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43 (2003) 4756

Targeting signal transduction

! s I. Orba ! nb, George Webera,*, Fei Shena, Tama b . Szabolcs Kokeny , Edith Olahb
Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, IN 46202-5119, USA b Department of Molecular Genetics, The National Institute of Oncology, Budapest H-1525, Hungary
a

Introduction Treatment of neoplastic diseases in human in the last decade of the twentieth century focused on three main modalities: (1) surgical procedures and their renements; (2) radiation therapies and their improvements; (3) chemotherapy with focus on blockers of purine and pyrimidine biosynthesis and inhibitors of DNA action and mitotic spindle formations. New Targets in Anticancer Drug Treatment Novel approaches in drug treatment mainly include those listed in Table 1. The most commonly used drugs in the therapy of metastatic breast cancer include anthracyclines, cyclophosphamide, methotrexate, 5-uorouracil, taxanes, capecitabine, platinum salts, vinorelbine, gemcitabine, mitoxantrone and etoposide. The new approaches are based on progress in studies of the biochemistry and molecular biology of cancer cells. The therapeutic usefulness of some of these approaches is not yet proven. Vaccines have been a long-standing hope and promise in the cancer eld but they have largely remained a promise.
Abbreviations: DAG, diacylglycerol; IMP DH, IMP dehydrogenase (EC 1.1.1.205); IP3, inositol-1,4,5trisphosphate; R-5-P, ribose-5-phosphate; PI, 1-phosphatidylinositol; PI kinase, 1-phosphatidylinositol 4kinase (EC 2.7.1.67); PIP, 1-phosphatidylinositol 4-phosphate; PIP kinase, PI 4-phosphate 5-kinase (EC 2.7.1.68); PIP2, 1-phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C (PIP phosphodiesterase, EC 3.1.4.11); TAD, thiazole-4-carboxamide adenine dinucleotide, TR, tiazofurin; Riba, ribavirin. *Corresponding author. Lab. for Indiana University School of Experimental Oncology Medicine, 699 West Drive RR337, Indianapolis, IN 46202-5119, USA. Tel.: +1-317-274-7921; fax: +1-317-274-3939. E-mail address: gweber1@iupui.edu (G. Weber). 0065-2571/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0065-2571(03)00021-9

48 Table 1 Cancer treatments Treatment Surgical procedures Radiation therapies Chemotherapy Anti-angiogenesis factors Anti-metastatic factors Anti-oncogenic factors Chemoprevention therapies Gene therapies Monoclonal antibodies

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Target Multiple Multiple; prostate, solid tumors in internal organs, lymphomas Multiple Multiple Multiple Multiple (leukemia, breast, colon, pancreatic, lung) Breast, head and neck Multiple (breast, ovarian, smallcell lung) Non-Hodgkins lymphoma, breast, colon, melanoma Melanoma, breast, colon pancreatic, ovarian, and others

Mechanism of action Lymphatic mapping, more selective in removing nodes Dose delivery with micro-precision Over two dozen new chemotherapy agents Growth of new blood vessels in tumors Dissolving tissue and boring holes Ras oncogene Tamoxifen, arimidex, antiestrogens, retinoids Infect cancer cells with normal tumor-suppressor genes Miniature guided missiles, against specic proteins on surface of cancer cells Vaccinating patients with antigens derived from tumors

Vaccines

Anti-angiogenesis factors are now in clinical evaluation. Anti-metastatic factors are of interest since cancer death is usually due to the spread of the disease from the primary site. Anti-oncogenic factors include those that target the expression of ras oncogene (tiazofurin) or the maturation of this oncogene (anti-farnesyltransferase). Some of the chemopreventive therapies have been tested extensively in the clinic; among these the value of tamoxifen has been well proven in patients with estrogenpositive breast carcinoma. Tamoxifen acts through binding to the estrogen receptors, thus depriving breast cancer cells from this hormonal stimulation. Other compounds that inhibit the aromatase enzyme block estrogen biosynthesis at a crucial step. In recent studies arimidex was even more effective than tamoxifen and had fewer side effects. Since this comparison is based on a 33-month evaluation, the long-term side effects of arimidex are not yet known. In hormone therapy, anti-estrogens (tamoxifen and others), aromatase inhibitors (arimidex, anastrozole and others) are tested. Ovariectomy and treatment with progestins and androgens are also used. Gene therapies have been tested in a number of studies in patients and although there are some clinical problems this program is ongoing in various centers. The use of monoclonal antibodies has been proposed and tested for many years and a clinical evaluation is in progress. Among the newer agents, tiazofurin, an inhibitor of ras expression, and blockers of ras maturation such as inhibitors of farnesyltransferase are in trial. Inhibitors of

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epidermal growth factor, tyrosine kinase blockers (Gleevec and others) are being tested. Flavopyridol, a modulator of cyclin-dependent kinase, is in clinical trial. Inhibitors of matrix metalloproteases are in trial, as well as liposomal doxorubicin. Additional Novel New Targets Include the Following Cell receptor targets. These include cell surface receptors: insulin-like growth factor receptor (IGF), integrins, the ErbB family of receptors and others. Intracellular receptors. These intracellular elements include the oncogenes ras and Raf and MAP kinase. Further possible targets are BCR-ABL, m-TOR, Src, protein kinase C, STAT proteins and JNK kinase. PI-3 kinase is suggested to be an interesting target. Nuclear transcription factors. These include steroid receptors, chiey estrogen and androgen receptors, c-myc and p53. Cell-cycle targets. These include cyclins, cyclin-dependent kinases and inhibitors of the kinases. Apoptosis targets. These include Bcl-2, p53, TRAIL and Fas. Targets inducing differentiation. These include retinoid and vitamin D nuclear steroid receptors and ras and c-myc oncogenes and other targets which can induce maturation. Such drugs include tiazofurin, ribavirin, retinoids, tamoxifen, quercetin, genistein and other avonoids. Further novel targets include telomerase, histone deacetylase and DNA methylation. Well-dened novel sensitive targets may be found in cancer cells in the pathway of signal transduction, focusing on the three-step conversion of phosphatidylinositol into the second messengers IP3 and DAG. The present overview will concentrate on the elevated activity of signal transduction, which should make it a good target, and on some of the drugs that down-regulate this pathway in cancer cells. Signal Transduction in Cancer Cells as Targets of Anticancer Drugs This paper will focus on drugs against signal transduction with emphasis on a comparison of the mechanism of action, molecular impact and oncolytic effectiveness of two purine nucleosides, tiazofurin and ribavirin. Opposing enzymes of signal transduction A key segment of signal transduction processes is located in the cell membrane. The business end of signal transduction involves the production from phosphatidylinositol (PI) of the second messengers, IP3 and DAG, through the activities of three synthetic enzymes, PI 4-kinase, PI 4,5-kinase and PLC. DAG activates protein kinase C in the membrane, whereas IP3 diffuses to the particulate fraction, combines with receptors and releases calcium from the internal stores of the cell. The three synthetic enzymes are opposed by two specic phosphatases which degrade PIP and PIP2 to PI (Weber et al., 1999).

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Evidence for elevated signal transduction activity in cancer cells Signal transduction activity is increased in animal and human cancer cells as revealed by the following observations. (1) Increased activities of the synthetic enzymes PI 4-kinase, PIP kinase and PLC. (2) Decreased activities of the degradative enzymes 5-phosphatase and 4-phosphatase. As a result there is an amplied capacity for IP3 production. (3) There is an increased concentration of the end product of the synthetic pathway, IP3 which correlates positively with growth rate and malignancy of the hepatomas. (4) When cancer cells in tissue culture display their proliferative capacity in lag and log phases, there are early sharp increases in the concentration of IP3 followed by the elevations in the activities of PI and PIP kinases (Weber et al., 1999). These observations were made from systematic research on a particularly meaningful biological model system: the chemically induced, transplantable rat hepatocellular carcinomas of different growth rates (Weber, 1983). The pattern also applies to other transplantable tumors in animals and to human primary neoplasms including leukemia and carcinomas of liver, colon, ovary and breast. These prominent alterations in the biochemical pattern confer selective advantages to cancer cells and some of these should provide sensitive targets to chemotherapy. Drugs Effective Against Production of Second Messengers Extensive evidence in tissue culture and in animals indicates that inhibitors of PI 4kinase and PI 4,5-kinase activities reduce the cellular concentration of the second messenger, IP3. These include inhibitors of the two kinases, quercetin and genistein. Also included is tamoxifen with an unclear mechanism of action. Important agents are the purine biosynthetic inhibitors, tiazofurin and ribavirin, which will be the main focus of this overview. Tiazofurin: Inhibitor of GTP Biosynthesis and More In our systematic studies we showed that in animal and human tumors the ratelimiting enzyme of de novo GTP biosynthesis, IMP DH, was markedly elevated. The increase in this enzymic activity paralleled the proliferation rate of the tumors in the hepatoma spectrum (Weber, 1983). Biological signicance of the elevated capacity to provide GDP and GTP The increased capacity of the guanylate synthetic pathway provides GDP, the substrate for ribonucleotide reductase which yields dGDP, and with the subsequent action of a kinase produces dGTP, an immediate precursor of DNA. It is interesting that whereas IMP DH is the rate-limiting enzyme of GTP biosynthesis the hepatic specic activity of ribonucleotide reductase is 10-fold lower than that of IMP DH. The reductase activity is markedly increased in a transformation- and progressionlinked fashion in the hepatoma spectrum (Weber, 1983). The transformation- and progression-linked increase in IMP DH and the reductase activities indicates a close linkage with the core of the expression of the neoplastic biochemical strategy.

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Biological signicance of GTP Whereas numerous studies centered on the functions and signicance of ATP, the biological importance of GTP was understood only recently (Weber et al., 1992). GTP is an important factor in protein and RNA production. It is particularly relevant here that GTP is a required co-factor of PLC, the enzyme immediately involved in the production of IP3 and DAG. GTP is also an activator of the ratelimiting enzyme of de novo CTP biosynthesis, CTP synthetase. Moreover, GTP is an activator of ribonucleotide reductase and thus promotes the conversion of GDP into dGTP. Therefore, a pronounced depression in the concentration of the GDP pools should reduce the biosynthesis of dGTP which, as the rate-limiting dNTP pool, would result in a limitation of DNA biosynthesis because of the emerging imbalance in the concentrations of the four dNTPs. It is interesting that GTP also has a role in carbohydrate metabolism because it is the required co-enzyme of phosphoenolpyruvate carboxykinase, the second key enzyme in hepatic gluconeogenesis. We showed that in starvation in the rat the hepatic activity of IMP DH is maintained in the face of a decrease of other enzyme activities and protein concentrations with the exception of the three other enzymes of gluconeogenesis. It is in line with these observations that in starvation the hepatic GTP concentration is preferentially maintained (Jackson et al., 1977). A number of nucleosides were prepared by Roland Robins, an outstanding nucleoside chemist, who took up our suggestion that IMP DH was a potentially sensitive target for chemotherapy. This background is described elsewhere (Weber, 1983). Among the compounds Robins synthesized were tiazofurin and ribavirin. These drugs were potent inhibitors for IMP DH and at NCI and other centers it was shown that various experimental tumors in animals were sensitive to TR action. On the other hand, ribavirin proved to be active against a broad spectrum of viruses. Tiazofurin and IMP DH Pre-clinical biochemical studies in our laboratory showed that TR killed hepatoma cells in culture with an LD50=5 mM and markedly inhibited growth of hepatoma 3924A in rats. We showed that a single intraperitoneal injection of TR in rats rapidly reduced the activity of IMP DH in the subcutaneously carried hepatomas followed by a marked depression of the pools of GDP and GTP and particularly in the concentration of dGTP (Lui et al., 1984). Subsequently, with my clinical associates we carried out clinical evaluation of TR action in leukemia (Tricot et al., 1990). This neoplastic disease was well suited to chemotherapeutic studies because we could sample leukocytes and blast cells during treatment, allowing us to follow the activity of IMP DH and the concentration of GTP and other relevant metabolites. Selectivity was provided by the fact that normal leukocytes converted very little of the TR to the active form, TAD, whereas a great deal of the infused drug was converted into TAD in the blast cells. Tiazofurin is a pro-drug which is converted in sensitive cells in two enzymatic steps to the active form TAD which inhibited IMP DH at the NAD/NADH ligand site on the enzyme molecule. Our clinical studies included sequential measurements of TR and TAD content in normal and leukemic leukocytes (Tricot et al., 1990).

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We puried IMP DH and in molecular biological studies we discovered type I and type II isoforms of IMP DH (Natsumeda et al., 1990). The type-I was constitutively expressed whereas type-II expression was markedly up-regulated and was chiey responsible for the increased total IMP DH activity we observed in cancer cells (Nagai et al., 1991). Subsequent studies showed that this increased IMP DH activity was due to an elevated amount of the enzyme and increased production of mRNA (Nagai et al., 1991). Such enzyme kinetic and immunological evidence was already shown for ve carbohydrate enzymes, two pyrimidine enzymes and three purine metabolic enzymes including ribonucleotide reductase. Thus, the evidence for a reprogramming of gene expression is based on studies on 10 key enzymes in three areas of metabolism (Weber, 1983). Tiazofurin down-regulates the expression of the ras and myc oncogenes Since GTP is a critical co-factor in the maturation and translocation into the membrane and the expression of the ras oncogene we tested with Dr. Olah the hypothesis that TR might reduce ras expression. In K-562 and OVCAR-5 cells addition of TR in low concentrations (510 mM) resulted in a down-regulation of ras expression and in induced differentiation (Olah et al., 1988). These in vitro observations agreed well with the increased maturation of blast cells and the downregulation of ras and myc oncogenes in the bone marrow of patients during TR treatment (Weber et al., 1991). The TR-induced differentiation of the blast cells in the patients was of particular interest because in our studies after completion of therapy the patients could be discharged without the need of antibiotic or other bone marrow protective therapy. This is in clear contrast to therapy with all other commonly used drugs which destroy and deplete the bone marrow in these leukemic patients (Tricot et al., 1990). Impact of tiazofurin on signal transduction activity in cancer cells TR has several major impacts on the signal transduction process. (1) TR, by limiting macromolecular biosynthesis, caused a decrease in activities of enzymes with short half-lives. We showed that among 10 examined enzymes in rat bone marrow cells PI and PIP kinases have the shortest half-lives, t1=2 12 min. Accordingly, a single injection of TR in the rat resulted in a major reduction in activities of PI and PIP kinases and in the concentration of IP3 in bone marrow cells. (2) The reduction of GTP concentration deprived PLC activity from this coenzyme which contributed to the dose- and time-dependent reduction of signal transduction activity as measured by IP3 concentration. The biological and biochemical impact of TR is summarized in Table 2. Ribavirin: Biochemical and Clinical Impact The molecular action of the nucleoside Riba depends, in part at least, on its conversion to Riba-monophosphate which inhibits IMP DH at the IMPXMP ligand site. The biochemical impact leads to a reduction of cellular guanylate

G. Weber et al. / Advan. Enzyme Regul. 43 (2003) 4756 Table 2 Molecular and clinical impact of tiazofurin Drug action 1. Chemotherapy 2. 3. 4. 5. 6. Non-targeted action Oncogene activity Differentiation Signal transduction Apoptosis Targets IMP DH GTP, dGTP Enzymes with rapid turnover* ras, myc Maturation PI and PIP kinase, IP3 Programmed cell death Impact

53

Target enzymeinhibited Target metabolitesdecreased Decreased enzyme amounts Down-regulated Up-regulated Down-regulated Activated

*Thymidine kinase, thymidylate synthase, guanine phosphoribosyltransferase, PI 4-kinase and PIP kinase.

concentrations. Because TR inhibits IMP DH at the NADNADH site and Riba inhibits at the IMPXMP ligand site of the same enzyme, these two nucleosides synergistically inhibit the conversion of IMP to guanylates and yield synergistic cytotoxicity in hepatoma cells (Natsumeda et al., 1988). TR was designed as an anti-cancer oncolytic agent whereas Riba was developed as an anti-viral drug. Riba is recognized as a wide-spectrum, potent anti-viral agent with a spectrum which includes the hepatitis C virus. The hepatitis C virus is the most common blood-borne virus in the United States where about 2% of the adult population is infected and the number of diagnosed cases is expected to continue to increase over the coming years. In patients who present with hepatitis C, Riba is effective in combination with interferon-a. Since hepatitis C infection is common in the far East and the number of patients steadily increases in the United States, the clinical signicance and the mechanism of action of Riba are of major public health signicance. It is also of importance that TR and Riba are synergistic in tissue culture against hepatoma cells because they might be useful in combination against hepatocellular carcinoma in human. It is also possible that in the treatment of hepatitis C administration of TR might improve the synergistic action of Riba and interferon-a. Kinetic studies on puried IMP DH indicated that RMP exerted the same inhibitory mechanism as XMP. However, the Ki values of RMP were two orders of magnitude lower than those of XMP. Thus, RMP interacts at the ligand site of IMP DH with higher afnity than the natural substrates and products (Yamada et al., 1988). Riba through its inhibition of IMP DH reduces cellular GTP concentrations. Because GTP is required for protein biosynthesis a decrease in GTP concentration reduces the activities of enzymes of short half-lives including PI 4-kinase and PIP 5kinase. Moreover, PLC absolutely requires GTP for functioning. Therefore, reducing GTP concentration leads to a decrease in signal transduction activity as measured by the concentration of IP3. Riba blocks the cell cycle at the G1 phase and was shown to cause apoptosis and induce differentiation. Evidence was obtained that in rats given TR i.p. the marked depression of IMP DH activity in the rat bone marrow was maintained by subsequent Riba injection

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(Prajda et al., 1993). When TR was followed by saline injections, IMP DH activity promptly returned to normal levels, whereas in Riba-treated rats, it remained at about 20% of the original values. Dose-response studies indicated that Riba was nearly as effective as TR in depressing IMP DH activity in the rat bone marrow. Because of their short half-lives, TR also lowered guanine phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase activities and subsequent Riba injections continued to depress these enzyme activities, whereas they rapidly returned to normal values in rats where TR treatment was followed by saline injections (Prajda et al., 1993). Both TR and Riba down-regulate the expression of the myc oncogene. TR is particularly effective in lowering expression of the ras oncogene in tissue culture and in leukemic blast cells of patients (Weber et al., 1991). Contrasting the biochemical and cytotoxic action of TR and Riba should be helpful for translation to sequential or synergistic chemotherapy. Riba in human ovarian carcinoma OVCAR-5 cells down-regulated signal transduction activity in a dose (IC50=65 mM) and time (t1=2 24 min) dependent fashion. In human leukemia K562 cells Riba in growth inhibition studies (10 mM) down-regulated expression of c-myc oncogene in a time- and dose-dependent fashion (t1=2 0.5 h) and caused apoptosis up to 44% and induced differentiation up to 8.9%. TR (10 mM) in K-562 cells depressed IMP DH activity to 50% at 20 h incubation and induced differentiation, measured by hemoglobin synthesis, by 50% in 30 h (Orban et al., 2002). In a patient suffering from chronic myelogenous leukemia in blast crisis and particularly sensitive to TR, this drug in peripheral blast cells reduced IMP DH activity with t1=2 30 min, GTP concentration with t1=2 6 h and expression of c-Kiras with t1=2 8 h. In OVCAR-5 cells in growth inhibition assay and in clonogenic assay TR yielded IC50=26 and LC50=17 mM, respectively. In estrogen-receptor-negative human breast carcinoma MDA-MB-435 cells IC50 for TR was 17 and LC50 was 4 mM. Since Riba is nearly as effective in inhibition of IMP DH, in reduction of IP3 concentration and in causing down-regulation of oncogene expression and since both caused induced differentiation and apoptosis they should be synergistic in their biochemical and oncolytic impact. In clinical trials in leukemic patients TR was given i.v. but in patients with hepatitis C Riba was administered by mouth. A protocol in cancer patients starting induction therapy with TR treatment might be consolidated and maintained by Riba. As outlined above the depression caused by TR and continued maintenance of low IMP DH activity caused by Riba were shown in rat bone marrow (Prajda et al., 1993). These considerations support a sequential chemotherapy protocol targeted against IMP DH. The results are relevant for translation also because the tested concentrations of TR and Riba are well within the plasma concentrations achievable in clinical treatment.

Summary This overview provides a concise survey of the main aspects of recent progress in the therapy of cancer. The emphasis is on new treatments with a focus on drugs

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targeted against the elevated signal transduction observed in all examined animal and human neoplasms. 1. The elevated activity of signal transduction in cancer cells should confer selective advantages to the tumor cell population. 2. The clinical and molecular impact of two clinically signicant nucleosides, tiazofurin and ribavirin, is compared. 3. Tiazofurin, developed as an anti-cancer agent, provides a multi-faceted impact on biochemical, molecular biology and clinical action. It is noteworthy that in tissue culture and in leukemic patients it yielded inhibition of IMP dehydrogenase activity, resulting in reduced concentrations of GTP and dGTP. In various systems tiazofurin down-regulated the expression of ras and myc, caused apoptosis and induced differentiation. The nal common path of tiazofurin action may well be the reduction of signal transduction activity in the cancer cells. 4. Ribavirin, was developed as an anti-viral drug with a spectrum which includes the hepatitis C virus. In patients with hepatitis C ribavirin is effective in combination with interferon-a. The molecular action of ribavirin also leads to reduction of cellular guanylate concentrations and down-regulation of myc oncogene expression. This drug yields apoptosis and induced differentiation. Ribavirin also down-regulated signal transduction activity. These actions were somewhat similar to those of tiazofurin. 5. Since the two drugs inhibit IMP dehydrogenase at two different ligand sites of the enzyme they may be used in a synergistic way in sequence where tiazofurin is administered intravenously, and ribavirin is given by mouth. The two drugs are also meaningful and interesting experimental tools.

Acknowledgements This investigation was supported by NIH grants including an Outstanding Investigator Grant from the National Cancer Institute. Further support was from the Ladies Auxiliary of the Veterans of Foreign Wars, Indiana Division to GW and ! chenyi project, NKFP1/48/2001 and by Hungarian Ministry of Education, Sze OTKA T030039 to EO.

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Natsumeda Y, Ohno S, Kawasaki H, Konno Y, Weber G, Suzuki K. Two distinct cDNAs for human IMP dehydrogenase. J Biol Chem 1990;265:52925. Natsumeda Y, Yamada Y, Yamaji Y, Weber G. Synergistic cytotoxic effect of tiazofurin and ribavirin in hepatoma cells. Biochem Biophys Res Commun 1988;153:3217. Olah E, Natsumeda Y, Ikegami T, Kote Z, Horanyi M, Szelenyi J, Paulik E, Kremmer T, Hollan SR, Sugar J, Weber G. Induction of erythroid differentiation and modulation of gene expression by tiazofurin in K562 leukemia cells. Proc Natl Acad Sci USA 1988;85:65337. Orban T, Olah E, Weber G. Ribavirin (RIBA) is antiproliferative and oncolytic; it down-regulates c-myc expression and signal transduction, causes apoptosis and induced differentiation. Proc Am Assoc Cancer Res 2002;43:967. Prajda N, Hata Y, Abonyi M, Singhal RL, Weber G. Sequential impact of tiazofurin and ribavirin on enzymic program of the bone marrow. Cancer Res 1993;53:59826. Tricot G, Jayaram HN, Weber G, Hoffman R. Tiazofurin: biological effects and clinical uses. Int J Cell Cloning 1990;8:16170. Weber G. Biochemical strategy of cancer cells and the design of chemotherapy: G.H.A. Clowes memorial lecture. Cancer Res 1983;43:346692. Weber G, Nagai M, Natsumeda Y, Eble JN, Jayaram HN, Paulik E, Zhen W, Hoffman R, Tricot G. Tiaozfurin down-regulates expression of c-Ki-ras oncogene in a leukemic patient. Cancer Commun 1991;3:616. Weber G, Nakamura H, Natsumeda Y, Szekeres T, Nagai M. Regulation of GTP biosynthesis. Adv Enzyme Regul 1992;32:5769. Weber G, Shen F, Yang H, Prajda N, Li W. Amplication of signal transduction capacity and downregulation by drugs. Adv Enzyme Regul 1999;39:5166. Yamada Y, Natsumeda Y, Weber G. Action of the active metabolites of tiazofurin and ribavirin on puried IMP dehydrogenase. Biochemistry 1988;27:21936.