolaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes. J Invest Dermatol 101, 118126. Hazan RB, Norton L. (1998). The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton. J Biol Chem 273, 90789084. Heilmann C, Spamer C, Leberer E, Gerok W, Michalak M. (1993). Human liver calreticulin. Characterisation and Zn2+-dependent interaction with phenyl-sapphires. Biochem Biophys Res Commun 193, 611616. Hein L, Kobilka BK. (1997). Adrenergic receptors. From molecular structure to in vivo function. Trends Cardiovasc Med 7, 137145. Heintzelman MB, Hassont, Mooseker MS. (1994). Multiple unconventional domains of the intestinal brush border cytoskeleton. J Cell Sci 107, 35353543. Heiska L, Kantor C, Parr T, Critchley DR, Vilja P, Gahmberg CG, Carpen O. (1996). Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to alphaactinin. J Biol Chem 271, 2621426219. Heizmann CW, Berchtold MW. (1987). Expression of parvalbumin and other calcium binding proteins in normal and tumour cells. A topical review. Cell Calcium 8, 141. Heizmann CW, Braun K. (1990). Calcium binding proteins: Molecular and functional aspects. In: The role of calcium in biological systems, ed. Anghileri IJ, CRC Press, Boca Raton, FL, pp. 2126. Heizmann CW, Braun K. (1992). Changes in Ca2+-binding proteins in human neurodegenerative disorders. Trends Neurosci 15, 259264. Heizmann CW, Cox JA. (1998). New perspectives on S100 proteins. A multifunctional Ca2+, Zn2+- and Cu2+-binding protein family. BioMetals 11, 383397. Heizmann CW, Hunziker W. (1990). Intracellular calcium binding molecules. In: Intracellular calcium regulation, ed. Bronner F, Alan R Liss, New York, pp. 211248. Heizmann CW. (1984). Parvalbumin, an intracellular calcium binding protein, distribution, properties and possible roles in mammalian cells. Experientia 40, 910921. Heizmann CW. (1996). Multistep calcium signalling in health and disease. Mol Cells 6, 629636. Helenius A, Trombetta ES, Hebert DN, Simon JF. (1997). Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol 7, 193200.
References
283
Hendey B, Maxeld FR. (1993). Regulation of neutrophil motility and adhesion by intracellular calcium transients. Blood Cells 19, 143164. Hendricks M, Weintraub H. (1981). Tropomyosin is decreased in transformed cells. Proc Natl Acad Sci USA 78, 56335637. Hendricks M, Weintraub H. (1984). Multiple tropomyosin polypeptides in chicken embryo broblasts. Mol Cell Biol 4, 18231833. Henzl MT, Shibasaki O, Comegys TH, Thalmann I, Thalmann R. (1997). Oncomodulin is abundant in the organ of Corti. Hearing Res 106, 105111. Henzl MT, Zhao HM, Saez CT. (1995). Self association of CPV3, an avian thymic parvalbumin. FEBS Lett 375, 137142. Hepler JR, Berman DM, Gilman AG, Kozasa T. (1997). The RGS4, GAIP are GTPaseactivating proteins for G (q alpha) and block activation of phospholipase C beta by gamma-thio-GTP-G (q alpha). Proc Natl Acad Sci USA 94, 428432. Herbert DN, Foellmer B, Helenius A. (1996). Calnexin and calreticulin promote folding, delay oligomerisation and suppress degradation of inuenza haemagglutinin in microsomes. EMBO J 15, 29612968. Herman JG, Merlo A, Mao L, Lapidus RG, Issa JPJ, Davidson NE, Sidransky D, Baylin SB. (1995). Inactivation of the CDKN2/P16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 55, 45254530. Hershberger ME, Tuan RS. (1998). Placental 57-kDa Ca2+-binding protein: Regulation of expression and function in trophoblast calcium transport. Dev Biol 199, 8092. Heubach JF, Hartwell R, Leswon M, Kraft T, Brenner B, Chalovich JM. (1997). Inhibition of crossbridge binding to actin by caldesmon fragments in skinned skeletal muscle bres. Biophys J 72, 12871294. Hidaka H, Mizutani A. (1993). Function and structure of calcium receptive protein, annexin and EF-hand protein. Calcyclin and its target protein, CAP-50. Biomed Res 14, 5355. Hildebrand R, Dilger I, Horlin A, Stutte JH. (1995). Urokinase plasminogen activator induces angiogenesis and tumour vessel invasion in breast cancer. Pathol Res Prac 191, 403409. Hinck L, Nelson WJ, Papkoff J. (1994). Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilising beta catenin binding to the cell adhesion protein cadherin. J Cell Biol 124, 729741. Hino M, Kurogi K, Okubo MA, Murata-Hori M, Hosoya H. (2000). Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells. Biochem Biophy Res Commun 271, 164169. Hirai S, Kawasaki H, Yaniv M, Suzuki K. (1991). Degradation of transcription factor, c-jun and c-fos, by calpain. FEBS Lett 287, 5761. Hirota S, Ito A, Nagoshi J, Takeda M, Kurata A, Takatsuka Y, Kohri K, Nomura S, Kitamura Y. (1995). Expression of bone matrix protein messenger ribonucleic acids in human breast cancers. Possible involvement of osteopontin in development of calcifying foci. Lab Invest 72, 6469. Hirschhorn RR, Aller P, Yuan ZA, Gibson CW, Baserga R. (1984). Cell cycle specic cDNAs from mammalian cells temperature sensitive for growth. Proc Natl Acad Sci USA 81, 60046008. Hisano T, Ono M, Nakayama M, Naito S, Kuwano M, Wada M. (1996). Increased expression of T-plastin gene in cisplatin-resistant human cancer cells. Identication by mRNA differential display. FEBS Lett 397, 101107. Hiscox S, Jiang WG. (1999). Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/beta catenin. J Cell Sci 112, 30813090.
284
Hitomi J, Kimura T, Kusumi E, Nakagawa S, Kuwabara S, Hatakeyama K, Yamaguchi K. (1998). Novel S100 proteins in human oesophageal epithelial cells. CAAF1 expression is associated with cell growth arrest. Arch Histol Cytol 61, 163178. Hitomi J, Maruyama K, Kikuchi Y, Nagasaki K, Yamaguchi K. (1996). Characterisation of a new calcium binding protein abundant in amniotic uid, CAAF2, which is produced by foetal epidermal keratinocytes during embryogenesis. Biochem Biophys Res Commun 228, 757763. Hiwasa T, Nakagawara A. (1998). Suppression of transformed phenotypes of Ha-ras-transformed NIH3T3 cells by caspase-2. Biochem Biophys Res Commun 250, 741746. Hixenbaugh EA, Goeckeler ZM, Papaiya NN, Wsolmerski RB, Silverstein SC, Huang AJ. (1997). Stimulated neutrophils induce myosin light chain phosphorylation and isometric tension in endothelial cells. Am J Physiol 42, H981H988. Ho S, Clipstone N, Timmerman L, Northrop J, Graef I, Fiorentino D, Nourse J, Crabtree GR. (1996). The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol 80, S40S45. Hoelting T, Duh QY, Clark OH, Herfarth C. (1997). Invasion and metastatic human follicular thyroid cancer is inhibited via antagonism of protein kinase C. Cancer Lett 119, 15. Hof PR, Nimchinsky EA, Celio MR, Bouras C, Morrison JH. (1993). Calretinin immunoreactive neocortical interneurons are unaffected in Alzheimers disease. Neurosci Lett 152, 145149. Hoffman S, Gopalakrishna R, Gundimeda U, Murata T, Spee C, Ryan SJ, Hinton DR. (1998). Verapamil inhibits proliferation, migration and protein kinase C activity in human retinal pigment epithelial cells. Exp Eye Res 67, 4552. Hoffmann HJ, Olsen E, Etzerodt M, Madsen P, Thorgersen HC, Kruse T, Celis JE. (1994). Psoriasin binds calcium and is up regulated by calcium to levels that resemble those observed in normal skin. J Invest Dermatol 103, 370375. Hohenadl C, Mann K, Mayer U, Timpl R, Paulsson R, Aeschlimann D. (1995). Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminase C-catalysed modication. J Biol Chem 270, 2341523420. Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lagman M, Shimbo T, Suthanthiran M. (1999). Cyclosporin induces cancer progression by a cell-autonomous mechanism. Nature 397, 530534. Holland PWH, Harper SJ, McVey JH, Hogan BLM. (1987). In vivo expression of mRNA for the Ca2+-binding protein SPARC (osteonectin) revealed by in situ hybridisation. J Cell Biol 105, 473482. Holtzer H, Hijikata T, Lin ZX, Zhang ZQ, Holtzer S, Protasi F, Franzini- Armstrong C, Sweeney L. (1997). Independent assembly of 1.6 m long bipolar MHC laments and I-Z-I bodies. Cell Structure Function 22, 8393. Holz FG, Bellmann C, Steffen H, Olle B, Huober J, Krastel H, Alexndridis E. (1997). Carcinoma associated retinopathy in the presence of breast carcinoma and a cervical carcinoid tumour. Ophthalmologie 94, 337342. Honda K, Yamada T, Endo R, Ino Y, Gotoh M, Tsuda H, Yamada Y, Chiba H, Hirohashi S. (1998). Actinin-4, a novel actin bundling protein associated with cell motility and cancer invasion. J Cell Biol 140, 13831393. Honore B, Rasmussen HH, Celis A, Leffers H, Madsen P, Celis JE. (1994). The molecular chaperones HSP28, GRP78, endoplasmin and calnexin exhibit strikingly different levels in quiescent keratinocytes as compared to their proliferating normal and transformed counterparts. cDNA cloning and expression of calnexin. Electrophoresis 15, 482490. Hoock TC, Peters LL, Lux SE. (1997). Isoforms of ankyrin-3 that lack the NH2-terminal repeats associate with mouse macrophage lysosomes. J Cell Biol 136, 10591070.
References
285
Horiuchi A, Nikaido T, Taniguchi S, Fujii S. (1999). Possible role of calponin h1 as a tumour suppressor in human uterine leiomyosarcoma. J Natl Cancer Inst 91, 790796. Horiuchi S, Koyanagi Y, Zhou YW, Miyamoto H, Tanaka Y, Waki M, Matsumoto A, Yamamoto M, Yamamoto N. (1994). Soluble interleukin-6 receptors released from T cell or granulocyte-macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism. Eur J Immunol 24, 19451948. Horowitz A, Clement-Chomienne O, Walsh MP, Tao T, Katsuyama H, Morgan K. (1996). Effects of calponin on force generation by single smooth muscle cells. Am J Physiol 270, H1858H1863. Hoseld CM, Elce JS, Davies PL, Jia ZC. (1999). Crystal structure of calpain reveals the structural basis of Ca2+-dependent protease activity and a novel mode of enzyme activation. EMBO J 18, 68806889. Hoshi T, Sasano H, Kato K, Yabuki N, Ohara S, Konno R, Asaki S, Toyota T, Tateno H, Nagura H. (1998). Immunohistochemistry of caspase-3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis. Anticancer Res 18, 43474353. Hseu MJ, Yen CH, Tzeng MC. (1999). Crocalbin: A new calcium binding protein that is also a binding protein for crotoxin, a neurotoxic phospholipase A2. FEBS Lett 445, 440444. Hseu MJ, Yen CY, Tseng CC, Tzeng MC. (1997). Purication and partial aminoacid sequence of a novel protein of the reticulocalbin family. Biochem Biophys Res Commun 239, 1822. Hsu JW, Hsu SL, Chu JJ, Liu MC, Chiang CD. (1997). Increased nm23:mts1 ratio inversely correlates with metastasis behaviour in human lung squamous carcinoma. Anticancer Res 17, 407411. Hu JR, Ferreira A, Van Eldik LJ. (1997). S100 induces neuronal cell death through nitric oxide release from astrocytes. J Neurochem 69, 22942301. Huang JW, Ming Z, Shreshtha P, Mori M, Ilg E, Schafer BW, Heizmann CW. (1996). Immunohistochemical evaluation of the Ca2+-binding S100 proteins S100A1, S100A2, S100A4, S100A6 and S100 in salivary gland tumours. J Oral Pathol Med 25, 547555. Huang SH, Wu JC, Chang KJ, Liaw KY, Wang SM. (1998). Distribution of the cadherincatenin complex in normal human thyroid epithelium and a thyroid carcinoma cell line. J Cell Biochem 70, 330337. Huang YH, Peng JX, Oberley LW, Domann FE. (1997). Transcriptional inhibition of manganese superoxide dismutase (SOD2) gene expression by DNA methylation of the 5 CpG island. Free Radical Biol Med 23, 314320. Hubaishy H, Jones PG, Bjorge J, Bellagamba C, Fitzpatrick S, Fujita DJ, Waisman DM. (1995). Modulation of annexin II tetramer by tyrosine phosphorylation. Biochemistry 34, 1452714534. Huber PAJ. (1997). Caldesmon. Int J Biochem Cell Biol 29, 10471051. Hulsken J, Birchmeier W, Behrens J. (1994). E-cadherin and APC compete for the interaction with beta catenin and the cytoskeleton. J Cell Biol 6, 20612069. Hunt TW, Fields TA, Casey PJ, Peralta EG. (1996). RCS10 is a selective activator of GTP alpha(i) GTPase activity. Nature 383, 175177. Hurlstone AF, Reid G, Reevers JR, Fraser J, Strathdee G, Rahilly M, Parkinson EK, Black DM. (1999). Analysis of the caveolin-1 gene at chromosome 7q31.1 in primary tumours and tumour-derived cell lines. Oncogene 18, 18811890. Huttelmaier S, Harbeck B, Steffens NO, Messerschmidt T, Illenberger S, Jockusch BM. (1999). Characterisation of actin binding properties of the vasodilator-stimulated phosphoprotein VASP. FEBS Lett 451, 6874.
286
Huttelmaier S, Mayboroda O, Harbeck B, Jarchau T, Jockusch BM, Rudiger M. (1998). The interaction of cell contact proteins VASP and vinculin is regulated by phosphotidylinositol-4,5-bisphosphate. Current Biol 8, 479488. Huttenlocher A, Palecek SP, Lu Q, Zhang WL, Mellgren RL, Lauffenburger DA, Ginsberg MH, Horwitz AF. (1997). Regulation of cell migration by the calcium-dependent protease calpain. J Biol Chem 272, 3271932722. Iacopino AM, Rhoten WB, Christakos S. (1990). Calcium binding protein (calbindin D28K) gene expression in the developing and ageing mouse cerebellum. Mol Brain Res 8, 283290. Iida N, Bourguignon LYW. (1996). A new splice variant of the inositol 1,4,5-trisphosphate receptor (IP3). Cell Signalling 6, 449. Ikeda H, Nakamura Y, Hiwasa T, Sakiyama S, Kuida K, Su MSS, Nakagawara A. (1997). Interleukin-1 beta converting enzyme (ICE) is preferentially expressed in neuroblastomas with favourable prognosis. Eur J Cancer 33, 20812083. Ikura M, Clore M Gronenborn AM, Zhu G, Klee CB, Bax A. (1992). Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632638. Ikura M. (1996). Calcium binding proteins and conformational response in EF-hand proteins. Trends Biochem Sci 21, 1417. Ilg E, Schafer BW, Heizmann CW. (1996). Expression pattern of S100 calcium binding proteins in human tumours. Int J Cancer 68, 325332. Ilian MA, Forsberg NE. (1992). Gene expression of calpains and their specic endogenous inhibitor, calpastatin, in skeletal muscle of fed and fasted rabbits. Biochem J 287, 163171. Ilian MA, Forsberg NE. (1994). A comparative study of gene expression of calpains, calpastatin, cathepsin D, and proteasome in tissues of fed and fasted rabbits. J Nutr Biochem 5, 529535. Imamura M, Endo T, Kuroda M, Tanaka T, Masaki T. (1988). Substructure and higher structure of chicken smooth muscle alpha-actinin molecule. J Biol Chem 263, 78007805. Imamura M, Masaki T. (1992). A novel non-muscle alpha-actinin. Purication and characterisation of chick lung alpha-actinin. J Biol Chem 267, 2592725933. Imamura M, Masaki T. (1994). Identication of a 115-kDa protein from muscle tissues. Expression of a novel non-muscle alpha-actinin in vascular endothelial cells. Exp Cell Res 211, 380390. Imamura M, Sakurai T, Ogawa Y, Ishikawa T, Goto K, Masaki T. (1994). Molecular cloning of low Ca2+-sensitive type non-muscle alpha-actinin. Eur J Biochem 223, 395401. Inagaki H, Lin KH, Maeda S, Saito T. (1996). Osteonectin gene expression in brotic liver. Life Sci 58, 927934. Inomato M, Hayashi M, Ohno-Iwashita Y, Tsubaki S, Saido TC, Kawashima S. (1996). Involvement of calpain in integrin-mediated signal transduction. Arch Biochem Biophys 328, 129134. Inoue K, Furihata M, Ohtsuki Y, Fujita Y. (1993). Distribution of S-100 protein positive dendritic cells and expression of HLA-DR in transitional cell carcinoma of the urinary bladder in relation to tumour progression and prognosis. Virchows Arch A 422, 351355. Iqbal K, Grundke-Iqbal I. (1996). Molecular mechanism of Alzheimers neurobrillary degeneration and therapeutic intervention. Ann NY Acad Sci 777, 132138. Irigoyen JP, Besser D, Nagamine Y. (1997). Cytoskeleton reorganisation induces the urokinase-type plasminogen activator gene via the ras/extracellular signal regulated kinase (ERK) signalling pathway. J Biol Chem 272, 19041909.
References
287
Irigoyen JP, Nagamine Y. (1999). Cytoskeletal reorganisation leads to induction of the urokinase-type plasminogen activator gene by activating FAK and src and subsequently the ras/erk pathway. Biochem Biophys Res Commun 262, 666670. Iruela-Arispe ML, Diglio CA, Sage EH. (1991a). Modulation of extracellular matrix proteins by endothelial cells undergoing angiogenesis in vitro. Arterioscler Thromb 11, 805815. Iruela-Arispe ML, Hasselaar P, Sage H. (1991b). Differential expression of extracellular matrix proteins is correlated with angiogenesis in vitro. Lab Invest 64,174186. Ishida-Yamamoto A, Eady RAJ, Underwood RA, Dale BA, Holbrook KA. (1994). Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis). Br J Dermatol 131, 767779. Ishikawa R, Yamashiro S, Matsumura F. (1989a). Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosin by 83 kDa non-muscle caldesmon. J Biol Chem 264, 74907497. Ishikawa R, Yamashiro S, Matsumura F. (1989b). Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon. J Biol Chem 264, 1676416770. Ishisaka R, Utsumi T, Yabuki M, Kanno T, Furuno T, Inoue N, Utsumi K. (1998). Activation of caspase-3-like protease by digitonin-treated lysosomes. FEBS Lett 435, 233236. Isobe T, Okuyama T. (1978). The aminoacid sequence of S100 protein (PAP 1-b protein) and its relation to the calcium binding proteins. Eur J Biochem 89, 379388. Isobe T, Okuyama T. (1981). The aminoacid sequence of the -subunit of bovine S100a protein. Eur J Biochem 116, 7986. Isobe T, Takahashi K, Okuyama T. (1984). S100A0() protein is present in neurones of the central and peripheral nervous system. J Neurochem 43, 14941496. Isom LL, De Jongh KS, Catterall WA. (1994). Auxiliary Subunits of voltage-gated ion channels. Neuron 12, 11831194. Issa LL, Leong GM, Eisman JA. (1998). Molecular mechanisms of vitamin D receptor action. Inammation Res 47, 451475. Issa JPJ, Zehnbauer BA, Kaufmann SH, Biel MA, Baylin SB. (1997). HIC 1 hypermethylation is a late event in haematopoietic neoplasms. Cancer Res 57, 16781681. Itagaki Y, Akagi K, Uda M, Tanaka Y. (1998). Role of intracellular calcium concentration on tumour cell death from hyperthermia. Oncology Reports 5, 135141. Ito A, Sato T, Ojima Y, Chen LC, Nagase H, Mori Y. (1991). Calmodulin differentially modulates the interleukin 1-induced biosynthesis of tissue inhibitor of metalloproteinase and matrix metalloproteinases in human uterine cervical broblasts. J Biol Chem 266, 1359813601. Ito T, Udaka N, Inayama Y, Kitamura H, Kaanisawa M. (1998). Hamster pulmonary endocrine cells with positive immunostaining for calbindin D28K. Histochem Cell Biol 109, 6773. Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, Narumiya S. (1999). An essential part for Rho-associated kinase in the transcellular invasion of tumour cells. Nature Med 5, 221225. Ivashkiv LB, Ayres A, Glimcher LH. (1994). Inhibition of IFN-gamma induction of class II MHC genes by cAMP and prostaglandins. Immunopharmacology 27, 6777. Iwasaki Y, Shiojima T, Kinoshita M. (1997). S100 prevents the death of motor neurones in new-born rats after sciatic nerve section. J Neurol Sci 151, 712. Iyengar R, Birnbaumer L. (1990a). G proteins Academic Press, New York, pp. 114. Iyengar R, Birnbaumer L. (1990b). Roles of G proteins and G protein subunits in signal transduction. Lymphokine Res 9, 533537.
288
Jaattela M, Wissing D, Kokholm K, Kallunki T, Egeblad M. (1998). HSP-70 exerts its antiapoptotic function downstream of caspase-3-like proteases. EMBO J 17, 61246134. Jackson-Grusby L, Swiergel J, Linzer D. (1987). A growth related mRNA in cultured mouse cells encodes a placental calcium binding protein. Nucl Acids Res 15, 66776690. Jacob K, Webber M, Benayahu D, Kleinman HK. (1999). Osteonectin promotes prostate cancer cell migration and invasion. A possible mechanism for metastasis to bone. Cancer Res 59, 44534457. Jacobs W, Mikkelsen T, Smith R, Nelson K, Rosenblum ML, Kohn EC. (1997). Inhibitory effects of CAI in glioblastoma growth and invasion. J Neuro-Oncol 32, 93101. Jadeski LC, Lala PK. (1999). Nitric oxide synthase inhibition by NG-nitro-L-arginine methyl ester inhibits tumour-induced angiogenesis in mammary tumours. Amer J Pathol 155, 13811390. Jain PT, Trump BF. (1997). Tamoxifen induces deregulation of [Ca2+] in human breast cancer. Anticancer Res 17, 11671174. James P, Vorherr T, Carafoli E. (1995). Calmodulin-binding domains just two faced or multifaceted. Trends Biochem Sci 20, 3842. Jamieson S, Barraclough R, Rudland PS. (1990a). Generation of metastatic variants by transfection of a non-metastatic rat mammary epithelial cell line with DNA from a metastatic rat mammary cell line. Pathobiology 58, 329342. Jamieson S, Barraclough R, Rudland PS. (1990b). Transfection of a non-metastatic diploid rat mammary epithelial cell line with the oncogenes or EJ-ras-1 and polyoma large Tantigen. Int J Cancer 46, 10711080. Janis RA, Silver PJ, Triggle DJ. (1987). Drug action and cellular calcium regulation. Adv Drug Res 16, 309538. Janssen KP, Eichinger L, Janmey PA, Noegel AA, Shliwa M, Witke W, Schleicher M. (1996). Viscoelastic properties of F-actin solutions in the presence of normal and mutated actinbinding proteins. Arch Biochem Biophys 325, 183189. Jayaraman T, Marks AR. (1997). T cells decient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis. Mol Cell Biol 17, 30053012. Jayaraman T, Ondrias K, Ondriasova E, Marks AR. (1996). Regulation of the inositol 1,4,5trisphosphate receptor by tyrosine phosphorylation. Science 272, 14921494. Jayaraman T, Ondriasova E, Ondrias K, Harnick DJ, Marks AR. (1995). The inositol 1,4,5trisphosphate receptor is essential for cell receptor signalling. Proc Natl Acad Sci USA 92, 60076011. Jendraschak E, Sage E. (1996). Regulation of angiogenesis by SPARC and angiostatin. Implications for tumour cell biology. Cancer Biol 7, 139146. Jeong SW, Ikeda SR, Wurster RD. (1999). Activation of various G-proteins coupled with receptors modulates Ca2+ channel currents via PTX-sensitive and voltage-dependent pathways in rat intracardiac neurones. J Autonomic Nerv Syst 76, 6847. Jiang M, Swann JW. (1997). Expression of calretinin in diverse neuronal populations during development of rat hippocampus. Neuroscience 81, 11371154. Jiang Z, Grange RW, Walsh MP, Kamm KE. (1997). Adenovirus-mediated transfer of the smooth muscle calponin gene inhibits proliferation of smooth muscle cells and broblasts. FEBS Lett 413, 441445. Jin JP, Wang K. (1991). Nebulin as a giant actin-binding template protein in skeletal muscle sarcomere. FEBS Lett 281, 93. Johnson DI, Pringle JR. (1990). Molecular characterisation of CDC2, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol 111, 143152.
References
289
Johnson GVW. (1992). Differential phosphorylation of tau by cyclic AMP dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II. Metabolic and functional consequences. J Neurochem 59, 20562062. Johnson RJ, Liu NG, Shanmugaratnam J, Fine RE. (1998). Increased calreticulin stability in differentiated NG-108-15 cells correlates with resistance to apoptosis induced by antisense treatment. Mol Brain Res 53, 104111. Johnson WC, Palczewski K, Gorczyca WA, Riazance-Lawrence JH. (1997). Calcium binding to recoverin. Implications for secondary structure and membrane association. Biochim Biophys Acta 1342, 164174. Jones LR, Suzuki YJ, Wang W, Kobayashi YM, Ramesh V, Franzini-Armstrong C, Cleemann L, Morad M. (1998). Regulation of Ca2+ signalling in transgenic mouse cardiac myocytes over-expressing calsequestrin. J Clin Invest 101, 13851393. Jones LR, Zhang L, Sanborn K, Jorgensen AO, Kelley J. (1995). Purication, primary structure, and immunological characterisation of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum. J Biol Chem 270, 3078730796. Jones NA, Rowlands DC, Johnson WEB, MacLennan ICM, Brown G. (1995). Persistent growth of Balb/C mouse plasmacytoma and human myeloma cell lines in the presence of phorbol myristate acetate is associated with the continued expression of LPA18 (stathmin). Haematol Oncol 13, 2943. Jones PA, Chandler L. (1986). DNA methylation, differentiation and cancer. Eur J Cell Biol 42, 4. Jones PA. (1986). DNA methylation and cancer. Cancer Res 46, 461466. Jones SL, Brown EJ. (1996). Fc gamma RII-mediated adhesion and phagocytosis induce Lplastin phosphorylation in human neutrophils. J Biol Chem 271, 1462314630. Jones SL, Wang J, Turck CW, Brown EJ. (1998). A role for the actin-bundling protein Lplastin in the regulation of leukocyte integrin function. Proc Natl Acad Sci USA 95, 93319336. Jordan J, Galindo MF, Miller RJ. (1997). Role of calpain and interleukin-1 beta converting enzyme-like proteases in the beta-amyloid-induced death of rat hippocampal neurones in culture. J Neurochem 68, 16121621. Joshi M, Pogue GP, Duncan RC, Lee NS, Singh NK, Atreya CD, Dwyer DM, Nakhasi HL. (1996). Isolation and characterisation of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity. Mol Biochem Parasitol 81, 5364. Jost M, Gerke V. (1996). Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II. Biochim Biophys Acta 1313, 283289. Jou TS, Stewart DB, Stappert J, Nelson WJ, Marrs JA. (1995). Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex. Proc Natl Acad Sci USA 92, 50675071. Juin P, Pelletier M, Oliver L, Tremlais K, Gregoire P, Meah K, Vallette FM. (1998). Induction of caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell free system. J Biol Chem 273, 1755917564. Jung D, Duclos F, Apostol B, Straub V, Lee JC, Allamand V, Venzke DP, Sunada Y, Moomaw CR, Leveille CJ, Slaughter CA, Crawford TO, McPherson JD, Campbell KP. (1996). Characterisation of -sarcoglycan, a novel component of the oligomeric sarcoglycan complex involved in limb girdle muscular dystrophy. J Biol Chem 271, 3232132329. Jungers P, Kuttenn F, Liote F, Pelisser C, Athea N, Laurent MC, Viriot J, Dougados M, Bach JF. (1985). Hormonal modulation in SLE. Arthritis Rheum 28, 12431250.
290
Kabbutat MHG, Vousden KH. (1997). Proteolytic cleavage of human p53 by calpain. A potential regulator of protein stability. Mol Cell Biol 17, 460468. Kabbutat MHG, Jones SM, Vousden KH. (1997). Regulation of p53 stability by mdm2. Nature 387, 299303. Kaczanbourgois D, Salles JP, Hullin F, Fauvel J, Moisand A, Duganeulat I, Berrebi A, Campistron G, Chap H. (1996). Increased content of annexin (P36) and p11 in human placenta brush border membrane vesicles during syncytiotrophoblast maturation and differentiation. Placenta 17, 669676. Kahana E, Gratzer WB. (1991). Properties of the spectrin-like structural element of smooth muscle alpha-actinin. Cell Motility Cytoskeleton 20, 242248. Kaibuchi K, Kuroda S, Fukata M, Nakagawa M. (1999). Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases. Curr Opin Cell Biol 11, 591596. Kakeya H, Onose R, Osada H. (1998). Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis. Cancer Res 58, 48884894. Kam E, Nirunsuksiri W, Hager B, Fleckman P, Dale BA. (1997). Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis. Br J Dermatol 137, 874882. Kamimura M, Tomita S, Kiuchi M, Fujiwara H. (1997). Tissue-specic and stage-specic expression of two silkworm ecdysone receptor isoforms. Ecdysteroid-dependent transcription in cultured anterior silk glands. Eur J Biochem 248, 786793. Karpova TS, Tatchel K, Cooper JA. (1995). Actin laments in yeast are unstable in the absence of capping protein or mbrin. J Cell Biol 131, 14831493. Karube A, Shidara Y, Hayasaka K, Maki M, Tanaka T. (1995). Suppression of calphobindin I (CPBI) production in carcinoma of uterine cervix and endometrium. Gynecol Oncol 58, 295300. Kass SU, Landsberger N, Wolffe AP. (1997). DNA methylation directs a time dependent repression of transcription initiation. Current Biol 7, 157165. Kato H. (1996). Expression and function of squamous cell carcinoma antigen. Anticancer Res 16, 21492153. Katusic ZS, Stelter AM. (1995). Type I inositol trisphosphate receptor gene is alternatively spliced in human cerebral arteries. Biochem Biophys Res Commun 214, 803809. Kaufmann SH. (1989). Induction of endonucleolytic DNA cleavage in human acute myelogenous leukaemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs. Cancer Res 49, 58705878. Kautiainen TL, Jones PA. (1986). DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture. J Biol Chem 261, 15941598. Kawahara A, Ohsawa Y, Matsumura H, Uchiyama Y, Nagata S. (1998). Caspase-independent cell killing by Fas-associated protein with death domain. J Cell Biol 143, 13531360. Kawamura S, Cox JA, Nef P. (1994). Inhibition of rhodopsin phosphorylation by nonmyristoylated recombinant recoverin. Biochem Biophys Res Commun 203, 121127. Kawamura S, Kuwata O, Yamada M, Matsuda S, Hisatomi O, Tokunaga F. (1996). Photoreceptor protein S26, a cone homologue of S-modulin in frog retina. J Biol Chem 271, 2135921364. Kawasaki H, Nakayama S, Kretsinger RH. (1998). Classication and evolution of EF-hand proteins. Biometals 11, 277295. Kayyali US, Zhang W, Yee AG, Seidman JG, Potter H. (1997). Cytoskeletal changes in the brain of mice lacking calcineurin A alpha. J Neurochem 68, 16681678.
References
291
Ke YQ, Jing C, Barraclough R, Smith P, Davies MPA, Foster CS. (1997). Elevated expression of calcium-binding protein p9Ka is associated with increasing malignant characteristics of rat prostate carcinoma cells. Int J Cancer 71, 832837. Kebers F, Lwealle J.M, Desreux J, Manaut C, Devy L, Foidart JM, Noel A. (1998). Induction of endothelial cell apoptosis by solid tumour cells. Exp Cell Res 240, 197205. Keegan L, Gill G, Ptashne M. (1986). Separation of DNA binding from the transcription activating function of a eukaryotic regulatory protein. Science 231, 699704. Keim D, Hailat N, Melhem R, Zhu X, Lascu I, Veron M, Strahler J. (1992). Proliferation related expression of p19/nm23 nucleoside diphosphate kinase. J Clin Invest 89, 919924. Keirsebilck A, Bonne S, Bruyneel E, Vermassen P, Lukanidin E, Mareel M, van Roy, F. (1998a). E-cadherin and metastasis (mts1/S100A4) expression levels are inversely regulated in two tumour cell families. Cancer Res 58, 45874591. Keirsebilck A, Bonne S, Staes K, Van Hengel J, Nollet F, Reynolds A, Van Roy F. (1998b). Molecular cloning of the p120(ctn) catenin gene (CTNND1): Expression of multiple alternatively spliced isoforms. Genomics 50, 129146. Keller D, Grover AK. (2000). Nerve growth factor treatment alters Ca2+ pump levels in PC12 cells. Neuroreport 11, 6568. Kelley CA, Adelstein RS. (1994). Characterisation of isoform diversity in smooth muscle myosin heavy chains. Canad J Physiol Pharmacol 72, 13511360. Kelm RJ, Mann KG. (1990). Human platelet osteonectin. Release surface expression and partial characterisation. Blood 75, 11051113. Kelm RJ, Swords NA, Orteo T, Mann KG. (1994). Osteonectin in matrix remodelling. A plasminogen-collagen complex. J Biol Chem 269, 3014730153. Kent J, Lee M, Schedl A, Boyle S, Fantes J, Powell M, Rushmere N, Abbott C, Van Heyningen V, Bickmore WA. (1997). The reticulocalbin gene maps to the WAGR region in human and to the small eye Harwell deletion in mouse. Genomics 42, 260267. Kerbel RS, Waghorne C, Man MS, Elliot B, Breitman ML. (1987). Alteration of the tumorigenic and metastatic properties of cells is associated with the process of calcium phosphate mediated DNA transfection. Proc Natl Acad Sci USA 84, 12631267. Kerkhoff C, Eue I, Sorg C. (1999a). The regulatory role of MRP8 (S100A8) and MRP14 (S100A9) in the transendothelial migration of human leukocytes. Pathobiology 67, 230232. Kerkhoff C, Klempt M, Kaever V, Sorg C. (1999b). The two calcium binding proteins S100A8 and S100A9 are involved in the metabolism of arachidonic acid in human neutrophils. J Biol Chem 274, 3267232679. Kerkhoff C, Klempt M, Sorg C. (1998). Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9). Biochim Biophys Acta 1448, 200211. Kerkhoff C, Vogl T, Nacken W, Sopalla C, Sorg C. (1999c). Zinc binding reverses the calciuminduced arachidonic acid binding capacity of the S100A8/A9 protein complexes. FEBS Lett 460, 134138. Khaitlina S, Hinssen H. (1997). Conformational changes in actin induced by its interaction with gelsolin. Biophys J 73, 929937. Khanna NC, Tokuda M, Waisman DM. (1987). Calregulin, purication, cellular localisation and tissue distribution. Meth Enzymol 139, 3650. Kido J, Yamauchi N, Ohishi K, Kataoka M, Nishikawa S, Nakamura T, Kadono H, Idedo D, Ueno A, Nonomura N, Okuyama A, Nagata T. (1997). Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro. J Cell Biochem 67, 248256.
292
Kihara H, Hiasa A, Yamamoto M, Katayama N, Kuno T, Ohtsuka K, Shiku H, Nishikawa M. (1998). Possible involvement of calcineurin in retinoic acid-induced inhibition of leukemic HL-60 cells. Int J Oncol 12, 629634. Kilby PM, Van Eldik LJ, Roberts GCK. (1997). Identication of the binding site on S100B protein for the actin capping protein CapZ. Protein Sci 6, 24942503. Kim SY, Ondhia N, Vidgen D, Malaval L, Ringuette M, Kainins VI. (1997). Spatio-temporal distribution of SPARC/osteonectin in developing and mature chicken retina. Exp Eye Res 65, 681689. Kim LT, Yamada KM. (1997). The regulation of expression of integrin receptors. Proc Soc Exp Biol Med 214, 123131. Kimura A, Ikeda H, Yasuda S, Yamaguchi K, Tsuji T. (1995). Brain tissue identication based on myosin heavy chain isoforms. Int J Legal Med 107, 193196. Kimura Y, Koga H, Araki N, Mugita N, Fujita N, Takeshima H, Nishi T, Yamashimaaa T, Saido T, Yamasaki T, Moritake K, Saya H, Nakao M. (1998). The involvement of calpaindependent proteolysis of the tumour suppressor NF2 (merlin) in schwannomas and meningiomas. Nature Med 4, 915922. Kincaid RL, Takayama H, Billingsley ML, Sitkovsky MV. (1987). Differential expression of calmodulin-binding proteins in lymphocytes B, lymphocytes T and thymocytes. Nature 330, 176178. Kinder-Mugge I, Herbacek I, Jantschitsch C, Micksche M, Trautinger F. (1996). Modication of growth and tumorigenicity in epidermal cell lines by DNA-mediated gene transfer of Mr 27,000 heat shock protein (hsp27). Cell Growth Differentiation 7, 11671174. King KB, Chubinskaya S, Reid DL, Madsen LH, Mollenhauer J. (1997). Absence of cell surface annexin V is accompanied by defective collagen matrix binding in Swarm rat chondrosarcoma. J Cell Biochem 65, 131144. Kiraly E, Celio MR. (1992). Ontogeny of calretinin in chick dorsal root ganglion neurones. Dev Brain Res 70, 149152. Kiraly E, Celio MR. (1993). Parvalbumin and calretinin in the avian thymus. Anat Embryol 188, 339344. Kirati H, Thirkill CE, Bilgic S, Eldem B, Kececi A. (1997). Paraneoplastic retinopathy associated with metastatic cutaneous melanoma of unknown primary site. Eye 11, 889892. Kishimoto J, Tsuchiya T, Cox H, Emson PC, Nakayama Y. (1998). Age related changes of calbindin D28K, calretinin, and parvalbumin mRNAs in the hamster brain. Neurobiol Ageing 19, 7782. Kishore U, Sontheimer RD, Sastry KN, Zappi EG, Hughes GRV, Khamashta MA, Reid KBM, Eggleton P. (1997). The systemic lupus erythematosus (SLE) disease autoantigen. Calreticulin inhibits C1q association with immune complexes. J Clin Exp Immunol 108, 181190. Kitamura Y, Shimohama S, Kamoshima W, Ota T, Matuoka Y, Nomura Y, Smith MA, Perry C, Whitehouse PJ, Taniguchi T. (1998). Alterations of proteins regulating apoptosis, Bcl2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32 in Alzheimers disease. Brain Res 780, 260269. Kitayama C, Sugimoto A, Yamamoto M. (1997). Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J Cell Biol 137, 13091319. Kiura S, Seppalainen AM, Salonen O, Kokkanen L, Somer H, Palo JNA. (1995). CNS abnormalities in patients with familial amyloidosis Finnish type (FAF). Amyloid Int J Exp Clin Invest 2, 2230.
References
293
Kizawa K, Tsuchimoto S, Hashimoto K, Uchiwa H. (1998). Gene expression of mouse S100A3, a cysteine-rich calcium binding protein, in developing hair follicle. J Invest Dermatol 111, 879886. Kizawa K, Uchiwa H, Murakami U. (1996). Highly expressed S100A3, a calcium binding protein, in human cuticle. Biochim Biophys Acta 1312, 9498. Klee B, Guerini D, Krinks MH, De Camilli P, Solinema M. (1990). Calcineurin. A major Ca2+/calmodulin regulated protein phosphatase in brain. Raven Press, Washington DC. Klee CB, Dracetta GF, Hubbard MJ. (1988). Calcineurin. Adv Enzymol 61, 149200. Klempt M, Melkonyan H, Nacken W, Wiesmann D, Holtkemper U, Sorg C. (1997). The heterodimer of the Ca2+-binding proteins MRP8 and MRP14 binds arachidonic acid. FEBS Lett 408, 8184. Kleymenova EV, Yuan XQ, LaBate ME, Walker CL. (1998). Identication of a tumour specic methylation site in the Wilms tumour suppressor gene. Oncogene 16, 713720. Kligman D, Hilt DC. (1988). S100 protein family. Trends Bio Sci 13, 437443. Kligman D, Marshak DR. (1985). Purication and characterisation of a neurite extension factor from bovine brain. Proc Natl Acad Sci USA 82, 71367139 Kline D, Kline JT. (1992). Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev Biol 149, 8089. Kline D. (1996). Activation of the mouse egg. Theriogenology 45, 8190. Klingelhofer J, Ambartsumian NS, Lukanidin EM. (1997). Expression of the metastasis associated mts1 gene during mouse development. Dev Dynamics 210, 8795. Klotz T, Block W, Jacobs G, Niggemann S, Engelmann U, Addicks J. (1999). Immunolocalisation of inducible and constitutive nitiric oxide synthase in human bladder cancer. Urology 54, 416419. Klotz T, Block W, Volberg C, Englmann U, Addicks K. (1998). Selective expression of inducible nitric oxide synthase in human prostate carcinoma. Cancer 82, 18971903. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. (1997). The release of cytochrome c from mitochondria. A primary site for bcl-2 regulation. Science 275, 11321136. Klug M, Blum JE, Ye QR, Berchtold MW. (1994). Intracellular Ca2+ and Ca2+-binding proteins in chemically transformed broblasts. Exp Cell Res 213, 313318. Knudsen KA, Soler AP, Johnson KR, Wheelock MJ. (1995). Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha- actinin. J Cell Biol 130, 6777. Ko SC, Cheon J, Kao CH, Gotoh A, Shirakawa T, Sikes RA, Karsenty G, Chung LWK. (1996). Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res 56, 46144619. Kobayashi M, Aita N, Hayashi S, Okada K, Ohta T, Hirose S. (1998). DNA supercoiling factor localises to puffs on polytene chromosomes in Drosophila melanogaster. Mol Cell Biol 18, 67376744. Kobayashi Y, Sakai H, Ikeda S, Kobayashi K, Kato Y, Mataki. (1998). Processing of NH2and COOH-terminal peptides of rat osteocalcin by cathepsin B and cathepsin L. J Bone Mineral Metab 16, 7280. Koch KW. (1995). Control of photoreceptor proteins by Ca2+. Cell Calcium 18, 314321. Koelle MR. (1997). A new family of G-protein regulators. The RGS proteins. Curr Opin Cell Biol 9, 143147. Kohama K, Okagaki T, Hayakawa K, Lin Y, Ishikawa R, Shimmen T, Inoue A. (1992). A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATPdependent interaction between actin and myosin. Biochem Biophys Res Commun 184, 12041211.
294
Kohn EC, Reed E, Sarosy G, Christian M, Link CJ, Cole K, Figg WD, Davis PA, Jacob J, Goldspiel B, Liotta LA. (1996). Clinical investigation of a cytostatic calcium inux inhibitor in patients with refractory cancers. Cancer Res 56, 569573. Kolakowski J, Karkucinska A, Dabrowska R. (1997). Calponin inhibits actin-activated MgATPase of myosin subfragment 1 (S1) without displacing S1 from its binding site on actin. Eur J Biochem 243, 624629. Kolega J, Kumar S. (1999). Regulatory light chain phosphorylation and the assembly of myosin II into the cytoskeleton of microcapillary endothelial cells. Cell Motility Cytoskeleton 43, 255268. Kolega J. (1998). Cytoplasmic dynamics of myosin II and IIB. Spatial sorting of isoforms in locomoting cells. J Cell Sci 111, 20852095. Komalavilas P, Lincoln TM. (1994). Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP dependent protein kinase. J Biol Chem 269, 87018707. Komalavilas P, Lincoln TM. (1996). Phosphorylation of the inositol 1,4,5-trisphosphate receptor. Cyclic GMP dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta. J Biol Chem 271, 2193321938. Kong DH, Suzuki A, Zou TT, Sakurada A, Kemp LW, Wakatsuki S, Yokoyama T, Yamakawa H, Furukawa T, Sato M, Ohuchi N, Sato S, Yin J, Wang SN, Abraham JM, Souza RF, Smolinski KN, Meltzer SJ, Horii A. (1997). PTEN1 is frequently mutated in primary endometrial carcinomas. Nature Genet 17, 143144. Korkotian E, Segal M. (1999). Release of calcium from stores alters the morphology of dendritic spines in cultured hippocampal neurones. Proc Natl Acad Sci USA 96, 1206812072. Kouchi Z, Saido TC, Ohyama H, Maruta H, Suzuki K, Tanuma S. (1997). The restrictive proteolysis of alpha fodrin to a 120-kDa fragment is not catalysed by calpains during thymic apoptosis. Apoptosis 2, 8490. Kozak A, Nikodijevic B, Yavin E, Guroff G. (1992). Intracellular calcium levels regulate the actions of nerve growth factor on calcium uptake in PC12 cells. J Neurosci Res 33, 3036. Kozumi T, Odani N, Okuyama T, Ichikawa A, Negishi M. (1995). Identication of a cisregulatory element for 12-prostaglandin J2-induced expression of the rat heme oxygenase gene. J Biol Chem 270, 2177921784. Kraut N, Frampton J, Graf T. (1995). REM-1, a putative direct target gene of the myb-ets fusion oncoprotein in haematopoietic progenitors, is a member of the recoverin family. Oncogene 10, 10271036. Krauze K, Makuch R, Stepka M, Dabrowska R. (1998). The rst caldesmon-like protein in higher plants. Biochem Biophys Res Commun 247, 576579. Krebs EG. (1989). Role of cyclic AMP dependent protein kinase in signal transduction. JAMA 262, 18151818. Kretsinger RH and Kockolds CE. (1973). Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem 248, 33133326. Kretsinger RH. (1980). Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem 8, 119174. Kretsinger RH. (1992). Calmodulin and myosin light chain kinase. How helices are bent. Science 258, 5051. Kretsinger RH. (1997). EF-hand embrace. Nature Struct Biol 4, 514516. Kriajevska M, Tarabykina S, Bronstein I, Maitland N, Lomonosov M, Hansen K, Georgiev G, Lukanidin E. (1998). Metastasis associated mts1 (S100A4) protein modulates protein kinase C phosphorylation of the heavy chain of non-muscle myosin. J Biol Chem 273, 98529856,
References
295
Krieg P, Schuppler M, Koesters R, Mincheva A, Lichter P, Marks F. (1997). Repetin (Rptn), a new member of the fused gene subgroup within the S100 gene family encoding a murine epidermal differentiation protein. Genomics 43, 339348. Krieger C, Wagey R, Linus R, Shaw CA. (1993). Activation of PKC reverses apparent NMDA receptor reduction in ALS. Neuroreport 4, 931934. Kruger M, Wright J, Wang K. (1991). Nebulin as a length regulator of thin laments of vertebrate skeletal muscles. Correlation of thin lament length, nebulin size, and epitope prole. J Cell Biol 115, 97107. Kumamoto T, Kleese WC, Cong J, Goll DE, Pierce PR, Allen RE. (1992). Localisation of the Ca2+-dependent proteinases and their inhibitor in normal, fasted, and denervated rat skeletal muscle. Anat Rec 232, 6077. Kumamoto T, Ueyama H, Sugihara R, Kominami E, Goll DE, Tsuda T. (1997). Calpain and cathepsin in the skeletal muscle of inammatory myopathies. Eur Neurol 37, 176181. Kumamoto T, Ueyama H, Watanabe S, Yoshioka K, Mike T, Goll DE, Ando M, Tsuda T. (1995). Immunohistochemical study of calpain and its endogenous inhibitor in the skeletal muscle of muscular dystrophy. Acta Neuropathol 89, 399403. Kuo TH, Liu BF, Yu YJ, Wuytack F, Raeymaekers L, Tsang W. (1997). Co-ordinated regulation of the plasma membrane calcium pump and the sarco(endo)plasmic reticular calcium pump gene expression by Ca2+. Cell Calcium 21, 399408. Kupprion C, Motamed K, Sage EH. (1998). SPARC (BM-40, osteonectin) inhibits the mitogenic effect of vascular endothelial growth factor on microvascular endothelial cells. J Biol Chem 273, 2963529640. Kurasawa S, Schulz RM, Kopf GS. (1989). Egg-induced modications of the zona pellucida of the mouse egg. Effects of microinjected inositol 1,4,5-trisphosphate. Dev Biol 133, 295304. Kurota H, Yamaguchi M. (1997). Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in renal cortex cytosol. Mol Cell Biochem 177, 239243. La Greca N, Hibbs AR, Riffkin C, Foley M, Tilley L. (1997). Identication of an endoplasmic reticulum-resident calcium-binding protein with multiple EF-hand motifs in asexual stages of Plasmodium falciparum. Mol Biochem Parasitol 89, 283293. Labait S, Gibson T, Lakey A, Leonard K, Zeviani M, Knight P, Wardale J, Trinick J. (1991). Evidence that nebulin is a protein ruler in muscle thin laments. FEBS Lett 282, 313316. Labeit S, Kolmerer B. (1995a). Titins. Giant proteins in charge of muscle ultrastructure and elasticity. Science 276, 293296. Labeit S, Kolmerer B. (1995b). The complete primary structure of human nebulin and its correlation to muscle structure. J Mol Biol 248, 308315. Lahita RG. (1993). Sex hormones as immune modulators of disease. Ann NY Acad Sci 685, 278287. Lahti JM, Xiang J, Heath LS, Campana D, Kidd VJ. (1995). PITSLRE protein kinase activity is associated with apoptosis. Mol Cell Biol 15, 111. Lai FA, Erickson HP, Rousseau E, Liu QY, Meissner G. (1988). Purication and reconstitution of the calcium release channel from skeletal muscle. Nature 331, 315319. Laing NG, Wiltons SD, Akkari PA, Dorosz S, Boundy K, Kneebone C, Blumbergs P, White S, Watkins H, Love DR, Haan E. (1995). A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nature Genet 9, 7579. Lakshmi MS, Parker C, Sherbet GV. (1993). Metastasis associated mts1 and nm23 genes affect tubulin polymerisation in B16 melanoma cells. A possible mechanism of their regulation of metastatic behaviour. Anticancer Res 13, 299304.
296
Lakshmi MS, Parker C, Sherbet GV. (1997). Expression of the transmembrane glycoprotein CD44 and metastasis associated 18A2/mts1 gene in B16 murine melanoma cells. Anticancer Res 17, 34513456. Lala PK, Graham CH. (1990). Mechanisms of trophoblast invasiveness and their control. The role of proteases and protease inhibitors. Cancer Metastasis Rev 9, 369379. Lala PK, Orucevi A. (1998). Role of nitric oxide in tumour progression. Lessons from experimental tumours. Cancer Metastasis Rev 17, 91106. Lally G, Faull RLM, Waldvogel HJ, Ferrari S, Emson PC. (1997). Calcium homeostasis in ageing. Studies on the calcium binding protein calbindin D28K. J Neural Transmission 194, 11071112. Lambert PA, Somers KD, Kohn EC, Perry RR. (1997). Antiproliferative and anti-invasive effects of carboxyamido-triazole on breast cancer cell lines. Surgery 122, 372378. Landar A, Caddell G, Chessher J, Zimmer DB. (1996). Identication of an S100A1/S100B target protein phosphoglucomutase. Cell Calcium 20, 279285. Landon F, Gache Y, Touitou H, Olomucki A. (1985). Properties of two isoforms of human blood platelet alpha-actinin. Eur J Biochem 153, 231237. Lane TF, Iruela-Arispe ML, Johnson RS, Sage EH. (1994). SPARC is a source of copperbinding peptides that stimulate angiogenesis. J Cell Biol 125, 174186. Lane TF, Sage EH. (1990). Functional mapping of SPARC peptides from two distinct Ca2+binding sites modulate cells shape. J Cell Biol 111, 30653076. Lane TF, Sage EH. (1994). The biology of SPARC, a protein that modulates cell-matrix interactions. Fed Am Soc Exp Biol J 8, 163172. Langen R, Isas JM, Hubbell WL, Haigler HT. (1998). A transmembrane form of annexin XII detected by site-directed spin labelling. Proc Natl Acad Sci USA 95, 1406014065. Lanier LM, Gates MA, Witke W, Menzies AS, Wehman AM, Macklis JD, Kwiatkowski D, Soriano P, Gertier FB. (1999). Mena is required for neurulation and commissure formation. Neuron 22, 313325. Lappalainen P, Drubin DG. (1997). Colin promotes rapid actin lament turnover in vivo. Nature 388, 7882. Larsson N, Melander H, Marklund U, Osterman O, Gullberg M. (1995). G2/M transition requires multisite phosphorylation of oncoprotein-18 by two distinct protein kinase systems. J Biol Chem 270, 1417514183. Latvala T, Puolakkainen P, Vesaluoma M, Tervo T. (1996). Distribution of SPARC protein (osteonectin) in normal and wounded feline cornea. Exp Eye Res 63, 579584. Laub M, Jennissen HP. (1991). Ubiquitination of endogenous calmodulin in rabbit tissue extracts. FEBS 294, 220233. Lawler S, Gavet O, Rich T, Sobel A. (1998). Stathmin over-expression in 293 cells affects signal transduction and cell growth. FEBS Lett 421, 5560. Lazebnik YA, Takahashi A, Moir RD, Goldman RD, Poirier GG, Kaufmann SH, Earnshaw WC. (1995). Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci USA 92, 90429046. Leavitt J, Latter G, Lutomski L, Goldstein D, Burbeck S. (1986). Tropomyosin isoform switching in tumorigenic human broblasts. Mol Cell Biol 6, 27212726. Leavitt J, Ng SY, Aebi U, Varma M, Latter G, Burbeck S, Kedes L, Gunning P. (1987a). Expression of transfected mutant -actin genes: Alterations of cell morphology and evidence of autoregulation in actin pools. Mol Cell Biol 7, 24572466. Leavitt J, Ng SY, Varma M, Latter G, Burbeck S, Gunning P, Kedes L. (1987b). Expression of transfected mutant -actin genes: Transitions towards the stable tumorigenic state. Mol Cell Biol 7, 24672476.
References
297
Ledda F, Bravo AI, Adris S, Bover L, Mordoh J, Podhajcer OL. (1997). The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma. J Invest Dermatol 108, 210214. Lee A, Wong ST, Gallagher D, Li B, Storm DR, Scheur T, Catterall WA. (1999). Ca2+/calmodulin binds to and modulates P/Q-type calcium channels. Nature 399, 155158. Lee HJ, Sorimachi H, Jeong SY, Ishiura S, Suzuki K. (1998). Molecular cloning and characterisation of a novel tissue-specic calpain predominantly expressed in the digestive tract. Biol Chem 379, 175183. Lee HY, Lee H. (1999). Inhibitory activity of nm23-H1 on invasion and colonisation of human prostate carcinoma cells is not mediated by its NDP kinase activity. Cancer Lett 145, 9399. Lee L, Klee SK, Evangelista M, Boone C, Pellman D. (1999). Control of mitotic spindle position by Saccharomyces cerevisiae formin Bni 1p. J Cell Biol 144, 947961. Lee S, Clark SA, Gill RK, Christakos S. (1994). 1,25-dihydroxy vitamin D3 and pancreatic beta cell function, vitamin D receptors, gene expression, and insulin secretion. Endocrinol 134, 16021610. Lee SC, Kim IG, Marekov LN, OKeefe EJ, Parry DAD, Steinert PM. (1993). The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium binding protein. A cornied cell envelope precursor and an intermediate lament associated (cross-linking) protein. J Biol Chem 268, 1216412176. Lee SW, Reimer CL, Oh P, Campbell DB, Schnitzer JE. (1998). Tumour cell growth inhibition by caveolin re-expression in human breast cancer cells. Oncogene 16, 13911397. Lee SW, Tomasetto C, Sager R. (1991). Positive selection of candidate tumour suppressor genes by subtractive hybridisation. Proc Natl Acad Sci USA 88, 28252829. Lee SW, Tomasetto C, Swisshelm K, Keyomarsi K, Sager R. (1992). Down-regulation of a member of the S100 gene family in mammary carcinoma cells and re-expression by azadeoxycytidine treatment. Proc Natl Acad Sci USA 89, 25042508. Leers MPG, Aarts MMJ, Theunissen PHMH. (1998). E-cadherin and calretinin: A useful combination of immunochemical markers for differentiation between mesothelioma and metastatic adenocarcinoma. Histopathology 32, 209216. Leevers SJ, Van Haesebroeck B, Watereld MD. (1999). Signalling through phophoinositide kinases. The lipids take centre stage. Curr Opin Cell Biol 11, 219225. Lehman W, Kaminer B. (1984). Identication of a thin lament linked 35,000 dalton protein in vertebrate smooth muscle. Biophys J 45, 109a. Lehman W. (1991). Calponin and the composition of smooth muscle thin laments. J Muscle Res Cell Motility 12, 221224. Lena JY, Legrand C, Faivre-Sarrailh C, Sarlieve LL, Ferraz C, Rabie A. (1994). High gelsolin content of developing oligodendrocytes. Int J Develop Neurosci 12, 375386. Lenz SE, Braunewell KH, Weise C, Nedlinachittka A, Gundelnger ED. (1996). The neuronal EF-hand ca2+-binding protein VILIP. Interaction with cell membrane and actin-based cytoskeleton. Biochem Biophys Res Commun 225, 10781083. Leonardos L, Butler LM, Hewett PJ, Zalewski PD, Cowled PA. (1999). The activity of caspase-3-like protease is elevated during the development of colorectal carcinoma. Cancer Lett 143, 2935. Lephart ED, Watson MA, Jacobson NA, Rhees RW, Ladle DR. (1997). Calbindin-D28K is regulated by adrenal steroids in hypothalamic tissue during prenatal development. Develop Brain Res 100, 117120. Lephart ED, Watson MA, Rhees RW, Ladle DR, Jacobson NA. (1997). Developmental expression of calretinin in the medial basal hypothalamus and amygdala from male and female rats. Neurosci Res 28, 269273.
298
Levine J, Willard M. (1981). Fodrin. Axonally transported polypeptides associated with the internal periphery of many cells. J Cell Biol 90, 631641. Levy YY, Lai EY, Remillard SP, Heintzelman MB, Fulton C. (1996). Centrin is a conserved protein that forms diverse associations with centrioles and MTOCS in Naegleria and other organisms. Cell Motility Cytoskeleton 33, 298323. Lewalle JM, Cataldo D, Bajou K, Lambert C, Foidart JM. (1998). Endothelial cell intracellular Ca2+ concentration is increased upon breast tumour cell contact and mediates tumour cell transendothelial migration. Clin Exptl Metastasis 16, 2129. Leygue E, Snell L, Hiller T, Dotzlaw H, Hole K, Murphy LC, Watson PH. (1996). Differential expression of psoriasin messenger RNA between in situ and invasive human breast carcinoma. Cancer Res 56, 46064609. Li E, Bestor TH, Jaenisch R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915926. Li G, Comte M, Wollheim CB, Cox JA. (1989). Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium. Biochem J 260, 771775. Li J, Simpson L, Takahashi M, Miliarcsis C, Myers MP, Tonks N, Parsons R. (1998). The PTEN/MMAC1 tumour suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res 58, 56675672. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SL, Pue J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 19431947. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. (1997). Cytochrome c a dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479489. Li S, Okamoto T, Chun M Sargiacomo M, Casanova JE, Hansen SH, Nishimoto I, Lisanti MP. (1995). Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J Biol Chem 270, 1569315701. Li YP, Chen W, Stashenko P. (1995). Characterisation of a silencer element in the rst exon of the human osteocalcin gene. Nucleic Acids Res 23, 50645072. Li ZH, Banik NL. (1995). The localisation of m-calpain in myelin. Immunocytochemical evidence in different areas of rat brain and nerves. Brain Res 697, 112121. Lian JB, Stein GS, Stein JL, Van Wijnen A, McCabe L, Banerjee C, Hoffmann H. (1996). The osteonectin gene promoter provides a molecular blueprint for regulatory mechanisms controlling bone tissue formation. Role of transcription factors involved in development. Connective Tissue Res 35, 6975. Lian JB, Tassinari M, Glowacki J. (1984). Resorption of implanted bone prepared from normal and warfarin-treated rats. J Clin Invest 73, 12231226. Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, Zheng Z, Bose S, Call KM, Tsou HC, Peacocke M, Eng C, Parsons R. (1997). Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nature Genet 16, 6467. Lin CS, Chang CH, Huynh T. (1997). The murine L-plastin gene promoter. Identication and comparison with the human L-plastin gene promoter. DNA Cell Biol 16, 916. Lin JJC, Helfman DM, Hughes SH, Chou CS. (1985). Tropomyosin isoforms in chicken embryo broblasts. Purication, characterisation, and changes in Rous sarcoma virus transformed cells. J Cell Biol 100, 692703. Lin KM, Wenegieme E, Lu PJ, Chen CS, Yin HL. (1997). Gelsolin binding to phosphatidyl inositol 4,5bisphosphate is modulated by calcium and pH. J Biol Chem 272, 2044320450.
References
299
Lin P, Le Niculescu H, Hofmeister R, McCaffery JM, Jin MJ. (1998). The mammalian calcium-binding protein, nucleobindin (CALNUC), is a Golgi resident protein,. J Cell Biol 141,15151527. Lin P, Yao Y, Hofmeister R, Tsien RY, Farquhar MG. (1999). Over-expression of calnuc (nucleobindin) increases agonist and thapsigargin releasable Ca2+ storage in the Golgi. J Cell Biol 145, 279289. Lindahl T, Satoh MS, Poirier GG, Klungland A. (1995). Post-translational modication of poly (ADP-ribose) polymerase induced by DNA breaks. Trends Biochem Sci 20, 405411. Lingle WL, Salisbury JL. (1997). Centrins and the cytoskeleton of the protist Holomastigotoides. Cell Motility Cytoskeleton 36, 377390. Linke WA, Stockmeier MR, Ivemeyer M, Hosser H, Mudel P. (1998). Characterising titins I-band Ig domain as an entropic spring. J Cell Sci 111, 15671574. Linse S, Forsen S. (1995). Determinants that govern high afnity calcium binding. Adv Second Messenger Phosphoprotein Res 30, 89151. Linse S, Thulin E, Gifford LK, Radzewsky D, Hagan J, Wilk RR, Akerfeldt KS. (1997). Domain organisation of calbindin D28K as determined from the association of six synthetic EF-hand fragments. Protein Sci 6, 23852396. Lisanti MP, Tang ZL, Scherer PE, Kubler E, Koleske AJ, Sargiacomo M. (1995). Caveolae, transmembrane signalling and cellular transformation. Mol Memb Biol 12, 121124. Liu BP, Chrazanowska-Wodnicka M, Burridge K. (1998). Microtubule depolymerisation induces stress bres, focal adhesions, and DNA synthesis via GTP-binding protein Rho. Cell Adhesion Commun 5, 249255. Liu CWA, Lee G, Jay DG. (1999). Tau is required for neurite outgrowth and growth cone assembly. Cell Motility Cytoskeleton 43, 232242. Liu GX, Sheng HF, Wu S. (1996). A study of the levels of calmodulin and DNA in human lung cancer cells. Br J Cancer 73, 899901. Liu J, Aoki M, Illa I, Wu CY, Fardeau M, Angelini C, Serrano C, Urtizberea JA, Hentati F, Ben-Hamida M, Bohlega S, Culper EJ, Amato AA, Bossie K, Oeltjen T, Bejaoui K, McKenna-Yasek D, Hosler BA, Schurr E, Arahata K, De Jong PH, Brown RH. (1998). Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nature Genetics 20, 3136. Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, Schreiber SL. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807815. Liu JH, Wei S, Burnete PK, Gamero AM, Hutton M, Djeu JY. (1999). Functional association of TGF beta receptor II with cyclin B. Oncogene 18, 269275. Liu KY, Li LM, Nisson PE, Bruber C, Jessee J, Cohen SN. (1999). Reversible tumorigenesis induced by deciency of vasodilator-stimulated phosphoprotein. Mol Cell Biol 19, 36963703. Liu L, Wang MS, Fisher AB, Zimmerman UJP. (1996). Involvement of annexin II in exocytosis of lamellar bodies from alveolar epithelial type II cells. Am J Physiol 14, L668L676. Liu N, Fine RE, Johnson RJ. (1993). Comparison of cDNAs from bovine brain coding for 2 isoforms of calreticulin. Biochim Biophys Acta 1202, 7076. Liu N, Siek G, Fine RE, Johnson RJ. (1996). Calreticulin expression in differentiated vs. undifferentiated cells. Mol Biol Cell 7, 3069. Liu TC, Lin PM, Chang JG, Lee JP, Chen TP. (2000). Mutation analysis of PTEN/MMAC1 in acute myeloid leukaemia. Amer J Haematol 63, 170175. Liu WG, James CD, Frederick L, Alderete BE, Jenkins RB. (1997). PTEN/MMAC1 mutations and EGFR amplication in glioblastomas. Cancer Res 57, 52545257.
300
Liu X, Zou H, Slaughter C, Wang X. (1997). DFF, a heterodimeric protein that functions down stream of caspase 3 to trigger DNA fragmentation during apoptosis. Cell 89, 175184. Liu XS, Li P, Widlak P, Zou H, Luo X, Garrard WT, Wang XD. (1998). The 40-kDa subunit DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc Natl Acad Sci USA 95, 84618466. Liu ZD, Brattain MG, Appert H. (1997). Differential display of reticulocalbin in the highly invasive cell line MDA-MB-435, versus the poorly invasive cell line MCF-7. Biochem Biophys Res Commun 231, 283289. Lledo PM, Somasundaram B, Morton AJ, Emson PC, Mason WT. (1992). Stable transfection of calbindin D28K into the GH3 cell line alters calcium currents and intracellular calcium homeostasis. Neuron 9, 943954. Lloyd BH, Platt-Higgins A, Rudland PS, Barraclough R. (1998). Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumour cells. Oncogene 17, 465473. Loetscher H, Deuschle U, Brockhaus M, Reinhardt D, Nelboeck P, Mous J, Grunberg J, Haass C, Jacobsen H. (1997). Presenilins are processed by caspase-type proteases. J Biol Chem 272, 2065520659. Lofvenberg L, Backman L. (1999). Calpain-induced proteolysis of beta spectrins. FEBS Lett 443, 8992. Longthorne V, Willams G. (1997). Caspase activity is required for commitment to Fasmediated apoptosis. EMBO J 16, 38053812. Lu KP, Osmani SA, Osmani AH, Means AR. (1993). Essential roles for calcium and calmodulin G2/M progression in Aspergillus nidulans. J Cell Biol 121, 621630. Lu S, Davies PJA. (1997). Regulation of the expression of tissue transglutaminase gene by DNA methylation. Proc Natl Acad Sci USA 94, 46924697. Lu YL, Lin YZ, La Pushin R, Cuevas B, Fand XJ, Yu SX, Davies MA, Khan H, Furui T, Mao ML, Zinner R, Hung MC, Steck P, Siminovitch K, Mills GB. (1999). The PTEN/MMAC1/TEP tumour suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 18, 70347045. Luecke H, Chang BT, Mailliard WS, Schlaepfer DD, Haigler HT. (1995). Crystal structure of the annexin XII hexamer and implications for bilayer insertion. Nature 378, 512515. Luegmayr E, Varga F, Glantschnig H, Frantzl-Zelmann N, Rumpler M, Ellinger A, Klaushofer K. (1998). 1, 25-dihyroxyvitamin D-3 and triiodothyronine stimulate the expression of a protein immunologically related to osteocalcin. J Histochem Cytochem 46, 477486. Lukowski S, Lecomte MC, Mira JP, Marin P, Gautero H, Russo-Marie F, Geny B. (1996). Inhibition of phospholipase D activity by fodrin. An active role for the cytoskeleton. J Biol Chem 271, 2416424171. Lukowski S, Mira JP, Zachowski A, Geny B. (1998). Fodrin inhibits phospholipases A2, C, and D by decreasing polyphosphoinositide cell content. Biochem Biophys Res Commun 248, 278284. Lundgren S, Carling T, Jjalm G, Juhlin C, Rastad J, Pihlgren U, Rask L, Akerstrom G, Hellman P. (1997). Tissue distribution of human gp330/megalin, a putative Ca2+-sensing protein. J Histochem Cytochem 45, 383392. Luo C, Shaw KTY, Raghavan A, Aramburu J, Garcia-Cozar F, Perrino BA, Hogan PG, Rao A. (1996). Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import. Proc Natl Acad Sci USA 93, 89078912. Luo XN, Mookerjee B, Ferrari A, Mistry S, Atweh GF. (1994). Regulation of phosphoprotein P18 in leukaemia cells. Cell cycle-regulated phosphorylation by p34 (cdc2) kinase. J Biol Chem 269, 1031210318.
References
301
Lutz W, Lindle WL, Salisbury JL. (1995). Phosphorylation of centrin during prophase and metaphase of mitosis. Mol Biol Cell 6, 239. Lynch MP, Stein JL, Stein GS, Lian JB. (1995). The inuence of type 1 collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts. Modication of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralisation. Exp Cell Res 216, 3545. Ma LG, Xu XD, Cui SJ, Sun DY. (1999). The presence of a heterotrimeric G protein and its role in signal transduction of extracellular calmodulin pollen germination and tube growth. Plant Cell 11, 13511363. Maas RL, Jepeal LI, Elfering SL, Holcombe RF, Morton CC, Eddy RL, Byers MG, Shows TB, Leder P. (1991). A human gene homologous to the formin gene residing at the murine limb deformity locus. Chromosomal location and RFLPS. Am J Hum Genet 48, 687695. Maas RL, Zeller R, Woychik RP, Vogt TF, Leder P. (1990). Disruption of formin-encoding transcripts in two mutant limb deformity alleles. Nature 346, 853855. Mabuchi K, Li B, Ip W, Tao T. (1997). Association of calponin with desmin intermediate laments. J Biol Chem 272, 2266222666. Machleidt T, Li WP, Liu PS, Anderson RGW. (2000). Multiple domains in caveolin-1 control its intracellular trafc. J Cell Biol 148, 1728. Mackrill JJ, Sesay AK, Lai FA. (1999). Autoimmune antigen megalin displays similarities with ryanodine receptor/Ca2+- release channel. Int J Mol Med 3, 625632. MacNeil S, Lakey T, Tomlinson S. (1985). Calmodulin regulation of adenylate cyclase activity. Cell Calcium 6, 213226. Maeda H, Koizumi M, Yoshimura K, Yamauchi T, Kawai T, Ogata E. (1997). Correlation between bone metabolic markers and bone scan in prostatic cancer. J Urol 157, 539543. Maehama T, Dixon JE. (1998). The tumour suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidyl 3,4,5trisphosphate. J Biol Chem 273, 1337513378. Maehama T, Dixon JE. (1999). PTEN. A tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol 9, 125128. Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumiya S. (1999). Signalling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895898. Maelandsmo GM, Florenes VA, Mellingsaeter T, Hovig E, Kerbel RS, Fodstad O. (1997). Differential expression patterns of S100A2, S100A4 and S100A6 during progression of human malignant melanoma. Int J Cancer 74, 464469. Maelandsmo GM, Hovig E, Skrede M, Engebraaten O, Florenes VA, Myklebost O, Grigorian M, Lukanidin E, Scanlon KJ, Fodstad O. (1996). Reversal of the in vivo metastatic phenotype of human tumour cells by an anti-CAPL (mts1) ribozyme. Cancer Res 56, 54905498. Magnetto S, Boissier S, Delmas PD, Clezardin P. (1999). Additive antitumour activities of taxoids in combination with the bisphosphonate ibandronate against invasion and adhesion of human breast carcinoma cells to bone. Int J Cancer 83, 263269. Mailliard WS, Haigler HT, Schlaepfer DD. (1996). Calcium-dependent binding of S100C to the N-terminal annexin I. J Biol Chem 271, 719725. Mailliard WS, Luecke H, Haigler HT. (1997). Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers. A chemical cross-linking study. Biochemistry 36, 90459050.
302
Majak K, Berdel B, Kowianski P, Dziewiatkowski J, Lipowska M, Morys J. (1998). Parvalbumin immunoreactivity changes in the thalamic reticular nucleus during maturation of the rat brain. Folia Neuropathologica 36, 714. Malchiodi-Albedi F, Ceccarini M, Winkelmann JC, Morrow JS, Petrucci TC. (1993). The 270 kDa splice variant of erythrocyte beta-spectrin (beta I sigma 2) segregates in vivo and in vitro to specic domains of cerebellar neurones. J Cell Sci 106, 6778. Malhotra R, Sim RB. (1989). Chemical and hydrodynamic characterisation of the human leukocyte receptor for complement subcomponent C1q. Biochem J 282, 625631. Malhotra R, Willis AC, Jensenius JC, Jackson J, Sim RB. (1993). Structure and homology of the human C1q receptor (Collectin receptor). Immunology 78, 341348. Malinda KM, Goldstein AL, Kleinman HK. (1997). Thymosin beta (4) stimulates directional migration of human umbilical vein endothelial cells. FASEB J 11, 474481. Malmendal A, Carlstrom G, Hambraeus C, Drakenberg T, Forsen S, Akke M. (1998). Sequence and context dependence of EF-hand loop dynamics. An N-15 relaxation study of a calcium-binding site mutant of calbindin D-9K. Biochemistry 37, 25862595. Manabe M, Oguin WM. (1994). Existence of trichohyalin-keratohyalin hybrid granules. Colocalisation of two major intermediate lament associated proteins in non-follicular epithelia. Differentiation 58, 6575. Manabe M, Yaguchi H, Butt KI, Oguin WM, Loomis CA, Sung TT, Ogawa H. (1996). Trichohyalin expression in skin tumours. Retrieval of trichohyalin antigenicity in tissues by microwave irradiation. Int J Dermatol 35, 325329. Mandelkow EM, Schweers O, Grewes G, Biernat J, Gustke N, Trinczek B, Mandelkow E. (1996). Structure, microtubule interactions, and phosphorylation of tau protein. Ann NY Acad Sci 777, 96106. Mani RS, Kay CM. (1993). Calcium-dependent regulation of the caldesmon heavy meromyosin interaction by caltropin. Biochemistry 32, 1121711223. Mani RS, Kay CM. (1995a). Effect of caltropin on caldesmon-actin interaction. J Biol Chem 270, 66586663. Mani RS, Kay CM. (1995b). Inuence of caltropin on the caldesmon induced polymerisation of G-actin. Biochem Biophys Res Commun 213, 349355. Mani RS, McCubbin WD, Kay CM. (1992). Calcium-dependent regulation of caldesmon by an 11 kDa smooth muscle calcium binding protein, caltropin. Biochemistry 31, 1189611901. Marcelli M, Cunningham GR, Walkup M, He ZI, Sturgis L, Kagan C, Mannucci R, Nicoletti I, Teng BB, Benner L. (1999). Signalling pathway activated during apoptosis of the prostate cancer cell line LNCaP. Over-expression of caspase 7 as a new gene therapy strategy for prostate cancer. Cancer Res 59, 382390. Marenholz I, Volz A, Ziegler A, Davies A, Ragoussis I, Korge BP, Mischke D. (1996). Genetic analysis of the epidermal differentiation complex (EDC) on human chromosome 1q21. Chromosomal orientation, new markers, and a 6-Mb YAC contig. Genomics 37, 295302. Marin J, Encabo A, Briones A, Garcia-Cohen EC, Alonso MJ. (1998). Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: Calcium pumps. Life Sci 64, 279303. Marino M, Zheng G, McCluskey RT. (1999). Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured Fisher rat thyroid cells. J Biol Chem 274, 1289812904. Markova NG, Marekov LN, Chipev CC, Gan SQ, Idler WW, Steinert PM. (1993). Prolaggrin is a major epidermal calcium binding protein. Mol Cell Biol 13, 613625. Marklund U, Larsson N, Gradin HM, Brattsand G, Gullberg M. (1996). Oncoprotein-18 is a phosphorylation responsive regulator of microtubule dynamics. EMBO J 15, 52905298.
References
303
Marklund U, Osterman O, Melander H, Bergh A, Gullberg M. (1994). The phenotype of a cdc2 kinase target site-decient mutant of oncoprotein-18 reveals a role of this protein in cell cycle control. J Biol Chem 269, 3062630635. Marks PW, Maxeld F. (1990). Transient increases in cytosolic free calcium appear to be required for the migration of adherent neutrophils. J Cell Biol 110, 4352. Mars WM, Zarnegar R, Michalopoulos GK. (1993). Activation of hepatocyte growth factor by the plasminogen activator uPA and tPA. Am J Pathol 143, 949958. Marsh D, Coulon V, Lunetta KL, Rocca-Serra P, Dahia PLM, Zheng ZM, Liaw D, Caron S, Duboue B, Lin AY, Richardson AL, Bonnetblanc JM, Bressieux JM, Cabarrot-Moreau A, Chompret A, Demange L, Eeles RA, Yahanda AM, Fearon ER, Fricker JP, Gorlin RJ, Hodgson SV, Huson S, Lacombe D, Le Prat F, Odent S, Toulouse C, Olopade OI, Sobol H, Tishler S, Woods CG, Robinson BG, Weber HC, Parsons R, Peacocke M, Longy M, Eng C. (1998a). Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Human Mol Genet 7, 507515. Marsh DJ, Dahia PL, Coulon V, Zheng Z, Dorion-Bonnet F, Call KM, Little R, Lin AY, Eeles RA, Goldstein AM, Hodgson SV, Richardson AL, Robinson BG, Weber HC, Longy M, Eng C. (1998b). Allelic imbalance, including PTEN/MMAC1, at the Cowden disease locus on 10q22-23, in hamartomas from patient with Cowden syndrome and germline PTEN mutation. Genes Chromosomes Cancer 21, 6169. Martel V, Guerci A, Humbert JC, Gregoire MT, Chery M, Lederlin P, Jonveaux P. (1997). De novo methylation of tumour suppressor genes CDKN2A and CDKN2B is a rare nding in B-cell chronic lymphocytic leukaemia. Br J Haematol 99, 320324. Martin SJ, Green DR. (1995). Protease activation during apoptosis. Death by 1000 cuts. Cell 82, 349352. Martin SJ, OBrien GA, Nishioka WK, McGahon AJ, Mahboubi A, Saido TC, Green DR. (1995). Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem 270, 64256428. Martin V, Ribieras S, Song Wang XG, Lasne Y, Frappart L, Rio MC. (1997). Involvement of DNA methylation in the control of the expression of an oestrogen-induced breast cancer associated protein (pS2). J Cell Biochem 65, 95106. Martin WH, Creutz CE. (1987). Chromobindin-A. A Ca2+ and ATP regulated chromafn granule binding protein. J Biol Chem 262, 29032810. Martin WH, Creutz CE. (1990). Interactions of the complex secretory vesicle binding protein chromobindin A with nucleotides. J Neurochem 54, 612619. Martin WH, Fromer E, Creutz CE. (1989). Chromobindin A, A Ca2+-dependent and ATPdependent chromafn granule-binding protein, is found in a variety of tissues and in yeast. Biochem Biophys Res Commun 165, 3742. Martinez J, Fuentes M, Cambiazo V, Santibanez JF. (1999). Bone extracellular matrix stimulates invasiveness of oestrogen-responsive human mammary MCF7 cells. Int J Cancer 83, 278282. Marttunen MB, Hietanen P, Tiitinen A, Ylikorkala O. (1998). Comparison of effects of tamoxifen and toremifene on bone biochemistry and bone mineral density in postmenopausal breast cancer patients. J Clin Endocrinol Metabolism 83, 11581162. Marty S, Carroll P, Cellerino A, Castren E, Staiger, V, Thoenen H, Lindholm D. (1996). Brain derived neurotropic factor promotes the differentiation of various hippocampal nonpyramidal neurones, including Cajal-Retzius cells, in organotypic slice cultures. J Neurosci 16, 675687.
304
Maruyama K, Kanemaki N, May JD. (1995). Thyroid hormones on embryo development and appearance of heavy chain isoforms in turkeys (Melagris gallopavo). Comparative Biochem Physiol 112, 109117. Masaki T, Tokuda M, Ohnishi M, Watanabe S, Fujimura T, Miyamoto K, Itano T, Matsui M, Arima K, Shirai M, Maeba T, Sogawa K, Konishi R, Taniguchi K, Hatanaka Y, Hatase O, Nishioka M. (1996). Enhanced expression of the protein kinase-substrate annexin I in human hepatocellular carcinoma. Hepatology 24, 7281. Mashima T, Naito M, Tsuruo T. (1999). Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis. Oncogene 18, 24232430. Masiakowski P, Shooter E. (1988). Nerve growth factor induces genes for two proteins related to a family of calcium-binding proteins. Proc Natl Acad Sci USA 85, 12771281. Mason IJ, Taylor A, Williams JG, Sage H, Hogan BL. (1986). Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell culture shock glycoprotein of Mr 43,000. EMBO J 5, 14651472. Matena K, Boehm T, Dear TN. (1998). Genomic organisation of mouse Capn5 and Capn6 genes conrms that they are a distinct calpain subfamily. Genomics 48, 117120. Mathiasen IS, Lademann U, Jaattela M. (1999). Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by Bcl-2 but does not involve known caspases or p53. Cancer Res 59, 48484856. Matsumoto T, Sowa Y, Ohtani-Fujita N, Tamaki T, Takenaka T, Kuribayashi K, Sakai T. (1998). P53-independent induction of Waf1/cip1 is correlated with osteoblastic differentiation of vitamin D-3. Cancer Lett 129, 6168. Matsumura F, Lin JJC, Yamashiro-Matsumura S, Hightower MJ, Topp WC. (1982). Changes in the species of tropomyosin localised in microlaments upon viral transformation. J Cell Biol 95, p.A284. Matsumura F, Yamashiro-Matsumura S, Lin JJC. (1983). Isolation and characterisation of tropomyosin-containing microlaments from cultured cells. J Biol Chem 258, 66366644. Matsumura F, Yamashiro-Matsumura S. (1985). Purication and characterisation of multiple isoforms of tropomyosin from rat cultured cells. J Biol Chem 260, 38513859. Matsuura M, Ikebe M. (1995). Requirement of the 2-headed structure for the phosphorylationdependent regulation of smooth muscle myosin. FEBS Lett 363, 246250. Matusubara S, Yamaji Y, Fujita T, Kanayama T, Yamadori I, Sato M, Fujita J, Shiotani T, Takahara J. (1996). Cancer associated retinopathy syndrome. A case of small cell lung cancer expressing recoverin immunoreactivity. Lung Cancer 14, 265271. Maurer P, Hohenadl C, Hohenester E, Gohring W, Timpl R, Engel J. (1995). The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collage IV. J Mol Biol 253, 347357. Maurer P, Hohenester E, Engel J. (1996). Extracellular calcium binding proteins. Curr Opin Cell Biol 8, 609617. Maurer P, Sasaki T, Mann K, Gohring W, Schwarzbauer JE, Timpl R. (1997). Structural and functional characterisation of the extracellular calcium binding protein BM-40/secreted protein, acid, rich in cysteine/osteonectin from the nematode Caenorhabditis elegans. Eur J Biochem 248. 209216. Mayer U, Aumailley M, Mann K, Timpl R, Engel J. (1991). Calcium-dependent binding of basement protein BM-40 (Osteonectin, SPARC) to basement membrane collagen type IV. Eur J Biochem 189, 141159.
References
305
McColl KS, Ghe HL, Zhong HY, Whitacre CM, Berger NA, Distelhorst CW. (1998). Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves bcl-2 regulated caspase activation. Mol Cell Endocrinol 139, 229238. McCurdy DW, Kim M. (1998). Molecular cloning of a novel mbrin-like cDNA from Arabidopsis thaliana. Plant Mol Biol 36, 2331. McGinnis JF, Austin B, Klisak I, Heizmann C, Kojis T, Sparkes RS, Bateman JB, Lerious V. (1995). Chromosomal assignment of the human gene for the cancer associated retinopathy protein (recoverin) to chromosome 17p13.1. J Neurosci Res 40, 165168. McGinnis JF, Stepanik PL, Jariangprasert S, Lerious V. (1997). Functional signicance of recoverin localisation in multiple retinal cell types. J Neurosci Res 50, 487495. McGinnis KM, Whitton MM, Gnegy ME, Wang KKW. (1998). Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase 3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis. J Biol Chem 273, 1999320000. McKinnon PJ, Kapsetaki M, Margolskee RF. (1996). The exon structure of the mouse Sc1 gene is very similar to the mouse SPARC gene. Genome Res 6, 10771083. McMahon A, Wong BS, Iacopino AM, Ng MC, Chi S, German DC. (1998). Calbindin D28K buffers intracellular calcium and promotes resistance to degeneration in PC12 cells. Mol Brain Res 54, 5663. McNally EM, Moreira ED, Duggan DJ, Lisanti MP, Lidov HGW, Vainzof M, Bonnemann CG, Passos-Bueno MR, Hoffman EP, Zatz M, Kunkel LM. (1998a). Caveolin-3 in muscular dystrophy. Human Mol Genetics 7, 871-877. McNally EM, Ly CT, Kunkel LM. (1998b). Human -sarcoglycan is highly related to sarcoglycan (adhalin), the limb girdle muscular dystrophy 2D gene. FEBS Lett 422, 2732. McNulty TJ, Taylor CW. (1999). Extracellular heavy metal ions stimulate Ca2+ mobilisation in lymphocytes. Biochem J 339, 551561. Means AR. (1988). Molecular mechanisms of action of calmodulin. Recent Progress Hormone Res 44, 223259. Means RA. (1994). Calcium, calmodulin and cell cycle regulation. FEBS Lett 347, 14. Meberg PJ, Ono S, Minamide LS, Takahashi M, Bamburg JR. (1998). Actin depolymerising factor and colin phosphorylation dynamics: Response to signals that regulate neurite extension. Cell Motility Cytoskeleton 39, 172190. Medvedeva MV, Kolobova EA, Wang PY, Gusev NB. (1996). Interaction of proteolytic fragments of calmodulin with caldesmon and calponin. Biochem J 315, 10211026. Megidish T, Takio K, Titani K, Iwabuchi K, Hamaguchi A, Igarashi Y, Hakomori S. (1999). Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: Heat shock proteins, glucose-regulated protein, protein disulphide isomerase, and calreticulin. Biochemistry 38, 33693378. Mehlmann LM, Mikoshiba K, Kline D. (1996). Redistribution and increase in cortical inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse oocyte. Develop Biol 180, 489498. Mehrara BJ, Rowe NM, Steinbrech DS, Dudziak ME, Saadeh PB, McCarthy JG, Gittes GK, Longaker MT. (1999). Rat mandibular distraction osteogenesis. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression. Plastic Reconstructive Surg 103, 536547. Melloni E, Michetti M, Salamino F, Pontremoli S. (1998a). Molecular and functional properties of a calpain activator protein specic for -isoforms. J Biol Chem 273, 1282712831.
306
Melloni E, Michetti M, Salamino F, Sparatore B, Pontremoli S. (1998b). Mechanism of action of a new component of the Ca2+-dependent proteolytic system in rat brain. The calpain activator. Biochem Biophys Res Commun 249, 583588. Mendis DB, Ivy GO, Brown IR. (1998). SPARC/osteonectin mRNA is induced in blood vessels following injury to the adult rat cerebral cortex. Neurochem Res 23, 11171123. Menice CB, Hulvershorn J, Adam LP, Wang CLA, Morgan KG. (1997). Calponin and mitogen-activated protein kinase signalling in differentiated vascular smooth muscle. J Biol Chem 272, 2515725161. Menz S, Bumann J, Jaworski E, Malchow D. (1991). Mutant analysis suggests that cyclic GMP mediates the cyclic AMP induced Ca2+ uptake in Dictyostelium. J Cell Sci 99,187191. Meredith J, Mu ZM, Saido T, Du XP. (1998). Cleavage of the cytoplasmic domain of the integrin beta (3) subunit during endothelial cell apoptosis. J Biol Chem 273, 1952519531. Mery L, Mesaeli N, Michalak M, Opas M, Lew DP, Krause KH. (1996). Over-expression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ inux. J Biol Chem 271, 93329339. Merzak A, Koochekpour S, Pilkington GJ. (1994a). CD44 mediates glioma cell adhesion and invasion in vitro. Cancer Res 54, 39883992. Merzak A, Parker C, Koochekpour S, Sherbet GV, Pilkington GJ. (1994b). Over-expression of the 18A2/mts1 gene and down-regulation of the TIMP-2 gene in invasive human glioma cell lines in vitro. Neuropathol App Neurobiol 20, 614619. Mesaeli N, Nakamura K, Zvaritch E, Dickie P, Dziak E, Krause KH, Opas M, MacLennan DH, Michalak M. (1999). Calreticulin is essential for cardiac development. J Cell Biol 144, 857868. Messerli M, Robinson KR. (1997). Tip localised Ca2+ pulses are coincident with peak pulsative growth rates in polles tubes of Lilium longiorum. J. Cell Sci 110, 12691278. Messier JM, Shaw L, Chafel M, Matsudaira P, Mercurio A. (1993). Fimbrin localised to an insoluble cytoskeletal fraction is constitutively phosphorylated on its head piece domain in adherent macrophages. Cell Motility Cytoskeleton 25, 223233. Meyer T, Brinck U. (1997). Expression of myogenic marker proteins in human leiomyosarcoma. APMIS 105, 793800. Miale IL, Sidman RL. (1961). An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp Neurol 4, 277296. Miano JM, Krahe R, Garcia E, Elliott JM, Olson EN. (1997). Expression, genomic structure and high resolution mapping to 19p13.2 of the human smooth muscle cell calponin gene. Gene 197, 215224. Michalowitz D, Halevy O, Oren M. (1990). Conditional inhibition of transformation and cell proliferation by a temperature sensitive mutant of p53. Cell 62, 671680. Michel M, Fleckman P, Dale BA. (1998). Calpain I is expressed in early normal skin development but is reduced in neonatal harlequin ichthyosis. J Invest Dermatol 110, 533. Michel M, Fleckman P, Smith LT, Dale BA. (1999). The calcium-activated protease calpain I is present in normal foetal skin and is decreased in neonatal harlequin ichthyosis. Br J Dermatol 141, 10171026. Middendorp S, Paoletti A, Schiebel E, Bornens M. (1997). Identication of a new mammalian centrin gene more closely related to Saccharomyces cerevisiae CDC31 gene. Proc Natl Acad Sci USA 94, 91419146. Milan D, Grifth J, Su M, Price E, McKeon F. (1994). The latch region of calcineurin B is involved in both immunosuppressantimmunophilin complex docking and phosphatase activation. Cell 79, 437447.
References
307
Milarsky K, Morimoto RI. (1986). Expression of human hsp70 during the synthetic phase of the cell cycle. Proc Natl Acad Sci USA 82, 95179521. Millake DB, Blanchard AD, Patel B, Critchley DR. (1989). The cDNA sequence of a human placental alpha-actinin. Nucleic Acids Res 17, 6725. Miller J, McLachlan AD, Klug A. (1985). Repetitive zinc binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J 4, 16091614. Miller JR, Moon RT. (1996). Signal transduction through beta catenin and specication of cell fate during embryogenesis. Genes Development 10, 25272539. Millevoi S, Trombitas K, Kolmerer B, Kostin S, Schaper J, Pelin K, Granzier H, Labeit S. (1998). Characterisation of nebulette and nebulin and emerging concepts of their role for vertebrate Z-discs. J Mol Biol 282, 111123. Mills JC, Stone NL, Erhardt J, Pittman RN. (1998). Apoptotic membrane blebbing in regulated by myosin light chain phosphorylation. J Cell Biol 140, 627636. Millward TA, Cron P, Hemmings BA. (1995). Molecular cloning and characterisation of a conserved nuclear serine (threonine) protein kinase. Proc Natl Acad Sci USA 92, 50225026. Millward TA, Heizmann CW, Schafer BW, Hemmings BA. (1998). Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins. EMBO J 17, 59135922. Milne JL, Coukell MB. (1991). A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors. J Cell Biol 112, 103110. Milner RE, Baksh S, Shemanko C, Carpenter MR, Smillie E, Vance JE, Opas M, Michalak M. (1991). Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. J Biol Chem 266, 71557165. Minami H, Tokumitsu H, Mizutani A, Watanabe Y, Watanabe M, Hidaka H. (1992). Specic binding of CAP-50 to calcyclin. FEBS Lett 305, 217219. Minami Y, Emori Y, Imajoh-Ohmi S, Kawasaki S, Suzuki K. (1988). Carboxyl terminal truncation and site directed mutagenesis of the EF-hand structure domain of the small subunit of rabbit calcium-dependent protease. J Biochem 104, 927933. Minetti C, Sotgia F, Egeo A, Donati MA, Volonte D, Galbiati F, Cordone G, Bricarelli FD, Lisanti MP, Zaara F. (1998). Mutations in the caveolin-3 gene cause autosomal dominant limb girdle muscular dystrophy. Nat Genet 18, 365368. Mino T, Yuasa U, Nakamura F, Naka M, Tanaka T. (1998). Two distinct actin-binding sites of smooth muscle calponin. Eur J Biochem 251, 262268. Minocherhomjee AM, Shattuck RL, Storm DR. (1988). Calmodulin-stimulated adenylate cyclase. In Calmodulin, eds. Cohen P, Klee CB, Elsevier, New York, pp 249263. Minota S, Cameron B, Welch WJ, Wineld JB. (1988). Autoantibodies to the constitutive 73kDa member of the HSP70 family of heat shock proteins in systemic lupus erythematosus. J Exp Med 168, 14751480. Mirre C, Monlauzuer L, Garcia M, Delgrossi MH, Le Bivic A. (1996). Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin. Am J Physiol 40, C887894. Mirzayans R, Famulski KS, Enns L, Fraser M, Paterson MC. (1995). Characterisation of the signal transduction pathway mediating gamma ray induced inhibition of DNA synthesis in human cells. Indirect evidence for involvement of calmodulin but not protein kinase C nor p53. Oncogene 11, 15971605.
308
Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. (1996). Genes encoding structural proteins of epidermal cornication and S100 calcium binding proteins from a gene complex (epidermal differentiation complex) on human chromosome 1q21. J Invest Dermatol 106, 989992. Mishra VS, Henske EP, Kwiatkowski DJ, Southwick FS. (1994). The human actin-regulatory protein cap G. Gene structure and chromosome location. Genomics 23, 560565. Mistry SJ, Atweh GF. (1999). Stathmin expression in immortalised and oncogene transformed cells. Anticancer Res 19, 573577. Mitchell RD, Simmerman HKB, Jones LR. (1988). Ca2+-binding effects on protein conformation and protein interactions of canine cardiac calsequestrin. J Biol Chem 263, 13761381. Mittermann I, Fetrow JS, Schaak DL, Almo SC, Kraft D, Heberle-Bors E, Valenta R. (1998). Oligomerisation of prolins from birch, man and yeast. Prolin, a ligand for itself? Sexual Plant Reproduction 11, 183191. Miyakoda M, Nakahata K, Suzuki K, Kodama S, Watanabe M. (1999). Heat-induced G1 arrest is dependent on p53 function but not on rb dephosphorylation. Biochem Biophys Res Commun 266, 377381. Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM. (1995). Integrin function. Molecular hierarchies of cytoskeletal and signalling molecules. J Cell Biol 131, 791805. Miyata S, Khan AM, Hatton GI. (1998). Co-localisation of calretinin and calbindin-D28K with oxytocin and vasopressin in rat supraoptic nucleus neurones. A quantitative study. Brain Res 785, 178182. Mizunuma M, Hirata D, Miyahara K, Tsuchiya E, Miyakawa T. (1998). The role of calcineurin and Mpk-1 in regulating the onset of mitosis in budding yeast. Nature 392, 303306. Mohiti J, Casell AM, Walker JH. (1995). Calcium induced relocation of annexin IV and annexin V in the human osteosarcoma cell line MG-63. Mol Memb Biol 12, 321329. Mohiti J, Caswell AM, Walker JH. (1997). The nuclear localisation of annexin V in the human osteosarcoma cell line MG-63 depends on serum factors and tyrosine kinase signalling pathway. Exp Cell Res 234, 98104. Mohri T, Kameshita I, Suzuki S, Hioki K, Tokunaga R, Takatani S. (1998). Rapid adhesion and spread of non-adherent colon cancer Colo201 cells induced by the protein kinase inhibitors, K252a and KT5720 and suppression of the adhesion by immunosuppressants FK506 and cyclosporin A. Cell Struct Funct 23, 255264. Mok SC, Chan WY, Wong KK, Muto MG, Berkowitz RS. (1996). SPARC, an extracellular matrix protein with tumour-suppressing activity in human ovarian epithelial cells. Oncogene 12, 18951901. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. (1998). A calcineurin dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215228. Moncman CL, Wang K. (1999). Functional dissection of nebulette demonstrates actin binding of nebulin-like repeats and Z-line targeting of SH3 and linker domains. Cell Motility Cytoskeleton 44, 122. Monteith GR, Wanigasekara Y, Roufogalis BD. (1998). The plasma membrane calcium pump, its role and regulation. New complexities and possibilities. J Pharmacol Toxicol Meth 40, 183190. Moog-Lutz C, Bouillet P, Regnier CH, Tomasetto C, Matei MG, Chennard MP, Anglard P, Rio MC, Basset P. (1995). Comparative expression of the psoriasin (S100A7) and S100C genes in breast carcinoma and co-localisation to human chromosome 1q21-q22. Int J Cancer 63, 297303.
References
309
Moore PB and Dedman JR. (1984). Calcimedins. Calcium-induced hydrophobic binding proteins. Ann N Y Acad Sci 435, 173175. Moore PB, Roisen F, Yorke G, Bird JWC. (1984). Calcimedins. Altered levels in both dystrophic chicken muscle. J Cell Biol 99, A433. Mor C, Gariboldi A, Bissi O, Concas D, Di Mattia D, Gajani MR, Gilardoni E, Liverani A, Mancini S, Santi C, Bartorelli A. (1997). On the compassionate use of UK101 in metastatic cancer. J Tumour Marker Oncol 12, 2937. Moreira ES, Vainzof M, Marie SK, Sertie AL, Zatz M, Passos-Bueno MR. (1997). The seventh form of autosomal recessive limb girdle muscular dystrophy is mapped to 17q11-12. Amer J Human Genetics 61, 151159. Morii N, Teru-Uchi T, Tominaga T, Kumagi N, Kozaki S, Ushikubi F, Narumiya S. (1992). A Rho gene product in human blood platelets. II Effects of the ADP ribosylation by botulinum C3 ADP ribosyltransferase on platelet aggregation. J Biol Chem 267, 2092120926. Morimoto AM, Berson AE, Fujii GH, Tneg DHF, Tavtigian SV, Bookstein R, Steck PA, Bolen JB. (1999). Phenotypic analysis of human glioma cells expressing the MMAC1 tumour suppressor phosphatase. Oncogene 18, 12611266. Morimoto AM, Tomlinson MG, Nakatani K, Bolen JB, Roth RA, Herbst R. (2000). The MMAC1 tumour suppressor phosphatase inhibits phospholipase C and integrin-lined kinase activity. Oncogene 19, 200209. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. (1997). Activation of beta catenin-Tcf signalling in colon cancer by mutations in beta catenin or APC. Science 275, 17871790. Morishi K, Huang DCS, Cory S, Adams JM. (1999). Bcl-2 family members do not inhibit apoptosis by binding the caspase activator apaf-1. Proc Natl Acad Sci USA 96, 96839688. Morishima-Kawashima M, Hasegawa M, Takio K, Suzuki M, Yoshida H, Titani K, Ihara Y. (1995). Proline-directed and non-proline-directed phosphorylation of PHF-tau. J Biol Chem 270, 823829. Moriya S, Yanagihara K, Fujita H, Kuzumaki N. (1994). Differential expression of HSP90, gelsolin and GST-Pi in human gastric carcinoma cell lines. Int J Oncol 5, 13471351. Morse SS, Moore PB. (1988). 67-K calcimedin (67-kDa) is distinct from p67 calelectrin and lymphocyte 68-kDa Ca2+-binding protein. Biochem J 251, 171174. Morton DM, Tchao R. (1994). Regulation of motility and cytoskeletal organisation of rat bladder carcinoma cells by cyclic AMP. Cell Motility Cytoskeleton 29, 375382. Mosavin R, Mellon WS. (1996). Post-transcriptional regulation of osteocalcin mRNA in clonal osteoblast cells by 1, 25-dihydroxy vitamin D3. Arch Biochem Biophys 332, 142152. Moses MA, Wiederschain D, Wu IM, Fernandez CA, Ghaziziadeh V, Lane WS, Flynn E, Sytkowski A, Tao T, Langer R. (1999). Troponin I is present in human cartilage and inhibits angiogenesis. Proc Natl Acad Sci USA 96, 26452650. Moursi AM, Damsky CH, Lull J, Zimmerman D, Doty SB, Aota SI, Globus RK. (1996). Fibronectin regulates calvarial osteoblast differentiation. J Cell Sci 109, 13691380. Moursi AM, Globus RK, Damsky CH. (1997). Interactions between integrin receptors and bronectin are required for calvarial osteoblast differentiation in vitro. J Cell Sci 110, 21872196. Mugita N, Kimura Y, Ogawa M, Saya H, Nakao M. (1997). Identication of a novel, tissuespecic calpain htra-3, a human homologue of the Caenorhabditis elegans sex determination gene. Biochem Biophys Res Commun 239, 845850. Mund A, Simson J, Rotheeld N. (1963). Effect of pregnancy on course of systemic lupus erythematosus. JAMA 183, 109112.
310
Mundy GR, Poser JW. (1983). Chemotactic activity of the gamma-carboxyglutamic acidcontaining protein in bone. Calcied Tissue Int 35, 164168. Munemitsu S, Albert I, Souza B, Rubinfeld B, Polakis P. (1995). Regulation of intracellular beta catenin levels by the adenomatous polyposis coli (APC) tumour suppressor protein. Proc Natl Acad Sci USA 92, 30463050. Munroe PB, Olgunturk RO, Fryns JP, Van Maldergem L, Ziereisen F, Yuksel B, Gardiner RM, Chung E. (1999). Mutations in the gene encoding the human matrix gla-protein cause Keutel syndrome. Nature Genetics 21, 142144. Muramatsu Y, Kamegai A, Shiba T, Shreshta P, Takai Y, Mori M, Ilg EC, Schafer BW, Heizmann CW. (1997). Histochemical characteristics of calcium binding S100 proteins and bone morphogenetic proteins in chondro-osseous tumours. Oncol Rep 4, 4953. Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M, Masaki H, Maruyama K, Tsutusumi Y, Moriguchi Y, Shibazaki Y, Tanaka Y, Iwasaka T, Inada M, Matsubara H. (1998). Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circulation Res 82, 13381348. Murata M, Peranen J, Schreiner R, Wieland F, Kurzchalia TV, Simons K. (1995). VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci USA 92, 1033910343. Murata T, Shinya N, Yamaguchi M. (1997). Expression of calcium binding protein regucalcin mRNA in the cloned human hepatoma cells HepG2. Stimulation by insulin. Mol Cell Biochem 175, 163168. Murata T, Yamaguchi M. (1998). Tissue-specic binding of nuclear factors to the 5-anking region of the rat gene for calcium binding protein regucalcin. Mol Cell Biochem 178, 305310. Murphy GA, Solski PA, Jillian SA, Dela-Ossa PP, DEustachio P, Der CJ, Rush MG. (1999). Cellular functions of TC10, a Rho family GTPase. Regulation of morphology, signal transduction and cell growth. Oncogene 18, 3831-3845. Murphy MA, Thirkill CE, Hart WM. (1997). Paraneoplastic retinopathy. A novel autoantibody reaction associated with small cell lung carcinoma. J Neuro-Ophthamol 17, 7783. Murphy TJ, Alexander RW, Girendling KK, Runge MS, Berstein KE. (1991). Isolation of a cDNA encoding the vascular type I angiotensin II receptor. Nature 351, 233236. Murphy-Ullrich JE, Lane TF, Pallero MA, Sage EH. (1995). SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca2+-binding EF-hand. J Cell Biochem 57, 341350. Murray BE, Ohlendieck K. (1998). Complex formation between calsequestrin and the ryanodine receptor in fast- and slow-twitch rabbit skeletal muscle. FEBS Lett 429, 317322. Murray NR, Davidson LA, Chapkin RS, Gustafson WC, Schattenberg DG, Fields AP. (1999). Over-expression of protein kinase C beta (II) induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J Cell Biol 145, 699711. Murray SS, Grisanti MS, Bentley GV, Kahn AJ, Urist MR, Murray EJB. (1997). The calpaincalpastatin system and cellular proliferation and differentiation in rodent osteoblastic cells. Exp Cell Res 233, 297309. Murtha P, Tindall DJ, Young CYF. (1993). Androgen induction of a human prostate specic kallikrein, HKLK2. Characterisation of an androgen responsive element in the 5 promoter region of the gene. Biochemistry 32, 64596464. Musaro A, McCullagh KJA, Naya FJ, Olson EN, Rosenthal N. (1999). IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-Atc1. Nature 400, 581593. Nagao S, Saido TC, Akita Y, Tsuchiya T, Suzuki K, Kwashima S. (1994). Calpain calpastatin interactions in epidermoid carcinoma KB cells. J Biochem 115, 11781184.
References
311
Nagaoka R, Minami N, Hayakawa K, Abe H, Obinata T. (1996). Quantitative analysis of low molecular weight G-actin binding proteins, colin and prolin, expressed in developing and degenerating chicken skeletal muscles. J Muscle Res Cell Motility 117, 463473. Nagata S, Golstein P. (1995). The Fas death factor. Science 267, 14491456. Nagata S. (1997). Apoptosis by death factor. Cell 88, 355365. Naguib RNG, Adams AE, Horne CHW, Angus B, Smith AF, Sherbet GV, Lennard TWJ. (1997). Prediction of nodal metastasis and prognosis in breast cancer. A neural model. Anticancer Res 17, 27352742. Naguib RNG, Albertazzi E, Cajone F, Leone BE, Lakshmi MS, Sherbet GV. (1998). Statistical and blind neural analyses of the expression of the metastasis associated genes h-mts1 (S100A4) and nm23 in carcinoma of the breast. Proc 20th Ann Int Conf IEEE/EMBS, Hong Kong, pp. 1426-1429. Naik UP, Patel PM, Parise LV. (1997). Identication of a novel calcium binding protein that interacts with the integrin IIb cytoplasmic domain. J Biol Chem 272, 46514654. Nair AK, Menon KMJ. (2000). Regulatory role of the 3 untranslated region of luteinising hormone receptor. Effect on mRNA stability. FEBS Lett 471, 3944. Naka M, Qing ZX, Sasaki T, Kise H, Tawara I, Hamaguchi S, Tanaka T. (1994). Purication and characterisation of a novel calcium binding protein, S100C from porcine heart. Biochim Biophys Acta 1223, 348353. Naka M, Yuasa U, Mino T, Tanaka T. (1998). Agonist induced phosphorylation of calponin in porcine coronary artery smooth muscle. Http://www.medic.mie.u.ac.jp Poster BK0211. Nakajo S, Okamoto M, Masuda Y, Sakai I, Ohsawa S, Nakaya K. (1996). Geranylgeraniol causes a decrease in levels of calreticulin and tyrosine phosphorylation of a 36-kDa protein prior to the appearance of apoptotic features in HL-60 cells. Biochem Biophys Res Commun 226, 741745. Nakamura N, Takenaga K. (1998). Hypomethylation of the metastasis-associated S100A4 gene correlates with gene activation in human colon adenocarcinoma cell lines. Clin Exptl Metastasis 16, 471479. Nakamura S, Kamihagi K, Satakeda H, Katayama M, Pan H, Okamoto H, Noshiro M, Takahashi K, Yoshihara Y, Shimmei M, Okada Y, Kato Y. (1996). Enhancement of SPARC (osteonectin) synthesis in arthritic cartilage. Increased levels in synovial uids from patients with rheumatoid arthritis and regulation by growth factors and cytokines in chondrocyte cultures. Arthritis Rheumatism 39, 539551. Nakayama H, Yasui W, Yokozaki H, Tahara E. (1993). Reduced expression of nm23 is associated with metastasis of gastric carcinomas. Japanese J Cancer Res 84, 184190. Nakayama T, Ohtsuru A, Nakao K, Shima M, Nakata K, Watanabe K, Ishii N, Kimura N, Nagataki S. (1992). Expression of in hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product. J Natl Cancer Inst 84, 13491354. Namba Y, Ito M, Zu YL, Shigesada K, Maruyama K. (1992). Human T-cell plastin bundles actin laments in a calcium-dependent manner. J Biol Chem 112, 503507. Nambu Y, Iannettoni MD, Orringer MD, Beer DG. (1998). Unique expression patterns and alterations in the intestinal protein villin in parry and metastatic pulmonary adenocarcinomas. Mol Carcinogenesis 23, 234242. Nanba Y, Nishida K, Yoshikawa T, Sato T, Inoue H, Kuboki Y. (1997). Expression of osteonectin in articular cartilage of osteoarthritic knees. Acta Med Okayama 51, 239243. Nardi MC, Giacomelli E, Dainese P, Fitchete-Laine AC, Faye L Baldan B, Navazio L, Mariani P. (1998). Ginkgo biloba expresses calreticulin, the major calcium-binding reticuloplasmic in eukaryotic cells. Botanica Acta 111, 6670.
312
Navazio L, Baldan B, Dainese P, Ames P, Damiani E, Margreth A, Mariani P. (1995). Evidence that spinach leaves express calreticulin but not calsequestrin. Plant Physiol 109, 983990. Navazio L, Sponga L, Dainese P, Fitchette-Laine AC, Faye L, Baldan B, Mariani P. (1998). The calcium binding protein calreticulin in pollen of Liriodendron tulipifera L. Plant Sci 131, 3542. Neamati N, Fernandez A, Wright S, Kiefer J, McConkey DJ. (1995). Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolate thymocyte nuclei. J Immunol 154, 37883795. Nelen MR, Van Staveren WC, Peeters EA, Hassel MB, Gorlin RJ, Hamm H, Lindboc CF, Fryns JP, Sijmons RH, Woods DG, Mariman ECM, Padberg GW, Kremer H. (1997). Germline mutations in the PTEN/MMAC1 gene in patients with Cowdens disease. Human Mol Genet 6, 13831387. Nelson JB, Lee WH, Nguyen SH, Jarrard DF, Rooks JD, Magnuson SR, Opgenorth TJ, Nelson WG, Bova GS. (1997). Methylation of the 5 CpG island of the endothelin B receptor gene is common in human prostate cancer. Cancer Res 57, 3537. Nelson MR, Chazin WJ. (1998). An interaction-based analysis of calcium-induced conformational changes in Ca2+-sensor proteins. Protein Sci 7, 270282. Nelson PS, Plymate SR, Wang K, True LD, Ware JL, Gan L, Liu AE, Hood L. (1998). Hevin, an anti-adhesive extracellular matrix protein, is down-regulated in metastatic prostate adenocarcinoma. Cancer Res 58, 232236. Nelson WJ, Shore EM, Wang AZ, Hammerton RW. (1990). Identication of a membrane cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin and fodrin in Madin-Darby canine kidney epithelial cells. J Cell Biol 110, 349357. Neri LM, Billi AM, Manzoli L, Rubbini S, Gjilmour RS, Cocco L, Martelli AM. (1994). Selective nuclear translocation of protein kinase C in Swiss 3T3 cells treated with IFG-I, PDGR and EGF. FEBS Lett 347, 6368. Neuberger T, Chakrabarti AK, Russel T, De Vries GH, Hogan EL, Banik NL. (1997). Immunolocalisation of cytoplasmic and myelin m-calpain in transfected Schwann cells. I. Effect of treatment with growth factors. J Neurosci Res 47, 521530. Neumann B, Barlow DP. (1996). Multiple roles for DNA methylation in gametic imprinting. Current Opinion Genet Develop 6, 159163. Newton RA. Hogg N. (1998). The human S100 MRP14 is a novel activator of the 2 integrin Mac-1 on neutrophils. J Immunol 160, 14271435. Ng HH, Bird A. (1999). DNA methylation and chromatin modication. Current Opinions Genet Develop 9, 158163. Nguyen TQ, Capra JD, Sontheimer RD. (1996). Calreticulin is transcriptionally up-regulated by heat shock, calcium and heavy metals. Mol Immunol 33, 379386. Nickerson J, Wells W. (1988). The microtubule associated nucleoside diphosphate kinase. J Biol Chem 259, 1129711304. Niemeier A, Willnow T, Dieplinger H, Jacobsen C, Meyer N, Hilpert J, Beisiegel U. (1999). Identication of megalin/gp330 as a receptor for lipoprotein(a) in vitro. Arterioscl Thrombosis Vasc Biol 19, 552561. Niesset JE, Redeld AR, Jin F, Knudsen KA, Johnson KR, Wheelock MJ. (1997). Characterisation of the interactions of alpha-catenin with alpha-actinin and beta-catenin/plakoglobin. J Cell Sci 110, 10131022. Nigro V, Moreira ED, Piluso G, Vainzof M, Belsito A, Politano L, Puca AA, Passos-Bueno MR, Zatz M. (1996). Autosomal recessive limb girdle muscular dystrophy, LGMD-2F, caused by a mutation in the -sarcoglycan gene. Nature Genetics 14, 195198.
References
313
Nikitenko DLL, Lloyd BH, Rudland PS, Fear S, Barraclough R. (2000). Localisation by in situ hybridisation of S100A4 (p9Ka) mRNA in primary human breast tumour specimens. Int J Cancer 86, 219228. Nirunsuksiri W, Presland RB, Brumbaugh SG, Dale BA, Fleckman P. (1995). Decreased prolaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control. J Biol Chem 270, 871876. Nirunsuksiri W, Zhang SH, Fleckman P. (1998). Reduced stability and bi-allelic, coequal expression of prolaggrin mRNA in keratinocytes cultured from subjects with ichthyosis vulgaris. J Invest Dermatol 110, 854861. Nischt R, Pottgiesser J, Krieg T, Mayer U, Aumailley M, Timpl R. (1991). Recombinant expression and properties of the human calcium binding extracellular matrix protein BM-40. Eur J Biochem 200, 529536. Nishi M, Kawata M, Azmitia EC. (1997). S100 beta promotes the extension of microtubule associated protein 2 (MAP2)-immunoreactive neurites retracted after colchicine treatment in rat spinal cord cultures. Neurosci Lett 229: 212214. Nishimura T, Goll DE. (1991). Binding of calpain fragments to calpastatin. J Biol Chem 266, 1184211850. No D, Yao TP, Evans RM. (1996). Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 93, 3346-3351. Nobes CD, Hall A. (1995). Rho, rac, and Cdc42 GTPases regulate the assembly of multimolecular local complexes associated with the actin stress bres, lamellipodia, and lopodia. Cell 81, 53. Nobes CD, Hall A. (1999). Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 144, 12351244. Noegel A, Witke W, Schleicher M. (1987). Calcium sensitive non-muscle alpha-actinin contains EF-hand structures and highly conserved regions. FEBS Lett 221, 391396. Noguchi M, Sarin A, Aman MJ, Nakajima H, Shores EW, Henkart PA, Leonard WJ. (1997). Functional cleavage of the common cytokine receptor gamma chain (gamma c) by calpain. Proc Natl Acad Sci USA 94, 1153411539. Noguchi M, Sarin A, Aman MJ, Nakajima H, Shores EW, Henkart PA, Leonard WJ. (1997). Functional cleavage of the common cytokine receptor chain (c) by calpain. Proc Natl Acad Sci USA 94, 1153411539. Nomura S, Wills AJ, Edwards DR, Heath JK, Hogan BLM. (1988). Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA revealed by in situ hybridisation. J Cell Biol 106, 441450. Norrby K. (1998). Nitric oxide suppresses bFGF- and IL-1-alpha-mediated but not BEGF 165-mediated angiogenesis in natively vascularised mammalian tissues. Acta Pathol Microbiol Immunol Scand 106, 11421148. North G. (1991). Cell cycle. Starting and stopping. Nature 351, 604605. Novy RE, Lin JLC, Lin CS, Lin JJC. (1993b). Human broblast tropomyosin isoforms. Characterisation of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in transformed cells. Cell Motility Cytoskeleton 25, 267281, Novy RE, Lin JLC, Lin JJC. (1991). Characterisation of cDNA clones encoding a human broblast caldesmon isoform and analysis of caldesmon expression in normal and transformed cells. J Biol Chem 266, 1691716924. Novy RE, Sellers Jr, Liu LF, Lin JJC. (1993a). In vitro functional characterisation of bacterially expressed human broblast tropomyosin isoforms and their chimeric mutants. Cell Motility Cytoskeleton 26, 248261. OKeefe EJ, Hamilton EH, Lee SC, Steinert P. (1993). Trichohyalin. A structural protein of hair, tongue, nail and epidermis. J Invest Dermatol 101, S65S71.
314
OKeefe SJ, Tamura J, Kincaid RL, Tocci MJ, ONeill EA. (1992). FK506-sensitive and CsAsensitive activation of the interleukin-2 promoter by calcineurin. Nature 357, 692694. ONeill KT, De Grado WF. (1990). How calmodulin binds its targets: Sequence independent recognition of amphiphilic -helices. Trends Biochem Sci 15, 5964. OShea JJ. (1997). Jaks, STATs, cytokine signal transduction, and immunoregulation. Are we there yet? Immunity 7, 111. OSullivan E, Kinnon C, Brickell P. (1999). Wiskott-Aldrich syndrome protein, WASP. Int J Biochem Cell Biol 31, 383387. Oancea E, Meyer T. (1998). Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307318. Oates AJ, Barraclough R. Rudland PS. (1997). The role of osteopontin in tumorigenesis and metastasis. Invasion Metastasis 17, 115. Oates AJ, Barraclough R, Rudland PS. (1996). The identication of osteopontin as a metastasis-related gene product in a rodent mammary tumour model. Oncogene 13, 97104. Obinata T, Nagaoka-Yasuda R, Ono S, Kusano K, Mohri K, Ohtaka Y, Yamashiro S, Okada K, Abe H. (1997). Low molecular weight G-actin binding proteins involved in regulation of actin assembly during myobrillogenesis. Cell Struct Funct 22, 181189. Odink K, Cerletti N, Bruggen J, Clerc RC, Tarcsay L, Zwadlo G, Gerhards G, Schlegel R, Sorg C. (1987). Two calcium binding proteins in inltrate macrophages of rheumatoid arthritis. Nature 330, 8082. Ogawa K, Yamada Y, Kishibe K, Ishizaki K, Tokusashi Y. (1999). Beta catenin mutations are frequent in hepatocellular carcinomas but absent in adenomas induced by diethylnitrosamine in B6C3F1 mice. Cancer Res 59, 18301833. Oguin WM, Sun TT, Manabe M. (1992). Interaction of trichohyalin with intermediate laments. Three immunologically dened stages of trichohyalin maturation. J Invest Dermatol 98, 2432. Ohguro HN, Ogawa K, Nakagawa T. (1999). Recoverin and hsc70 are found as autoantigens in patients with cancer-associated retinopathy. Invest Ophthalmol Visual Sci 40, 8289. Ohno S, Emori Y, Imajoh S, Kawasaki H, Kisaragi M, Suzuki K. (1984). Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calciumbinding protein. Nature 312, 566570. Ohno S, Minoshima S, Kudoh J, Fukuyama R, Shimizu Y, Ohmi-Imajoh S, Shimizu N, Suzuki K. (1990). Four genes for the calpain family locate on four distinct human chromosomes. Cytogenet Cell Genet 53, 225229. Ohta H, Sasaki T, Naka M, Hiraoka O, Miyamoto C, Furuichi Y, Tanaka T. (1991). Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle. FEBS Lett 295, 9396. Ohtsu M, Sakai N, Fujita H, Kashiwagi M, Gasa S, Shimizu S, Eguchi Y, Tsujimoto Y, Sakiyama Y, Kobayashi K, Kuzumaki N. (1997). Inhibition of apoptosis by the actin regulatory protein gelsolin. EMBO J 16, 46504656. Ohtsuka H, Yajima H, Maruyama K, Kimura S. (1997). Binding of the N-terminal 63-kDa portion of connectin/titin to alpha-spectrin as revealed by the yeast two-hybrid system. FEBS Lett 401, 6567. Okada H, Danoff TM, Fischer A, Lopez-Guisa JM, Strutz F, Neilson EG. (1998). Identication of a novel cis-acting element for broblast specic transcription of the FSP1 gene. Amer J Physiol 44, F306F314. Okada H, Danoff TM, Kalluri R, Neilson EG. (1997). Early role of FSP1 in epithelialmesenchymal transformation. Amer J Physiol 42, F563F574.
References
315
Okami K, Wu L, Riggins G, Cairns P, Goggins M, Evron E, Halachmi N, Ahrendt SA, Reed AL, Hilgers W, Kern SE, Koch WM, Sidransky D, Jen J. (1998). Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumours. Cancer Res 58, 509511. Okamoto T, Schlegel A, Scherer PE, Lisanti MP. (1998). Caveolins, a family of scaffolding proteins for organising preassembled signal complexes at the plasma membrane. J Biol Chem 273, 54195422. Okamoto-Inoue M, Kamada S, Kimura G, Taniguchi S. (1999). The induction of smooth muscle alpha actin in a transformed rat cell line suppresses malignant properties in vitro and in vivo. Cancer Lett 142, 173178. Okuse K, Matsuoka I, Kurihara K. (1997). Tissue specic methylation occurs in the essential promoter element of the tyrosine hydroxylase gene. Mol Brain Res 46, 197207. Olcese R, Qin N, Schneider T, Neely A, Wei X, Stefani E, Birnbaumer L. (1994). The amino terminus of a calcium channel subunit sets rates of channel inactivation independently of the subunits effect on activation. Neuron 13, 14331438. Old WM, Ahn NG, Resing KA. (1997). PKC delta is a prolaggrin kinase and may be involved in solubilisation of prolaggrin during epidermal differentiation. FASEB J 11, 2913. Oliferenko S, Paiha K, Harder T, Gerke V, Schwarzler C, Schwarz H, Beug H, Gunthert U, Huber LA. (1999). Analysis of CD44-containing lipid rafts. Recruitment of annexin II and stabilisation by the actin cytoskeleton. J Cell Biol 146, 843854. Oliva, D., Venturella, S., Passantino, R., Feo, S. and Giallonogo, A. (1995). Conserved alternative splicing in the 5-untranslated region of the muscle specic enolase gene. Primary structure of messenger RNAs, expression and inuence of secondary structure on translation efciency. Eur J Biochem 232, 141149. Olivo C, Vanni C, Mancini P, Silengo L, Torrisi MR, Tarone G, De Filippi P, Eva A. (2000). Distinct involvement of Cdc42 and RhoA GTPases in actin organisation and cell shape in untransformed and dbl oncogene transformed NIH3T3 cells. Oncogene 19, 14281436. Ollendorf Y, Szepetowski P, Matel MG, Gaudray P, Birnbaum D. (1992). New gene in the homologous human 11q13-q14 and mouse 7F chromosomal regions. Mammalian Genome 2, 195200. Olsen E, Rasmussen HH, Celis JE. (1995). Identication of proteins that are abnormally regulated in differentiated cultured human keratinocytes. Electrophoresis 16, 22412248. Onischenko A, Chenard MP, Lefebvre O, Bruyneel E, Rio MC. (1996). Defective tumour vascularisation induced by metastasin I expression. Invasion Metastasis 16, 160168. Onishi H, Maita T, Matsuda G, Fujiwara K. (1992). Interaction between the heavy and the regulatory light chains in smooth muscle myosin subfragment 1. Biochemistry 31, 12011210. Ono S, Abe H, Obinata T. (1996). Disorganisation of actin laments induced by overexpression of colin in mammalian cultured cells. Mol Biol Cell 7, 1175. Ono S, Minami N, Abe H, Obinata T. (1994). Characterisation of a novel colin isoform that is predominantly expressed in mammalian skeletal muscle. J Biol Chem 269, 1528015286. Ono Y, Shimada H, Sorimachi H, Richard I, Saido TC, Beckmann JS, Ishiura S, Suzuki K. (1998). Functional defects of a muscle-specic calpain, p94, caused by mutations associated with limb girdle muscular dystrophy type 2A. J Biol Chem 273, 1707317078. Opas M, Dziak E, Fliegel L, Michalak M. (1991). Regulation of expression and intracellular distribution of calreticulin, a major calcium-binding protein of non-muscle cells. J Cell Physiol 149, 160171.
316
Opas M, Szewczenko-Pawlikowski M, Jess GK, Mesaeli N, Michalak M. (1996a). Calreticulin modulates cell adhesiveness via regulation of vinculin expression. J Cell Biol 135, 19131923. Opas M, Tharin S, Milner RE, Michalak M. (1996b). Identication and localisation of calreticulin in plant cells. Protoplasma 191, 164171. Orlando RA, Rader K, Authier F, Yamazaki H, Posner BI, Bergeron JJM, Farquhar MG. (1998). Megalin is an endocytic receptor for insulin. J Amer Soc Nephrol 9, 17591766. Orrenius S, Ankarcrona M, Nocotera P. (1996). Mechanisms of calcium-related cell death. Adv Neurol 71, 137151. Orucevic A, Bechberger J, Green AM, Shapiro RA, Billiar TR, Lala PK. (1999). Nitric oxide production by murine mammary adenocarcinoma cells promotes tumour cell invasiveness. Int J Cancer 81, 889896. Ostergaard M, Wolf H, Orntoft TF, Celis JE. (1999). Psoriasin (S100A7). A putative urinary marker for the follow-up of patients with bladder squamous cell carcinomas. Electrophoresis 20, 349354. Ostergard HL, Trowbridge IS. (1991). Negative regulation of CD45 protein in tyrosine phosphatase activity by ionomycin in T-cells. Science 253, 14231425. Ostwald TJ, MacLennan DH. (1974). Isolation of a high-afnity calcium-binding protein from sarcoplasmic reticulum. J Biol Chem 249, 974979. Otto-Bruc A, Fariss RN, Haeseleer F, Huang J, Buczylko J, Surgucheva I, Baehr W, Milan AH, Palczewski K. (1997). Localisation of guanylate cyclase-activating protein 2 in mammalian retinas. Proc Natl Acad Sci USA 94, 47274732. Ou WJ, Thomas DY, Bell AW, Bergeron JJM. (1992). Casein kinase II phosphorylation of signal sequence receptor-alpha and the associated membrane chaperone calnexin. J Biol Chem 267, 2378923796. Oxvig C, Springer TA. (1998). Experimental support for a -propeller domain in integrin subunits and a calcium binding site on its lower surface. Proc Natl Acad Sci USA 95, 48704875. Ozawa M, Muramatsu T. (1993). Reticulocalbin, a novel endoplasmic reticulum resident Ca2+binding protein with multiple EF-hand motifs and a carcobxyl terminal HDEL sequence. J Biol Chem 268, 699705. Ozawa M. (1995a). Structure of the gene encoding mouse reticulocalbin, a novel endoplasmic reticulum resident Ca2+-binding protein with multiple EF-hand motifs. J Biochem 118, 154160. Ozawa M. (1995b). Cloning of a human homologue of mouse reticulocalbin reveals conservation of structural domains in the novel endoplasmic reticulum resident Ca2+-binding protein with multiple EF-hand motifs. J Biochem 117, 11131119. Pacaud M, Derancourt J. (1993). Purication and further characterisation of macrophage 70kDa protein, a calcium-regulated actin-binding protein identical to L-plastin. Biochemistry 32, 34483455. Palacek SP, Huttenlocher A, Horwitz AF, Lauffenburger DA. (1998). Physical and biochemical regulation of integrin release during rear detachment of migrating cells. J Cell Sci 111, 929940. Pan T, Coleman JE. (1991). Sequential assignments of the H-1-NMR resonances of Zn(II)2 and CD-113(II)2 derivatives of the DNA-binding domain of the GAL4 transcription factor reveal a novel structural motif for specic DNA recognition. Biochemistry 30, 42124222.
References
317
Pang JT, Lloyd SE, Woodling C, Farren B, Pottinger B, Harding B, Leigh SE, Pook MA, Benham FJ, Gillett GT, Taggart RT, Thakker RV. (1996). Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of -calpain as the multiple endocrine neoplasia type 1 gene. Human Genet 97, 732741. Papkoff J, Aikawa M. (1998). Wnt-1 and HGF regulate GSK3 beta activity and beta catenin signalling in mammary epithelial cells. Biochem Biophys Res Commun 247, 851958. Pappas I, Parnavelas JG. (1997). Neurotropins and basic broblast growth factor induce the differentiation of calbindin-containing neurones in the cerebral cortex. Exp Neurol 144, 302314. Pappas IS, Parnavelas JG. (1998). Basic broblast growth factor promotes the generation and differentiation of calretinin neurones in the rat cerebral cortex in vitro. Eur J Neurosci 10, 14361445. Pappas IS, Sophianos D, Tzartos S, Tsiftsoglou AS. (1996). Expression of memory, differentiation and repression of c-myc and p53 genes in human RD/TE-671 cells induced by a ureido derivative of pyridine (UDP-4). Cell Growth Differentiation 7, 797809. Paramio JM, Navarro M, Segrelles C, Gomez-Casero E, Jorcano JL. (1999). PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 18, 74627468. Park YK, Yang MH, Park HR. (1996). The impact of osteonectin for differential diagnosis of osteogenic bone tumours. Skeletal Radiology 25, 1317. Parker C, Lakshmi MS, Piura B, Sherbet GV. (1994a). Metastasis associated mts1 gene expression correlates with increased p53 expression. DNA Cell Biol 13, 343351. Parker C, Sherbet GV. (1992). Modulators of intracellular Ca2+ and the calmodulin inhibitor W7 alter the expression of metastasis associated genes MTS1 and NM23 in metastatic variants of the B16 murine melanoma. Melanoma Res 2, 337343. Parker C, Whittaker PA, Usmani BA, Lakshmi MS, Sherbet GV. (1994b). Induction of 18A2/mts1 gene expression and its effects on metastasis and cell cycle control. DNA Cell Biol 13, 10211028. Parker C, Whittaker PA, Weeks RJ, Thody AJ, Sherbet GV. (1991). Modulators of metastatic behaviour alter the expression of metastasis associated mts1 and nm23 genes in metastatic variants of the B16 murine melanoma. Clin Biotechnol 3, 217222. Parker CA, Takahashi K, Tang JX, Tao T, Morgan KG. (1998). Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle of the ferret. J Physiol (London) 508, 187198. Parminder AH, Murakami A, Inana G, Berson EL, Dryja TP. (1997). Evaluation of the human gene encoding recoverin in patients with retinitis pigmentosa or an allied disease. Invest Ophthalmol Visual Sci 38, 704709. Pascale A, Etcheberrigaray R. (1999). Calcium alterations in Alzheimers disease. Pharmacol Res 39, 8188. Patel S, Joseph SK, Thomas AP. (1999). Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium 25, 247264. Paunio T, Kangas H, Kiuru S, Palo J, Peltonen L, Syvanen AC. (1997). Tissue distribution and levels of gelsolin mRNA in normal individuals and patients with gelsolin related amyloidosis. FEBS Lett 406, 4955. Payne AM, Downes SM, Bessat DAR, Taylor R, Holder GE, Warren MJ, Bird AC, Bhattacharya SS. (1998). A mutation in guanylate cyclase activator 1A (GUCA1A) in an autosomal dominant cone dystrophy pedigree mapping to a new locus on chromosome 6p21. Human Mol Genetics 7, 273277.
318
Pearlstone JR, Sykes BD, Smilie LB. (1997). Interactions of structural C and regulatory N domains of troponin C with repeated motifs of troponin I. Biochemistry 36, 76017606. Pedrocchi M, Schafer BW, Mueller H, Eppenberger U, Heizmann CW. (1994). Expression of Ca2+-binding proteins of the S100 family in malignant human breast cancer cell lines and biopsy samples. Int J Cancer 57, 684690. Peerschke EIB, Ghebrehiwet B. (1987). Human blood platelets possess specic binding sites for C1q. J Immunol 138, 15371541. Peerschke EIB, Malhotra R, Ghebrehiwet B, Reid KMB, Willis AC, Sim RB. (1993). Isolation of a human endothelial cell C1q receptor (C1q-R). J Leukocyte Biol 53, 179184. Peifer M, Rauskolb C, Williams M, Riggleman B, Wieschaus E. (1991). The segment polarity gene armadillo interacts with the wingless signalling pathway in both embryonic and adult pattern formation. Development 111, 10291043. Peifer M. (1995). Cell adhesion and signal transduction. The armadillo connection. Trends Cell Biol 5, 224229. Pelham HR, Brown DD. (1980). A specic transcription factor than can bind either the 5S RNA gene or 5S RNA. Proc Natl Acad Sci USA 77, 41704174. Pelin K, Hiipela P, Donner K, Sewry C, Akkari PA, Wilton SD, Wattanasirichaigoon W, Bang ML, Centner T, Hansfeld F, Odent S, Fardeau M, Urtizberea JA, Muntoni F, Dubowitz V, Beggs AH, Laing NG, Labeit S, De La Chapelle A, Wallgren-Pettersson C. (1999). Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proc Natl Acad Sci USA 96, 23052310. Pellegata NS, Antoniono RJ, Redpath JL, Stanbridge EJ. (1996). DNA damage and p53mediated cell cycle arrest: A re-evaluation. Proc Natl Acad Sci USA 93, 1520915214. Penniston JT, Enyedi A. (1998). Modulation of the plasma membrane Ca2+ pump. J Membrane Biol 165, 101109. Perara LP, Waldmann TA. (1998). Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE. Proc Natl Acad Sci USA 95, 1430814313. Periasamy M, Reed TD, Liu LH, Ji Y, Loukianov E, Paul RJ, Nieman ML, Riddle T, Duffy JJ, Doetschman T, Lorenz JN, Shull GE. (1999). Impaired cardiac performance in heterozygous mice with a null mutation in the sarco (endo)-plasmic reticulum Ca2+ATPase isoform 2 (SERCA2) gene. J Biol Chem 274, 25562562. Perkins C, Kim CN, Fang GF, Bhalla KN. (1998). Over-expression of apaf-1 promotes apoptosis of untreated and paclitaxel- or etoposide-treated HL-60 cells. Cancer Res 58, 45614566. Perrino BA, Fong YL, Brickey DA, Saitoh Y, Ushio Y, Fukunaga K, Miyamoto E, Soderling TR. (1992). Characterisation of the phosphatase activity of a Baculovirus-expressed calcineurin A isoform. J Biol Chem 267, 1596515969. Petersen OH, Cancela JM. (1999). New Ca2+-releasing messengers. Are they important in the nervous system? Trends Neurosci 22, 488494. Peterson BZ, De Maria CD, Yue DT. (1999). Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 22, 549558. Peterson HF, Self AJ, Garrett MD, Just I, Aktories K, Hall A. (1990). Microinjection of recombinant p21 Rho induces rapid changes in cell morphology. J Cell Biol 111, 10011007. Pug BR, Reiter RE, Nelson JB. (1999). Caveolin expression is decreased following androgen deprivation in human prostate cancer cell lines. Prostate 40, 269273. Philippova MP, Bochkov VN, Stambolsky DV, Tkachuk VA, Resink TJ. (1998). T-cadherin and signal transducing molecules co-localise in caveolin-rich membrane domains of vascular smooth muscle cells. FEBS Lett 429, 207210.
References
319
Phillips DJ, De Kretser DM. (1998). Follistatin. A multifunctional regulatory protein. Frontiers Neuroendocrinol 19, 287322. Pietromonaco SF, Seluja GA, Elias L. (1995). Identication of enzymatically active Ca2+/calmodulin dependent protein kinase in centrosomes of haematopoietic cells. Blood Cells Molecules Dis 21, 3441. Pinhasi-Kimhi O, Michalowitz D, Ben-Zeev A, Oren M. (1986). Specic interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature 320, 182185. Pittenger MF, Kazzaz JA, Helfman DM. (1994). Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol 6, 96104. Plaitakis A. (1990). Glutamate dysfunction and selective motor neurone degeneration in amyotrophic lateral sclerosis. A hypothesis. Ann Neurol 28, 38. Podhajcer OL, Wolf C, Lefebvre O, Segain JP, Rouyer N, Stoll I, Rio MC, Chambon P, Basset P. (1996). Comparative expression of the SPARC and stromelysin-3 genes in mammary tumours. Breast 5, 1320. Podsypanina K, Ellenson LH, Nemes A, Gu JG, Tamura M, Yamada KM, Cordon-Cardo C, Catoretti G, Fisher PE, Parsons R. (1999). Mutations of PTEN/MMAC1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci USA 96, 15631568. Pohl J, Winder SJ, Allen BG, Walsh MP, Sellers JR, Gerthoffer WT. (1997). Phosphorylation of calponin in airway smooth muscle. Amer J Physiol 16, L115L123. Polans AS, Witkowska D, Haley TL, Amundson D, Baizer L, Adamus G. (1995). Recoverin, a photoreceptor-specic calcium binding protein, is expressed by the tumour of a patient with cancer associated retinopathy. Proc Natl Acad Sci USA 92, 91769180. Pollard TD, Almo S, Quirk S, Vinson V, Lattman EE. (1994). Structure of actin-binding proteins. Insights about function at atomic resolution. Annu Rev Cell Biol 10, 207249. Polly P, Carlsberg C, Eisman JA, Morrison NA. (1996). Identication of a vitamin D-3 response element in the bronectin gene that is bound by a vitamin D-3 receptor homodimer. J Cell Biochem 60, 322333. Polyakov AA, Huber PAJ, Marston SB, Gusev NB. (1998). Interaction of isoforms of S100 protein with smooth muscle caldesmon. FEBS Lett 422, 235239. Popov S, Yu K, Kozasa T, Wilkie TM. (1997). The regulators of G protein signalling (RGS) domain of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro. Proc Natl Acad Sci USA 94, 72167220. Porn-Ares MI, Ares MPS, Orrenius S. (1998). Calcium signalling and the regulation of apoptosis. Toxicol in vitro 12, 539543. Porter PL, Sage EH, Lane TF, Funk SE, Gown AM. (1995). Distribution of SPARC in normal and neoplastic human tissue. J Histochem Cytochem 43, 791800. Potter JD, Johnson JD. (1982). Troponin. In Calcium and cell function, ed. Cheung WY, Academic Press, London, pp. 145173. Potter KA, Mier JW, Maki M, Herman IM. (1998). Calpain regulates actin remodelling during cell spreading. J Cell Biol 141, 647662. Pragnall M, De Waard M, Mori Y, Tanabe T, Snutch TP, Campbell KP. (1994). Calcium channel beta subunit binds to a conserved motif in the i-ii cytoplasmic linker of the alpha (1)-subunit. Nature 368, 6770. Prasad GL, Masuelli L, Raj MHG, Harindranath N. (1999). Suppression of src-induced transformed phenotype by expression of tropomyosin-1. Oncogene 18, 20272031. Prasad SC, Thraves PK, Dritschilo A, Kuettel MR. (1997). Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells. Electrophoresis 18, 629637.
320
Prassler J, Stocker S, Marriott G, Heidecker M, Kellermann J, Gerisch G. (1997). Interaction of a Dictyostelium member of the plastin/mbrin family with actin laments and actinmyosin complexes. Mol Biol Cell 8, 8395. Presland RB, Bassuk JA, Kimball JR, Dale BA. (1995). Characterisation of two distinct calcium binding sites in the amino terminus of human prolaggrin. J Invest Dermatol 104, 218223. Price P. (1992). Gla-containing proteins of mineralised tissues. In: Chemistry and biology of mineralised tissues, eds. Slavkin H, Price P., Elsevier/North Holland, Amsterdam, pp. 169176. Price PA, Baukol SA. (1980). 1,25-dihydroxy vitamin D3 increases synthesis of vitamin Kdependent bone protein by osteosarcoma cells. J Biol Chem 255, 1166011663. Prostko CR, Zhang C, Hait WN. (1997). The effects of altered cellular calmodulin expression on the growth and viability of C6 glioblastoma cells. Oncol Res 9, 1317. Purcell L, Gruia-Gray J, Scanga S, Ringuette M. (1993). Developmental anomalies of Xenopus embryos following microinjection of SPARC antibodies. J Exp Zool 265, 153164. Putney JW. (1997). Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium entry. Cell Calcium 21, 257261. Qu Q, Perala-Heape M, Kapanen A, Dahllund J, Salo J, Vaananen HK, Harkdonen P. (1998). Oestrogen enhances differentiation of osteoblasts in mouse marrow culture. Bone 22, 201209. Quadroni M, James P, Carafoli E. (1994). Isolation of phosphorylated calmodulin from rat liver and identication of the in vivo phosphorylation sites. Biol Chem 269, 1611616122. Quadroni M, LHostis EL, Corti C, Myagkikh I, Durussel I, Cox J, James P, Carafoli E. (1998). Phosphorylation of calmodulin alters its potency as an activator of target enzymes. Biochemistry 37, 65236532. Radotra B, McCormick D, Crockard A. (1994). CD44 plays a role in adhesive interactions between glioma cells and extracellular matrix components. Neuropathol App Neurobiol 20, 399405. Rahman MM, Iida H, Shibata Y. (1997). Expression and localisation of annexin V and annexin VI during limb bud formation in the rat foetus. Anat Embryol 195, 3139. Ramaswamy S, Nakamura N, Vazquez F, Batt DB, Perera S, Roberts TM, Sellers WR. (1999). Regulation of G1 progression by the PTEN tumour suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci USA 96, 21102115. Ramchandani S, Bhattacharya SK, Cervone N, Szyef M. (1999). DNA methylation is a reversible biological signal. Proc Natl Acad Sci USA 96, 61076112. Rammes A, Roth J, Goebeler M, Klempt M, Hartmann M, Sorg C. (1997). Myeloid-related protein MRP8 and MRP14, calcium-binding proteins of the S100 family, are secreted by activated monocytes via a novel, tubulin-dependent pathway. J Biol Chem 272, 94969502. Ranta-Knuuttila T, Puolakkainen P, Kiviluoto T, Watanabe S, Sato N, Kivilaakso E. (1998). Calcium/calmodulin-dependent pathway plays a second messenger role in signal transduction of migration in cultured gastric epithelial cells. Gastroenterology 114, G1088. Rao A, Luo C, Hogan PG. (1997). Transcription factors of the NFAT family. Regulation and function. Ann Rev Immunol 15, 707747. Rao JY, Jin YS, Zheng QL, Cheng J, Tai J, Hemstreet GP. (1999). Alterations of the actin polymerisation status as an apoptotic morphological effector in HL-60 cells. J Cell Biochem 75, 686697.
References
321
Rasmussen C, Garen C, Brining S, Kincaid RL, Means RL, Means AR. 1994). The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. EMBO J 13, 25452552. Rayment I, Holden HM, Whittaker M, Yohn CB, Lorenz M, Holmes KC, Milligan RA. (1993). Structure of the actin-myosin complex and its implications for muscle contraction. Science 261, 5865. Raynal P, Pollard HB, Srivastava M. (1997). Cell cycle and post-transcriptional regulation of annexin expression in IMR-90 human broblasts. Biochem J 322, 365371. Reddy D, Pollock A, Clark SA, Sooy K, Vasavada RC, Stewart AF, Honeyman T, Christakos S. (1997). Transfection and over-expression of the calcium binding protein calbindin D28K results in a stimulatory effect on insulin synthesis in a rat cell line (RIN 104638). Proc Natl Acad Sci USA 94, 19611966. Redfern CPF. (1997). Molecular mechanisms of retinoid function. In Retinoids: Their physiological function and therapeutic potential, ed. Sherbet GV, Jai Press Inc., Greenwich, Conn. and London, 1997, pp. 3577. Redmon JB, Olson LK, Armstrong MB, Greene MJ, Robertson RP. (1996). Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells. J Clin Invest 98, 27862793. Reed JC. (1997). Double identity for proteins of the bcl-2 family. Nature 387, 773776. Reed MJ, Vernon RB, Abrass IB, Sage EH. (1994). TGF-beta 1 induces the expression of type I collagen and SPARC, and enhances contraction of collagen gels, by broblasts from young and aged donors. J Cell Physiol 158, 169179. Reeves RH, Yao JB, Crowley MR, Buck S, Zhang XF, Yarowsky P, Hearhart JD, Hilt DC. (1994). Astrocytosis and axonal proliferation in the hippocampus of S100B transgenic mice. Proc Natl Acad Sci USA 91, 53595363. Regalado SP, Nambu Y, Iannettoni MD, Orringer MD, Beer DG. (1998). Abundant expression of the intestinal protein villin in Barretts metaplasia and oesophageal adenocarcinomas. Mol Carcinogenesis 22, 182189. Reifergerste R, Grimm S, Albert S, Lipski N, Heimback G, Hofbauer A, Pugfelder GO, Quack D, Reichmuth C, Shug B, Zinsmaier KE, Buchner S, Buchner E. (1993). An invertebrate calcium binding protein of the calbindin subfamily. Protein structure, genomic organisation and expression pattern of the calbindin-32 gene of Drosophila. J Neurosci 13, 21862198. Reinhard M, Griehl K, Abel K, Haffner C, Jarchau T, Hoppe V, Jockusch BM, Walter U. (1995a). The proline-rich focal adhesion and microlament protein VASP is a ligand for prolins. EMBO J 14, 15831589. Reinhard M, Halburgge M, Scheer U, Wiegand C, Jockusch BM, Walter U. (1992). The 46/50kDa phosphoprotein VASP puried from human platelets is a novel protein associated with actin laments and focal contacts. EMBO J 11, 20632070. Reinhard M, Jouvenal K, Tripier D, Walter U. (1995b). Identication, purication, and characterisation of zyxin-related protein that binds the focal adhesion and microlament protein VASP (vasodilator-stimulated phosphoprotein). Proc Natl Acad Sci USA 92, 79567960. Reinhard M, Rudiger M, Jockusch BM, Walter U. (1996). VASP interaction with vinculin. A recurring theme of interactions with proline-rich motifs. FEBS Lett 399, 103107. Resing KA, Al-Alawi N, Blomquist C, Fleckman P, Dale BA. (1993b). Independent regulation of cytoplasmic processing stages of the intermediate lament-associated protein laggrin and role of Ca2+ in the second stage. J Biol Chem 268, 2513925145. Resing KA, Johnson RS, Walsh KA. (1993a). Characterisation of protease processing sites during conversion of rat prolaggrin to laggrin. Biochemistry 32, 1003610045.
322
Resing KA, Thulin C, Whiting K, Alalawi N, Mostad S. (1995). Characterisation of prolaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis. J Biol Chem 270, 2819328198. Reymond A, Volorio S, Merla G, Al Maghtheh M, Zuffardi O, Bulfone A, Ballabio A, Zollo M. (1999). Evidence for interaction between human PRUNE and nm23-H1 NDP kinase. Oncogene 51, 72447252. Ribeiro CMP, Reece J, Putney DW. (1997). Role of cytoskeleton in calcium signalling in NIH3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+](i) signalling, but not for capacitative calcium entry. J Biol Chem 272, 2655526561. Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Hillaire D, Passos-Bueno M, Zatz M, Tischeld JA, Fardeau M, Jackson CE, Cohen D, Beckmann JS. (1995). Mutations in the proteolytic enzyme calpain 3 cause limb girdle muscular dystrophy 2A. Cell 81, 2740. Richardson DS, Allen PD, Kelsey SM, Newland AC. (1999). Effects of PARP inhibition on drug and Fas-induced apoptosis in leukemic cells. Adv Exptl Med Biol 457, 267279. Rider V, Foster RT, Evans M, Suenaga R, Abdou NI. (1998). Gender differences in autoimmune diseases. Oestrogen increases calcineurin expression in systemic lupus erythematosus. Clin Immunol Immunopathol 89, 171180. Ridinger K, Ilg EC, Niggli FK, Heizmann CW, Schafer BW. (1998). Clustered organisation of S100 genes in human and mouse. Biochem Biophys Acta 1448, 254263. Ridley AJ, Hall A. (1992). The small GTP-binding protein Rho regulates the assembly of focal adhesion and actin stress bres in response to growth factors. Cell 70, 389399. Riedy MC, Brown MC, Molloy CJ, Turner CE. (1999). Activin A and TGF- stimulate phosphorylation of focal adhesion proteins and cytoskeletal reorganisation in rat aortic smooth muscle cells. Exp Cell Res 251, 194202. Riegman PHJ, Vietstra RJ, Van Der Korput JAGM, Brinkmann AO, Trapman J. (1991). The promoter of the prostate specic antigen gene contains a functional androgen responsive element. Mol Endocrinol 5, 19211930. Riggleman B, Wieschaus E, Schedl P. (1989). Molecular analysis of the armadillo locus. Uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene. Genes Develop 3, 96113. Riggleman B, Schedl P, Wieschaus E. (1990). Spatial expression of the Drosophila segment polarity gene armadillo is post-transcriptionally regulated by wingless. Cell 63, 549560. Rivero F, Koppel B, Peracino B, Bozzaro S, Siegert F, Weijer CJ, Schleicher M, Albrecht R, Noegel AA. (1996). The role of the cortical cytoskeleton. F-actin cross-linking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development. J Cell Sci 109, 26792691. Robinson JA, Harris SA, Riggs BL, Spelsberg TC. (1997). Oestrogen regulation of human osteoblastic cell proliferation and differentiation. Endocrinology 138, 29192927. Rogers JH, Resibois A. (1992). Calretinin and calbindin D28K in rat brain. Patterns of partial co-localisation. Neuroscience 51, 843865. Rogers JH. (1987). Calretinin: a gene for a novel calcium binding protein expressed principally in neurones. J Cell Biol 105, 13431353. Romagnolo B, Molina A, Leroy G, Blin C, Proteux A, Thomasset M, Van de Walle, Kahn A, Perret C. (1996). Oestradiol-dependent uterine leiomyomas in transgenic mice. J Clin Invest 98, 777784. Romero F, Fischer S. (1996). Structure and function of vav. Cell Signalling 8, 545553. Rooke K, Briquet-Laugier V, Xia YR, Lusis AJ, Doolitle MH. (1997). Mapping of the gene for calreticulin (Calr) to mouse chromosome 8. Mammalian Genome 8, 870871.
References
323
Roseman BJ, Bollen A, Hsu J, Lamborn K, Israel MA. (1994). Annexin II marks astrocytic brain tumours of high histological grade. Oncol Res 6, 561567. Rosemblit N, Moschella MC, Ondriasova E, Gutstein DE, Ondrias K, Marks AR. (1999). Intracellular calcium release channel expression during embryogenesis. Develop Biol 206, 163177. Rosenblatt S, Bassuk JA, Alpers CE, Sage EH, Timpl R, Preissner KT. (1997). Differential modulation of cell adhesion by interaction between adhesive and counter-adhesive proteins: Characterisation of the binding of vitronectin to osteonectin (BM40, SPARC). Biochem J 324, 311-319. Rosengard AM, Kutsch HC, Shearn A, Biggs JR, Barker E, Margulies IMK, King CR, Liotta LA, Steeg PS. (1989). Reduced nm23/awd protein in tumour metastasis and aberrant Drosophila development. Nature 342, 177180. Roth AF, Sullivan DM, Davis NG. (1998). A large PEST-like sequence directs the ubiquitination, endocytosis, and vacuolar degradation of the yeast a-factor receptor. J Cell Biol 142, 949961. Rowlands DC, Harrison RF, Jones NA, Williams A, Husbscher SG, Brown G. (1995). Stathmin is expressed by proliferating hepatocytes during liver regeneration. J Clin Pathol 48, M88M92. Roy LM, Gittinger CK, Landreth GE. (1989). Characterisation of epidermal growth factor receptor associated with cytoskeleton of A431 cells. J Cell Physiol 140, 295304. Roy LM, Gittinger CK, Landreth GE. (1991). Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor. J Cell Physiol 146, 6372. Roy, N., La Flamme, G. and Raymond, V. (1992). 5 untranslated sequences modulate rapid mRNA degradation mediated by 3 AU-rich element in v-/c-fos recombinants. Nucleic Acids Res 20, 57535762. Royds JA, Stephenson TJ, Rees RC, Shorhouse AJ, Silcocks PB. (1993). Nm23 protein expression in ductal in situ and invasive human breast carcinoma. J Natl Cancer Inst 85, 727731. Rudland PS, Platt-Higgins A, Renshaw C, West CR, Winstanley JHR, Robertson L, Barraclough R. (2000). Prognostic signicance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer. Cancer Res 60, 15951603. Rudnicka-Nawrot M, Surgucheva I, Humes JD, Haeseleer F, Sokal I, Crabb JW, Baehr W, Palczewski K. (1998). Changes in biological activity and folding of guanylate cyclaseactivating protein 1 as a function of calcium. Biochemistry 37, 248257. Ryhanen S. Pirskanen A, Jaaskelainen T, Maenpaa PH. (1997). State of methylation of human b osteocalcin gene in bone-derived and other cell types, J Cell Biochem 66, 404412. Ryoo HM, Hoffmann HM, Beumer T, Frenkel B, Towler DA, Stein GS, Stein JL, Van Wijnen AJ, Lian JB. (1997). Stage specic expression of Dix-5 during osteoblast differentiation. Involvement in regulation of osteocalcin gene expression. Mol Endocrinol 11, 16811694. Sabry JH, Moores SL, Ryan S, Zang JH, Spudich JA. (1997). Myosin heavy chain phosphorylation sites regulate myosin localisation during cytokinesis in live cells. Mol Biol Cell 8, 26052615. Sachs LM, Lebrun JJ, De Luze A, Kelly PA, Demeneix BA. (1997). Tail regression, apoptosis and thyroid hormone regulation of myosin heavy chain isoforms in Xenopus tadpoles. Mol Cell Endocrinol 131, 211219. Sacks DB, Davis HW, Crimmins DL, McDonald JM. (1992). Insulin-stimulated phosphorylation of calmodulin. Biochem J 286, 211216.
324
Sacks DB, Mazus B, Joyal JL. (1995). The activity of calmodulin is altered by phosphorylation. Modulation of calmodulin function by the site of phosphate incorporation. Biochem J 312, 197204. Safer D, Chowrashi PK. (1997). Beta thymosins from marine invertebrates. Primary structure and interaction with actin. Cell Motility Cytoskeleton 38, 163171. Safer D, Sosnick TR, Elzinga M. (1997). Thymosin beta-4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 36, 58065816. Safer D. (1992). The interaction of actin with thymosin beta 4. J Muscle Res Cell Motility 13, 269271. Sage EH, Bassuk JA, Yost JC, Folkman MJ, Lane TF. (1995). Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca2+-binding EF-hand sequence. J Cell Biochem 57, 127140. Sage EH. (1992). Modulation of endothelial cell shape by SPARC doe not involve chelation of extracellular Ca2+ and Mg2+. Biochem Cell Biol 70, 5662. Sage H, Johnson C, Bornstein P. (1984). Characterisation of a novel serum albumin-binding glycoprotein secreted by endothelial cells in culture. J Biol Chem 259, 39934007. Sage H, Vernon RB, Decker J, Funk S, Luisa M, Iruela-Arispe L. (1989a). Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J Histochem Cytochem 37, 819929. Sage H, Vernon RB, Funk SE, Everitt EA, Angello J. (1989b). SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca2+dependent binding to the extracellular matrix. J Cell Biol 109, 341356. Saido TC, Sorimachi H, Suzuki K. (1994). Calpain. New perspectives in molecular diversity and physiological-pathological involvement. FASEB J 8, 814822. Saido TC, Suzuki H, Yamazaki H, Tanoue K, Suzuki K. (1993). In situ capture of -calpain activation in platelets. J Biol Chem 268, 74227426. Sainsbury RJC, Farndon JR, Harris AL, Sherbet GV. (1987a). Epidermal growth factor receptors on human breast cancer. Br J Cancer 72, 186188. Sainsbury RJC, Needham GK, Farndon JR, Malcolm AJ, Harris, AL. (1987b). Epidermal growth factor receptor status as predictor of early recurrence and death from breast cancer. Lancet 1, 13981402. Saitoh K, Takahashi H, Yamamoto M, Kishi H, Parsons PG. (1996). Expression of metastasis suppressor gene product, nm23 protein, is not inversely correlated with tumour progression in human malignant melanoma. Histopathology 29, 497505. Sakane F, Imai S, Kai M, Wada I, Kanoh H. (1996). Molecular cloning of a novel diacylglycerol kinase isozyme with a pleckstrin homology domain and a C-terminal tail similar to those of the EPH family of protein tyrosine kinases. J Biol Chem 271, 83948401. Sakane F, Kanoh H. (1997). Diacylglycerol kinase. Int J Biochem Cell Biol 29, 11391143. Sakane F, Yamada K, Kanoh H, Yokoyama C, Tanabe T. (1990). Porcine diacylglycerol kinase sequence has zinc nger and EF-hand motifs. Nature 344, 345348. Sakuntabhai A, Burge S, Monk S, Hovnanian A. (1999a). Spectrum of novel ATP2A2 mutations in patients with Dariers disease. Human Mol Genet 8, 16111619. Sakuntabhai A, Ruiz-Perez V, Carter S, Jacobsen N, Burge S, Monk S, Smith M, Munro CS, ODonovan M, Craddock N, Kucherlapati R, Rees JL, Owen M, Lathrop GM, Monaco AP, Strachan T, Hovnanian A. (1999b). Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nature Genet 21, 271277. Salazar R, Bell SE, Davis GE. (1999). Co-ordinate induction of the actin cytoskeletal regulatory proteins gelsolin, vasodilator-stimulated phosphoprotein, and prolin during capillary morphogenesis in vitro. Exp Cell Res 249, 2232.
References
325
Salgia R, Hedges TR, Rizk M, Reimer RH, Skarin AT. (1998). Cancer-associated retinopathy in a patient with non-small cell lung cancer. Lung Cancer 22, 149152. Salih MA, Orhii PB, Kalu DN. (1997). Growth hormone stimulates calcium absorption and the expression of mRNAs for intestinal calbindin-D9K, vitamin D receptor and IGF-I in aged ovariectomised rats. J Bone Mineral Res 12, F443. Samowitz WS, Powers MD, Spirio LN, Nollet F, van Roy F, Slattery ML. (1999). Beta catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas. Cancer Res 59, 14421444. Sampson-Johannes A, Wang W, Shtivelman E. (1996). Colonisation of human lung grafts in SCID-hu mice by human colon carcinoma cells lines. Int J Cancer 65, 864869. Sanada K, Kokame K, Yoshizawa T, Takao T, Shimonishi Y, Kukada Y. (1995). Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation. J Biol Chem 270, 1545915462. Sanada K, Shimizu F, Kameyama K, Haga K, Haga T, Fukuda Y. (1996). Calcium bound recoverin targets rhodopsin to membranes to inhibit rhodopsin phosphorylation. FEBS Lett 384, 227230. Sandberg K, Ji H, Clark AJL, Shapira H, Catt KJ. (1992). Cloning and expression of a novel angiotensin II receptor subtype. J Biol Chem 267, 94559458. Sanders MA, Salisbury JL. (1994). Centrin plays an essential role in microtubule severing during agellar excision in Chlamydomonas reinhardtii. J Cell Biol 124, 795805. Sanger JM, Golla R, Safer D, Choi JK, Yu KR, Sanger JW, Nachmias VT. (1995). Increasing intracellular concentrations of thymosin beta 4 in PTK2 cells. Effects on stress bres, cytokinesis and cell spreading. Cell Motility Cytoskeleton 31, 307322. San Jose E, Benguria A, Geller P, Villalobo A. (1992). Calmodulin inhibits epidermal growth factor receptor tyrosine kinase. J Biol Chem 267, 1523715245. Sanson B, White P, Vincent JP. (1996). Uncoupling cadherin-based adhesion from wingless signalling in Drosophila. Nature 383, 627630. Sarin A, Nakajima H, Henkart PA. (1995). A protease-dependent TCR-induced death pathway in mature lymphocytes. J Immunol 154, 58065812. Sartor CI, Strawderman MH, Lin XH, Kish KE, McLaughlin PW. (1997). Rate of PSA rise predicts metastatic versus local recurrence after denite radiotherapy. Int J Radiation Oncol Biol Physics 38, 941947. Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y, Inagami T. (1991). Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature 351, 230233. Sasaki T, Gohring W, Mann K, Maurer P, Hohenester E, Knauper V, Murphy G, Timpl R. (1997). Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its afnity for collagens. J Biol Chem 272, 92379243. Sasaki-Iwaoka H, Maruyama K, Endoh H, Komori T, Kato S, Kawashima H. (1999). A transacting enhances modules oestrogen-mediated transcription of reported genes in osteoblasts. J Bone Mineral Res 14, 248255. Sastry M, Ketchem RR, Crescenzi O, Weber C MJ, Hidaka H, Chazin WJ. (1998). The threedimensional structure of Ca2+-bound calcyclin. Structure 6, 223231. Sata M, Matsuura M, Ikebe M. (1996). Characterisation of the motor and enzymatic properties of smooth muscle long S1 and short HMM. Role of the 2-headed structure on the activity and regulation of myosin motor. Biochemistry 35, 1111311118. Sato M, Iga H, Yoshioka N, Fukui K, Kawamata H, Yoshida H, Hirota S, Kitamura Y. (1997). Emergence of osteoblast-like cells in a neoplastic human salivary cancer cell line after treatment with 22-oxa-1, 25-dihydroxy vitamin D3. Cancer Lett 115, 149160.
326
Sato M, Yasui N, Nakase T, Kawahata H, Sugimoto M, Hirota S, Kitamura Y, Nomura S, Ochi T. (1998). Expression of bone matrix proteins mRNA during distraction osteogenesis. J Bone Mineral Res 13, 12211231. Sato T, Sawaji Y, Matsui N, Sato H, Seiki M, Mori Y, Ito A. (1999). Heat shock suppresses membrane type 1 matrix metalloproteinase production and progelatinase activation in human brosarcoma HT-1080 cells and thereby inhibits cellular invasion. Biochem Biophys Res Commun 265, 189193. Savarese DMF, Russel JT, Fatatis A, Liotta LA. (1992). Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumour cell motility. J Biol Chem 267, 2192821935. Saville MK, Houslay MD. (1994). Phosphorylation of calmodulin on tyr(99) selectively attenuates the action of calmodulin antagonists on type-1 cyclic nucleotide phosphodiesterase activity. Biochem J 299, 863868. Sawan A, Lascu I, Veron M, Anderson JJ, Wright C, Horne CHW, Angus B. (1994). NDPK/NM23 expression in human breast cancer in relation to relapse, survival, and other prognostic factors An immunohistochemical study. J Pathol 172, 2734. Schafer BW, Heizmann CW. (1996).The S100 family of EF-hand calcium binding proteins: functions and pathology. Trends Biochem Sci 21, 134139. Schafer BW, Wicki R, Engelkamp D, Mattei MG, Heizmann CW. (1995). Isolation of a YAC clone covering a cluster of 9 S100 genes on human chromosome 1q21. Rationale for a new nomenclature of the S100 calcium binding protein family. Genomics 25, 638643. Schafer DA, Cooper JA. (1995). Control of actin assembly at lament ends. Annu Rev Cell Dev Biol 11, 497518. Scherer PE, Lederkremer GZ, Williams S, Fogliano M, Baldini G, Lodish HF. (1996). Cab45, novel Ca2+-binding protein localised to the Golgi lumen. J Cell Biol 13, 257268. Schiebel E, Bornens M. (1995). In search of a function for centrins. Trends Cell Biol 5, 197201. Schiffmann SN, Cheron G, Lohof A, DAlcantara P, Meyer M, Parmentier M, Schurmans S. (1999). Impaired motor co-ordination and Purkinje cell excitability in mice lacking calretinin. Proc Natl Acad Sci USA 96, 52575262. Schindl M, Wallraff E, Deubzer B, Witke W, Gerisch G, Sackmann E. (1995). Cell-substrate interactions and locomotion of Dictyostelium wild type and mutants defective in three cytoskeletal proteins. A study using quantitative reection interference contrast microscopy. Biophys J 68, 11771190. Schlaepfer DD, Bode HR, Haigler HT. (1992a). Distinct cellular expression pattern of annexins in Hydra vulgaris. J Cell Biol 118, 911928. Schlaepfer DD, Fisher DA, Brandt ME, Bode HR, Jones JM, Haigler HT. (1992b). Identication of a novel annexin in Hydra vulgaris. Characterisation, cDNA cloning, and protein kinase C phosphorylation of annexin XII. J Biol Chem 267, 95299539. Schlaepfer DD, Haigler HT. (1990). Expression of annexins as a function of cellular growth. J Cell Biol 111, 229238. Schlaepfer DD, Jones J, Haigler HT. (1992c). Inhibition of protein kinase C by annexin V. Biochemistry 31, 18861891. Schlosser K, Scigalla P. (1997). Biochemical markers as surrogates in clinical trials in patients with metastatic bone disease and osteoporosis. Scand J Clin Lab Invest 57, 2128. Schneider J, Pollan M, Jimenez E, Marenbach K, Martinez N, Volm M, Marx D, Meden H. (2000). nm23-H1 expression denes a high-risk subpopulation of patients with early stage epithelial ovarian carcinomas. Br J Cancer 82, 16621670.
References
327
Schoenwaelder SM, Yuan YP, Cooray P, Salem HH, Jackson SP. (1997). Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alpha(IIb)beta(3) (platelet glycoprotein IIb/IIIa) and the cellular retraction of brin clots. J Biol Chem 272, 16941702. Schotte P, Van Criekinge W, Van de Craen M, Van Loo G, Desmedt M, Grooten J, Cornelissen M, De Ridder L, Van de Kerckhove J, Fiers W, Van den Abeele P, Beyaert R. (1998). Cathepsin B-mediated activation of the pro-inammatory caspse-11. Biochem Biophys Res Commun 251, 379387. Schuchel A, Wiltscheck R, Eichinger L, Schleicher M, Holak TA. (1995). Structure of severin domain 2 in solution. J Mol Biol 247, 2127. Schuler GD and several others (1996). A gene map of the human genome. Science 274, 540546. Schuller H, Orloff M, Reznik GK. (1991). Anti-proliferative effects of the Ca2+/calmodulin antagonist B859-35 and the Ca2+-channel blocker verapamil on human lung cancer. Carcinogenesis 12, 23012303. Schwabe JWR, Rhodes D. (1991). Beyond zinc ngers. Steroid receptors have a novel structural motif for DNA recognition. Trends Biochem Sci 16, 291296. Schwaller B, Celio MR, Hunziker W. (1995). Alternative splicing of calretinin messenger RNA leads to different forms of calretinin. Eur J Biochem 230, 424430. Schwaller B, Herrmann B. (1997). Regulated redistribution of calretinin in WiDr cells. Cell Death Differentiation 4, 325333. Schwaller B, Meyer-Monard S, Gander JC, Pugin P, Celio MR, Ludwig C. (1998). The calcium binding protein calretinin 22K is detectable in serum and specic cells of cancer patients. Anticancer Res 18, 36613667. Schwarzbauer JE, Spencer CS. (1993). The Caenorhabditis elegans homologue of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol Biol Cell 4, 941952. Sconzo G, Palla F, Agueli C, Spinelli G, Giudice G, Cascino D, Geraci F. (1999). Constitutive hsp70 is essential to mitosis during early cleavage of Paracentrotus lividus embryos. The blockage of constitutive hsp70 impairs mitosis. Biochem Biophys Res Commun 260, 143149. Seemann J, Weber K, Gerke V. (1996). Structural requirements for annexin I-S100C complex formation. Biochem J 319, 123129. Segal M. (1995). Morphological alterations in dendritic spines of rat hippocampal neurones exposed to N-methyl-D-aspartate. Neurosci Lett 193, 7376. Selinfreund RH, Barger SW, Welsh MJ, van Eldik LJ. (1990). Antisense inhibition of glial S100 production results in alterations in cell morphology, cytoskeletal organisation and cell proliferation. J Cell Biol 111, 20212028. Semple-Rowland SL, Gorczyca WA, Buczylko J, Helekar BS, Ruiz CC, Subbaraya I, Palczewski K, Baehr W. (1996). Expression of GCAP1 and GCAP2 in the retinal degeneration (rd) mutant chicken retina. FEBS Lett 385, 4752. Semprini S, Capon F, Bovolenta S, Bruscia E, Pizzuti L, Fabrizi G, Schietroma C, Zambruno G, Dallapiccola B, Novelli G. (1999). Genomic structure, promoter characterisation and mutational analysis of the S100A7 gene. Exclusion of a candidate for familial psoriasis susceptibility. Human Genetics 104, 130134. Semsarian C, Wu MJ, Ju YK, Marciniec T, Yeoh T, Allen DG, Harvey RP, Graham RM. (1999). Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway. Nature 400, 576581.
328
Sengupta A, Kabat J, Novak M, Wu Q, GrundkeIqbal I, Iqbal K. (1998). Phosphorylation of tau at both threonine 231 and serine 262 is required for maximal inhibition of its binding to microtubules. Arch Biochem Biophys 357, 299309. Senin II, Zargarov AA, Alekseev AM, Gorodovikova EN, Lipkin VM, Philippov PP. (1995). N-myristoylation of recoverin enhances its efciency as an inhibitor of rhodopsin. FEBS Lett 376, 8790. Sequier JM, Hunziker W, Andressen C, Celio MR. (1990). Calbindin D28K protein and messenger RNA localisation in rat brain. Eur J Neurosci 2, 11181126. Shago M, Flock G, Hagesteijn CYL, Woodside M, Grinstein S, Giguere V, Dedhar S. (1997). Modulation of the retinoic acid and retinoid X receptor signalling pathways in P19 embryonal carcinoma cells by calreticulin. Exp Cell Res 230, 5060. Shah GV, Rayford W, Noble MJ, Austenfeld M, Weigel J, Vamos S, Mebust WK. (1994). Calcitonin stimulates growth of human prostate cancer cells through receptor-mediated increase in cyclic adenosine 3,5-monophosphates and cytoplasmic Ca2+ transients. Endocrinology 134, 596602. Shah GV, Rayford W, Noble MJ, Austenfeld M, Weigel J, Vamos S, Mebust WK. (1994). Calcitonin simulates growth of human prostate cancer cells through receptor mediated increase in cyclic adenosine 3,5-monophosphates and cytoplasmic Ca2+ transients. Endocrinology 134, 596602. Shah V, Toruner M, Haddad F, Cadelina G, Papapetropoulos A, Choo K, Sessa WC, Gorszmann RJ. (1999). Impaired endothelial nitric oxide synthase activity is associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology 117, 12221228. Shankar G, Stern PH. (1993). Evaluation of the role of the second messenger systems in tumour necrosis factor stimulated resorption of foetal rat limb bones. Bone 14, 871876. Shapiro MA, Fitzsimmons SP, Clark KJ. (1999). Characterisation of a B cell surface antigen with homology to the S100 protein MRP8. Biochem Biophys Res Commun 263, 1722. Shaul PW, Anderson RGW. (1998). Role of plasmalemmal caveolae in signal transduction. Amer J Physiol 275, L843L851. Sherbet GV, Cajone F, Albertazzi E, Lakshmi MS. (1996). Induction of heat shock protein HSP28 and down-regulation of 18A2/mts1 gene are associated with inhibition of proliferation of B16 murine melanoma cell lines. Proc Amer Assoc Cancer Res 37, 74. Sherbet GV, Cajone F. (2000). S1004 calcium binding protein co-operates with p53 and stathmin in cell cycle progression. Proc Amer Assoc Cancer Res 41, 430. Sherbet GV, Lakshmi MS. (1997a). Retinoid and growth factor signal transduction. In: Retinoids: Their physiological function and therapeutic potential, ed. Sherbet GV, Jai Press Inc, Greenwich, Conn. and London, pp. 141159. Sherbet GV, Lakshmi MS. (1997b). The genetics of cancer. Genes associated with cancer invasion, metastasis and cell proliferation. Academic Press, London and New York. Sherbet GV, Lakshmi MS. (1998). S100A4 (mts1) calcium binding protein in cancer growth, invasion, and metastasis. Anticancer Res 18, 24152422. Sherbet GV, Parker C, Usmani BA, Lakshmi MS. (1995). Epidermal growth factor receptor status correlates with cell proliferation related 18A2/mts1 gene expression in human carcinoma cell lines. Ann NY Acad Sci 768, 272276. Sherbet GV. (1982). The biology of tumour malignancy. Academic Press, London and New York. Sherbet GV. (1987). The metastatic spread of cancer. Macmillan, Basingstoke and London. Sheterline P, Sparrow JC. (1994). Actin. Protein Prole 1, 1121.
References
329
Shi DL, Launay C, Fromentoux V, Feige J, Boucaut JC. (1994). Expression of broblast growth factor receptor-2 splice variants is developmentally and tissue-specically regulated in the amphibian embryo. Dev Biol 164, 173182. Shiba E, Kim SJ, Kambayashi JI, Kawamura I, Lacey E, Manda T, Shimomura K, Taguchi T, Takai SI. (1997). Mechanisms of growth inhibition by calpain inhibitor in MCF-7 cells. Anticancer Res 17, 19191923. Shiba E, Kim SS, Fujitani M, Kambayashi J, Kawamura I. (1996). Possible involvement of calpain in the growth of oestrogen-receptor positive breast cancer cells. Anticancer Res 16, 773777. Shiba H, Fujita T, Doi N, Nakamura S, Nakanishi K, Takemoto T, Hino T, Noshiro M, Kawamoto T, Kurihara H, Kato Y. (1998). Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, bronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol 174, 194205. Shiba H, Nakamura S, Shirakawa M, Nakanishi K, Okamoto H, Satakeda H, Noshiro M, Kamihagi K, Katayama M, Kato Y. (1995). Effect of basic broblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcication in cultures of human pulp cells. Dev Biol 170, 457466. Shibata M, Ohoka T, Mizuno S, Suzuki K. (1993). Characterisation of a 64-kDa protein phosphorylated during chemotactic activation with IL-8 and FMLP of human polymorphonuclear leukocytes. 1. Phosphorylation of a 64-kDa protein and other proteins. J Leukocyte Biol 54, 19. Shibata M, Yamakawa Y, Ohoka T, Mizuno S, Suzuki K. (1993). Characterisation of a 64kDa protein phosphorylated during chemotactic activation with IL-8 and FMLP of human polymorphonuclear leukocytes. 2. Purication and aminoacid analysis of phosphorylated 64-kDa protein. J Leukocyte Biol 54, 1016. Shields DC, Banik NL. (1998a). Putative role of calpain in the pathophysiology of experimental optic neuritis. Exp Eye Res 67, 403410. Shields DC, Banik NL. (1998b). Up-regulation of calpain activity and expression in experimental allergic encephalomyelitis. A putative role for calpain in demyelination. Brain Res 794, 6874. Shields DC, Schaecher KE, Goust JM, Banik NL. (1999b). Calpain activity and expression are increased in splenic inammatory cells associated with experimental allergic encephalomyelitis. J Neuroimmunol 99, 112. Shields DC, Schaecher KE, Saido TC, Banik NL. (1999a). A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA 96, 1148611491. Shields DC, Tyor WR, Deibler GE, Banik NL. (1998b). Increased calpain expression in experimental demyelinating optic neuritis. An immunocytochemical study. Brain Res 784, 299304. Shields DC, Tyor WR, Deibler GE, Hogan EL, Banik NL. (1998a). Increased calpain expression in activated glial and inammatory cells in experimental allergic encephalomyelitis. Proc Natl Acad Sci USA 95, 57685772. Shih CL, Linse KD, Wang K. (1998). Identication of molecular contacts between nebulin and calmodulin. Biophys J 74, p.A349. Shimohama S, Tanino H, Fujimoto S. (1999). Changes in caspase expression in Alzheimers disease. Comparison with development and ageing. Biochem Biophys Res Commun 256, 381384.
330
Shimomura A, Ogawa Y, Kitani T, Fujisawa H, Hagiwara M. (1996). Calmodulin dependent protein kinase II potentiates transcriptional activation through activating transcription factor-1 but not cAMP response element binding protein. J Biol Chem 271, 1795717960. Shin GT, Khanna A, Ding RC, Sharma VK, Lagman M, Li BG, Suthanthiran M. (1998). In vivo expression of transforming growth factor beta (1) in humans. Stimulation by cyclosporin. Transplantation 65, 313318. Shinomiya H, Hagi A, Fukuzumi M, Mizobuchi M, Hirata H, Utsumi S. (1995). Complete primary structure and phosphorylation site of the 65-kDa macrophage protein phosphorylated by stimulation with bacterial lipopolysaccharide. J Immunol 154, 34713478. Shiozaki H, Kadowsaki T, Doki Y, Inoue M, Tamura S, Oka H, Iwagawa T, Matsui S, Shimaya K, Takeichi M, Mori T. (1995). Effect of epidermal growth factor on cadherin-mediated adhesion in human oesophageal cancer cell line. Br J Cancer 71, 250258. Shirakawa T, Ko SC, Gardner TA, Cheon J, Miyamoto T, Gotoh A, Chung LWK, Kao CH. (1998). In vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapy. Cancer Gene Ther 5, 274280. Shiroo M, Oppenheim JJ, Matsushima K. (1988). IL-1, TNF and PMA induce phosphorylation of a common cytosolic 65-kDa protein in human peripheral blood mononuclear cells (PBMC). FASEB J 2, p.A1830. Shizukuda Y, Shaoquing T, Yokota R, Ware JA. (1999). Vascular endothelial growth factorinduced endothelial cell migration and proliferation depend on a nitric oxide-mediated decrease in protein kinase delta activity. Circ Res 85, 247256. Shrestha P, Muramatsu Y, Kudeken W, Mori M, Takai Y, Ilg EC, Schafer BW, Heizmann CW. (1998). Localisation of Ca2+-binding S100 proteins in epithelial tumours of the skin. Virchows Arch Pathol 432, 53-59. Siever DA, Erickson HP. (1997). Extracellular annexin II. Int J Biochem Cell Biol 29, 12191223. Sihag RK, Shea TB, Wang FS. (1996). Spectrin-actin interaction is required for neurite extension in NE2a/dl neuroblastoma cells. J Neurosci Res 44, 430437. Sim RB, Arlaud GJ, Colomb M. (1979). C1 inhibitor-dependent dissociation of human complement component C1 bound to immune complexes. Biochem J 179, 449457. Simbulan-Rosenthal CM, Rosenthal DS, Iyer S, Boulares AH, Smulson ME. (1998). Transient poly (ADP-ribosyl)ation of nuclear proteins and role of poly (ADP-ribose) polymerase in the early stages of apoptosis. J Biol Chem 273, 1370313712. Simbulan-Rosenthal CM, Rosenthal DS, Iyer S, Boulares H, Smulson ME. (1999). Involvement of PARP and poly (ADP-ribosyl)ation in the early stages of apoptosis and DNA replication. Mol Cell Biochem 193, 137148. Simionescu N. (1993). Cellular aspects of transcapillary exchange. Physiol Rev 63, 15361579. Simon M, Vincent C, Haftek M, Girbal E, Sebbag M, Gomes-Daudrix V, Serre G. (1995). The rheumatoid arthritis-associated autoantibodies to laggrin label the brous matrix of the cornied cells but not the prolaggrin containing keratohyalin granules in human epidermis. Clin Exptl Immunol 100, 9098. Sims NA, White CP, Sunn KL, Thomas GP, Drummond ML, Morrison NA, Eisman JA, Gardiner EM. (1997). Human and murine osteocalcin gene expression. Conserved tissue restricted expression and divergent responses to 1, 25-dihydroxy vitamin D-3 in vivo. Molec Endocrinol 11, 16951708. Singh JN, Atreya CD, Nakhasi HL. (1994). Identication of calreticulin as a rubella virus RNA binding protein. Proc Natl Acad Sci USA 91, 1277012774. Singhal RL, Prajda N, Weber G. (1997). Intracellular signalling: Phosphatidylinositol lipid metabolism. Ind J Biochem Biophys 34, 2528.
References
331
Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ. (1999). Ordering the cytochrome cinitiated caspase cascade. Hierarchical activation of caspases 2,3,6,7,8, and 10 in a caspase 9-dependent manner. J Cell Biol 144, 281292. Small JV. (1995). Getting the actin laments straight. Nucleation-release or treadmilling. Trends Cell Biol 5, 5254. Smith MJ, Koch GLE. (1989). Multiple zones in the sequence of calreticulin. EMBO J 8, 35813586. Smyth CJ, Marron MB, Twohig JMGJ, Smith SGJ. (1996). Fimbrial adhesins: Similarities and variations in structure and biogenesis. FEMS Immunol Med Microbiol 16, 127139. Snyder SH, Supattapone S. (1989). Isolation and functional characterisation of an inositol trisphosphate receptor from brain. Cell Calcium 10, 337342. Soderling JA, Reed MJ, Corsa A, Sage EH. (1997). Cloning and expression of murine SC1, a gene product homologous to SPARC. J Histochem Cytochem 45, 823835. Soldes OS, Kuick RD, Thompson IA, Hughes SJ, Orringer MB, Iannettoni MD, Hanash SM, Beer DG. (1999). Differential expression of Hsp27 in normal oesophagus, Barretts metaplasia and oesophageal adenocarcinomas. Br J Cancer 79, 595603. Someren JS, Faber LE, Klein JD, Tumlin JA. (1999). Heat shock proteins 70 and 90 increase calcineurin activity in vitro through calmodulin-dependent and independent mechanisms. Biochem Biophys Res Commun 260, 619625. Sommer B, Bickel M, Hofstetter W, Wetterwald A. (1996). Expression of matrix proteins during the development of mineralised tissues. Bone 19, 371380. Song KS, Li SW, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP. (1996). Co-purication and direct interaction of ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purication of caveolae membranes. J Biol Chem 271, 96909697. Sorimachi H, Kinbara K, Kimura S, Takahashi M, Ishiura S, Sasagawa N, Sorimachi N, Shimada H, Tagawa K, Maruyama K, Suzuki K. (1995). Muscle specic calpain, p94, responsible for limb girdle muscular dystrophy 2A, associated with connectin through IS2, a p94-specic sequence. J Biol Chem 270, 3115831162. Sormunen R, Eskelinen S, Leong ASY. (1997). Fodrin immunolocalisation in epithelial tumours. Applied Immunohistochem 5, 179184. Soroush V, Gurevitch AW. (1997). Dariers disease associated with multiple caf-au-lait macules. Cutis 59, 193195. Spada A, Lania A, Mantovani S, Ballare E. (1997). Cyclic AMP and calcium in the transduction of hypothalamic neurohormone action in human pituitary tumours. Hormone Res 47, 235239. Spector NL, Mehlen P, Ryan C, Hardy L, Samson W, Levine H, Nadler LM, Fabre N, Arrigo AP. (1994). Regulation of the 28 kDa heat shock protein by retinoic acid during differentiation of human leukaemic HL-60 cells. FEBS Lett 337, 184188. Spector NL, Ryan C, Samson W, Levine H, Nadler LM, Arrigo AP. (1993). Heat shock protein is a unique marker of growth arrest during macrophage differentiation of HL-60 cells. J Cell Physiol 156, 619625. Spector NL, Samson W, Ryan C, Gribben J, Urba W, Welch WJ, Nadler LM. (1992). Growth arrest of human lymphocytes B is accompanied by induction of the low molecular weight mammalian heat shock protein (HSP28). J Immunol 148, 16681673. Spencer MJ, Tidball JG, Anderson LVB, Bushby KMD, Harris JB, Passos-Bueno MR, Somer H, Vainzof M, Zatz M. (1997). Absence of calpain 3 in a form of limb-girdle muscular dystrophy (LGMD-2A). J Neurol Sci 146, 173178.
332
Spyracopoulos L, Li MX, Sia SK, Gagne SM, Chandra M, Solaro RJ, Sykes BD. (1997). Calcium-induced structural transition in the regulatory domain of human cardiac troponin C. Biochemistry 36, 1213812146. Squier MKT, Miller ACK, Malkinson AM, Cohen JJ. (1994). Calpain activation in apoptosis. J Cell Physiol 159, 229237. Srinivasan G, Campbell E, Bashirelahi N. (1995). Androgen, oestrogen, and progesterone receptors in normal and ageing prostates. Microscopy Res Technique 30, 293304. Srivastsa PJ, Cliby WA, Keeney GL, Dodson MK, Suman VJ, Roche PD, Podratz, KC. (1996). Elevated nm23 protein expression is correlated with diminished progression-free survival in patients with epithelial ovarian cancer. Gynecol Oncol 60, 363372. Staal A, Geertsma-Kleinekoort WMC, Van den Bemd GJCM, Buurman CJ, Birkenhager JC, Pols HAP, Van Leeuwen JPTM. (1998). Regulation of osteocalcin production and bone resorption by 1, 25-dihydroxyvitamin D3 in mouse long bones. Interaction with the bonederived growth factor TGF-beta and IGF-1. J Bone Mineral Res 13, 3643. Stabler SM, Ostrowski LL, Janicki SM, Monteiro MJ. (1999). A myristoylated calcium binding protein that preferentially interacts with the Alzheimers disease presenilin 2 protein. J Cell Biol 145, 12771292. Stahl JA, Leone A, Rosengard AM, Porter L, Richter-King C, Steeg PS. (1991), Identication of a second human nm23 gene, nm23-H2. Cancer Res 51, 445449. Staiger CJ, Gibbon BC, Kovar DR, Zonia LE. (1997). Prolin and actin-depolymerising factor: Modulators of actin organisation in plants. Trends Plant Sci 2, 275281. Stankewich MC, Tse WT, Peters LL, Chng Y, John KM, Stabach PR, Devarajan P, Morrow JS, Lux SE. (1998). A widely expressed III spectrin associated with Golgi and cytoplasmic vesicles. Proc Natl Acad Sci USA 95, 1415814163. Staubli F, Klein A, Rentsch JM, Hameister H, Berchtold MW. (1995). Structure and chromosomal localisation of the mouse oncomodulin gene. Mammalian Genome 6, 769777. Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DHF, Tavtigian SV. (1997). Identication of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23 that is mutated in multiple advanced cancers. Nature Genet 15, 356362. Steeg PS, Bevilacqua G, Kopper L, Thorgierson VP, Talmadge JE, Liotta L A, Sobel ME. (1988). Evidence for novel gene associated with low tumour metastatic potential. J Natl Cancer Inst 80, 200204. Steeg PS, Bevilacqua G, Pozzatti R, Liotta LA, Sobel ME. (1988). Altered expression of nm23, a gene associated with low tumour metastatic potential, during adenovirus 2 ElA inhibition of experimental metastasis. Cancer Res 48, 65506554. Stenman UH, Leinonen J, Zhang WM, Finne P. (1999). Prostate specic antigen. Seminars Cancer Biol 9, 8393. Stephenson MA, Castro CY, Gondo MM, Carlson NR, Cagle PT. (1998). Nm23/nucleoside diphosphate kinase A expression in 412 lung carcinomas. Lack of correlation with tumour progression, metastasis or prognosis. Modern Pathol 11, p. 180. Stewart N, Hicks GG, Paraskevas F, Mowat M. (1995). Evidence for a second cell cycle block at G2/M by p53. Oncogene 10, 109115. Steyger PS, Burton M, Hawkins JR, Schuff NR, Baird RA. (1997). Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs. Int J Develop Neurosci 15, 417432. Stoeva S, Horger S, Voelter W. (1997). A novel beta thymosin from the sea urchin. Extending the phylogenetic distribution of beta thymosin from mammals to echinoderms. J Peptide Sci 3, 282290.
References
333
Stoll SW, Zhao XP, Elder JT. (1998). EGF stimulates transcription of CaN19 (S100A2) in HaCaT keratinocytes. J Invest Dermatol 111, 10921097. Stradal T, Kranewitter W, Winder SJ, Gimona M. (1998). CH domains revisited. FEBS Lett 431, 134137. Strandjord TP, Sage EH, Clark JG. (1995). SPARC participates in the branching morphogenesis of developing foetal rat lung. Amer J Respiratory Cell Mol Biol 13, 279287. Stringa E, Filanti C, Giunciuglio D, Albini A, Manduca P. (1995). Osteoblastic cells from rat long bone. A. Characterisation of their differentiation in culture. Bone 16, 663670. Stryer L. (1991). Visual excitation and recovery. J Biol Chem 266, 1071110714. Stuart GR, Lynch NJ, Lu J, Geick A, Moffat BE, Sim RB, Schwaeble WJ. (1996). Localisation of the C1q site within C1q receptor/calreticulin. FEBS Lett 397, 245249. Stull JT, Tansey MG, Tang DC, Word RA, Kamm KE. (1993). Phosphorylation of myosin light chain kinase. A cellular mechanism for Ca2+ desensitisation. Mol Cell Biochem 128, 229237. Sudo T, Hidaka H. (1999). Characterisation of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis. FEBS Lett 444, 1114. Suetsugu S, Miki H, Takenawa T. (1998). The essential role of prolin in the assembly of actin for microspike formation. EMBO J 17, 65166526. Sumi T, Matsumoto K, Takai Y, Nakamura T. (1999). Colin phosphorylation and actin cytoskeletal dynamics regulated by Rho- and Cdc42 activated LIM-kinase 2. J Cell Biol 147, 15191532. Summers MF, South TL, Kim B, Hare DR. (1990). High resolution structure of an HIV zinc nger-like domain via new NMR-based distance geometry approach. Biochemistry 29, 329340. Sun HQ, Wooten DC, Janmey PA, Yin HL. (1994). The actin side-binding domain of gelsolin also caps actin laments. Implications for actin lament severing. J Biol Chem 269, 94739479. Sun PQ, Enslen H, Myung PS, Mauarer RA. (1994). Differential activation of CREB by Ca2+/calmodulin dependent phosphorylation of a site that negatively regulates activity. Genes Dev 8, 25272539. Sung V, Gilles C, Murray A, Clarke R, Aaron AD, Azumi N, Thompson EW. (1998). The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype. Exp Cell Res 241, 273284. Supattapone S, Worley PF, Baraban JM, Snyder SH. (1988). Solubilisation, purication, and characterisation of an inositol trisphosphate receptor. J Biol Chem 263, 15301534. Sutko JL, Airey JA. (1996). Ryanodine receptor Ca2+ release channels. Does diversity in form equal diversity in function? Physiol Rev 76, 10271071. Suzuki K, Saido TC, Hirai S. (1992). Modulation of cellular signals by calpain. Ann NY Acad Sci 674, 218227. Suzuki T, Suzuki Y, Hanada K, Hashimoto A, Redpath JL, Stanbridge EJ, Nishijima, M, KitagawaT. (1998). Reduction of caveolin-1 expression in tumorigenic cell hybrids. J Biochem 124, 383388. Swaroop A, Hogan BL, Francke U. (1988). Molecular analysis of the cDNA for human SPARC/osteonectin/BM-40. Sequence, expression, and localisation of the gene to chromosome 5q 31-q33. Genomics 2, 3747. Szewczenko-Pawlikowski M, Dziak E, McLaren MJ, Michalak M, Opas M. (1997). Heat shock-regulated expression of calreticulin in retinal pigment epithelium. Mol Cell Biochem 177, 145152. Szymanski PT, Tao T. (1997). Localisation of protein regions involved in the interaction between calponin and myosin. J Biol Chem 272, 1114211146.
334
Tabernero L, Taylor DA, Chandross RJ, Van Berkum MFA, Means AR, Quiocho FA, Sack JS. (1997). The structure of a calmodulin mutant with a deletion in the central helix: Implications for molecular recognition and protein binding. Structure 5, 613622. Tachikui H, Navet AF, Ozawa M. (1997). Identication of the Ca2+-binding domains in reticulocalbin, an endoplasmic reticulum resident Ca2+-binding protein with multiple EFhand motifs. J Biochem 121, 145149. Taillon BE, Jarvik JW. (1995). Central helix mutations in the centrosome associated EF-hand protein centrin. Protoplasma 189, 203215. Takadera T, Ohyashiki T. (1997). Apoptotic cell death and caspase 3 (CPP32) activation induced by calcium ionophore at low concentrations and their prevention by nerve growth factor in PC12 cells. Eur J Biochem 249, 812. Takahashi H, Yamaguchi M. (1997). Stimulatory effect of regucalcin on ATPdependent calcium transport in rat liver plasma membranes. Mol Cell Biochem 168, 149153. Takahashi K, Hiwada K, Kokubu T. (1986). Isolation and characterisation of a 34-kDa calmodulin and F-actin binding protein from chicken gizzard. Biochem Biophys Res Commun 141, 2026. Takemura H, Hughes AR, Thastrup O, Putney JW. (1989). Activation of calcium entry by the tumour promoter thapsigargin in parotid acinar cells. J Biol Chem 264, 1226612271. Takenaga K, Nakmura Y, Endo H, Sakiyama S. (1994a). Involvement of S100-related calcium binding protein pEL98 (mts1) in cell motility and tumour cell invasion. Japanese J Cancer Res 85, 831839. Takenaga K, Nakamra Y, Sakiyama S. (1994b). Cellular localisation of PEL98 protein, an S100-related calcium binding protein, in broblasts and its tissue distribution analysed by monoclonal antibodies. Cell Struc Funct 19, 133141. Takenaga K, Nakamura Y, Sakiyama S, Hasegawa Y, Sato K, Endo H. (1994b). Binding of PEL98 protein, an S100 related calcium-binding protein to non-muscle tropomyosin. J Cell Biol 124, 757768. Takenaga K, Nakamura Y, Sakiyama S. (1997a). Expression of antisense RNA to S100A4 encoding S100 related calcium-binding protein suppresses metastatic potential of high metastatic Lewis lung carcinoma cells. Oncogene 14, 331337. Takenaga K, Nakanishi H, Wada K, Suzuki M, Matsuzaki O, Matsuura A, Endo H. (1997b). Increased expression of S100A4, a metastasis associated gene in human colorectal adenocarcinoma. Clin Cancer Res 3, 23092316. Takenaga K. (1999). Suppression of metastasis-associated S100A4 gene expression by gamma interferon in human colon adenocarcinoma cells. Br J Cancer 80, 127132. Takita H, Kuboki Y. (1995). Conformational changes of bovine bone osteonectin induced by interaction with calcium. Calcied Tissue Int 56, 559565. Takizawa T, Takizawa T, Arai S, Kizawa K, Uchiwa H, Sasaki I, Inoue T. (1999). Ultrastructural localisation of S100A3, a cysteine-rich calcium binding protein, in human scalp hair shafts revealed by rapid freezing immunocytochemistry. J Histochem Cytochem 47, 525532. Talgham SA, Adamo HP. (1999). Substitution of the N-terminal segment of the plasma membrane Ca pump isoform 4 by that of isoform 1 results in a functional chimeric enzyme. Biochim Biophys Acta 1419, 7177. Tamura G, Yin J, Wang S, Fleisher AS, Zou TT, Abraham JM, Kong DH, Smolinski KN, Wilson KT, James SP, Silverberg SG, Nishizuka s, Terashima M, Motoyama T, Meltzer SJ. (2000). E-cadherin gene promoter hypermethylation in primary human gastric carcinomas. J Natl Cancer Inst 92, 569573.
References
335
Tamura M, Gu JG, Danen EHJ, Takino T, Miyamoto S, Yamada KM. (1999a). PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 274, 2069320703. Tamura M, Gu JG, Takino T, Yamada KM. (1999b). Tumour suppressor PTEN inhibition of cell invasion, migration and growth. Differential involvement of focal adhesion kinase and p130 (Cas). Cancer Res 59, 442449. Tamura M, Gu JG, Tran H, Yamada KM. (1999c). PTEN gene and integrin signalling in cancer. J Natl Cancer Inst 91, 18201828. Tan FC, Goll DE, Otsuka Y. (1988). Some properties of the millimolar Ca2+-dependent proteinase from bovine cardiac muscle. J Mol Cell Cardiol 20, 983997. Tan JQ, Vorum H, Larsen CG, Madsen P, Rasmussen HH, Gesser B, Etzerodt M, Honore B, Celis JE, Thestrup-Pedersen K. (1996). Psoriasin. A novel chemotactic protein. J Invest Dermatol 107, 510. Tan JY, Sidhu G, Greco MA, Ballard H, Wieczorek R. (1998). Villin, cytokeratin 7, and cytokeratin 20 expression in pulmonary adenocarcinoma with ultrastructural evidence of microvilli with rootlets. Human Pathol 29, 390396. Tan MJ, Heizmann CW, Guan KL, Schafer BW, Sun Y. (1999). Transcriptional activation of the human S100A2 promoter by wild-type p53. FEBS Lett 445, 265268. Tanaka J, Sobue K. (1994). Localisation and characterisation of gelsolin in nervous tissues. Gelsolin is specically enriched in myelin forming cells. J Neurosci 14, 10381052. Tanaka M, Mullauer L, Ogiso Y, Fujita H, Moriya S, Furuuchi K, Harabayashi T, Shinohara N, Koyanagi T, Kuzumaki N. (1995). Gelsolin. A candidate for suppressor of human bladder cancer. Cancer Res 55, 32283232. Tang JX, Szymanski PT, Janmey PA, Tao T. (1997). Electrostatic effects of smooth muscle calponin on actin assembly. Eur J Biochem 247, 432440. Tang MS, Zheng JB, Denissenko MF, Pfeifer GP, Zheng Y. (1999). Use of UvrABC nuclease to quantify benzo[a]pyrine diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene. Carcinogenesis 20, 10851089. Tang W, Bunting NM, Zimmerman GA, McIntyre TM, Prescott SM. (1996). Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates. J Biol Chem 271, 1023710241. Tao P, Coleman JE. (1990). GAL4 transcription factor is not a zinc nger but forms a Zn(II)cys6 binuclear cluster. Proc Natl Acad Sci USA 87, 20772081. Tarcsa E, Marekov LN, Andeoli J, Idler WW, Candi E, Chung SI, Steinert PM. (1997). The fate of trichohyalin. Sequential, post-translational modications by peptidyl-arginine deiminase and transglutaminases. J Biol Chem 272, 2789327901. Tashiro H, Blazes MS, Wu R, Cho KR, Bose S, Wang SI, Li J, Parsons R, Ellenson LH. (1997). Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynaecological malignancies. Cancer Res 57, 39353940. Tavakkol A, Zouboulis CC, Duell EV, Voorhees JJ. (1994). A retinoic acid-inducible skinspecic gene (RIS-1 psoriasin). Molecular cloning and analysis of gene expression in human skin in vivo and cultured skin cells in vitro. Mol Biol Reports 20, 7583. Temm-Grove CJ, Jockusch BM, Weinberger RP, Schevzov G, Helfman DM. (1998). Distinct localisation of tropomyosin isoforms in LLC-PK1 epithelial cells suggests specialised function at cell-cell adhesion. Cell Motility Cytoskeleton 40, 393407. Tenner AJ, Cooper NR. (1981). Identication of cell types in human peripheral blood that bind C1q. J Immunol 126, 11741179. Tesmer JJG, Berman DM, Gilman AG, Sprang SR. (1997). Structure of RGS4 found to AIF4activated G (I alpha 1). Stabilisation of the transition state for GTP hydrolysis. Cell 89, 251261.
336
Thalmann I, Shibasaki O, Comegys TH, Henzl MT, Senarita M, Thalmann R. (1995). Detection of a -parvalbumin isoform in the mammalian inner ear. Biochem Biophys Res Commun 215, 142147. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. (1990). Thapsigargin, a tumour promoter, discharges intracellular Ca2+ stores by specic inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87, 24662470. Theobald J, Hanby A, Patel K, Moss SE. (1995). Annexin VI has tumour suppressor activity in human A431 squamous epithelial carcinoma cells. Br J Cancer 71, 786788. Theobald J, Smith PD, Jacob SM, Moss SE. (1994). Expression of annexin VI in A431 carcinoma cells suppresses proliferation. A possible role for annexin VI in cell growth regulation. Biochim Biophys Acta 1223, 383390. Theopold U, Pinter M, Daffre S, Tryselius Y, Friedrich P, Nassel DR, Hultmark D. (1995). Calpa, a Drosophila homologue specically expressed in a small set of nerve, midgut and blood cells. Mol Cell Biol 15, 824834. Theriot JA. (1997). Accelerating on a treadmill. ADF/colin promotes rapid actin lament turnover in the dynamic cytoskeleton. J Cell Biol 136, 11651168. Thomas WG, Pipolo L, Quin HW. (1999). Identication of a Ca2+/calmodulin-binding domain within the carboxyl terminus of the angiotensin II (AT-1A) receptor. FEBS Lett 455, 367371. Thomas WG. (1999). Regulation of angiotensin II type 1 (AT-1) receptor function. Regulatory Peptides 79, 923. Thompson TC. (1998). Metastasis-related genes in prostate cancer. The role of caveolin-1. Cancer Metastasis Rev 17, 439442. Thomsen LL, Miles DW. (1998). Role nitric oxide in tumour progression. Lessons from human tumours. Cancer Metastasis Rev 17, 107118. Thornberry NA, Lazebnik Y. (1998). Caspases. Enemies within. Science 281, 13121316. Thornberry NA. (1997). The caspase family of cysteine proteases. Br Med Bull 53, 478490. Thulin CD, Walsh KA. (1995). Identication of the amino-terminus of human laggrin using differential LC/MS techniques. Implications for prolaggrin processing. Biochemistry 34, 86878692. Tilney LG, Bonder EM, Coluccio LM, Moosekar MS. (1983). Actin from thyone sperm assembles only one end of an actin lament. A behaviour regulated by prolin. J Cell Biol 97, 112124. Timmerman LA, Clipstone NA, Ho SN, Northrop JP, Crabtree GR. (1996). Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression. Nature 383, 837840. Togashi H, Emala CW, Hall IP, Hirshman CA. (1998). Carbachol-induced actin reorganisation involves G(1) activation of Rho in human airway smooth muscle cells. Am J Physiol 18, L803L809. Tolkacheva T, Chan AML. (2000). Inhibition of h-ras transformation by the PTEN/MMAC1/TEP1 tumour suppressor gene. Oncogene 19, 680689. Tominaga T, Sugie K, Hirata M, Morii N, Fukata J, Uchida A, Imura H, Narumiya S. (1993). Inhibition of PMA-induced, LFA-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, Rho. J Cell Biol 120, 15291537. Tomono M, Toyoshima K, Ito M, Amano H, Kiss Z. (1998). Inhibitors of calcineurin block expression of cyclins A and E induced by broblast growth factor in Swiss 3T3 broblasts. Arch Biochem 353, 374378. Tomono M, Toyoshima K, Ito M, Amano H. (1996). Calcineurin is essential for DNA synthesis in Swiss 3T3 broblasts. Biochem J 317, 675680.
References
337
Tong G, Shepherd D, Jahr CE. (1995). Synaptic desensitisation of NMDA receptors by calcineurin. Science 267, 15101512. Tonini GP, Casalaro A, Cara A and Di Martino D. (1991). Inducible expression of calcyclin, a gene with strong homology to S100 protein, during neuroblastoma cell differentiation and its prevalent expression in Schwann-like cell lines. Cancer Res 51, 17331737. Tozawa K, Sakurada S, Kohri K, Okkamoto T. (1995). Effects of anti-nuclear factor kappa B reagents in blocking adhesion of human cancer cells to vascular endothelial cells. Cancer Res 55, 41624167. Trave G, Lacombe PJ, Pfuhl M, Saraste M, Pastore A. (1995). Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands. EMBO J 14, 49224931. Tremble PM, Lane TF, Sage EH, Werb Z. (1993). SPARC, a secreted protein associated with morphogenesis and tissue remodelling, induces expression of metalloproteinases in broblasts through a novel extracellular matrix-dependent pathway. J Cell Biol 121, 14331444. Trigatti B, Anderson RGW, Gerber G. (1999). Identication of caveolin-1 as a fatty acid binding protein. Biochem Biophys Res Commun 255, 3439. Trinick J. (1994). Titin and nebulin. Protein rulers in muscle? Trends Biochem Sci 19, 405409. Tripet B, Van Eyk JE, Hodges RS. (1997). Mapping of a second actin tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+ dependent regulation of muscle contraction. J Mol Biol 271, 728750. Truman JW. (1996). Steroid receptors and nervous system metamorphosis in insects. Dev Neurosci 18, 87101. Tsien RW, Lipscombe D, Madison DV, Bley KR, Fox AP. (1988). Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 11, 431437. Tsitsilonis OE, Bekris E, Voutsas IF, Baxevanis CN, Markopoulos C, Papadopoulou SA, Kontzoglou K, Stoeva S, Gogas J, Voelter W, Papaichail M. (1998). The prognostic value of alpha thymosins in breast cancer. Anticancer Res 18, 15011508. Tsou HC, Ping XL, Xie XX, Gruener AC, Zhang H, Nini R, Swisshelm K, Sybert V, Diamond TM, Sutphen R, Peacocke M. (1998). The genetic basis of Cowdens disease. Three novel mutations in PTEM/MMAC1/TEP1. Human Genet 102, 467473. Tsunamoto K, Todo S, Imashuku S, Kato K. (1988). Induction of S100 protein by 5-bromo 2-deoxyuridine in human neuroblastoma cell lines. Cancer Res 48, 170174,. Tsunoda Y. (1993). Receptor operated Ca2+ signalling and cross talk in stimulus secretion coupling. Biochim Biophys Acta 1154, 105156. Tsuzuki S, Toyama-Sorimachi N, Kitamura F, Tobita Y, Miyasaka M. (1998). FK506 (tacrolimus) inhibits extravasation of lymphoid cells by abrogating VLA-4/VCAM-1-mediated transendothelial migration. FEBS Lett 430, 414418. Tuck AB, Arsenault DM, OMalley FP, Hota C, Ling MC, Wilson SM, Chambers AF. (1999). Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene 18, 42374246. Tuckwell DS, Brass A, Humphries MJ. (1992). Homology modelling of integrin EF-hands. Evidence for widespread use of a conserved cation-binding site. Biochem J 285, 325331. Tulchinsky E, Ford HL, Kramerov D, Reshetnyak E, Grigorian M, Zain S, Lukanidin E. (1992). Transcriptional analysis of the mts1 gene with specic references to 5 anking sequences. Proc Natl Acad Sci USA 89, 91469150. Tulchinsky E, Gregorian MS, Ebralidze AK, Milshina NI, Lukanidin EM. (1990). Structure of gene mts1, transcribed in metastatic tumour cells. Gene 87, 219223.
338
Tulchinsky E, Grigorian M, Tkatch T, Georgiev G, Lukanidin E. (1995). Transcriptional regulation of the mts1 gene in human lymphoma cells. The role of DNA methylation. Biochim Biophys Acta 1261, 243248. Tulchinsky EM, Georgiev GP, Lukanidin EM. (1996). Novel AP-1 binding site created by DNA methylation. Oncogene 12, 17371745. Tumlin JA. (1997). Expression and function of calcineurin in the mammalian nephron. Physiological roles, receptor signalling, and ion transport. Am J Kidney Dis 30, 884895. Tuominen H, Sormunen R, Kallioinen M. (1996). Non-erythroid spectrin (fodrin) in cutaneous tumours. Diminisedin cell membranes, increased in the cytoplasm. Br. J. Dermatol 135, 576580. Tuvia S, Buhusi M, Davis L, Reedy M, Bennett V. (1999). Ankyrin-B is required for intracellular sorting of structurally diverse CA2+ homeostasis proteins. J Cell Biol 147, 9951007. Tyska MJ, Duppuis DE, Guilford WH, Patlak JB, Waller GS, Trybus KM, Warshaw DM, Lowey S. (1999). Two heads of myosin are better than one for generating force and motion. Proc Natl Acad Sci USA 96, 44024407. Tzima E, Trotter PJ, Orchard MA, Walker JH. (1999). Annexin V binds to the actin-based cytoskeleton at the plasma membrane of activated platelets. Exp Cell Res 251, 185193. Uchio E, Standord M, Hasan A, Satoh S, Ohno S, Shinnick T, Van der Zee R, Mizushima Y, Lehner T. (1998). HSP-derived peptides inducing uveitis and IgG and IgA antibodies. Exp Eye Res 67, 719727. Ueda Y, Wang MC, OU BR, Huang J, Elce J, Tanaka K, Ichihara A, Forsberg NE. (1998). Evidence for the participation of the proteasome and calpain in early phases of muscle cell differentiation. Int J Biochem Cell Biol 30, 679694. Uemura T, Liu YK, Feng Y, Nemoto A, Yabe T, Ushida T, Miyamoto H, Tateishi T. (1997). The role of sialoproteins in recognition of bone surface by osteoblasts via integrins. Mater Sci Eng 4, 303309. Ueyama H, Kumamoto T, Fujimoto S, Murakami T, Tsuda T. (1998). Expression of three calpain isoform genes in human skeletal muscle. J Neurol Sci 155, 163169. Uray NJ, Gona AG, Sexton PS. (1998). Calbindin immunoreactivity in Purkinje cells of the bullfrog cerebellum during thyroxin-induced metamorphosis. Brain Behaviour Evolution 51, 284290. Usul A, Kato K, Sasa H, Minaguchi K, Abe T, Murase M, Tanaka M, Takeuchi E. (1990). S100A0 protein in serum during acute myocardial infarction. Clin Chem 36, 639641. Uzawa M, Grams J, Madden B, Toft D, Salisbury JL. (1995). Identication of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation. Dev Biol 171, 5159. Vaamonde CA, Mayers P, Darvish M, Thiery G, Bergeron M. (1996). Protection against gentamicin nephrotoxicity in the streptozotocin-induced diabetic rat is related to a loss of cationic drug membrane receptor gp330 (megalin). J Am Soc Nephrol 7, p.A3006. Vaghy PL, Fang J, Wu WR, Vaghy LP. (1998). Increased caveolin-3 levels in mdx mouse muscles. FEBS Lett 431, 125127. Vakkala M, Lahteenmaki K, Raunio H, Paakko P, Soini Y. (1999b). Apoptosis during breast carcinoma progression. Clin Cancer Res 5, 319324. Vakkala M, Paakko P, Soini Y. (1999a). Expression of caspases 3, 6, and 8 is increased in parallel with apoptosis and histological aggressiveness of the breast lesion. Br J Cancer 81, 592599. Valente AJ, Abramov MA, Peasrson DW, Clark RA. (1996). Identication of a retinoblastoma control element in the promoter of human calreticulin. Mol Biol Cell 7, 3071.
References
339
Vallen EA, Ho W, Winey M, Rose MD. (1994). Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organising centre in Saccharomyces cerevisiae. Genetics 137, 407422. Van Aelst L, DSouza-Schory C. (1997). Rho GTPases and signalling networks. Genes Devel 11, 22952322. Van Antwerp DJ, Verma IM. (1996). Signal induced degradation of I kappa B alpha. Association with NF-kappa B and the PEST sequence in I kappa B alpha are not required. Mol Cell Biol 16, 60376045. Van Asselt EJ, Dijkstra BW. (1999). Binding of calcium in the EF-hand of Escherichia coli lytic transglycosylase Slt35 is important for stability. FEBS Lett 458, 429435. Van Bergen en Henegouwen PMP, Deze LHK, Dekroon J, Van Damme H, Verkleij AJ, Boonstra J. (1989). Ligand induced association of epidermal growth factor receptor to cytoskeleton of A431 cells. J Cell Biochem 39, 455465. Van Compernolle K, Goethals M, Huet C, Louvard D, Van de Kerchkove J. (1992). G-actin to F-actin modulation by a single aminoacid substitution in the actin-binding site of actobindin and thymosin beta 4. EMBO J 11, 47394746. Van de Craen M, De Jonghe C, Van den Brande I, Declercq W, Van Gassen G, Van Criekinge W, Vanderhoven I, Fiers W, Van Broeckhoven C, Hendriks L, Vandenabeele P. (1999). Identication of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases. FEBS Lett 445, 149154. Van de Kerckhove J, Bauw G, Van Compernolle K, Honore B, Celis J. (1990). Comparative 2-dimensional gel analysis and micro-sequencing identied gelsolin as one of the most prominent down-regulated markers of transformed human broblasts and epithelial cells. J Cell Biol 111, 95102. Van de Wetering M, Cavallo R, Dooijes D, Van Beest M, Van Es J, Loureiro J, Ypma A, Hursh D, Jones T, Bejsovec A, Peifer M, Mortin M, Clevers H. (1997). Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88, 788799. Van den Bos C, Roth J, Koch HG, Hartmann M, Sorg C. (1996). Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca2+-dependent translocation from cytoplasm to membranes and cytoskeleton. J Immunol 156, 12471254. Van der Kooi AJ, De Visser M, Van Meegen M, Ginjaar HB, Van Essen AJ, Jennekens FGI, Jongen PJH, Leschot NJ, Bolhuis PA. (1998). A novel -sarcoglycan mutation causing childhood onset, slowly progressive limb girdle muscular dystrophy. Neuromusc Dis 8, 305308. Van der Zee R, Anderton SM, Prakken BJ, Paul AGAL, Van Eden W. (1998). T cell responses to conserved bacterial heat shock protein epitopes induce resistance in experimental autoimmunity. Immunology 10, 3541. Van Ginkel PR, Gee RL, Walker TM, Du DN, Heizmann CW, Polans AS. (1998). The identication and differential expression of calcium binding proteins associated with ocular melanoma. Biochim Biophys Acta 1448, 290298. Van Groningen JJM, Cornelissen IMAH, Van Muijen GNP, Bloemers HPJ, Swart GWM. (1996). Simultaneous suppression of progression marker genes in the highly malignant human melanoma cell line BLM after transfection with the adenovirus-5 E1A gene. Biochem Biophys Res Commun 225, 808816. Van Leeuwen JEM, Kearse KP. (1996). The related molecular chaperones calnexin and calreticulin differentially associate with nascent T-cell antigen receptor proteins within the endoplasmic reticulum. J Biol Chem 271, 2534525349.
340
Van Nhieu GT, Ben Zeev A, Sansonetti PJ. (1997). Modulation of bacterial entry into epithelial cells by association between vinculin and the Shigella IipaA invasion. EMBO J 16, 27172729. Van Roy F, Vleminckx K, Vakaet L Jr, Berx G, Fiers W, Mareel M. (1992). The invasion suppressor role of E-cadherin. Contribution Oncol 44, 108126. Van Troys M, Dewite D, Goethals M, Carlier MF, Van de Kerckchove J, Ampe C. (1996). The actin-binding site of thymosin beta-4 mapped by mutational analysis. EMBO J 15, 201210. Van Troys M, Dewitte D, Verschelde JL, Goethals M, Van de Kerckhove J, Ampe C. (1997). Analogous F-actin binding by colin and gelsolin segment 2 substantiates their structural relationship. J Biol Chem 272, 3275032758. Van Troys M, Van de Kerckhove J, Ampe C. (1999). Structural modules in actin-binding proteins. Towards a new classication. Biochim Biophys Acta 1448, 323348. Vanags DM, Porn-Ares MI, Coppola S, Burgess DH, Orrenius S. (1996). Protease involvement in fodrin cleavage and phosphatidyl serine exposure in apoptosis. J Biol Chem 271, 3107531085. Vandolah FM, Ramsdell JS. (1996). Maitotoxin, a calcium-channel activator, inhibits cell cycle progression through the G1/S and G2/M transitions and prevents CDC2 kinase activation in GH(4)C(1) cells. J Cell Physiol 166, 4956. Varadi G, Lory P, Schultz D, Varadi M, Schwartz A. (1991). Acceleration of activation and inactivation by the subunit of the skeletal muscle calcium channel. Nature 352, 159162. Varga F, Rumpler M, Luegmayr E, Fratzl-Zelman N, Glantschnig H, Klaushofer K. (1997). Triiodothyronine, a regulator of osteoblastic differentiation. Depression of histone H4, attenuation of c-fos/c-jun, and induction of osteocalcin expression. Calcied Tissue Int 61, 404411. Velazquez E, Wieczorek R, Tan J, de Davilla MTG, Theise ND, Greco MA. (1998). Villin expression in foetal liver, hepatoblastomas and hepatocellular carcinomas. Modern Pathol 11, p. A157. Venkatesh K, Hasan G. (1997). Disruption of IP3 receptor gene of Drosophila affects larval metamorphosis and ecdysone release. Curr Biol 7, 500509. Verghese-Nikolakaki S, Apostolikas N, Livaniou E, Ithakissios DS, Evangelatos GP. (1996). Preliminary ndings on the expression of thymosin beta-10 in human breast cancer. Br J Cancer 74, 14411444. Verma AK, Paszty K, Filoteo AG, Penniston JT, Enyedi A. (1999). Protein kinase C phosphorylates plasma membrane Ca2+ pump isoform 4a at its calmodulin-binding domain. J Biol Chem 274, 527531. Vial E, Castellazzi M. (2000). Down-regulation of the extracellular matrix protein SPARC in v-src and v-jun transformed chick embryo broblasts contributes to tumour formation in vivo. Oncogene 19, 17721782. Vier J, Linsinger G, Hacker G. (1999). Cytochrome c is dispensable for Fas-induced caspase activation and apoptosis. Biochem Biophys Res Commun 261, 7178. Vincent C, Simon M, Sebbag M, Girbal-Neuhauser E, Durieux JJ, Cantagrel A, Fournie B, Mazieres B, Serre G. (1998). Immunoblotting detection of autoantibodies to human epidermis laggrin. A new diagnostic test for rheumatoid arthritis. J Rheumatol 25, 838846. Vishwanatha JK, Chiang YP, Kumbl DK, Hollingsworth MA. (1993). Enhanced expression of annexin II in human pancreatic carcinoma cells and primary pancreatic cancer. Carcinogenesis 14, 25752579.
References
341
Vito P, Ghayur T, DAdamio L. (1997). Generation of anti-apoptotic presenilin-2 polypeptides by alternative transcription, proteolysis, and caspase-3 cleavage. J Biol Chem 272, 2831528320. Vogt TMM, Welsh J, Stolz W, Kullmann F, Jung B, Landthaler M, McClelland M. (1997). RNA nger printing displays UVB-specic disruption of transcriptional control in human melanocytes. Cancer Res 57, 35543561. Volm M, Koomagi R. (2000). Prognostic relevance of c-myc and caspase-3 for patients with non-small cell lung cancer. Oncol Reports 7, 9598. Von den Driesch P, Modschiedler K, Vorum H, Celis JE. (1998). Psoriasin enhances neutrophil adhesion to epidermal cells without affecting CD11B/CD18 expression. J Invest Dermatol 110, 648. Vorum H, Liu XD, Madsen P, Rasmussen HH, Honore B. (1998). Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity. Biochim Biophys Acta 1386, 121131. Vorum H, Madsen P, Rasmussen HH, Etzerodt M, Svendsen I, Celis JE, Honore B. (1996). Expression of divalent cation binding properties of the novel chemotactic inammatory protein psoriasin. Electrophoresis 17, 17871796. Vouyiouklis DA, Brophy PJ. (1997). A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system. J Neurochem 69, 9951005. Wachsman JT. (1997). DNA methylation and the association between genetic and epigenetic changes. Relation to carcinogenesis. Mutation Res 375, 18. Wada I, Kai M, Imai S, Sakane F, Kanoh H. (1996). Translocation of diacylglycerol kinase alpha to the nuclear matrix of rat thymocytes and peripheral T-lymphocytes. FEBS Lett 393, 4852. Wagey R, Krieger C, Shaw CA. (1997). Abnormal dephosphorylation effect on NMDA receptor regulation in ALS spinal cord. Neurobiol Dis 4, 350355. Wainfan E, Poirier LA. (1992). Methyl groups in carcinogenesis. Effects of DNA methylation and gene expression. Human Genetic 52, 2071s2077s. Walensky LD, Snyder SH. (1995). Inositol 1,4,5-trisphosphate receptors selectively localised to the acrosome of mammalian sperm. J Cell Biol 130, 857869. Walisser JA, Thies RL. (1999). Poly (ADP-ribose) polymerase inhibition in oxidant-stressed endothelial cells prevents oncosis and permits caspase activation and apoptosis. Exp Cell Res 251, 401413. Walsh MP, Horowitz A, Clement-Chomienne O, Andrea JE, Allen BG, Morgan KG. (1996). Protein kinase C mediation of Ca2+-indepenent contraction of vascular smooth muscle. Biochem Cell Biol 74, 485502. Walter J, Schindzielorz A, Grunberg J, Haass C. (1999). Phosphorylation of presenilin-2 requires its cleavage by caspases and retards progression of apoptosis. Proc Natl Acad Sci USA 96, 13911396. Wang CC, Chan DC, Leder P. (1997). The mouse formin (Fmn) gene: Genomic structure, novel exons, and genetic mapping. Genomics 39, 303311. Wang FS, Wolenski JS, Cheney RE, Mooseker MS, Jay DG. (1996). Function of myosin V in lopodial extension of neuronal growth cones. Science 273, 660663. Wang K, Knipfer M, Huang QQ, Van Heerden A, Hsu LCL, Guttierez G, Quian XL, Stedman H. (1996). Human skeletal nebulin sequence encodes a blueprint for thin lament architecture. Sequence motifs and afnity proles of tandem repeats and terminal SH3. J Biol Chem 271, 43044314. Wang K, McCarter R, Wright J, Beverly J, Mitchell R. (1991). Regulation of skeletal muscle stiffness and elasticity by titin isoforms. A test of the segmental extension model of resting tension. Proc Natl Acad Sci USA 88, 71017105.
342
Wang KL, Roufogalis BD. (1999). Ca2+/calmodulin stimulates GTP binding to the ras-related protein Ral-A. J Biol Chem 274, 1452514528. Wang MG, Yi H, Guerini D, Klee CB, McBride OW. (1996). Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21->q22 and 2p16->p15 respectively. Cytogen Cell Genet 72, 236241. Wang NP, Wan BC, Skelly M, Frid MG, Glukhova MA, Koteliansky VE, Gown AM. (1997). Antibodies to novel myoepithelium-associated proteins distinguish benign lesions and carcinomas in situ from invasive carcinoma of the breast. Appl Immunochem 5, 141151. Wang S, Moerman EJ, Jones RA, Thweatt R, Goldstein S. (1996). Characterisation of IGFBP3, PAI-1 and SPARC mRNA expression in senescent broblasts. Mech Ageing Devel 92, 121132. Wang YL. (1985). Exchange of actin subunits at the leading edge of living broblasts. Possible role of treadmilling. J Cell Biol 101, 597602. Wang YZ, Christakos S. (1995). Retinoic acid regulates the expression of the calcium-binding protein, calbindin D28K. J Endocrinol 9, 15101521. Warren RH, Gordon E, Azarnia R. (1985). Tropomyosin in peripheral rufes of cultured rat kidney cells. Eur J Cell Biol 38, 245253. Waser M, Masaeli N, Spencer C, Michalak M. (1997). Regulation of calreticulin gene expression by calcium. J Cell Biol 138, 547557. Wasilenko WJ, Cooper J, Palad AJ, Somers KD, Blackmore PF, Rhim JS, Wright GL, Schellhammer PF. (1997). Calcium signalling in prostate cancer cells: Evidence for multiple receptors and enhanced sensitivity to bombesin/GRP. Prostate 30, 167173. Wasilenko WJ, Palad AJ, Somers KD, Blackmore PF, Kohn EC, Rhim JS, Wright GL, Schellhammer PF. (1996). Effects of calcium inux inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumour cells. Int J Cancer 68, 259264. Wasserman RH, Taylor AN. (1966). Vitamin D3-induced calcium binding protein in chick intestinal mucosa. Science 152, 791793. Watanabe H, Imaizumi M, Ojika T, Abe T, Hida T, Kato K. (1994). Evaluation of biological characteristics of lung cancer by the human 28 kDa vitamin D-dependent calcium binding protein calbindin D28K. Japan J Clin Oncol 24, 121127. Watanabe N, Madaule P, Reid T, Ishizaki T, Watanabe G, Kakizuka A, Saitao Y, Nakao K, Jockusch BM, Narumiya S. (1997). P140mDia, a mammalian homologue of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for prolin. EMBO J 16, 30443056. Watanabe Y, Usuda N, Minami H, Morita T, Tsugane S, Ishikawa R, Kohama K, Yomida Y, Hidaka H. (1993). Calvasculin, a factor affecting the microlament assemblies in rat broblasts transfected by src gene. FEBS Lett 324, 5155. Watanabe Y, Usuda N, Tsugane S, Kobayashi R, Hidaka H. (1992). Calvasculin, an encoded protein from messenger RNA termed pEL-98, 18A2, 42A or p9Ka, is secreted by smooth muscle cells in culture and exhibits Ca2+-dependent binding to 36 kDa microbrilassociated glycoprotein. J Biol Chem 267, 1713617140. Watson N, Linder ME, Druey KM, Kehri JH, Blumer KJ. (1996). RGS family members. GTPase-activating proteins for heterotrimeric G-protein alpha subunits. Nature 383, 172175. Watson PH, Leygue ER, Murphy LC. (1998). Psoriasin (S100A7). Int J Biochem Cell Biol 30, 567571. Webber MM, Waghray A, Bello D. (1995). Prostate specic antigen, a serine protease, facilitates human prostate cancer cell invasion. Clin Cancer Res 1, 10891094.
References
343
Weber G, Shen F, Prajda N, Yeh YA, Yang HY, Herenyiova M, Look KY. (1996). Increased signal transduction activity and down-regulation in human cancer cells. Anticancer Res 16, 32713281. Weichmann AF. (1996). Recoverin in cultured human retinoblastoma cells. Enhanced expression during morphological differentiation. J Neurochem 67, 105110. Weis K, Grifths G, Lamond AI. (1994). The endoplasmic reticulum calcium binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl terminal his-asp-glu-leu motif. J Biol Chem 269, 1914219150. Weisenhorn DMV, Weruagaprieto E, Celio MR. (1996). Calretinin immunoreactivity in organotypic cultures of the rat cerebral cortex. Effects of serum deprivation. Exp Brain Res 108, 101112. Well M, Jacobson MD, Raff MC. (1998). Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes. J Cell Sci 111, 27072715. Welsch CF, Zhu D, Bourguignon LYW. (1995). Interaction of CD44 variant isoforms with hyaluronic acid and the cytoskeleton in human prostate cells. J Cell Physiol 164, 605612. Weng LP, Smith WM, Dahia PLM, Ziebold U, Gil E, Lees JA, Eng C. (1999). PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death. Cancer Res 59, 58085814. Weterman MAJ, Stoopen GM, Van Muijen GNP, Kuznicki J, Ruiter DJ. (1992). Expression of calcyclin in human melanoma cell lines correlates with metastatic behaviour in nude mice. Cancer Res 52, 12911296. Weterman MAJ, Van Muijen GNP, Bloemers HPJ, Ruiter DJ. (1993). Expression of calcyclin in human melanocytic lesions. Cancer Res 53, 60616066. Whitaker M, Patel R. (1990). Calcium and cell cycle control. Dev 108, 525542. White TK, Zhu, Tanzer ML. (1995). Cell surface calreticulin as a putative mannoside lectin which triggers mouse melanoma cell spreading. J Biol Chem 270, 1592615929. Wicki R, Franz C, Scholl FA, Heizmann CW, Schafer BW. (1997). Repression of the candidate tumour suppressor gene S100A2 in breast cancer is mediated by site-specic hypermethylation. Cell Calcium 22, 243254. Wicki R, Franz C, Scholl FA, Heizmann CW, Schafer BW. (1997). Repression of the candidate tumour suppressor gene S100A2 in breast cancer is mediated by site specic hypomethylation. Cell Calcium 22, 243254. Wicki R, Marenholz I, Mischke D, Schafer BW, Heizmann CW. (1996a). Characterisation of the human S100A12 (calgranulin C, p6, CAAF1, CGRP) gene, a new member of the S100 gene cluster on chromosome 1q21. Cell Calcium 20, 459464. Wicki R, Schafter BW, Erne P, Heizmann CW. (1996b). Characterisation of the human and mouse cDNAs coding for S100A13, a new member of the S100 protein family. Biochem Biophys Res Commun 227, 594599. Willams JP, Jo H, Sacks DB, Crimmings DL, Thoma RS, Hunnicutt RE, Radding W, Sharma RK, McDonald JM. (1994). Tyrosine-phosphorylated calmodulin has reduced biological activity. Arch Biochem Biophys 315, 119126. Willis FL, McCubbin WD, Kay CM. (1994). Smooth muscle calponin-caltropin interaction. Effect on biological activity and stability of calponin. Biochemistry 33, 55625569. Wineld JB. (1989). Stress proteins, arthritis, and autoimmunity. Arthritis Rheumatism 32, 14971504. Winnard RG, Gerstenfeld LC, Toma CD, Franceschi RT. (1995). Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts. J Bone Mineral Res 10, 19691977.
344
Witke W, Podtelejnikov AV, Di Nardo A, Sutherland JD, Gurniak CB, Dotti C, Mann M. (1998). In mouse brain prolin I and prolin II associate with regulators of the endocytic pathway and actin assembly. EMBO J 17, 967976. Witke W, Sharpe AH, Hartiwig JH, Azuma T, Stossel TP, Kwiatkowski DJ. (1995). Haemostatic, inammatory, and broblast responses are blunted in mice lacking gelsolin. Cell 81, 4151. Wnuk W. (1988). Calcium binding response to troponin C and the regulation of muscle contraction. A comparative approach. In Calcium and calcium binding proteins. Molecular and functional aspects, eds. Gerday C, Bolis L, Gilles R, Springer Verlag, Berlin, pp. 4468. Wojcik C, Stoklosa T, Giermasz A, Golab J, Zagozdzon R, Kawiak J, Wilk S, Komar A, Kaca A, Malejczyk J, Jakobisiak M. (1997). Apoptosis induced in L1210 leukaemia cells by an inhibitor of chymotrypsin-like activity of the proteasome. Apoptosis 2, 455462. Wolf G, Ziyadeh FN, Stahl RAK. (1999). Angiotensin II stimulates expression of transforming growth factor beta receptor type II in cultures mouse proximal tubular cells. J Mol Med 77, 556564. Wolfe SE, Morris SJ. (1999). Dopamine D-2 receptor isoforms expressed AtT20 cells differentially couple to G proteins to acutely inhibit high voltage-activated calcium channels. J Neurochem 73, 23752382. Wolfrum U. (1995). Centrin in the photoreceptor cells of mammalian retinae. Cell Motility Cytoskeleton 32, 5564. Wong HN, Ward MA, Bell AW, Chevet E, Bains S, Blackstock WP, Solari R, Thomas DY, Bergeron JJM. (1998). Conversed in vivo phosphorylation of calnexin at casein kinase II sites as well as protein kinase C-directed kinase site. J Biol Chem 273, 1722717235. Wood WR, Seftor EA, Lotan D, Nakajima M, Misiorowski RM, Seftor REB, Lotan R, Hendrix MJC. (1990). Retinoic acid inhibits human melanoma tumour cell invasion. Anticancer Res 10, 423432. Wrana JL, Overall CM, Sodek J. (1991). Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human broblasts by transforming growth factor beta. Comparison of transcriptional and post-transcriptional control with bronectin and type I collagen. Eur J Biochem 197, 519528. Wu XF, Kocher B, Wei Q, Hammer JA. (1998). Myosin Va associates with microtubule-rich domains in both interphase and dividing cells. Cell Motility Cytoskeleton 40, 286303. Xanthoudakis S, Roy S, Rasper D, Hennessey T, Aubin Y, Cassady R, Tawa P, Ruel R, Rosen A, Nicholson DW. (1999). HSP60 accelerated the maturation of pro-caspase 3 by upstream activator proteinases during apoptosis. EMBO J 18, 20492056. Xia LQ, Stoll SW, Liebert M, Ethier SP, Carey T, Esclamado R, Carroll W, Johnson TM, Elder JT. (1997). CaN19 expression in benign and malignant hyperplasias of the skin and oral mucosa. Evidence for a role in regenerative differentiation. Cancer Res 57, 30553062. Xiang J, Lahti JM, Grenet J, Easton J, Kidd VJ. (1994). Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms. J Biol Chem 269, 1578615794. Xiao GZ, Wang D, Benson MD, Karsenty G, Franceschi RT. (1998). Role of the alpha 2integrin in osteoblast specic gene expression and activation of the osf2 transcription factor. J Biol Chem 273, 32998832994. Xie HQ, Johnson GVW. (1998). Calcineurin inhibition prevents calpain-mediated proteolysis of tau in differentiated PC12 cells. J Neurosci Res 53, 153164.
References
345
Xie RL, Long GL. (1995). Role of N-linked glycosylation in human osteonectin. Effect of carbohydrate removal by N-glycanase and site directed mutagenesis of structure and binding of type V collagen. J Biol Chem 270, 2321223217. Xie RL, Long GL. (1996). Elements within the rst 17 aminoacids of human osteonectin are responsible for binding to type V collagen. J Biol Chem 271, 81218125. Xuan JW, Hota C, Shiegeyama Y, DErricod JA, Somerman MJ, Chambers AF. (1995). Sitedirected mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration. J Cell Biochem 57, 680690. Yabe D, Nakamura T, Kanazawa N, Tashiro K, Honjo T. (1997). Calumenin, a Ca2+-binding protein retained in the endoplasmic reticulum with a novel carboy-terminal sequence HDEF. J Biol Chem 272, 1823218239. Yam XX, Wiechmann AF. (1997). Early expression of recoverin in a unique population of neurones in the human retinal. Anat Embryol 195, 5163. Yamada K, Sakane F, Matsushima N, Kanoh H. (1997). EF-hand motif of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different afnities and conformational changes. Biochem J 321, 5964. Yamaguchi M, Isogai M, Shimada N. (1997). Potential sensitivity of hepatic specic protein regucalcin as a marker of chronic liver injury. Mol Cell Biochem 167, 187190. Yamaguchi M, Kanayama Y. (1996). Calcium binding protein regucalcin inhibits deoxyribonucleic acid synthesis in the nuclei of regenerating rat liver. Mol Cell Biochem 162, 121126. Yamaguchi M, Makino R, Shimokawa N. (1996). The 5 end sequences and exon organisation in rat regucalcin gene. Mol Cell Biochem 165, 145150. Yamaguchi M. (1998). Role of calcium binding protein regucalcin in regenerating rat liver. J Gastroenterol Hepatol 13, S106S112. Yamaguchi R, Maki M, Hatanaka M, Sabe H. (1994). Unphosphorylated and tyrosine phosphorylated forms of a focal adhesion protein, paxillin, are substrates for calpain II in vitro. Implications for the possible involvement of calpain II in mitosis-specic degradation of paxillin. FEBS Lett 356, 114116. Yamakita Y, Yamashiro S, Matsumura F. (1996). Phosphorylation of myosin light chain during serum stimulation. Mol Biol Cell 7, 1146. Yamamoto S, Nakamura M. (1996). Calnexin. Its molecular cloning and expression in the liver of the frog, Rana rugosa. FEBS Lett 387, 2732. Yamamura T, Hitomi J, Nagasaki K, Suzuki M, Takahashi E, Saito S, Tsukada T, Yamaguchi K. (1996). Human CAAF1gene. Molecular cloning, gene structure, and chromosome mapping. Biochem Biophys Res Commun 221, 356360. Yamashita N, Kosaka K, Ilg EC, Schafer BW, Heizmann CW, Kosaka T. (1997). Selective association of S100A6 (calcyclin)-immunoreactive astrocytes with the tangential migration pathway of subventricular zone cells in the rat. Brain Res 778, 388392. Yamazaki M, Ishodoh K, Suga Y, Saido T, Kawashima S, Suzuki K, Kominami E, Ogawa H. (1997). Cytoplasmic processing of human prolaggrin by active -calpain. Biochem Biophys Res Commun 235, 652656. Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, Nishida E, Mizuno K. (1998). Colin phosphorylation by LIM-1 kinase and its role in rac-mediated actin reorganisation. Nature 393, 809812. Yan XX, Wiechmann AF. (1997). Early expression of recoverin in a unique population of neurones in the human retina. Anatomy Embryol 195, 5163. Yang TX, Terada Y, Nonoguchi H, Tomita K, Marumo F. (1995). Expression of messenger RNAs for different IP3 receptors in rat kidneys. Amer J Physiol 37, F1046F1052.
346
Yap AS, Niessen CM, Gumbiner BM. (1998). The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p129(ctn). J Cell Biol 141, 779789. Ye LH, Hayakawa K, Kishi H, Imamura M, Nakamura A, Okagaki T, Takagi T, Iwata A, Tanaka T, Kohama K. (1997). The structure and function of the actin-binding domain of myosin light chain kinase of smooth muscle. J Biol Chem 272, 3218232189. Yeh KT, Chang JG, Chen YJ, Chen ST, Yu SY, Shih MC, Perng LI, Wang JC, Tsai M, Chang CP. (2000). Mutation analysis of the putative tumour suppressor gene PTEN/MMAC1 in hepatocellular carcinoma. Cancer Investigation 18, 123139. Yew DT, Luo DB, Heizmann CW, Chan WY. (1997). Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum. Dev Brain Res 103, 3745. Yiyu F, Venema VJ, Venema RC, Tsai N, Ali Behzadian M, Caldwell RB. (1999). VEGFinduced permeability increase is mediated by caveolae. Invest Ophthalmol Visual Sci 40, 157167. Yoon GS, Lee H, Jung Y, Yu E, Moon HB, Song K, Lee I. (2000). Nuclear matrix of calreticulin in hepatocellular carcinomas. Cancer Res 60, 11171120. Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R, Penninger JM, Mak TW. (1998). Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739750. Yost JC, Sage EH. (1993). Specic interaction of SPARC with endothelial cells is mediated through a carboxyl terminal sequence containing a calcium binding EF-hand. J Biol Chem 268, 2579025796. Young LH, Yang XQ, Voigt JM. (1996). Alteration of gene expression in rat mammary tumours induced by N-methyl-N-nitrosourea. Mol Carcinogenesis 15, 251260. Young P, Ferguson C, Banuelos S, Gautel M. (1998). Molecular structure of the sarcomeric Z-disk. Two types of titin interactions lead to an asymmetrical sorting of alpha-actinin. EMBO J 17, 16141624. Youyiouklis DA, Brophy PJ. (1997). A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system. J Neurochem 69, 9951005. Yu Y, Little JB. (1998). P53 is involved but not required for ionising radiation-induced caspase activation and apoptosis in human lymphoblast cell lines. Cancer Res 58, 42774281. Zacharias DA, Kappen C. (1999). Developmental expression of the four plasma membrane calcium ATPase (PMCA) genes in the mouse. Biochim Biophys Acta 1428, 397405. Zauli G, Visani G, Bassini A, Caramelli E, Ottaviani E, Bertolaso L, Bertagnolo V, Borgatti P, Capitani S. (1996). Nuclear translocation of protein kinase C and protein kinase C isoforms in HL-60 induced to differentiate along granulocytic lineage by all-trans retinoic acid. Br J Haematol 93, 542550. Zeitler H, Ko Y, Glodny B, Totzke G, Oppenheimer M, Sachinidis A, Vetter H. (1997). Cell cycle arrest in G0/G1 phase of growth factor induced endothelial cell proliferation by various calcium channel blockers. Cancer Detection Prevention 21, 332339. Zeller R, Haramis AG, Zuniga A, McGuigan C, Dono R, Davidson G, Chabanis S. (1999). Formin denes a large family of morphoregulatory genes and functions in establishment of the polarising region. Cell Tissue Res 296, 8593. Zhang HT, Craft P, Scott Pae, Ziche M, Weich HA, Harris AL, Bicknell R. (1995). Enhancement of tumour growth and vascular density by transfection of vascular endothelial growth factor into MCF7 human breast carcinoma cells. J Natl Cancer Inst 87, 213219.
References
347
Zhang JY, Zhang SB, Murtha PE, Zhu W, Hou SSM, Young CYF. (1997). Identication of two novel cis-elements in the promoter of the prostate specic antigen gene that are required to enhance androgen receptor mediated activation. Nucleic Acids Res 25, 31433150. Zhang L, Kelley J, Schmeisser G, Kobayashi YM, Jones LR. (1997). Complex formation between junctin, triadin, calsequestrin and ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem 272, 2338923397. Zhang M, Tanaka T, Ikura M. (1995). Calcium induced conformational transition revealed by the solution structure of apo-calmodulin. Nat Structural Biol 2, 758767. Zhang MJ, Vogel JH. (1997). Interaction of a partial calmodulin binding domain of caldesmon with calmodulin. Prot Peptide Lett 4, 291297. Zhang P, Steinberg BM. (2000). Overexpression of PTEN/MMAC1 and decreased activation of Akt in human papilloma virus-infected laryngeal papillomas. Cancer Res 60, 14571462. Zhang PJ, Harris KR, Brooks JJ. (1997). Villin, a marker for GI neuroendocrine tumours (NET). Lab Invest 76, p.1015. Zhang Q, Salter RD. (1998). Distinct patterns of folding and interactions with calnexin and calreticulin in human class I MHC proteins with altered N-glycosylation. J Immunol 160, 831837. Zhang YK, Fujita N, Tsuruo T. (1999). Caspase-mediated cleavage of p21 (waf1/cip1) converts cancer cells from growth arrest to undergoing apoptosis. Oncogene 18, 11311138. Zhao XQ, Naka M, Muneyuki M, Tanaka T. (2000). Ca2+-dependent inhibition of actinactivated myosin ATPase activity by S100C (S100A11), a novel member of the S100 protein family. Biochem Biophys Res Commun 267, 7779. Zheng G, Marino M, Zhao J, McCluskey RT. (1998). Megalin (gp330). A putative endocytic receptor for thyroglobulin (Tg). Endocrinology 139, 14621465. Zheng JP, Rudra-Ganguly N, Miller GJ, Moffatt KA, Cote RJ, Roy-Burman P. (1997). Steroid hormone induction and expression patterns of L-plastin in normal and carcinomatous prostate tissues. Amer J Pathol 150, 20092018. Zheng TS, Schosser SF, Dao T, Hingorani R, Crispe IN, Boyer JI, Flavell RA. (1998). Caspase3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo. Proc Natl Acad Sci USA 95, 1361813623. Zhivotovsky B, Burgess DH, Vanags DM, Orrenius S. (1997). Involvement of cellular proteolytic machinery in apoptosis. Biochem Biophys Res Commun 230, 481488. Zhou M, Shi Y, Alsediary S, Faird NR. (1993). High levels of nm23 gene expression in advanced stage of thyroid carcinoma. Br J Cancer 68, 385388. Zhu JK, Hasegawa PM, Bressan RA, Niu XM. (1996). Multiple transcripts of a calcium binding protein gene from Atriplex nummularia are differentially regulated by developmental and environmental stimuli. Physiol Plantarum 97, 499506. Zhu N, Pewitt EB, Cai XY, Cohn EB, Lang S, Chen R, Wang Z. (1998). Calreticulin. An intracellular Ca2+-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells. Endocrinology 139, 43374344. Zhu N, Wang Z. (1999). Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells. Cancer Res 59, 18961902. Zhu W, Murtha PE, Young CY. (1995). Calpain inhibitor-induced apoptosis in human prostate adenocarcinoma cells. Biochem Biophys Res Commun 214, 11301137. Zhuang SB, Mani RS, Kay CM, Wang CLA. (1995). Interaction between caltropin and the C-terminal region of smooth muscle caldesmon. Biochem Biophys Res Commun 209, 1217.
348
Zigmond SH. (1993). Recent quantitative studies of actin lament turnover during cell locomotion. Cell Motility Cytoskeleton 25, 309316. Zimmer DB, Chessher J, Wilson GL, Zimmer WE. (1997). S100A1 and S100B expression and target proteins in type 1 diabetes. Endocrinology 138, 51765183. Zimmer DB, Cornwall EH, Landar A, Song W. (1995). The S100 protein family. History, function and expression. Brain Res Bull 37, 417429. Zimmer DB, Cornwall EH, Reynolds PD, Donald CM. (1998). S100A1 regulates neurite organisation, tubulin levels, and proliferation in PC12 cells. J Biol Chem 273, 47054711. Zimmer DB, Landar A. (1995). Analysis of S100A1 expression during skeletal muscle and neuronal cell differentiation. J Neurochem 64, 27272736. Zine A, Hadi A, Romand R. (1995). Fimbrin expression in the developing rat cochlea. Hearing Res 87, 165169. Zingg JM, Jones PA. (1997). Genetic and epigenetic aspects of DNA methylation and carcinogenesis. Carcinogenesis 18, 869882. Zou H, Henzel W, Liu X, Lutschg A, Wang X. (1997). Apaf-1, a human protein homologous to C. elegans, participates in cytochrome c-dependent activation of caspase 3. Cell 90, 405413. Zu Y, Kohno M, Kubota I, Nishida E, Hanaoka M, Namba Y. (1990). Characterisation of interleukin-2 stimulated 65-kDa phosphoprotein in human T-cells. Biochemistry 29, 10551062. Zucker RS, Steinhardt RA. (1978). Prevention of the cortical reaction in fertilised sea urchin eggs by injection of calcium chelating ligands. Biochim Biophys Acta 541, 459466. Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H. (1999). Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399, 159162.
Index
A
ABP, see Amyloid -protein ACTH, see Adrenocorticotropic hormone -actinin, 51, 103 Actionmyosin interaction, 117 Actomyosin assembly, 112 ACV, see Acyclovir Acyclovir (ACV), 61, 62 Adenomatous polyposis coli (APC) protein, 104 Adenylyl cyclase, 22 Adhesion cellsubstratum, 58 intracellular, 58 -mediating proteins, 59, 94 Adrenal cortex, osteonection immunoreactivity in invasive tumours of, 193 Adrenocorticotropic hormone (ACTH), 24 Alzheimers disease calcineurin in, 136, 140 calretinin and, 133 caspases and neuronal loss in, 177 CBD-immunoreactive neurones in, 129 Amyloidosis, gelsolin expression in, 42 Amyloid precursor protein (APP), 178, 179 -Amyloid precursor protein (APP), 141 Amyloid -protein (ABP), 141 Androgen, 241 Androgen receptors (ARs), 101 Angina pectoris, 222 Angiotensin signals, 76 Annexin(s), 35 II, 208 cell cycle-related expression of, 39 function, PKC-mediated regulation of, 36 in human foreskin broblasts, 37 interaction between S100A11 and, 240 Antibodies, anti-keratin, 204 Antichymotrypsin, 242 Anti-keratin antibodies, 204 APC protein, see Adenomatous polyposis coli protein Apoptosis calpains in, 158 caspase-mediated, 173 dexamethasone-induced, 18 PARP as marker of, 172 APP, see Amyloid precursor protein APP, see -Amyloid precursor protein Arabidopsis thaliana, 96 Arachidonic acid, binding of, 67 ARs, see Androgen receptors Aspergillus nidulans, 136 Astrocytomas, expression of S100A3 studied in human, 225 Atriplex nummularia, 146 Autoimmune conditions, 53 Autoimmune diseases, 84
B
BAE cells, see Bovine aortic endothelial cells Barretts adenocarcinoma, villin expressed in, 45 Barretts metaplasia, 45, 216 Basement membrane (BM), 183 Basic broblast growth factor (bFGF), 76 BDNF, see Brain-derived neurotropic factor Becker muscular dystrophy (BMD), 165 Benign prostatic hyperplasia (BPH), 100 bFGF, see Basic broblast growth factor Bifonazole, 78 4,5-Bisphosphate, 10 Bladder, squamous cell carcinoma of, 238 BM, see Basement membrane BMD, see Becker muscular dystrophy Bombesin, 19 Bone disease, metastatic, 60 metabolism, osteocalcin in, 54 scans, 61 Bovine aortic endothelial (BAE) cells, 189 349
350 BPH, see Benign prostatic hyperplasia Bradykinin, 19 Brain -derived neurotropic factor (BDNF), 125 osteonection immunoreactivity in invasive tumours of, 193 Breast cancer(s), 70, 228 aggressiveness of, 243 ER-negative, 193 ER/PgR-negative, 232 ibandronate treatment of metastatic, 61 prognosis, marker for predicting, 233 carcinomas, NDP kinase expression in, 231 ductal carcinoma of, 98 epithelium, neoplastic transformation of, 111 osteonection immunoreactivity in invasive tumours of, 193 Butyrate, 127
Calcium Signalling in Cancer Calcimedin, 31 Calcineurin, in cell proliferation, cell adhesion, and cell spreading, 135144 calcineurin in Alzheimers disease, 140141 calcineurin in cell proliferation and adhesion-related phenomena, 136140 effects of calcineurin on cell adhesion and motility, 138140 putative role of calcineurin in cell cycle progression, 136138 calcineurin in immunosuppression, 141144 molecular features of calcineurin, 135136 Calcium /calmodulin-dependent protein kinases (CAMPKs), 13 capacitative energy of, 10 Calcium-binding proteins (CBPs), 1, 5 cell proliferation, 245 EF-hand, 29 gelsolin, 40 molecular conguration of, 65 natural classication, 2933 neural, 79 neuronal, 131 non-EF-hand, 3031 photoreceptor-specic, 32 posttranslational changes of, 66 S100 family of, 200 Calcium signalling pathway, 528 architectural aspects of signal transduction machinery, 2528 cyclic AMP in calcium signaling, 2125 deregulation of inositol 1,4,5trisphosphate pathway, 1820 homeostatis of cell calcium, 510 deregulation of calcium homeostasis as primary event in carcinogenesis, 910 plasma membrane Ca2+-ATPase pump, 57 sarcoplasmicendoplasmic reticulum Ca2+-ATPase pump, 78 voltaged gated calcium channels, 89 inositol phosphates in calcium signal transduction, 1618
C
Cadherin, 246 catenin complexes, forms of, 212 as marker for serous carcinoma of ovary, 134 Caenorhabditis elegans, 89, 102, 154 Caherin, 107 CAI, see Carboxyamido-triazole Calbindin D-9K (CBD-9K), 125 D-28K (CBD), 125 immunoreactivity, 126 neuroprotective function of, 129 Calbindin, structure and biology of, 125130 calbindin expression in embryonic development and with ageing, 126127 calbindin expression and metastatic phenotype, 129130 calbindin in neuronal populations, 125 neural cell lineage and regulation of calbindin expression, 125126 neuroprotective function of calbindin, 129 physiological function of calbindin, 127128
Index phospholipid signalling, 1011 protein kinase C and isoforms in signal transduction, 1416 protein kinase C pathway, 1314 PTEN phosphatase in regulation of lipid signalling, 1113 ryanodine and related receptors in calcium mobilisation, 2021 Calcyclin, 64, 235 Caldesmon, 121 Calelectrin, 31 Calmodulin (CaM) family, of calcium binding proteins, 7585 calmodulin and physiological function, 7579 calmodulin and cell proliferation, 7778 calmodulin-mediated signal transduction, 7677 calmodulin in neoplasia, 7879 structure and mode of action of calmodulin, 7576 guanylate cyclase-activating proteins, 85 recoverin subfamily of neural calcium binding proteins, 7985 G-protein signalling pathway, 7980 mode of action of recoverin, 82 post-translational modication of recoverin, 8283 recoverin and cancer-associated retinopathy, 8385 recoverin and function, 8082 Calmyrin, 33 Calnexin, 49 Calpains, in normal and aberrant cell physiology, 153167 calpain family of calcium-binding proteins, 153154 calpains in cancer growth and progression, 162163 calpains in cell proliferation and apoptosis, 158160 calpains in cell spreading and migration, 160161 calpains in intergrin-mediated cell adhesion and signal transduction, 161162 calpains in muscular dystrophy, 165167
351 association of calpains with Duchenne muscular dystrophy, 165166 calpains and limb girdle muscular dystrophy, 166167 calpains in myelodegenerative diseases, 163165 involvement of calpains in development and differentiation, 157158 molecular organisation of calpains, 154155 regulation of physiological events by proteolytic function,155157 Calponin calcium-dependent binding to, 209 homology (CH), 96, 122 myosin-ATPase inhibition by, 124 Calreticulin, 30, 35, 51 Calretinin, 32, 33, 131134 alternatively spliced isoforms, 131132 expression in cell proliferation and differentiation, 133 hormonal regulation of, 132 as marker for serous carcinoma of ovary, 134 possible neuroprotective property, 133 as potential tumour marker, 133134 regulation of calretinin expression, 132133 Calsequestrin, 30, 53 Caltropin, 200 Calumenin, 149 CaM, see Calmodulin family, of calcium binding proteins CAMPKs, see Calcium/calmodulindependent protein kinases cAMP response element binding protein (CREB), 23 cAMP response elements (CREs), 25 Cancer, see also specic types -associated retinopathy (CAR), 83 caveolin expression in, 28 gelsolin in, 42 involvement of mbrin in, 100 markers, experimental, 233 prognosis, relationship between EGFr expression and, 232 progression osteonectin and, 196 potential role of thymosins in, 95 relation of actinin to, 108
352 S100A4 expression as marker of, 244 S100P in, 241 Capase-9, cytochrome c-mediated activation of, 170 Capping protein (CP), 97 CAR, see Cancer-associated retinopathy Carboxyamido-triazole (CAI), 9 Caspases, in apoptosis, cell migration, proliferation, and neoplasia, 169179 caspase-mediated apoptosis and cell growth inhibition in tumour expansion, 173176 caspase-mediated proteolysis of fodrin, 176177 caspases in apoptotic cell death, 169172 caspases and neuronal loss in Alzheimers disease, 177179 poly (ADP-ribose) polymerase as marker of apoptosis, 172173 Caveolin, 28, 98 CBD, see Calbindin D-28K CBD-9K, see Calbindin D-9K CBPs, see Calcium-binding proteins Cellsubstratum adhesion, 58 Central nervous system (CNS), 1 Centrins, 145147 cGMP, see Cyclic guanine monophosphate CH, see Calponin homology Chemoattractants, 210 Chinese hamster ovary (CHO) cell line, 161 Chlamydomonas, 145, 146 CHO cell line, see Chinese hamster ovary cell line Clotrimazole, 78 CNS, see Central nervous system Colin, 88 Collagen, binding of osteonectin to, 191 Colon cancer metastatic, 163 model, 43 Colorectal neoplasms, S100A4 expression in, 243 Contractile proteins, structure of, 87124 actin component of contractile machinery of cell, 8793 actin isoforms, 8788
Calcium Signalling in Cancer colin in regulation of actin dynamics, 8890 interaction of formin with prolin and Rhp GTPases, 9293 prolin in regulation of actin dynamics, 90 regulation of actin dynamics, 88 Rho GTPases in actin dynamics and signal transduction, 9092 mbrin family of actin-binding proteins, 96110 -actinin, 102 -actinin isoforms, 103 actinins in cell adhesion, motility, and signal transduction, 104 cadherincatenin complex in signal transduction and cell adhesion, 104110 function of -actinin, 104 function of mbrin in cytoskeletal organisation, 9799 involvement of mbrin in cancer, 100101 modulation of actin dynamics and cancer cell dissemination, 101102 molecular features of mbrin, 9697 molecular structure of -actinin, 102103 regulation of mbrin expression, 99100 myosin laments, 110117 actomyosin assembly, 112115 myosin heavy chain isoforms, 111112 myosin light chain phosphorylation and function, 115117 role of thymosin family actin-binding proteins in actin dynamics, 9396 effects of thymosins on cell proliferation, 9394 expression of thymosins in embryonic development, 9495 potential role of thymosins in cancer progression, 9596 sequestration of actin by thymosins, 93 thymosins and cell motility and differentiation, 94
Index troponins and tropomyosins in regulation of muscle cell contraction, 118124 calponin, 122124 caltropin-mediated reversal of myosinATPase inhibition by caldesmon and calponin, 124 regulatory role of caldesmon, 121122 regulatory role of troponins and tropomyosins in muscle contraction, 118119 tropomyosin isoforms in benign and malignant cells, 119121 Corticosterone, 127 CP, see Capping protein CREB, see cAMP response element binding protein CREs, see cAMP response elements Crocalbin, 149 Cyclic AMP, in calcium signalling, 21 Cyclic guanine monophosphate (cGMP), 16 Cyclosporin, 141 Cytochalasin D, 18, 211 Cytokinesis, 93, 217 Cytosolic fatty acids, 237 Duchenne muscular dystrophy (DMD), 165
353
E
EAE, see Experimental allergic encephalomyelitis EC, see Endothelial cell Ecdysone, 18 ECM, see Extracellular matrix EDC, see Epidermal differentiation complex EFABP, see Epidermal-type fatty acidbinding protein EF-hand calcium binding proteins, 6374 alternatively spliced variants of S100A4, 6869 calcium binding and molecular conguration of calcium binding proteins, 6568 functional signicance of alternatively spliced isoforms, 6970 molecular organisation, 6364 regulation of expression of S100 family genes, 7174 DNA methylation in cancer, 7273 regulation of gene expression by DNA methylation, 72 regulation of S100 gene transcription by methylation, 74 transcriptional regulation of S100 genes, 71 structure and organisation of S100 family genes, 68 EGF, see Epidermal growth factor EGFr, see Epidermal growth factor receptors Embryonic stem (ES) cells, 51 Endoplasmic reticulum, 5, 46 Endothelial cell (EC), 27 Endothelial nitric oxide synthase (eNOS), 26 Enhancer element, 74 eNOS, see Endothelial nitric oxide synthase Enzymes, activation of target, 75 Epidermal differentiation complex (EDC), 202 Epidermal growth factor (EGF), 15, 224
D
DAG, see1,2-Diacylglycerol Dariers disease (DD), 7 DD, see Dariers disease Demyelination process, autoimmunemediated, 164 Dexamethasone, 18, 172 -inducible promoter, 206 S100A4 transfectants exposed to, 226 1,2-Diacylglycerol (DAG), 13, 14, 32 Dictyostelium discoideum, 22, 44, 96, 102 Dimethyl sulphoxide (DMSO), 19 DMD, see Duchenne muscular dystrophy DMSO, see Dimethyl sulphoxide DNA binding of transcription factors to, 67 damage, 72 fragmentation, 170, 174 hypermethylation, 73 methylation, in cancer, 72 Drosophila melanogaster, 42, 102, 150, 231 Drugimmunophilin complex, 141
354 Epidermal growth factor receptors (EGFr), 7677 Epidermal-type fatty acid-binding protein (EFABP), 237 Epithelialmesenchymal transformation, 214, 226 ERK, see Extracellular signal-related receptor kinases ERs, see Oestrogen receptors ES cells, see Embryonic stem cells Experimental allergic encephalomyelitis (EAE), 164 Extracellular matrix (ECM), 186 protein synthesis, 197 remodelling, 214 Extracellular signal-related receptor kinases (ERK), 77 Ezrin, 108
Calcium Signalling in Cancer in cancer, 42 expression, in amyloidosis, 42 Gene regulation, modes of, 71 therapy, with cytotoxic drugs, 62 Genetic activation, signal transduction and, 2 Genistein, 104 GFAP, see Glial brillary acidic protein Ginkgo biloba L., 46 GI tract, osteonection immunoreactivity in invasive tumours of, 193 Glial cell-derived neurotropic factor (GDNF), 132 Glial brillary acidic protein (GFAP), 207 Glutamate, 114, 206 Glycoprotein(s) adhesion mediating, 59 transmembrane, 246 G-protein -coupled receptors, 26 signalling pathway, 79 Growth hormone, 127 GRP, see Gastrin-releasing peptide GTP, see Guanosine triphosphate GTPase, see Guanosine triphosphatase Guanosine diphosphate (GDP), 80 Guanosine triphosphatase (GTPase), 80 Guanosine triphosphatase activating proteins (GAP), 80 Guanosine triphosphate (GTP), 77 Guanylate cyclase, 32, 81, 85
F
FH, see Formin homology Fibroblast(s) annexin in human foreskin, 37 -specic protein 1 (FSP1), 214 Fibronectin (FN), 57, 60 Fimbrins, 96, 100 FK506, 141 FLG, see Prolaggrin FN, see Fibronectin Fodrin calpain-specic degradation of, 164 inuence of cell adhesion and migration by, 176 Follicle-stimulating hormone (FSH), 25 Follistatin, 183 Formin homology (FH), 92 FSH, see Follicle-stimulating hormone FSP1, see Fibroblast-specic protein 1
H
HaileyHailey disease, 8 Heat shock protein (HSP), 84, 138, 216, 217 Heme oxygenase (HO), 229 HIV-1, see Human immunodeciency virus HO, see Heme oxygenase HPV, see Human papilloma virus HSP, see Heat shock protein Human immunodeciency virus (HIV-1), 137 Human papilloma virus (HPV), 156 Human umbilical vein endothelial cells (HUVEC), 175 HUVEC, see Human umbilical vein endothelial cells
G
GAP, see Guanosine triphosphatase activating proteins Gastrin-releasing peptide (GRP), 19 GDNF, see Glial cell-derived neurotropic factor GDP, see Guanosine diphosphate Gelation factor, 109 Gelsolin, 30, 40, 43
Index Hyaluronic acid, 211 Hydra vulgaris, 35 Hyperthermia, 220, 221, 229 Hypomethylation, 72
355 dystrophy Limb girdle muscular dystrophy (LGMD), 28, 165, 166, 167 Liriodendron tulipifera L., 46 LOH, see Loss of heterozygosity Long terminal repeats (LTR), 137 Loss of heterozygosity (LOH), 12 LTR, see Long terminal repeats Lung, osteonection immunoreactivity in invasive tumours of, 193 Lymphocyte proliferation, inhibition of, 143 Lymphokine-activated killer (LAK) cells, 98 Lymphoma, B-cell diffuse large cell, 175
I
Ibandronate treatment, of metastatic breast cancer, 61 ICE, see Interleukin-1-converting enzymes IF, see Intermediate laments IGF, see Insulin-like growth factor Inammatory diseases, S100A8 and S100A9 proteins in, 239 Inositol phosphates, in calcium signal transduction, 16 Insulin-like growth factor (IGF), 49 Integrin receptor, cell surface, 210 Interblastomere adhesion, 107 Interleukin, 229 Interleukin-1-converting enzymes (ICE), 169 Intermediate laments (IF), 87 Intracellular adhesion, 58 Invasion suppressor gene, 59
M
MadinDarby bovine kidney (MDBK), 127 Major histocompatibility complex genes, 25 Mammalian calpains, 153 Matrilysin promoter, 109 Matrix metalloproteinases, 10, 187 5-MC, see 5-Methylcytosine MCBK, see MadinDarby bovine kidney Mechanoenzymes, 110 Melanocyte-stimulating hormone (MSH), 22 Melanomas, 234, 243 Merlin, 108 Mesotheliomas, cadherin and calretinin as marker for, 134 Metalloproteinases, tumour-associated, 195, 213 Metastasis gene concept, 199, 241 Metastatic dissemination, osteotropism of, 60 N-Methyl-D-aspartate (NMDA), 135, 206 5-Methylcytosine (5-MC), 72 N-Methyl-N-nitrosourea (NMU), 236 MHC, see Myosin heavy chain Microtubules, 91, 217 MLC, see Myosin light chain MLCK, see Myosin light chain kinase MMTV, see Murine mammary tumour virus Moesin, 108 Monocytes, transendothelial migration of, 239 Moorens ulcer, 200 mRNA(s)
J
Jurkat T lymphocytes, 171
K
Kallikrein-1, 242 Keratin lament aggregation, 32 Keratinocyte differentiation, prolaggrin in, 202 Keutel syndrome, 55 Kidney, osteonectin immunoreactivity in invasive tumours of, 193
L
LAK cells, see Lymphokine-activated killer cells Laminin, cooperative functioning of in osteoblast migration, 59 Leiomyosarcomas, expression of calponin in, 124 Leischmania donovani RNA, 47 LGMD, see Limb girdle muscular
356 expression, up-regulation of calcineurin, 143 ICE, 174 tropomyosin, 119 MS, see Multiple sclerosis MSH, see Melanocyte-stimulating hormone Multiple sclerosis (MS), 164 Murine mammary tumour virus (MMTV), 219 Muscular dystrophy, calpains in, 165 Myelin basic protein, 163 Myelodegenerative diseases, 163 Myocardial infarction, 222 Myosin association with cytoskeleton, 22 -ATPase enzyme, 240 heavy chain (MHC), 110, 111 light chain (MLC), 63, 64, 110 kinase (MLCK), 76 phosphorylation, 115, 116, 117
Calcium Signalling in Cancer NFTs, see Neurobrillary tangles NGF, see Nerve growth factor Nitric oxide inhibition of focal adhesion by, 27 synthase (NOS), 10, 27, 81 Nitrogen-activated protein kinase, 11 NMDA, see N-Methyl-D-aspartate NMU, see N-Methyl-N-nitrosourea Non-EF-hand calcium binding proteins, 3031, 3562 annexins, 3540 annexins in cancer growth and progression, 3839 annexins in morphogenesis and differentiation, 3940 biological functions, 3638 structure, 3536 calreticulin and functional diversity, 4653 calreticulin and calnexin as molecular chaperones, 4950 calreticulin in cell adhesion, 5051 calreticulin in cell proliferation and differentiation, 50 calreticulin in intracellular calcium storage, 4849 calreticulin in neoplasia, 5152 immunological implications of calreticulin function, 5253 intracellular distribution of calreticulin, 48 phosphorylation of calreticulin, 4748 regulation of calreticulin expression, 4647 structure and molecular features of calreticulin, 46 calsequestrin and intracellular calcium storage, 5354 gelsolin family of calcium-binding proteins, 4045 gelsolin in cancer, 4243 galsolin in embryonic development and morphogenesis, 4142 gelsolin expression in amyloidosis, 42 gelsolin in severing and capping of actin laments, 4041 severin and cytoskeletal reorganisation, 44
N
Naegleria gruberi, 145 NCAM, see Neural cell adhesion molecules NCBPs, see Neural calcium-binding proteins N-Desmethyltamoxifen, 79 NDP, see Nucleoside diphosphate Nebulin, 114 Nemaline myopathy, 115 Neovascularisation, 191 Nerve growth factor (NGF), 5 Neural calcium-binding proteins (NCBPs), 79 Neural cell adhesion molecules (NCAM), 206 Neurite extension factor, 206 Neurodegenerative disease, implication of calpains in, 158 Neurobrillary tangles (NFTs), 140 Neurobromatosis type 2 (NF2), 155 Neuronal calcium-binding protein, 131 Neurones, calcium homeostasis regulation in, 125 Neurotransmission, calcineurin and, 136 Neurotropin-3 (NT3), 126 NF2, see Neurobromatosis type 2
Index villin in differentiation and neoplasia, 4445 osteocalcin in bone metabolism and osteotropism of cancer, 5462 biology of osteocalcin, 5455 calcium-binding properties of osteocalcin, 55 osteocalcin in cell proliferation and differentiation, 5760 osteocalcin gene structure and function, 5556 osteotropism of metastatic dissemination, 6062 regulation of osteocalcin by vitamin D3, 56 Non-small cell lung carcinoma (NSCLC), 198 NOS, see Nitric oxide synthase NSCLC, see Non-small cell lung carcinoma Nt3, see Neurotropin-3 Nucleoside diphosphate (NDP), 230 Nucleotide metabolism, 1
357 Osteonectin, 184, 247 cancer progression and, 196 expression, correlation between angiogenesis and modulation of, 192 immunoreactivity, 193 inhibition of cell spreading by, 190 molecule, functional domains of, 185 silencer element (OSE), 55 Osteonectin, in cell function and behaviour, 183198 effects of osteonectin on angiogenesis, 191192 functions and functional domains of osteonectin, 184 modulation of cell proliferation by osteonectin, 190191 modulation of cellular adhesion, cell shape, and motility by osteonectin, 188190 molecular structure of osteonectin, 183184 osteonectin in embryonic development and differentiation, 187188 osteonectin expression in cancer development and progression, 193196 osteonetcin homologues and putative tumour suppressor properties, 197198 osteonetcin involvement in other disease states, 196197 osteonetcin in remodelling of extracellular matrix, 186187 regulation of osteonectin expression, 184186 Osteopontin-transfectant cells, 194 OT, see Oxytocin Ovariectomy, expression of CBD gene transcripts and, 128 Ovary calretinin as marker for serous carcinoma of, 134 carcinomas, nm23-H1 in epithelial, 231 osteonection immunoreactivity in invasive tumours of, 193 Oxytocin (OT), 132
O
OA, see Osteoarthritis Odontoblast differentiation, 185 17-Oestradiol, 79 Oestrogen receptors (ERs), 79, 101, 159 OM, see Oncomodulin Oncogenes, overexpression of, 73 Oncogenic retroviral genes, 120 Oncomodulin (OM), 182 Open reading frame (ORF), 68, 150 ORF, see Open reading frame Ornithogalum virens, 122 OSE, see Osteonectin silencer element Osteoarthritis (OA), 196 Osteoblast migration, cooperative functioning of laminin and FN in, 59 Osteocalcin, 30, 35 in bone metabolism, 54 in cell proliferation and differentiation, 57 functions of, 60 gene transcription, 55 Osteoclasts, localisation of mbrin in, 97
358
Calcium Signalling in Cancer Proliferating cell nuclear antigen (PCNA), 38 Proline, 114 Prostaglandin, 229, 230 Prostaglandin E (PGE), 25 Prostate adenocarcinoma cells, apoptosis of by calpain inhibitors, 159 cancer cell lines, protection of, 52 carcinomas, Gleeson grade in, 95 ductal carcinoma of, 98 Proteases, activation of by calcium signalling, 159 Proteasome, 166 Protein(s) actin-binding, 88, 92, 93, 176 adenomatous polyposis coli, 104 adhesion-mediating, 94 amyloid precursor, 178, 179 -amyloid precursor, 141 armadillo, 105 CaM binding domains of target, 75 capping, 97 EF-hand, 67 folding, 84 glial brillary acidic, 207 guanosine triphosphatase activating, 80 guanylate cyclase-activating, 85 heat shock, 84, 216 intracellular, 36 linking plasma membrane with actin cytoskeleton, 108 mobilisation, requirement for, 166 myelin basic, 163 myobrillar, 165 ras family, 26 receptor, 22 retroviral, 68 Rho, 107 S100, 214, 217 stress-induced, 138 tau, 179 Proteinases, 10 Protein kinase A (PKA), 13, 99 Protein kinase C (PKC), 13, 14 -mediated regulation, of annexin function, 36 pathway, 13 Prothoracicotropic hormone (PTTH), 18
P
PA, see Plasminogen activator Paired helical laments (PHF), 140 Paracentrotus lividus, 216 PARP, see Poly (ADP-ribose) polymerase Parvalbumin (PV), 64, 125, 181182 Paxillin, 162 PCNA, see Proliferating cell nuclear antigen PDGF, see Platelet-derived growth factor PGE, see Prostaglandin E PgR, see Progesterone receptors Phenylephrine, 123 PHF, see Paired helical laments Phorbol 12-myristate 13-acetate (PMA), 99 Phosphatidylinositol (PI), 10 Phosphoglucomutase, 208 Phosphoinositide-3 kinase (PI3K), 11 Phospholipase C (PLC), 10 Phospholipid signalling, 10 Phototransduction, 81 Physarum polycephalum, 40 PI, see Phosphatidylinositol PI3K, see Phosphoinositide-3 kinase PKA, see Protein kinase A PKC, see Protein kinase C Plasma membrane Ca2+-ATPase extrusion pump (PMCA), 5 Plasminogen activator (PA), 186 Platelet -derived growth factor (PDGF), 18 integrin, 178 PLC, see Phospholipase C PMA, see Phorbol 12-myristate 13-acetate PMCA, see Plasma membrane Ca2+-ATPase extrusion pump PMN, see Polymorphonuclear leukocytes Poly (ADP-ribose) polymerase (PARP), 171, 172 Polymorphonuclear leukocytes (PMN), 99, 115 Prolaggrin (FLG), 199, 201 abnormal expression of, 204 genomic and molecular features of, 203 in keratinocyte differentiation, 202 monomers, 204 Prolin, 90, 92 Progesterone receptors (PgR), 101 Programmed cell death, 159
Index Psoriasin, 200, 236 PTEN abnormalities, 12 germline mutations, 12 overexpression, 13 PTTH, see Prothoracicotropic hormone PV, see Parvalbumin RV, see Rubella virus RXR, see Retinoid X receptor Ryanodine receptor (RyR), 20, 53 RyR, see Ryanodine receptor
359
S
S100 family genes, structure and organisation of, 68 S100 proteins, 199244 biological properties of S100A7, 236239 S100A7 in neoplastic disease, 238239 S100A7 in skin pathology, 237238 structure and molecular properties of S100A7, 236237 cell cycle-related expression of, 217 effects of S100 proteins on cell deformity and cellular morphology, 205222 cell adhesion and invasive potential of cancer cells, 210212 cell cycle-related expression of S100 proteins, 217219 postulated mechanism of cell cycle control by S100A4, 219222 S100 proteins in cell proliferation, 214217 S100 proteins in remodelling of extracellular matrix, 213214 potential value of S100 proteins as markers of cancer progression and prognosis, 243244 S100 proteins in cell differentiation, motility, and cancer invasion, 202205 prolaggrin in keratinocyte differentiation, 202205 trichohyalin, 205 S100A isoforms, 222 S100A2 as putative tumour suppressor, 223224 S100A3 expression in cell differentiation and neoplasia, 224225 molecular features of S100A3, 224225 S100A3 expression in cell differentiation and human gliomas, 225 S100A4 in cancer development and
R
RA, see Rheumatoid arthritis Radixin, 108 Rana rugosa, 46 Rapamycin, 141 Ras family proteins, 26 RBP, see Retinol-binding protein RCN, see Recoverin Receptor proteins, 22 tyrosine kinases (RTKs), 26 Recoverin (RCN), 79, 80 cancer-associated retinopathy and, 83 mode of action of, 82 rhodopsin kinase complex, 83 Repetin, 201, 203 Reticulocalbin family, of EF-hand proteins, 149151 molecular features of reticulocalbin homologues, 150 putative functions of reticulocalbin and homologues, 150151 Retinitis pigmentosa (RP), 81, 85 Retinoblastoma gene product, 17 susceptibility regulatory element, 50 Retinoic acid, 60, 209 Retinoid X receptor (RXR), 56 Retinol-binding protein (RBP), 21 Retroviral proteins, 68 Reverse transcriptase polymerase chain reaction (RT-PCR), 69 Rheumatoid arthritis (RA), 84, 196, 204 Rhodopsin kinase, 81 Rho protein, 107 Rod domain, 103 RP, see Retinitis pigmentosa RTKs, see Receptor tyrosine kinases RT-PCR, see Reverse transcriptase polymerase chain reaction Rubella virus (RV), 47
360 progression, 225235 clinical potential of S100A4 as marker for cancer prognosis, 230 S100A4 expression and metastatic potential of cancers, 225230 S100A4 in human breast cancer, 230234 S100A4 in other forms of human cancer, 234235 S100A6 in cancer, 235236 S100A8 and S100A9 proteins in inammatory diseases, 239 S100A11 and possible modes of function, 239240 S100P in cancer progression, 241243 Saccharomyces cerevisiae, 38, 90, 97, 145 pombe, 136 Sarcoplasmicendoplasmic reticulum Ca2+ATPase pump (SERCA), 5 SCCA, see Squamous cell lung carcinoma antigen Schizossaccharomyces pombe, 112 Sciatic nerve regeneration, stimulation of by S100B, 215 SCLC, see Small cell lung carcinoma SDKs, see Sphingosine-dependent kinases Secreted protein acidic, rich in cysteine (SPARC), 183 SERCA, see Sarcoplasmicendoplasmic reticulum Ca2+ATPase pump Serine protease, kallikrein-like, 242 /threonine kinase, 11 Severin, 30 Shigella exneri, 100, 109 Signal transduction genetic activation and, 2 machinery, architectural aspects of, 25 Skin neoplasms, 7 pathology, S100A7 in, 237 tumours, trichohyalin expression in, 205 SLE, see Systemic lupus erythematosus Small cell lung carcinoma (SCLC), 83 SmithMagenis syndrome (SMS), 1, 41 SMS, see SmithMagenis syndrome SPARC, see Secreted protein acidic, rich in cysteine
Calcium Signalling in Cancer Sphingosine-dependent kinases (SDKs), 4748 Squamous cell carcinoma, bladder, 238 Squamous cell lung carcinoma antigen (SCCA), 162 Stathmin expression, up-regulation of, 221 promoter, down-regulation of, 220 Steroid(s) calbindin expression and, 129 hormone, 18 Systemic lupus erythematosus (SLE), 52, 144
T
Talin, 51 Tau protein, 179 T-cell antigen receptor (TCR), 49, 141 signal transduction cascade, in SLE, 144 TCR, see T-cell antigen receptor Testicular hormones, 101 Thapsigargin, 172 THH, see Trichohyalin Thioridazine, 78 Threonine phosphatase, 137 Thrombospondin, 192 Thymidine kinase (TK), 55 Thymosins effects of on cell proliferation, 93 potential role of in cancer progression, 95 Thyroid carcinoma, human anaplastic, 107 hormones, 57, 67 Tissue plasminogen activator (tPA), 186 Titin, 114 TK, see Thymidine kinase T-lymphocytes, IP3R1-decient, 17 TNF, see Tumour necrosis factor Topoisomerase II, 150 tPA, see Tissue plasminogen activator Transcription factors, activator of, 156 Transfection studies, 11 Trichohyalin (THH), 201 expression, in skin tumours, 205 genomic and molecular features of, 203 Triticum aestivum, 96
Index Tropolyosin, down-regulation of, 42 Tropomyosins, 118 Troponin, 114, 118 Troponin C, 63 Tubulin monomers, 208 Tumour necrosis factor (TNF), 24 progression, expression of gelsolin and severin in, 44 suppressor gene, 73 S100A2 a putative, 223 Tyrosine kinase inhibitors, 137
361 VDRE, see VD response element VD3 receptor (VDR), 56 VD response element (VDRE), 56 VEGF, see Vascular endothelial growth factor Verapamil, 9, 10, 22, 23 VGCCs, see Voltage-gated calcium channels Villin, in differentiation and neoplasia, 44 Vinculin, 51 Vitamin D3 (VD3), 56 Vitamin D3 receptor, 67 Voltage-gated calcium channels (VGCCs), 6, 8 VP, see Vasopressin
U
UMUC-2, human bladder cancer cell line, 43 Urokinase-type plasminogen activator, 10
W
WAS, see Wiskott-Aldrich syndrome WASP, see Wiskott-Aldrich syndrome protein WHO, see World Health Organization Wiskott-Aldrich syndrome (WAS), 91 gene mutation, 92 protein (WASP), 91 World Health Organization (WHO), 235
V
Valine, 114 Vascular cell adhesion molecule (VCAM), 139 Vascular endothelial growth factor (VEGF), 27, 28 Vasodilator-stimulated phosphoprotein (VASP), 101 Vasopressin (VP), 132 VASP, see Vasodilator-stimulated phosphoprotein VCAM, see Vascular cell adhesion molecule VD3, see Vitamin D3 VDR, see VD3 receptor
Y
Yeast budding process, 92
Z
Zinc nger transcription factors, 225

Calcium Signalling in Cancer-Sh Erbet

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Calcium Signalling in Cancer-Sh Erbet

Uploaded by

Copyright:

Available Formats

Calcium Signalling in Cancer