Chapter 2Cell Proliferation, Differentiation, and Apoptosis

Michael Andreeff, MD, PhD, David W Goodrich, MD, and Arthur B Pardee, MD.

The biology of cell division, differentiation, and apoptosis is exceedingly similar in both normal and cancer
cells. The cancer cell differs from its normal counterpart in that it is aberrantly regulated. Cancer cells
generally contain the full complement of biomolecules that are necessary for survival, proliferation,
differentiation, cell death, and expression of many cell-type-specific functions. Failure to regulate these
functions properly, however, results in an altered phenotype and cancer.

Four cellular functions tend to be inappropriately regulated in a neoplasm. First, the normal constraints on
cellular proliferation are ineffective. Second, the differentiation program can be distorted. The tumor cells
may be blocked at a particular stage of differentiation, or they may differentiate into an inappropriate or
abnormal cell type. Third, chromosomal and genetic organization may be destabilized such that variant
cells arise with high frequency. Some variants may have increased motility or enzyme production that
permits invasion and metastases. Finally, the tightly regulated cell death program (apoptosis) may be
dysregulated.

To comprehend the biology of cancer, it is necessary to understand how these functions are controlled in
normal cells and how they become uncontrolled in cancer cells. This chapter focuses on the biology of cell
proliferation, differentiation, and apoptosis and how these functions are linked in the development of
neoplasia.

Proliferation

Tumor Growth and Cell Proliferation In Vivo

Fundamentally, cancer is a disease of accumulation of clonal cells. Abnormal cell proliferation is

necessary, although often insufficient, for tumorigenesis. It is the increase in tumor cell number, and thus
tumor burden, that ultimately accounts for the adverse effects on the host. Indeed, the goal of most current
cancer therapy is to reduce the number of tumor cells and to prevent their further accumulation. To better
accomplish this goal, a more complete description of the unique characteristics of tumor cell proliferation is
required. This task is made difficult by the fact that the mechanisms that underlie tumor and normal cell
proliferation are very similar. In this section, we will review current understanding of the complex molecular
mechanisms involved in the regulation of cell proliferation with particular emphasis on the aberrations in
this system that occur in malignant cells.

From the perspective of biologic evolution, it is obvious that cells within a multicellular organism like
humans have an intrinsic proliferative potential that is in vast excess of that required to meet the
requirements of normal growth and development. Cellular life has evolved from single-celled organisms
selected for their ability to replicate in a minimum amount of time, within the constraints imposed by the
biochemical processes of cell division. Standard laboratory strains of bacteria, for example, can divide
every 20 minutes under optimal conditions. The constraints of cell division imposed on multicellular
organisms are greater since replication must be carried out with absolute fidelity to maintain the integrity of
the organism. Even so, normal human cells can divide as often as once or twice a day in vivo. A cell
dividing once a day would generate a cell number equal to the total number of cells in an adult human in
less than 2 months. Clearly, then, human cells have inherited a surplus of proliferative capacity from their
unicellular ancestors. Multicellular organisms must evolve mechanisms to restrain this proliferative capacity
to appropriate times and places. The key in understanding tumor cell proliferation, then, is to characterize
these mechanisms and to understand how they fail during tumorigenesis.

The rate of cell proliferation within any population of cells depends on three parameters: (a) the rate of cell
division (Tc), (b) the fraction of cells within the population undergoing cell division (growth fraction), and (c)
the rate of cell loss from the population due to terminal differentiation or cell death (see next section). Tc
represents the time it takes to complete a cell division cycle. The cell division cycle can be divided into two
functional phases, S and M phases, and two preparatory phases, G1 and G2 (Fig. 2.1). S phase is defined
as the phase in which the DNA is replicated. Under normal circumstances, the time it takes a typical
human cell to complete S phase is about 8 hours and is invariant. Fully replicated chromosomes are
segregated to each of the two daughter nuclei by the process of mitosis during M phase. The length of M
phase is about 1 hour and is also normally invariant. G1 phase precedes S phase, whereas G2 phase
precedes M phase. G1 and G2 phases are required for the synthesis of cellular constituents needed to
support the following phase and ultimately to complete cell division. In mammalian cells, the length of G2
phase is about 2 hours. The length of G1 phase is highly variable and can range from about 6 hours to
several days or longer. The varying length of G1 phase accounts for most of the difference in Tc between
different cell types or between cells growing under different conditions.

Figure 2.1

The cell cycle. When a cell is not synthesizing DNA (S phase) or completing mitosis (M phase), it is
commonly termed as being a G (gap) phase. Normal cells are capable of resting in a nondividing (more...)

Cell Cycle Control

A successful cell division cycle requires the orderly and unidirectional transition from one cell cycle phase
to the next. Certain events must be completed before others are begun. For example, beginning mitosis
before the completion of DNA replication would obviously be deleterious to the cell. In theory, the ordering
of cell cycle events may be accomplished in a manner analogous to the substrate-product relationship of a
metabolic, biochemical pathway.1 The product of one reaction serves as the substrate and is thus required
for the next reaction. Hence, regulation of the system is inherent in the biochemical events of the process
itself. The prevailing view, however, is that the timing and ordering of cell cycle transitions is dependent on
separate positive and negative regulatory circuits. The regulatory circuits enforce a series of checkpoints,
allowing passage only after completion of critical cell cycle events. Two classes of regulatory circuits exist,
intrinsic and extrinsic. Intrinsic regulatory pathways are responsible for the precise ordering of cell cycle
events. Since the length of S, G2, and M phases in mammalian cells is relatively invariant, the transitions
between these phases are controlled predominantly by intrinsic regulatory pathways. Extrinsic regulatory
pathways function in response to environmental conditions or in response to detected cell cycle defects.
Both types of regulatory circuits can use the same checkpoints. We will focus our attention on the extrinsic
regulatory circuits where differences between normal and neoplastic cells are observed.

Passage of the cell cycle checkpoints ultimately requires the activation of intracellular enzymes known as
cyclin-dependent kinases (CDKs). CDKs are extremely well conserved through evolution. CDKs exist in all
eukaryotic cells from fungi to plants to mammals. In fact, CDKs from human cells can functionally
substitute for the enzymes in yeast. The structural and functional conservation of these enzymes through
evolution suggests that they are centrally important for the cell cycle in all eukaryotic cells. The
requirement for these enzymes for cell cycle transitions has been amply documented, particularly in
organisms like yeast that are amenable to genetic manipulation.1 Since activation of CDKs is the central
event in cell cycle transitions, it is not surprising that their activity is exquisitely regulated at several levels.2
The active CDK holoenzyme is composed of a catalytic subunit and the cyclin regulatory subunit. One level
of regulation is that each cyclin protein is synthesized at a particular stage of the cell cycle. For example,
cyclin D is synthesized during G1, cyclin E is synthesized in late G1, cyclin A is synthesized during S and
G2 phases, and cyclin B is synthesized in G2 and M phases (Fig. 2.2). Therefore, a given catalytic subunit
cannot become active until an appropriate cyclin is synthesized. Upon synthesis, a cyclin can assemble
with an appropriate catalytic subunit. However, this complex requires phosphorylation on threonine by
another regulated kinase, the CDK-activating kinase or CAK. CAK is itself a CDK composed of cyclin H
and CDK7 proteins. Hence the levels of CAK influence the activity of assembled CDKs. Another level of
regulation is deactivation of the CDK by phosphorylation of its ATP binding site by yet another regulated
kinase activity. This kinase activity is unusual in that it has dual specificity for both tyrosine and threonine.
A CDK deactivated by phosphorylation of its ATP binding site can be reactivated by a dual specificity
phosphatase of the Cdc25 family. In fact, dephosphorylation by these phosphatases may be the rate-
limiting step in triggering cell cycle transitions. Another level of regulation is the presence of a diverse
family of proteins known as cyclin-dependent kinase inhibitors, or CKIs, that can block activation of CDKs.
Two distinct classes of CKIs have been described. One class inhibits multiple CDKs and includes
p21CIP1, p27KIP1, and p57KIP2. The other class specifically inhibits cyclin D/CDK4 or 6 CDKs and
includes p16INK4, p15INK4B, p18INK4C, and p19INK4D. The synthesis, degradation, and activity of these
CKIs are regulated in response to both mitogenic and antimitogenic signals. For example, cell cycle
regulation by cell-cell contact or transforming growth factor-� (TGF-�) is mediated by p27KIP1.3,4 Once
activated, the CDKs that drive the transition into a particular cell cycle phase often need to be deactivated
before completion of that phase and transition to the ensuing phase. For example, the CDKs required for
initiation of mitosis also prevent exit from mitosis and into G1 phase. The final level of CDK regulation
involves their specific degradation in precise order. It is now generally understood that ubiquitin-mediated
proteolysis is responsible for this regulation as well as the regulation of a host of other cell cycle
regulators.5 Hence, synthesis, post-translational modification, and programed degradation all contribute to
the regulation of CDKs.

Figure 2.2

Cyclin-dependent kinase regulation of cell cycle transitions. A. The phases of the cell division cycle are
shown. Transition from one phase to the next requires transit of a (more...)

The G0 to S Checkpoints

As discussed above, the time it takes to progress through the S, G2, and M phases of the cell cycle is
relatively invariant. The length of G1 phase on the other hand is variable. In addition, cells can exit the cell
cycle for extended periods of time and mammalian cells do so during the G1 phase of the cell cycle. Cells
that have exited the cell cycle are said to be in a G0 state, or “quiescent.” Most cells in adults are in G0.
This absence from the cell division cycle can be temporary or permanent, as is the case with terminally
differentiated cells like neurons. Cells in G0 can be very active functionally and metabolically, and
proliferation of G0 cells can be initiated by changes in cell density, the presence of mitogens or growth
factors, or the supply of nutrients. These cells then enter the cell cycle, beginning a sequence of events
that culminates in cell division. Hence, the G0/G1 to S phase transition is highly regulated, and the result of
this regulation, by and large, determines the Tc and growth fraction of a population of cells.

Like other cell cycle transitions, the transition from G0 to S phase as cells re-enter the cell cycle is
regulated by two major checkpoints: competence and the restriction point (R). These checkpoints are
located approximately 12 and 2 hours before the start of the S phase, respectively. At least three growth
factors, provided in serum, are required sequentially to transit these checkpoints following resumption of
proliferation of fibroblasts: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and
insulin-like growth factor (IGF-1). As mentioned above, extracellular TGF-� has the opposite effect. It
inhibits the growth of various epithelial cells by modulating expression of CKIs. Paracrine production of
TGF-� could limit growth of both normal and cancer cells, and experimental models suggest that it may
play a role in the regression of breast cancers in response to hormonal or drug therapies.6 Once the R
point has been passed, the cell is committed to a round of cell division. The cell now completes S, G2, and
M phases without the need for growth factors or even additional protein synthesis. Once initiated, the cell
cycle is not free running; the competence and restriction checkpoints must be passed in each subsequent
G1 phase, thus requiring the continued presence of growth factors. The switching of cells back and forth
between quiescence and cycling depends on extracellular conditions and is regulated differently in normal
and tumor cells.

Growth factor receptors are complex, large proteins that span the plasma membrane. They have a specific
domain that recognizes the growth factor on the outside of the cell, and their cytoplasmic portion may have
an enzymatic function, such as a protein tyrosine kinase. Binding of a growth factor or ligand to its receptor
can induce transmission of a signal to the cytoplasm through activation of the kinase.7 The next step is a
transduction of the cytoplasmic signal to the cell nucleus. This is accomplished by a heterogeneous group
of molecules known as second messengers and includes various proteins that are phosphorylated by
kinases, small molecules such as inositol phosphates and cyclic AMP, and ions, including Ca++, H+, and
Zn++. Within the nucleus, genes are then activated in response to these second messengers. As an
illustration of this general scheme, upon binding of EGF to the extracellular domains of its receptor,
autophosphorylation occurs in the intracellular domain of the protein. This phosphorylated domain
facilitates the formation of a protein complex containing Grb2 and Sos. This complex activates the Ras
protein by catalyzing exchange of Ras-bound GDP for GTP. The GTP-bound form of Ras activates the c-
Raf kinase. This kinase then triggers a phosphorylation cascade ultimately activating mitogen-activated
protein kinase. This kinase may phosphorylate and regulate transcription factors such as Jun, Fos, and
Myc.

A variety of proteins are produced during G1 after cells leave quiescence. Some are enzymes that expand
metabolic functions lost by G0 cells, such as those providing energy, and more ribosomes are made for
rapid protein synthesis. Others have so-called housekeeping functions that keep both quiescent and
growing cells in metabolic balance. Only a few proteins appear to be key regulatory molecules. For
example, enzymes are required for the synthesis of isoprenoids, which are necessary for activity of the
Ras oncogene, and for the synthesis of polyamines, which have many functions including ionic binding to
nucleic acids. The Ras oncogene product is synthesized as a precursor protein that requires post-
translational processing to become biologically active and capable of transforming mammalian cells.
Farnesylation appears to be a critical modification of the Ras protein, and drugs that inhibit farnesyl-protein
transferase can block Ras-dependent transformation. These agents have been proposed as a potential
new class of therapy for cancer.8 Enzymes involved in the synthesis of DNA, such as thymidine kinase and
DNA polymerase, as well as histones, are synthesized just prior to the S phase. These enzyme molecules
relocate at the beginning of DNA synthesis, moving from the cytoplasm into the nucleus. A variety of
experiments show that DNA is made by a high-molecular-weight, multienzyme complex.9,10 This complex
contains many enzymes known to be involved in the process of DNA replication, but its size and other
features are still a matter of debate. The onset of DNA replication has been investigated recently with in
vitro systems, and these studies reveal that the synthesis of helicase enzymes, which possess DNA-
unwinding ability, may provide the final factor for initiating the S phase.11 After DNA synthesis has
commenced, cell growth becomes relatively independent of external controls. The daughter cells, now in
the G1 phase, will then either pass through another cycle or arrest in a quiescent G0 state, depending,
once more, on external conditions. If these conditions are not adequate, the cell will become arrested
before it reinitiates DNA synthesis.

How is re-entry into the cell cycle from G0 ultimately controlled? Like other cell cycle transitions, activation
of CDKs are required. G0 cells are devoid of significant CDK activity. In the presence of mitogenic growth
factors, expression of D-type cyclins (cyclins D1, D2, and D3) is stimulated and continues throughout G1
phase as long as the growth factors are present.12 D-type cyclins complex with either CDK4 or CDK6
catalytic subunits to form a holoenzyme modified by CAK. All of the relevant substrates for cyclin D/CDK4
or 6 have probably not been enumerated. However, one important substrate is likely the retinoblastoma
tumor-suppressor protein (Rb). Rb is constitutively expressed and constrains cells from progressing
through the G1 phase of the cell cycle.13 Rb complexes with many cellular proteins including the E2F
transcription factors. When in complex with E2F, Rb represses transcription from E2F-dependent
promoters. Upon phosphorylation by cyclin D/CDK4 or 6,14 Rb loses its ability to restrain the cell cycle.15
This response is presumably because it can no longer complex with E2F and represses E2F-dependent
transcription.16 The E2F family contains at least five members (E2F-1 through E2F-5). The E2F proteins
function as transcriptional activators when in heterodimeric complex with one of the E2F-related proteins
DP-1, 2, or 3. The heterodimeric complex binds a specific DNA sequence and activates transcription from
the promoters of many genes important for S phase including dihydrofolate reductase, DNA polymerase α,
and thymidine kinase.17 Perhaps most importantly, E2F influences the expression of cyclin E.18 In fact, of
many E2F-dependent genes, cyclin E is the only one deregulated upon loss of Rb in normal cells.19 Cyclin
E expression begins in late G1 phase and complexes with CDK2. Cyclin E/CDK2 activity is necessary and
sufficient for the start of S phase.20 Forced expression of cyclin E/CDK2 activity can trigger the start of S
phase in the absence of Rb phosphorylation and derepression of other E2F-dependent genes, suggesting
that cyclin E is the primary target for regulation by cyclin D/CDK4 or 6, Rb, and E2F.21,22

DNA Damage-Induced Checkpoints

When nuclear DNA is damaged, normal cells initiate a response that includes cell cycle arrest, apoptotic
cell death, and transcriptional induction of genes involved in DNA repair. Induction of apoptosis is an
important response to DNA damage and is discussed in detail below. Normal cells in G1 phase prior to the
R point will arrest in G1 phase upon sensing DNA damage. This arrest is presumably induced to prevent
the replication of damaged DNA. Replication of damaged DNA can result in the incorporation of heritable
genetic mutations. If cells are past the R point or within S phase, DNA replication is slowed, again to allow
time for DNA repair. If cells sense DNA damage while in G2 phase, a G2 cell cycle arrest will occur.
Different types of DNA damage can interfere with normal mitosis, resulting in heritable genetic mutations or
cell death.

The tumor-suppressor genes ATM and p53 play an important part in responses to damaged DNA.23,24 For
example, cells containing mutations in p53 fail to arrest in G1 or undergo apoptosis efficiently upon
irradiation. Cells containing mutations in ATM are also deficient for cell cycle arrest as well as some forms
of DNA repair. The p53 protein functions as a transcription factor by binding specific DNA sequences and
regulating transcription from promoters containing those sequences. In normal cells, DNA damage induces
an increase in p53 levels by inhibiting the normal rapid turnover of the protein. The p53 protein is normally
targeted for ubiquitin-dependent proteolysis by association with the Mdm2 protein. This association is
inhibited by phosphorylation of p53 on specific amino-terminal residues that is triggered by DNA damage.25
Phosphorylation on these amino terminal residues also facilitates dephosphorylation or acetylation of p53
carboxy-terminal residues. These modifications increase the affinity of p53 for its DNA binding site by
distinct mechanisms26,27 and hence increase its ability to activate transcription. The transcription of a
number of genes can be affected by activation of p53. However, the ability of p53 to directly increase
expression of p21CIP1 is probably important for p53-dependent G1 cell cycle arrest observed upon DNA
damage. As discussed above, p21CIP1 is a CKI that can inhibit the activity of multiple CDKs, including
cyclin D/CDK4 or 6 as well as cyclin E/CDK2. In summary, DNA damage generates a signal that can
activate p53 by post-translational modification. Increased p53 activity upregulates p21CIP1, which
prevents activation of CDKs, required for the G1 to S transition.

ATM is the gene whose mutation is responsible for ataxiatelangiectasia. Immunodeficiency, progressive
cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects, and cancer predisposition characterize this
disease. ATM encodes a protein containing a phosphatidyl-inositol 3-kinase-like domain, implicating it in
signal transduction. Like p53 mutant cells, mutant ATM cells are defective in the G1/S checkpoint activated
after radiation-induced DNA damage. This defect is attributable to the lack of p53 activation that normally
occurs, suggesting that ATM may participate in the same pathway as p53. ATM protein, and the related
ATR protein, can, in fact, associate with and phosphorylate p53 at its amino-terminal sites.28,29 The p53-
directed kinase activity of the ATM protein is itself activated by DNA damage. ATM protein, therefore,
contributes to the activation and stabilization of p53 by phosphorylating amino-terminal sites during the
radiation-induced DNA damage response. ATM protein may play a role in sensing DNA damage and
generating the DNA damage signal.

Environmental agents like radiation or DNA-damaging chemicals most commonly induce DNA damage.
Rarely, DNA damage can be generated by mistakes in the normal execution of the cell cycle. However, a
form of DNA damage eventually occurs in all normal cells as they suffer replicative senescence. Normal
cells have a limited replicative lifespan both in vivo and in vitro; a cell can undergo only a finite number of
cell divisions. This limit is thought to be imposed, at least in part, by levels of telomerase activity.30
Telomerase is an enzymatic activity within cells that is required to maintain the integrity of DNA ends.31
DNA polymerases involved in DNA replication synthesize DNA in the 5′ to 3′ direction and require a primer
and template. The requirement for a primer ensures that some genetic information will be lost from the 5′
end of DNA during each round of DNA replication. Telomerase adds DNA of a particular sequence to the
ends of DNA without the need for a separate primer or template, thus protecting cells from loss of genetic
information. Telomeric DNA also protects chromosomes from degradation or recombination. Without
telomeric DNA, chromosomes become unstable. As normal cells become senescent, they lose telomerase
activity and their cell division cycle is arrested. This cell cycle arrest may be mediated by the DNA damage
checkpoint since shortened telomeric DNA is associated with DNA strand breaks that may be sensed as
damaged DNA. Consistent with this hypothesis, shortening of telomeric DNA triggers a p53-dependent cell
cycle arrest by accumulation of single stranded DNA.32

Checkpoint Defects in Tumor Cells

To maintain tissue homeostasis and to support normal development, each organ maintains tight controls
over Tc, growth fraction, and cell loss. Physiologic stimuli can alter these parameters in normal tissues,
leading to increased tissue growth, but this growth will cease when the stimulus is withdrawn or a new
steady state is achieved. In contrast to normal cells, however, tumor cells continue to proliferate even in
the absence of proliferative signals. Although tumor cells proliferate under inappropriate conditions, they do
not necessarily proliferate faster than normal cells. In fact, some normal tissues grow faster than cancers
under physiologic conditions (Table 2.1). Biopsy samples from normal, inflammatory, and neoplastic
lesions of the lung, cervix, vocal cord, or pharynx have been analyzed for the rate of cell proliferation;
these studies showed that benign inflammatory lesions can grow over 20 times faster than cancer in a
discrete time and place.33–35 Similarly, rapid proliferation of human lymphoid cells is induced by
immunostimulants, and growth kinetics of these cells are similar to those observed in high-grade
lymphomas.36 So, it is not simply rapid growth at a single time and place that distinguishes neoplasia but
rather growth that is not restrained to appropriate times and places.

Table 2.1

Growth Parameters of Human Neoplasms and Normal Tissues.

It generally is believed that neoplastic cells multiply exponentially during the early phases of tumor cell
growth. As the tumor mass increases, however, the rate of growth declines. Measuring tumor growth over
time describes a curve with an exponential increase in the early period, then a flattening out of the growth
rate over time (i.e., Gompertzian curve).37 Several mechanisms have been invoked to explain this change
in growth rate with larger tumors: (a) decrease in the growth fraction, (b) increase in cell loss (i.e.,
exfoliation, necrosis), (c) nutritional depletion of tumor cells resulting from outgrowth of available blood
supply, or (d) lengthening of Tc. Experimental tumor models suggest that cell cycle time changes only
slightly when tumor growth decreases.38 Under adverse conditions, tumor cells often leave the growth
fraction and enter a nongrowing state (G0 or prolonged G1) (see Fig. 2.1), although these same cells can
re-enter the division cycle when conditions improve or when stimulated by growth factors. Therefore, the
mass doubling time of tumors is correlated with the growth fraction (Table 2.2).

Table 2.2

Correlation between Mass Doubling Time and Growth Fraction.

The biochemistry of growth appears to be very similar qualitatively in tumor and normal cells.39 Despite
numerous efforts, universal differences in biochemical machinery have not yet been discovered between
normal and tumor cells. The fundamental difference probably lies in a relaxation of the regulation of cell
growth.9,38 For example, normal cells generally are quiescent at physiologic levels of growth factors,
whereas related tumor cells are able to proliferate under these conditions. In some experimental models,
tumor cells proliferate in the absence of or at very low levels of growth factors. Further, fibroblast-derived
tumor cells are less sensitive than normal cells to the presence of other cells in their immediate vicinity.
Normal cells typically cease proliferation when the in vitro culture becomes confluent, but tumor cells can
reach several-fold higher densities in culture. Also, cells of normal solid tissue lie on a secreted
extracellular matrix (ECM) that is composed of various proteins that stimulate cell growth.40 Tumor cells
often are partly or completely independent of ECM for optimal growth, and they may secrete little matrix
material.41

What molecular defects bring about the relaxed growth requirements in neoplastic cells? Defects can occur
at several levels. For example, limiting growth factors may not be needed because tumor cells
inappropriately produce their own (i.e., an autocrine mechanism). Alternatively, receptors may be produced
in excess, as is the case for EGF receptors in numerous clinical tumors, leading to adequate stimulation at
the low growth factor concentrations found in vivo. Moreover, mutations that alter intracellular signaling
mechanisms may bypass growth factor dependence. Mutated forms of proto-oncogenes and inactivated
tumor-suppressor genes can activate growth in these ways. We will focus here on defects that occur in cell
cycle regulatory proteins that enforce the checkpoints discussed above.

Like normal cells, the transit of cell cycle checkpoints in cancer cells ultimately requires the activation of
CDKs. Due to the complexity of CDK regulation, defects leading to inappropriate activation of CDKs can
occur at several levels. The overexpression of cyclin D1 has been detected in many human cancers due to
gene amplification or translocation of the cyclin D1 gene.42 The cyclin D1 gene is located on chromosome
11q13. This chromosomal region is amplified in a wide variety of human cancers including small-cell lung
tumors (10%), primary breast cancers (13%), bladder cancer (15%), esophageal carcinoma (34%), and
squamous cell carcinoma of the head and neck (43%) among others.43 Of course, other potential
oncogenes could be contained within the amplified region. However, cyclin D1 is likely important since its
expression is consistently elevated in these tumors. Cyclin D1 overexpression can also be observed in
tumors, such as sarcomas, colorectal tumors, and melanomas, without amplification of the gene. In some
cases, cyclin D1 expression is activated by chromosomal translocation. In parathyroid adenoma, Motokura
and colleagues.44 have identified cyclin D1 as being translocated to the parathyroid hormone gene, thereby
deregulating cyclin D1 expression. Translocation of the cyclin D1 gene with immunoglobulin heavy chain
gene transcriptional control elements has also been observed in B cell lineage mantle cell lymphomas.
Cyclin D1 is a growth factor responsive cyclin that plays an important role in regulating the G0/S
checkpoint. Deregulated expression of cyclin D1 could inappropriately increase cyclin D1/CDK4 activity
and drive transit of the checkpoint even in the absence of growth factors. Direct evidence that forced
expression of cyclin D1 can facilitate tumorigenesis has been obtained from transgenic mice in which
overexpression of cyclin D1 has been targeted to the mammary epithelium. These mice develop ductal
hyperproliferation and eventual mammary tumor formation.45

CDK activation can also be accomplished by inactivation of CKIs. Genetic mutation of CKI genes has also
been observed frequently in human cancer. In this scenario, loss of a CKI relieves one constraint on the
activation of CDKs and provides a proliferation stimulus. In particular, the INK4 locus within chromosomal
region 9p21 is one of the most frequently mutated areas in human cancers.46 This locus is also frequently
methylated in some tumor types including bladder cancer and leukemia. Extensive methylation of DNA
prevents efficient transcription of genes within the methylated region, thus silencing gene expression.
Three proteins are encoded by the INK4 locus including the CKIs p16INK4a and p15INK4b as well as
p19ARF (see below). It is likely that p16INK4a is a bona fide tumor-suppressor gene since many of the
mutations detected in tumors specifically target expression of this protein, and because germline mutations
that specifically map to p16INK4a have been detected in kindreds with familial melanoma and pancreatic
adenocarcinoma. In addition, mutations in CDK4 that prevent binding with p16INK4a, thus relieving it of
p16-mediated inhibition, have also been found in melanoma-prone families. Loss of p16INK4a may
facilitate activation of cyclin D1/CDK4 or 6, which is likely to affect regulation of the G0/S checkpoint.
Mutation of other CKIs in human cancer is rare, suggesting that they may be required for execution of the
cell cycle. However, expression of p27KIP1 is inversely correlated with clinical outcome in a limited number
of cancers, including melanoma and carcinoma of the oral cavity.

CDK activation also requires dephosphorylation of inhibitory threonine/tyrosine phosphorylation sites by

the Cdc25 family of dual specificity phosphatases. In vitro evidence exists that the Cdc25 family memers
are potential oncogenes.47 Forced expression of Cdc25 can cooperate with Ha-Ras or loss of Rb to induce
oncogenic transformation of primary cells. Overexpression of Cdc25 has also been detected in some
primary human tumors. Cdc25A may be a direct transcriptional target for the myc oncogene.48
Inappropriately high Cdc25 levels may provide an oncogenic stimulus by inappropriately activating CDK
activity.

One of the most important genes involved in human cancer is the Rb tumor-suppressor gene. An
interesting feature of retinoblastoma is that close to 40% of cases are hereditary, and susceptibility to
retinoblastoma is inherited as a simple autosomal dominant trait with high (90%) penetrance. The simple
genetics of retinoblastoma has provided the means to molecularly clone the gene responsible; mutational
inactivation of both alleles of Rb is necessary and sufficient for retinoblastoma.49 Mutation of Rb is
observed at high frequency in osteosarcoma and soft-tissue sarcoma as well. Rb mutations can also be
detected in a wide variety of clinically important cancers including carcinoma of the breast, prostate,
bladder, kidney, liver, pancreas, cervix, and lung, as well as leukemia. Further, expression of wild-type Rb
cDNA in cancer cells can inhibit their tumorigenicity.50 As mentioned above, cyclin D/CDK4 or 6
phosphorylation, which, in turn, is regulated by p16INK4a, inhibits Rb function. This finding suggests that
these three proteins function in the same biochemical pathway (Fig. 2.3). Support for this functional
interrelation comes from the observation that deregulation of any one of these proteins greatly decreases
the likelihood of detecting defects in the other proteins. For example, tumor cells that lose p16INK4a or
overexpress cyclin D1 generally retain wild-type Rb. Cells lacking wild-type Rb typically express normal
levels of cyclin D1 and p16INK4a. In addition, induction of cell cycle arrest by forced expression of
p16INK4a only occurs in cells that contain functional Rb. If mutations in any of the members of this
pathway are considered, disruption of this p16INK4a/cyclin D1/CDK4 or 6/Rb pathway may occur in most
human cancers. Since this pathway is important for regulation of the G0 to S phase transition, it has a
major influence on the growth fraction of normal tissues.

Figure 2.3

The Rb and p53 growth control pathways. Underphosphorylated and active Rb in complex with
transcription factors like E2F represses the transcription of genes required for entry into S phase. (more...)

The p53 gene is the most frequently mutated gene in human cancer.51 Germ line p53 mutation is involved
in the cancer-prone Li-Fraumeni syndrome.52 Mice lacking p53 due to genetically engineered disruption are
also cancer prone. Wild-type p53 is critically important for operation of the DNA damage-induced
checkpoint (see above). Upon sensing DNA damage, p53 is activated, resulting in either G1 cell cycle
arrest or apoptosis. These responses either allow time for the cell to repair the damage or to rid the body of
cells with damaged DNA. Loss of p53 function, therefore, decreases genomic stability. Loss of genomic
stability can increase the accumulation of additional genetic mutations required for neoplastic
transformation. The Mdm2 gene encodes a protein that binds p53 and targets it destruction by the
ubiquitin-proteosome pathway. Too much Mdm2 protein may be analogous to p53 inactivation since any
p53 synthesized would be rapidly degraded. Mdm2 was originally identified as an oncogene amplified in a
spontaneously transformed mouse cell line. Overexpression of Mdm2 mediated by gene amplification can
also be detected in human cancer, particularly sarcoma.53 Interestingly, the p19ARF protein encoded by
the INK4a locus also regulates p53 function.54 The p19ARF protein can bind Mdm2 and prevent Mdm2
from targeting p53 for degradation. Consistent with the ability of p19ARF to activate p53, forced expression
of p19ARF can cause a p53-dependent cell cycle arrest. As discussed previously, mutations of the INK4
locus that inactivate p19ARF, as well as p16INK4a, are commonly observed in human cancer. Inactivation
of p19ARF may contribute to tumorigenesis since Mdm2-mediated degradation of p53 would be
unimpeded. The functional interrelation between p19ARF, Mdm2, and p53 defines another cell cycle
checkpoint control pathway (see Fig. 2.3.) Deficiencies in this pathway also play a vital role in neoplastic
transformation.

Although cancer cells use the same cell cycle machinery as normal cells, the cell cycle checkpoints in
tumor cells are relaxed. Of the scores of proto-oncogenes and tumor-suppressor genes that have been
identified to date, most function in signal transduction pathways that mediate mitogenic stimulation. These
signal transduction pathways eventually converge on the cell cycle checkpoint that controlsthe G0/G1 to S
phase transition and activate appropriate CDKs.Influencing the transit of this checkpoint has a major
influence on the proliferation of normal and tumor cells by affecting both Tc and growth fraction. Despite
the number and variety of these genes involved in signal transduction, relaxation of the G1/G0 to S
checkpoint controls in tumor cells is mediated, for the most part, by disruption of two pathways, the Rb and
p53 growth control pathways. These two genes, individually, are the most frequently mutated in human
cancer cells. Disruption of the Rb or p53 pathways probably occurs in virtually every human cancer.

Differentiation

Most, if not all, tumor cells show abnormalities in differentiation (i.e., anaplasia). The anaplasia of tumors
can provide insights into their etiology, degree of malignancy, prognosis, and sensitivity to therapeutic
intervention by differentiation- or maturation-inducing agents. These differences in phenotype arise from
differences in gene expression, not in gene content. The genes expressed by a particular cell only
comprise approximately 10 to 20% of the coding capacity of the genome. In humans, there are over
100,000 genes that code for proteins; however, an individual cell generally expresses only 10,000 to
20,000 genes. Genes expressed by a particular cell depend on its embryonic lineage, developmental stage
of the organism, tissue and cellular environment, and functions that the cell must fulfill. The mechanisms
that regulate gene expression are incompletely understood; however, they most certainly entail the
sequential action of cell-type-specific or cell-lineage-specific transcription factors that repress or activate
the differentiation-specific genes. Programs of gene expression generally are instituted early in
embryogenesis and sequentially altered as development proceeds.55,56

Some genes are expressed by many, if not all, cell types. These “housekeeping” genes generally encode
proteins that participate in basic or universal cellular functions. Other genes that are expressed only in
specific cell types and/or stages of development are said to be cell-type- or differentiation-specific genes.
Thus, the expression of specific gene products marks both the cell lineage and the stage of differentiation.

Differentiation and Cell Proliferation

Differentiation begins shortly after the first few cell divisions that follow fertilization. Throughout
development, and in adult organisms, the ability of a cell to proliferate is intimately connected to its state of
differentiation. Adult tissues generally express a variety of factors that act to maintain both the proliferation
and the differentiation status of the cells. These include secreted molecules, transmembrane receptors,
intracellular signaling molecules, and transcription factors. For example, myoD57 and c/EBP-a58 are nuclear
factors that activate the transcription of muscle- and adipocyte-specific genes, respectively; in addition,
both proteins are potent inhibitors of cell proliferation.

In early embryos, cell proliferation is the primary means by which the cell mass increases. As the organism
develops, however, proliferation becomes restricted. Some differentiated cells continue to proliferate, but
others irreversibly lose this ability. Embryonic cells often display traits that confer on them a selective
growth advantage over that of an adult cell. They proliferate vigorously, are capable of extensive migration,
secrete factors that increase the local supply of blood, and produce enzymes capable of degrading
basement membranes. These traits also are characteristic of tumor cells, including the ability to increase
local blood supply (i.e., angiogenesis). Recent data suggest that tumor angiogenesis is an important,
negative prognostic indicator for carcinomas and leukemias.59,60 Angiogenesis is now being investigated as
a potential target for cancer therapy.61 Thus, in adult organisms, mutations or conditions that activate
portions of embryonic programs for gene expression or inactivate portions of the adult program can
produce cells with many properties of malignant tumor cells.62

Stem Cells

Stem cells have the capacity for both self-renewal (i.e., proliferation without a change in phenotype) and
differentiation (i.e., changing into a new phenotype). Some stem cells have already undergone
considerable differentiation, so further differentiation is restricted to a single cell type or lineage. Other
stem cells are multipotent and differentiate into a variety of cell types (i.e., hematopoietic stem cells). It has
been difficult to demonstrate cells in adults that are totipotent (i.e., capable of differentiating into most or all
of the cell types that comprise the organism), but the recent cloning of animals from mature cells
demonstrates the persistence of stem cell characteristics even in fully differential cells.63 Also, recently,
neuronal stem cells were shown to produce a variety of blood cell types64 and adult human mesenchymal
stem cells that are present in adult marrow were shown to have the potential to differentiate to lineages of
mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma.65 Conversely,
primitive hematopoietic stem cells can give rise to muscle cells.68

In general, stem cell differentiation results in two types of changes: the expression of specialized,
differentiation-specific gene products and a partial or complete restriction of the cell’s capacity for further
proliferation. It then follows that another mechanism by which tumor cells might arise is through mutations
that render a stem cell partly or wholly unable to differentiate.

Some cells, particularly in adults, are terminally differentiated. These cells are irreversibly blocked in their
ability to proliferate, although they may perform specialized functions for a long period of time. Tumors of
terminally differentiated cells are not found. Thus, tumors of mature muscle or nerve cells do not occur,
although tumors of less differentiated myoblastic or neuronal stem cells do. Cell proliferation appears to be
incompatible with the expression of a terminally differentiated program of gene expression. Thus,
irreversible arrest of cell division and expression of the terminally differentiated phenotype are
interdependent.

In many tissues, continuous proliferation is restricted to a subpopulation of cells, the stem cells, which
undergo self-renewal, as well as differentiation, into cell types with a more restrictive proliferative potential.
It then follows that mutations or conditions that interfere with the differentiation of stem cells will result in
unbalanced proliferation and, thus, uncontrolled growth of the tissue. Mutations that drive proliferation are
associated with an accumulation and overgrowth of less-differentiated cells in the tissue. A common
feature of tumor cells is their failure to differentiate terminally under appropriate conditions either in vivo or
in culture.67–69

Extracellular Factors That Control Differentiation

During embryogenesis and in a number of adult tissues, differentiation depends on external factors. These
include insoluble factors such as ECM and both the proximity and type of neighboring cells as well as a
growing list of soluble factors. In model systems, differentiation can be induced by a variety of biologic
agents and drugs (Table 2.3.). Both the ECM and differentiation-promoting soluble factors may be
produced in an autocrine or paracrine fashion.

Table 2.3

Induction of Differentiation in Culture.

Cell-cell and cell-ECM interactions are important for both the induction and maintenance of differentiation
in several cell lineages. Although our understanding at a molecular level of insoluble factors is still
incomplete, progress has been made in identifying key molecules and pathways through which these
factors act. In the case of the ECM, specific cell surface receptors bind to particular components of the
ECM.70 It now appears that the binding of an ECM component to its cellular receptor activates an
intracellular signal transduction pathway that is analogous to the signaling pathways that have been
identified for polypeptide GFs and growth inhibitors. Tumor cells often lose their ability to sense the ECM or
neighboring cells.71

The soluble factors that regulate differentiation can be broadly classified into those that bind to cell surface
receptors and those that freely cross the plasma membrane and bind to cytoplasmic or nuclear receptors.
The first class includes molecules such as the fibroblast growth factors (FGFs) (TGF-� and TGF-�) and
hematopoietic factors such as colony-stimulating factor-1 (CSF-1), granulocyte colony-stimulating factor
(G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Flt-3
Ligand, and the interleukins. These are all polypeptides, and many were first identified as GFs or growth
inhibitors. It now is clear, however, that these factors have a multitude of effects depending on the target
cells and the cellular microenvironment.72

For example, basic FGF was identified as a fibroblast mitogen in brain and pituitary extracts, but recent
data suggest that FGF induces mesodermal differentiation in early embryos, is angiogenic, and is a
survival factor for endothelial cells.73,74 FGF also inhibits the differentiation of some cells. Terminal
differentiation into mature myotubes cannot occur unless it is withdrawn from proliferating myoblasts.
Similarly, TGF-� was first identified as a stimulator of anchorageindependent growth in mesenchymal cells
and later as an inhibitor of epithelial cell proliferation.75,76 Like FGF, TGF-� stimulates the differentiation of
some cells (i.e., keratinocytes or intestinal epithelial cells) but inhibits differentiation in others (i.e.,
myoblasts or preadipocytes). In some human tumor cells cultured in vitro or in athymic mice, TGF-� both
inhibits tumor growth and promotes a more differentiated phenotype in the remaining cells. Other studies
suggest that TGF-� induces the expression of one or more inhibitors of cyclin-dependent protein kinases
(e.g., p21, p27, p16); inhibition of these kinases, in turn, prevents the phosphorylation and, thus,
inactivation of the RB protein, thereby inhibiting cell proliferation.77–80
The membrane-permeable regulators of differentiation include retinoic acid (i.e., vitamin A) and its
derivatives (RA).81 There is strong evidence that concentration gradients of RA are critical for the
morphogenesis of some tissues in the early embryo.63 RA can stimulate or inhibit growth and differentiation
depending on the cell type. In general, RA is required for the differentiation of many epithelial cells. It
diffuses freely into cells, whereupon it binds to specific nuclear protein receptors (i.e., the retinoic acid
receptors [RARs]). In addition, other nuclear proteins, called RAR coregulators, have been found that
interact with RARs and modulate their actions in various cell types.82 These differences may explain why
specific cells and tissues differ in their responses to RA. Some differentiation-specific genes that are
regulated by RA contain sequences specific to the initiation of transcription,83 to which RA-RAR complexes
bind and thereby activate transcription.63,84 The sex steroids, estrogen and testosterone, may regulate
differentiation by similar mechanisms.

Tumor cells often produce factors that affect both growth and differentiation. Basic FGF can confer
neoplastic properties when expressed in an inappropriate cell type (e.g., a fibroblast). In addition,
inappropriate expression of FGF by one cell may stimulate the growth and affect the differentiation of
neighboring cell types.74

Intracellular Regulators

External factors and intrinsic programs of gene expression control cellular differentiation. In either case, the
expression of differentiation-specific genes generally is under the control of a small number of master
regulatory genes. Genes recently have been identified that are potential “master regulators” of
developmental stages and differentiation-specific gene expression. The most globally acting master
regulatory genes are known as homeotic genes, which were first identified as genetic loci that determined
the developmental and spatial fates of cells in embryos of the fruit fly Drosophila. Similar genes have been
identified in the genomes of higher organisms, including humans. Individual homeotic genes are expressed
at different times during development and also are expressed in different adult tissues. Some homeotic
genes code for extracellular factors, whereas others code for nuclear proteins that are probably
transcriptional regulatory factors. Homeotic genes regulate programs of differentiation as opposed to
individual differentiation-specific genes. They appear to act by initiating cascades of gene expression that
involve regulatory genes having a more restricted range of actions.85,86 Some homeotic genes may function
as tumor suppressors in normal tissues; others may promote tumorigenesis when mutated or deregulated.
Mutations in homeotic genes may reactivate portions of an embryonic program of gene expression or
suppress portions of an adult program of gene expression.87–89

DNA Methylation

In many cases, cells must go through one or more rounds of DNA replication before they can differentiate.
This requirement may be because there often is a need to modify the pattern of DNA methylation before
differentiation begins. DNA methylation in eukaryotes involves addition of a methyl group to the carbon/5
position of the cytosine ring.90 Changes in DNA methylation commonly are introduced during DNA
replication. The methylation of DNA on specific cytosine residues is believed to contribute to the changes
in gene expression that occur during development. Presumably, DNA methylation affects gene expression
because the transcriptional regulatory proteins that bind to methylated DNA differ from those that bind to
unmethylated DNA. Many neoplastic tissues are hypomethylated relative to their normal counterparts,91,92
and, indeed, pharmacologic agents that alter the pattern of DNA methylation induce differentiation in a
number of cultured cell lines. DNA methylation is probably not a universal mechanism for differentiation,
however, and some cells can be induced to differentiate with either minimal or no change in cell cycle
progression.93

Differentiation and Cancer Therapy

Analysis of differentiation by tumor cells often provides valuable information for both the diagnosis and
therapy of human cancers. As tumor cells grow and die, they can release glycoproteins and other products
similar to those of fetal tissues, and these oncofetal products can be detected in serum or other body fluids
to assist in diagnosis, follow-up, and selection of therapies. Examples include estrogen receptors and α-
lactalbumin in breast cancer, prostate-specific antigen and prostatic acid phosphatase in prostate cancers,
and myoglobin and desmin in sarcomas.94
Elevations of these markers in the serum often predict relapse of the neoplasm before any sign by routine
examination or radiographic tests. In general, the specificity of such markers for a given neoplasm is poor
because minor elevations also occur with inflammatory and other benign conditions or with several types of
neoplasms. In carcinoma of unknown primary site, tissue markers for neuroendocrine differentiation select
for a subgroup of patients with improved response to chemotherapy.95

Some tumor cells can be induced to differentiate terminally. This has been shown most extensively in
cultured cell lines (see Table 2.3) but also in experimental animals.80,96,97 After tumor cells have been
induced to undergo terminal differentiation, their ability to grow as a tumor often is stably suppressed. In
contrast to most anticancer drugs, which have nonspecific toxicity to both normal and cancer cells, drug-
induced differentiation can be demonstrated with agents (or drug levels) that exert minimal effects on
normal cells.96 These observations have stimulated increased interest in clinical applications of
differentiating agents to provide therapeutic gain with minimal toxicity.139

A number of agents known to induce differentiation in various model systems have been used clinically
(see Table 2.3). Some are useful only in a particular type of tumor; for example, estrogens and androgens
have been useful in treating some breast, prostate, and gynecologic tumors, providing that tumor cells
express the appropriate nuclear receptor. Other differentiation-inducing drugs have been more widely
studied. For example, high doses of RA, hexamethylene bisacetamide (HMBA), or 5-azacytidine, which is
an inhibitor of DNA methylation, can induce differentiation and inhibit the growth of several types of tumors
in laboratory models.97 HMBA was found to be active in patients with myelodysplastic syndromes,98 and
complete remission was achieved with direct evidence of terminal differentiation of leukemic cells to clonal
granulocytes.

In fresh cultures of human promyelocytic leukemia, retinoid-induced differentiation, similar to the effects
seen in passaged leukemia cell lines, has been observed.99 All-trans-retinoic acid has been used in clinical
therapy of promyelocytic leukemia with promising results,100,101 and retinoids have been used in combination
with interferon-α to produce responses in patients with squamous cell cancer of the skin or cervix.102
Because differentiating agents may inhibit tumor cell growth by multiple mechanisms, it is difficult to prove
specific differentiating actions of these agents when used in patients.101,103 For example, retinoids not only
induce differentiation in leukemia; they also downregulate anti-apoptotic genes.104 An exception are studies
in leukemias where sequential samples are readily available, differentiation markers are well established,
and the clonality of differentiated cells can be ascertained by methods such as molecular cytogenetics
(FISH).99

Retinoids and other differentiating agents also are used in clinical trials to prevent cancer in patients with
premalignant lesions or a high risk for developing cancer of the breast, cervix, colon, skin, lung, or oral
cavity. Early results are encouraging, including the reversal of oral leukoplakia and prevention of second
neoplasms in patients with treated squamous cell carcinoma of the head and neck.103,105 Greater knowledge
about the molecular basis for the control of differentiation should lead to more accurate predictions,
however, as well as rational design of therapies for controlling tumor growth by manipulating the state of
differentiation.97,106,107

Apoptosis

Programmed cell death (PCD), also termed apoptosis, is the necessary mechanism complementary to
proliferation that ensures homeostasis of all tissues. It has been estimated that 50 to 70 billion cells perish
each day in the average adult because of PCD,108 a process by which, in a year, each individual will
produce and eradicate a mass of cells equal to its entire body weight. This process needs to be highly
regulated since defects in the apoptotic machinery will lead to extended cell survival and may contribute to
neoplastic cell expansion. Extended cell survival also creates a permissive environment for genetic
instability and accumulation of mutations. Furthermore, defects in apoptotic pathways confer resistance to
chemotherapy, radiation, and immune-mediated cell destruction.

Three major pathways have been elucidated so far, which all result in the activation of caspase-3, a
cysteine proteinase that cleaves substrates after aspartic acid (asp) residues. One is the
mitochondrial/cytochrome C pathway, largely mediated through Bcl-2 family members, which results in
activation of Apaf-1, caspase-9, and then caspase-3 (Fig. 2.4). The second signals ligation of members of
the TNF-receptor family (e.g., Fas, TRAIL receptors) and activates caspase-8 and subsequently caspase-
3. Finally, granzyme B (a cytolytic T-cell product) directly cleaves and activates several caspases, resulting
in apoptosis.

Figure 2.4

Mitochondrial (MC)/Bcl-2 (right) and death receptor pathways to apoptosis.

Apoptosis is a genetically determined process. Cell death during development of the nematode C. elegans
involves the molecules CED-3 and CED-4, which are required for cell death, and CED-9, which protects
cells from death. In mammals, CED-3 homologs constitute a family of cystein proteases with aspartate
specificity, formerly called the ICE (interleukin-1alpha-converting enzyme) family and now designated
caspases,109 which are the key effector proteins of apoptosis in mammalian cells.110 The discovery that
human Bcl-2 has functional and structural similarity to CED-9 demonstrated that programed cell death in
mammalian cells occurs by a highly conserved mechanism as apoptosis in the nematode.111,112

The PCD cascade can be divided into several stages (see Fig. 2.4). Multiple signaling pathways lead from
death, triggering extracellular or intracellular agents to a central control and an execution stage. In this
stage, the activation of CED-3/caspases occurs, which leads to the characteristic “apoptotic” structural
lesions accompanying cell death: cytoplasmic and chromatin condensation and DNA fragmentation. Many
environmental, pharmacologic, or physiologic stimuli can trigger apoptosis, a selection of which is listed in
Table 2.4.

Table 2.4

Proteins Involved in the Regulation of Apoptosis.

Central Role of Caspases in Apoptosis

Caspases are zymogens: they exist as inactive polypeptides that can be activated by removal of the
regulatory prodomain and assembled into the active heteromeric protease. Currently, the caspase family
consists of 13 members (see Table 2.4). They encompass a death domain (DD), a death effector domain
(DED), and a caspase-recruitment domain (CARD).113 The DD is present in members of the TNF receptor
family and is involved in the early events of the signaling pathway. The DED and CARD are critical in the
downstream portion of the pathways by recruiting caspases to the plasma membrane before their
activation. Recent studies have shown that the apoptotic cascade triggered by cytochrome C and dATP is
mediated by binding of caspase-9 to Apaf-1 through CARD/CARD interactions.114 Caspase-9 becomes
activated and, in turn, activates and cleaves caspase-3. NMR spectorscopy data provide evidence that
basic/acidic surface polarity in the CARD domain is highly conserved and may represent a general mode
for CARD/CARD interaction.115 Caspase-9 deletion in knockout mice prevents activation of caspase-3 in
embryonic brains in vivo, leading to perinatal death with a markedly enlarged and malformed cereburm.116
Caspase-9-deficient thymocytes show resistance to dexamethasone but not to Fas-mediated apoptosis,
implicating a functional diversification of caspase cascades, depending on the external stimulus.

Caspases can be grouped into three subfamilies based on their specificities. Group I, or ICE, subfamily of
caspases (caspase-1, -4, and -5) prefer the tetrapeptide sequence WEHD and are believed to play a role
mainly in inflammation, whereas members of group II (caspases-2, -3, and -7) and group III (caspases-6,
-8, -9, and -10) display specificity for DEXD and (I/L/V)EXD, respectively, and are mainly involved in
apoptosis.117–119 The finding that caspases-8 and -10 each contain two N-terminal located DEDs that enable
them to associate with death receptors has placed these two caspases upstream in the apoptotic activation
pathway.120–122 In turn, caspase-3 appears to be a downstream central executioner123–125 that can directly
process pro-caspases-2, -6, -7, and -9.126,127 Findings by several groups have revealed that the activation of
caspase-3 requires the Ced-4 homolog, Apaf-1, and pro-caspase-9, as well as dATP and cytochrome C.128–
130
Hence, caspase-9 is upstream in the pathway and is regulated by Bcl-2 family genes. For murine
caspase-11 and -12, no human counterparts have been described so far. Recently, the isolation of human
caspase-13 (ERICE) from the ICE subfamily was reported.131 The demonstration that activation of ERICE is
mediated by caspase-8 has supported a potential downstream role for active ERICE in caspase-8–
mediated cell death.

There is also evidence that cell death can proceed in the absence of caspases, perhaps through
alterations in the mitochondrial membrane permeability transition (PT).132,133

Substrates of caspases

Activated caspases cleave numerous targets resulting in the so-called “death of a thousand cuts.” Caspase
targets include cytoskeleton proteins (nuclear lamins, actin, gelsolin), regulators of DNA repair (poly ADP-
ribose polymerase [PARP]), degradation of nuclear DNA by activation of DNA-dependent protein kinase,
and deactivation of the inhibitor of caspase-activated deoxyribonuclease protein (ICAD)134; RNA splicing
(U1 protein), nuclear mitotic apparatus protein (NuMA), and cell cycle proteins (including Rb and p21-
activated kinase [PAK]).135 Caspases are also involved in extracellular apoptotic events including the
cleavage of apoptotic bodies and exposure of phosphatidylserine on the cell surface. Caspase-dependent
cleavage of p21136 and Rb137 during DNA-damage-induced apoptosis provides one of the potential links
between apoptosis and cell cycle progression. Recent studies demonstrated that Bcl-2, Bcl-XL, and XIAP
proteins are also substrates for caspase cleavage.138–140 Cleavage of these proteins releases a C-terminal
product that lacks the BH4 domain and acts as a death effector. Hence, once caspase activation has been
initiated, proteins that inhibit apoptosis are functionally inactivated and converted into peoapoptotic
efectors. Since drug resistance is associated with an inability of tumor cells to undergo apoptosis, direct
activation of caspases in cancer cells may be an effective strategy to kill resistant cells. One of the
proposed approaches is to induce intracellular cleavage of caspase-1 or caspase-3 by a nontoxic, lipid-
permeable, dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs.141 Using this
chemically induced dimerization, it was possible to induce rapid apoptosis in a Bcl-XL-independent manner.
Obviously, caspase activators should be selective for cancer cells. Whether these approaches can be
realized is presently uncertain.

Natural Inhibitors of Caspases

The function of caspases, even after activation by cleavage, is subject to inhibition by other physiologic
caspase inhibitors, thereby preventing unwanted or accidental proteolysis. Alterations in the expression or
function of these proteins may confer resistance of tumor cells to the apoptotic stimuli. Viral proteins
including CrmA (inhibitor of caspase-1 and -8) and p35 (which inhibits almost all caspases) were the first
described caspase inhibitors.142 The decoy protein, FLICE-inhibitory protein (FLIP), which prevents the
binding of FLICE to its cofactor FADD, inhibits caspase-8 FADD-like interleukin-1B-converting enzyme
(FLICE), which is required for its activation. The recent discovery of the CARD domain-containing protein
ARC (apoptosis repressor with caspase recruitment domain) suggests the existence of decoy proteins for
other caspases. A new family of proteins known as IAPs (for inhibitors of apoptosis proteins) was identified
via homology with the baculovirus IAP genes and includes IAP1, IAP2, NAIP, XIAP, and survivin.142–147
Survivin seems to preferentially target caspase-9. XIAP, c-IAP1, and c-IAP2 prevent the proteolytic
processing of pro-caspases-3, -6, and -7 by preventing the conformational changes of pro-caspase-9
required for downstream activation.148,149 Additionally, active caspase-3 function is directly inhibited by the
binding of cleaved caspase-3 or -7 by XIAP, c-IAP1, c-IAP2, and survivin. These findings suggest that the
ratio of caspases to IAPs is likely to be critical. However, since IAPs function by blocking caspase
activation, they may not be able to prevent cell death induced by caspase-independent mechanisms.

Loss of IAP-related genes may cause cell death in mammalian cells and certain disorders, that is, NAIP
mutations are observed in two-thirds of patients with spinal muscular atrophy144 c-IAP2 and a novel gene
MLT are rearranged in the t(11,18)150 found in MALT lymphomas, potentially conferring a survival
advantage to lymphoma cells and in some cases rendering them immune to Fas-induced apoptosis.151

A new human gene survivin, has been described that encodes a structurally unique IAP apoptosis inhibitor
that is undetectable in terminally differentiated adult tissues but prominently expressed in transformed cell
lines and in all of the most common human cancers of lung, colon, pancreas, prostate, and breast and in
high-grade non-Hodgkin’s lymphomas.152 Survivin is the first apoptosis inhibitor that is selectively
expressed in the G2-M cell cycle-phase and directly associates with mitotic spindle microtubules.153
Inhibition of the survivin-tubulin interaction by microtubule-disrupting agents such as vincristine or
nocodazole or mutagenesis of the caspase-binding BIR domain154 results in increased caspase-3 activity
and induction of apoptosis in G2-M-synchronized cells. Therefore, survivin appears to be a novel apoptotic
guardian of a cell cycle checkpoint. High levels of survivin expression are associated with poor clinical
outcome in neuroblastoma and colon and gastric cancers.155–157 Intriguingly, the coding strand of survivin is
extensively complementary to that of effector cell protease receptor-1 (EPR-1),158 although they are coded
from separate genes located at 17q25.159 The finding that downregulation of survivin by overexpression of
ERP-1 in vitro increases apoptosis and inhibits growth of transformed cells has supported a potential role
for endogenous ERP-1 as a natural antisense160 and survivin as a potential new target for apoptosis-based
therapy.

Death Receptors

Cells require both internal and external means of regulating the activation of caspases and the death
machinery. Cell surface death receptors can, depending on other contextual events, transmit apoptosis
signals in response to external stimuli such as death ligands, growth factor withdrawal, or
chemotherapeutic agents. Death receptors belong to the tumor necrosis factor (TNF) receptor family and
have a characteristic cysteine-rich extracellular domain161 and a homologous cytoplasmic “death domain”162
that initiates apoptotic signaling inside the cell. These receptors can induce apoptotic cell death within
hours after ligand binding and may exert their apoptogenic effects differentially in diverse cell types,163
depending on downstream signaling.

Fas/Fas Ligand

Fas ligand (FasL) is a type II membrane protein predominantly expressed in activated T cells. It is cleaved
by a metalloproteinase to produce a soluble form. Recent data indicate that the membrane-bound form of
FasL is functional, whereas shedding of soluble FasL inhibits cytotoxicity and may prevent the killing of
healthy bystander cells by cytotoxic T cells.164 Downregulation of Fas receptors and killing of activated T-
lymphocytes through the constitutive expression of Fas-ligand on tumor cells has been suggested as a
mechanism for pathologic suppression of immune surveillance.165 Such “immune privilege” has been
demonstrated in melanomas and colon cancers.166,167 Binding of FasL to Fas (CD95) or cross-linking Fas
with agonistic antibodies results in receptor trimerization.162,168 Adapter proteins (FADD/MORT1 and RAIDD)
bind to DD via their own DDs.169–172 A separate DED (of FADD/MORT1) binds to the prodomain of the
caspase-8 (FLICE/MACH) and thereby links of the Fas death inducing signaling complex (DISC) with
proteases173,174 and thereby apoptosis. Another pathway involves the Fas DD175 binding protein Daxx, which,
in turn, activates the c-Jun NH2-terminal kinase (JNK), the JNK kinase kinase ASK1 (apoptosis signal-
regulating kinase 1), and Bcl-2; however, the importance of this pathway is uncertain. Observations from
several studies176–178 suggest that a functional Fas pathway requires intact p53 and thus provide a potential
mechanism for p53-mediated resistance of cancer cells to chemotherapy. A p53-binding sequence has
been identified in the Fas promoter179 and gene restoration therapy with p53 results in upregulation of
Fas.178

The CD95 system is an important regulator of T-cell cytotoxicity that is involved in the killing of mature T
cells after immune response and killing of targets by cytotoxic T cells and natural killer cells. A frame shift
mutation that renders cells resistant to Fas-mediated apoptosis has been found in adult T-cell leukemia.
This finding has suggested that mutation of Fas gene may be one of the mechanisms in the progression of
ATL.180 Enthusiasm for the clinical use of Fas as a target is dampened by the observation that anti-Fas
antibody induces rapid (within hours) death of mice from fulminant hepatic toxicity.181,182 Soluble Fas L may
be less liver toxic but induces less apoptosis. This finding may explain why high soluble FasL levels found
in many cancers are not associated with toxicity.183 As a consequence, no trials in humans are underway.
Inhibitors of death receptor signaling

Downstream regulatory factors can suppress Fas/FasL death signaling. FLIP (for FLICE-inhibitory
proteins) and several viral homologs v-FLIP184 interact with the adapter protein FADD, inhibiting the
interaction of FLICE with death receptors (CD95 death receptor169 TRAMP185–187 and TRAIL-R) and thereby
protecting cells against death-receptor-induced apoptosis. This finding may contribute to the oncogenicity
of several FLIP-encoding viruses. The human cellular homologue, designated FLIP,188 is predominantly
expressed in muscle and lymphoid tissues. High levels of FLIP protein are also detectable in melanoma
cell lines and in primary malignant melanomas but not in normal melanocytes, indicating that FLIP
upregulation probably occurs during tumorigenesis. Downregulation of FLIP with actinomycin D correlates
with acquisition of TRAIL sensitivity in resistant melanomas.189 A novel cell-surface expressed gene toso
appears to interfere with caspase-8 processing,190 therefore inhibiting Fas-mediated apoptosis in T cells.
The FAIM (Fas inhibitory molecule) isolated from B-cells191 is another molecule that produces substantial,
but not complete, resistance against Fas-mediated apoptosis. Fas was also recently implicated in the
development of the multidrug resistance phenotype192 involving the MDR1 gene product P-glycoprotein (P-
gp).

TRAIL and Its Receptors

TRAIL (“TNF-related apoptosis inducing ligand” or APO2-L) is a molecule that binds to a different family of
death-inducing receptors DR4, DR5. These receptors bind to and activate caspases through FLICE2
(FADD-like interleukin-1�-converting enzyme2). Subsequently, nonsignaling decoy receptors (DcR1,
DcR2) were identified in normal human tissues but not in most cancer cell lines examined. Their
recognition of TRAIL may prevent TRAIL from binding to functional TRAIL receptors, therefore blocking
and not transducing the cell death signal. At this point in time, the definitive role(s) of TRAIL in apoptosis
remains to be determined since the presence of “protective” TRAIL receptors does not correspond to
resistance or sensitivity to TRAIL-mediated apoptosis in some systems.189 The fact that DR4 and DR5 are
expressed in many tumors, whereas DcR1 and DcR2 are expressed predominantly in normal tissues,
suggests that TRAIL could differentially induce apoptosis in tumor cells, but exceptions to this paradigm
already exist.

TRAIL has been evaluated as a possible therapeutic agent and appears to have more promise than Fas.
Distinguishing TRAIL from FasL is the observation that TRAIL seems to only induce apoptosis in malignant
cell lines and not normal cell lines. In melanoma cell lines, TRAIL induces apoptosis.193,194 Recombinant
soluble TRAIL induced significant apoptosis in myeloid and lymphoid cell lines and decreases in viability
were observed in 20% of samples from patients with hematologic malignancies.195 Among glioma cell lines,
which preferentially express DR4 and 5, but not the decoy receptors, 10/12 cell lines were sensitive to
TRAIL.196 In breast cancer, TRAIL induced >90% apoptosis in only 1/8 cell lines.197

A variety of factors may affect TRAIL sensitivity. There does not appear to be synergism with FasL, and
neither ATRA nor MDR1 affects sensitivity.195 P53 status also appears unrelated to TRAIL sensitivity;
however, high Bcl-2 levels inhibit sensitivity.196 Among sensitive melanoma cell lines, the levels of
DR4/DR5 correlated with sensitivity in one study193 but not in another.194 Resistance to TRAIL was shown to
be secondary to loss of cell surface expression secondary to either gene loss (4/9 lines) or because it was
trapped in the cytoplasm.198 Resistance has also been correlated with high levels of expression of FLIP, the
TRAIL inhibitor, in resistant melanoma cell lines;194 however, a correlation was not observed in all studies.
Expression of the inhibitory receptor TRID was also reported.197 Stimulation of cells with CD40-CD40L
leads to downregulation of TRAIL and upregulation of TNF and Fas to promote B cell survival.199
Combined, these data suggest that TRAIL is capable of inducing apoptosis in malignancies, including
those of hematologic origin, but that multiple mechanisms of resistance likely affect sensitivity to TRAIL.
Modification of the molecule by the introduction of a leucine zipper promotes multimerization. This modified
molecule, LZ-TRAIL, increases killing of human breast cancer cell lines and mouse cell lines and confers a
survival advantage to mice injected with the breast cancer cell line MDA-231. In an important distinction
from FasL, no hepatotoxicity was observed.200

Similar to many of the other apoptosis-inducing molecules discussed below, preclinical data suggest that
the greatest efficacy with TRAIL may be in its combination with conventional chemotherapy.
Structure and Function of BCL-2-Related Proteins

The Bcl-2 family of proteins consists of both inhibitors and promoters of PCD. There are four important Bcl-
2 structural homology motifs: BH1, BH2, and BH3, present in both the anti- and pro-survival subfamilies,
and BH4, present only among antiapoptotic proteins.201–204 Most antiapoptotic proteins contain BH1 and
BH2, and those closely resembling Bcl-2 contain all four domains. The proapoptotic proteins form two
subfamilies. The Bax group includes Bax, Bak, and Bok (Mtd), resembles Bcl-2, and contains BH1, 2, and
3 domains. The BH3 domain group encompasses seven family members (Bik, Blk, Hrk, BNIP3, BimL, Bad,
Bid, EGL-1) that possess only the BH3 domain, which is essential for their function.

Many Bcl-2-family proteins can associate with each other through a complex network of homo- and
heterodimers205 that depend on interactions between the BH1, BH2 or BH3 domains.201,206 Bcl-2 forms
homodimers or heterodimers with Bax, Bcl-XL, Bcl-XS, Mcl-1, and BAD.205–208 The ratio of antiapoptotic
versus proapoptotic dimers is important in determining resistance of a cell to apoptosis, but, in most cases,
the functional significance of these interactions has not been explored. However, mutational analysis
studies and concerns about the methods used to determine these interactions have raised questions about
the requirement for direct protein-protein interactions between the anti- and proapoptotic BCL2 family
members.209–211 Also, recent analyses of cells expressing various levels of Bcl-2 and Bax have revealed that
the degree of protection against apoptosis correlates with the amount of Bcl-2 that is free of Bax, rather
than the number of Bcl-2-Bax heterodimers.212 Deletion mutants of Bcl-2 lacking the BH4 domain, which
permits interaction with proteins, including Bag-1, Raf-1, calcineurin, p53-binding protein, and Nip1-3,
exhibit either loss of function or dominant-inhibitory activity and thereby paradoxically promote
apoptosis.213–215

Post-transcriptional modifications: Phosphorylation of BAD and Bcl-2.

Post-translational changes of Bcl-2 and BAD can affect protein-protein interactions and subsequent
activity. Phosphorylation of BAD by PKA or Akt216 results in decreased apoptosis.217 Alternatively,
dephosphorylation of BAD by the Ca2+-activated protein phosphatase calcineurin enhances BAD
heterodimerization with Bcl-XL and promotes apoptosis.218 The function of Bcl-2 also appears to be
modulated by phosphorylation; however, the consequence of this effect remains controversial. Studies
have demonstrated increased resistance to apoptosis when Bcl-2 is phosphorylated on serine 70 (by
PKCα) in response to IL-3, erythropoietin, or bryostatin.219–222 Furthermore, enforced phosphorylation of Bcl-
2 by bryostatin in low-Bcl-2-expressing REH cells induced a >10-fold increase in resistance to drug-
induced cell death.221 In contrast, other studies have suggested that phosphorylation results in the loss of
function and proapoptotic ability. In this context, the administration of taxol or other microtubule-damaging
drugs to leukemia, lymphoma, and breast and prostate cancer cell lines induces serine phosphorylation of
Bcl-2 and cell death.223–226 The finding that the c-Jun N-terminal kinase may also be a Bcl-2 kinase227
provides a hypothetical mechanism for the downstream events following JNK/SAPK pathway activation by
several proapoptotic stimuli.228,229 Intriguingly, overexpression of the kinase ASK-1 or the Fas-binding
protein Daxx leads to the activation of JNK/SAPK and apoptosis.175,230 These findings have supported a link
between Fas-induced apoptosis and Bcl-2 checkpoint control. Since there are five serine and three
threonine amino acids present in the loop domain of Bcl-2 where phosphorylation occurs, differences in the
site of phosphorylation or the kinase acting on Bcl-2 may account for the diametrically opposed effects of
Bcl-2 phosphorylation on apoptosis induction that have been reported.231–233

Other means of modulating the function of Bcl-2 have been observed. Cleavage of the loop domains of
Bcl-2 and Bcl-XL by caspases138,234 following Fas triggering, growth factor withdrawal, alphavirus infection,
and etoposide treatment139,235–237 has been associated with degradation by the ubiquitin-dependent
proteasome complex and loss of antiapoptotic potency.238 Therefore, the ratio of cleaved versus uncleaved
protein may be another mechanism for regulating the decision of cells to undergo apoptosis that could be
exploited therapeutically. In addition, the ratio may affect the susceptibility of tumor cells to attempts to
induce apoptosis. The demonstration that mimicking phosphorylation of certain Bcl-2 phosphorylation sites
abolishes ubiquitin-dependent degradation and confers resistance against induction of apoptosis has
supported the inability of the proteasome to degrade phosphorylated Bcl-2.239

Mitochondria and Apoptosis

Mitochondria isolated from cells induced to undergo apoptosis can stimulate apoptosis-like destruction of
naive nuclei, whereas mitochondria purified from Bcl-2 overexpressing cells fail to confer this effect.240
Interestingly, chemical inducers of mitochondrial megapore opening can induce mitochondria derived from
normal cells to liberate factors that result in the apoptosis-like destruction of nuclei.240 Recently, this effect
has been attributed to the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria
and thereby activation of proteases (see Fig. 2.4).241,242 Two general mechanisms for release of caspase-
activating proteins from mitochondria have been proposed: one involves an osmotic dysequilibrium that
leads to an expansion of the matrix space, organelle swelling, rupture of the outer membrane, and
eventually necrosis; the other envisions opening of channels in the outer membrane, release of
cytochrome c into the cytosol, and activation of caspases resulting in apoptosis.132 A key function of Bcl-2-
like proteins is to somehow retain cytochrome c in the mitochondria.243 Models using synthetic lipid
membranes in vitro support this idea, but at present there is no direct evidence for in vivo channel
formation or ion-channel activity of Bcl-2 or Bcl-XL. Also, it remains to be determined whether the
suppression of cytochrome c release by Bcl-2 reflects the ability of Bcl-2 either to block Bax channels or to
transport cytochrome c back to the mitochondria.243,244 However, the demonstration that Bcl-2 mutants that
lack their C-terminal TM domain, and therefore inefficiently associate with mitochondria, retain partial
antiapoptotic activity has suggested that membrane targeting of Bcl-2 is not absolutely critical for its
function.207

Although caspase activation usually occurs downstream of mitochondrial permeability transition, recent
data indicate that caspases can also induce dissipation of the mitochondrial inner transmembrane potential
and therefore act upstream of mitochondria.245,246 These observations suggest that caspases and
mitochondria can engage in a circular self-amplification loop that could accelerate or coordinate the
apoptotic response. Studies in yeast have provided compelling evidence that Bax-like proteins mediate
caspase-independent death via channel-forming activity, which could promote the mitochondrial
permeability transition or puncture the mitochondrial outer membrane.132 Bax can form pores in artificial
membranes in vitro.247 In contrast, recombinant Bcl-2 or Bcl-XL inhibits opening of the purified megachannel
reconstituted into liposomes as well as megachannel opening in cells and isolated mitochondria.248 Bax
triggers a rapid caspase-dependent apoptosis, but in the presence of caspase inhibitors, a slower
nonapoptotic cell death without DNA fragmentation occurs.249 Bax-induced cytochrome c release could
result in disruption of electron transport with loss of ATP and generation of reactive oxygen species that
cause caspase-independent cell death.

The proapoptotic protein Bid is cleaved by activated caspase-8 in Fas signaling pathway (see Fig. 2.4).250
The finding that truncated Bid translocates from the cytosol to mitochondria and induces cytochrome c
release and loss of membrane potential has provided a link between death-receptor-mediated and
mitochondrial pathways.133

In conclusion, mitochondrial damage is likely to be critical in controlling cell death, either by release of
proteins that trigger caspase activation or by disruption of electron transport followed by slow, nonapoptotic
cell death.

Apoptosis Activating Factors (Apafs)

Of the Bcl-2/Bcl-XL-binding proteins in C elegans, CED-4, which serves to recruit caspases via its N-
terminus CARD251 domain and activate them,252 is probably the most important.252–258 Zou and colleagues
have isolated apoptosis activating factors (Apafs)259 that result in cleavage and activation of caspase-9.
Apaf-1 resembles CED-4 with an amino-terminal CARD domain that can bind directly to caspases251 and a
large domain containing 12 WD-40 repeats at the C-terminus thought to interact with Apaf-2/cytochrome c.
ATP may activate Apaf-1 by binding to the nucleotide-binding p-loop motif. An essential role for Apaf-1 in
the Bcl-2-regulated pathway is supported by observations of reduced apoptosis in the brain and striking
craniofacial abnormalities with impaired processing of caspases-2, -3 and -8 in Apaf-1 knockout mice.260,261
Remarkably, cells from Apaf-1 –/– mice are refractory to apoptotic stimuli controlled by Bcl-2 but respond
normally to signals from “death receptors.”

In conclusion, CED-4 or human Apafs are likely to be critical for Bcl-2 function in regulation of the
caspases.
Taken together, an intriguing network of genes controlling proliferation, differentiation, and apoptosis has
evolved that provides new targets for cancer therapy that were unimaginable only a few years ago.

References
1. Hartwell L H, Weinert T A. Checkpoints: controls that ensure the order of cell cycle events.
Science. 1989;246:629–634. [PubMed]

2. Morgan D O. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell
Dev Biol. 1997;13:261–291. [PubMed]

3. Polyak K, Lee M -H, Erdjument-Bromage H, Koff A, Roberts J M, Tempst P, Massague J. Cloning

of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular
antimitogenic signals. Cell. 1994;78:59–66. [PubMed]
4. Polyak K, Kato J Y, Solomon M J, Sherr C J, Massague J, Roberts J M, Koff p27Kip1, a cyclin-Cdk
inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes
Dev. 1994;8:9–22. [PubMed]

5. Koepp D M, Harper J W, Elledge S J. How the cyclin became a cyclin: regulated proteolysis in the
cell cycle. Cell. 1999;97:431–434. [PubMed]

6. Zugmaier G, Lippman M E. Effects of TGF beta on normal and malignant mammary epithelium.
Ann N Y Acad Sci. 1990;593:272–275. [PubMed]

7. Druker B J, Mamon H J, Roberts T M. Oncogenes, growth factors, and signal transduction. N Engl
J Med. 1989;321:1383–1391. [PubMed]

8. Gibbs J B, Oliff A, Kohl N E. Farnesyltransferase inhibitors: Ras research yields a potential cancer
therapeutic. Cell. 1994;77:175–178. [PubMed]

9. Pardee A B. G1 events and regulation of cell proliferation. Science. 1989;246:603–608. [PubMed]

10. Tubo R A, Berezney R. Pre-replicative association of multiple replicative enzyme activities with the
nuclear matrix during rat liver regeneration. J Biol Chem. 1987;262:1148–1154. [PubMed]

11. Challberg M D, Kelly T J. Animal virus DNA replication. Ann Rev Biochem. 1989;58:671–717.
[PubMed]

12. Matsushime H, Roussel M F, Ashmun R A, Sherr C J. Colony-stimulating factor 1 regulates novel

cyclins during the G1 phase of the cell cycle. Cell. 1991;65:701–713. [PubMed]

13. Goodrich D W, Wang N P, Qian Y W, Lee E Y, Lee W H. The retinoblastoma gene product
regulates progression through the G1 phase of the cell cycle. Cell. 1991;67:293–302. [PubMed]

14. Matsushime H, Ewen M E, Strom D K. et al. Identification and properties of an atypical catalytic
subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell. 1992;71:323–334. [PubMed]

15. Connell-Crowley L, Harper J W, Goodrich D W. Cyclin D1/Cdk4 regulates retinoblastoma protein-

mediated cell cycle arrest by site-specific phosphorylation. Mol Biol Cell. 1997;8:287–301.
[PubMed]

16. Nevins J R, Leone G, DeGregori J, Jakoi L. Role of the Rb/E2F pathway in cell growth control
[review] J Cell Physiol. 1997;173:233–236. [PubMed]

17. Wu C L, Zukerberg L R, Ngwu C, Harlow E, Lees J A. In vivo association of E2F and DP family
proteins. Mol Cell Biol. 1995;15:2536–2546. [PubMed]
18. Geng Y, Eaton E N, Picon M. et al. Regulation of cyclin E transcription by E2Fs and
retinoblastoma protein. Oncogene. 1996;12:1173–1180. [PubMed]

19. Hurford R K J, Jr, Cobrinik D, Lee M H, Dyson N. pRB and p107/p130 are required for the
regulated expression of different sets of E2F responsive genes. Genes Dev. 1997;11:1447–1463.
[PubMed]

20. Ohtsubo M, Theodoras A M, Schumacher J, Roberts J M, Pagano M. Human cyclin E, a nuclear

protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995;15:2612–2624. [PubMed]

21. Leng X, Connell-Crowley L, Goodrich D, Harper J W. S-Phase entry upon ectopic expression of
G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation. Curr Biol.
1997;7:709–712. [PubMed]

22. Lukas J, Herzinger T, Hansen K. et al. Cyclin E-induced S phase without activation of the pRb/E2F
pathway. Genes Dev. 1997;11:1479–1492. [PubMed]

23. Amundson S A, Myers T G, Fornace A J, Jr Roles for p53 in growth arrest and apoptosis: putting
on the brakes after genotoxic stress. Oncogene. 1998;17:3287–3299. [PubMed]

24. Hoekstra M F. Responses to DNA damage and regulation of cell cycle checkpoints by the ATM
protein kinase family. Curr Opin Genet Dev. 1997;7:170–175. [PubMed]

25. Unger T, Juven-Gershon T, Moallem E. et al. Critical role for Ser20 of human p53 in the negative
regulation of p53 by Mdm2. EMBO J. 1999;18:1805–1814. [PubMed]

26. Sakaguchi K, Herrera J E, Saito S. et al. DNA damage activates p53 through a phosphorylation-
acetylation cascade. Genes Dev. 1998;12:2831–2841. [PubMed]

27. Waterman M J, Stavridi E S, Waterman J L, Halazonetis T D. ATM-dependent activation of p53

involves dephosphorylation and association with 14-3-3 proteins. Nature Genet. 1998;19:175–178.
[PubMed]

28. Banin S, Moyal L, Shieh S. et al. Enhanced phosphorylation of p53 by ATM in response to DNA
damage. Science. 1998;281:1674–1677. [PubMed]

29. Khanna K K, Keating K E, Kozlov S. et al. ATM associates with and phosphorylates p53: mapping
the region of interaction. Nature Genet. 1998;20:398–400. [PubMed]

30. de Lange T. Activation of telomerase in a human tumor. Proc Natl Acad Sci U S A. 1994;91:2882–
2885. [PubMed] [Free Full text in PMC]

31. Buchkovich K J. Telomeres, telomerase, and the cell cycle. Prog Cell Cycle Res. 1996;2:187–195.
[PubMed]

32. Saretzki G, Sitte N, Merkel U, Wurm R E, von Zglinicki T. Telomere shortening triggers a p53-
dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments. Oncogene.
1999;18:5148–5158. [PubMed]

33. Fabrikant J I, Cherry J. The kinetics of cellular proliferation in normal and malignant tissues. V.
Analysis of labeling indices and potential tissue doubling times in human tumor cell populations. J
Surg Oncol. 1969;1:23–47. [PubMed]

34. Fukuda K, Iwasaka T, Hachisuga T. et al. Immunocytochemical detection of S-phase cells in

normal and neoplastic cervical epithelium by anti-BrdU monoclonal antibody. Anal Quant Cytol
Histol. 1990;12:135–138. [PubMed]
35. Teodori L, Trinca M L, Goehde W. et al. Cytokinetic investigation of lung tumors using the anti-
bromodeoxyuridine (BUdR) monoclonal antibody method: comparison with DNA flow cytometric
data. Int J Cancer. 1990;45:995–1001. [PubMed]

36. Carlsson M, Totterman T H, Matsson P, Nilsson K. Cell cycle progression of B-chronic lymphocytic
leukemia cells induced to differentiate by TPA. Blood. 1988;71:415–421. [PubMed]

37. Tannock I. Cell kinetics and chemotherapy: a critical review. Cancer Treat Rep. 1978;62:1117–
1133. [PubMed]

38. Fingert HJ, Campisi J, Pardee AB. Molecular biology and biochemistry of cancer. In Gynecologic
Oncology. Edited by R Knapp, R Berkowitz. New York: Macmillan, 1991: p 30.

39. Weber G. Biochemical strategy of cancer cells and the design of chemotherapy: G. H. A. Clowes
Memorial Lecture. Cancer Res. 1983;43:3466–3492. [PubMed]

40. Juliano R. Cooperation between soluble factors and integrin-mediated cell anchorage in the control
of cell growth and differentiation. Bioessays. 1996;18:911–917. [PubMed]

41. Liotta L A. Tumor invasion and metastases—role of the extracellular matrix: Rhoads Memorial
Award lecture. Cancer Res. 1986;46:1–7. [PubMed]

42. Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in
human cancer. Adv Cancer Res. 1996;68:67–108. [PubMed]

43. Sherr C J. Cancer cell cycles. Science. 1996;274:1672–1677. [PubMed]

44. Motokura T, Bloom T, Kim H G. et al. A novel cyclin encoded by a bcl1-linked candidate oncogene.
Nature. 1991;350:512–515. [PubMed]

45. Wang T C, Cardiff R D, Zukerberg L. et al. Mammary hyperplasia and carcinoma in MMTV-cyclin
D1 transgenic mice. Nature. 1994;369:669–671. [PubMed]

46. Kamb A. Cyclin-dependent kinase inhibitors and human cancer. Curr Top Microbiol Immunol.
1998;227:139–148. [PubMed]

47. Galaktionov K, Lee A K, Eckstein J. et al. CDC25 phosphatases as potential human oncogenes.
Science. 1995;269:1575–1577. [PubMed]

48. Galaktionov K, Chen X, Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature.
1996;382:511–517. [PubMed]

49. Goodrich D W, Lee W H. The molecular genetics of retinoblastoma. Cancer Surveys. 1990;9:529–
554. [PubMed]

50. Lee W -H. The molecular basis of cancer suppression by the retinoblastoma gene. Princess
Takamatsu Symp. 1989;20:159–170. [PubMed]

51. Prives C, Hall P A. The p53 pathway. J Pathol. 1999;187:112–126. [PubMed]

52. Akashi M, Koeffler H P. Li-Fraumeni syndrome and the role of the p53 tumor suppressor gene in
cancer susceptibility. Clin Obstet Gynecol. 1998;41:172–199. [PubMed]

53. Oliner J D, Kinzler K W, Meltzer P S, George D L, Vogelstein B. Amplification of a gene encoding a

p53-associated protein in human sarcomas. Nature. 1992;358:80–83. [PubMed]
54. Sharpless N E, DePinho R A. The INK4A/ARF locus and its two gene products. Curr Opin Genet
Dev. 1999;9:22–30. [PubMed]

55. Gurdon J B. Embryonic induction—molecular prospects. Development. 1987;99:285–306.

[PubMed]

56. Jan Y N, Jan L Y. HLH proteins, fly neurogenesis, and vertebrate myogenesis. Cell. 1993;75:827–
830. [PubMed]

57. Sorrentino V, Pepperkok R, Davis R L, Ansorge W, Philipson L. Cell proliferation inhibited by

MyoD1 independently of myogenic differentiation. Nature. 1990;345:813–815. [PubMed]

58. Umek R M, Friedman A D, McKnight S L. CCAAT-enhancer binding protein: a component of a

differentiation switch. Science. 1991;251:288–292. [PubMed]

59. Weidner N, Carroll P R, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with
metastasis in invasive prostate carcinoma. Am J Pathol. 1993;143:401–409. [PubMed]

60. Weidner N, Semple J P, Welch W R, Folkman J. Tumor angiogenesis and metastasis —correlation
in invasive breast carcinoma. N Engl J Med. 1991;324:1–8. [PubMed]

61. Bernstein L R, Liotta L A. Molecular mediators of interactions with extracellular matrix components
in metastasis and angiogenesis. Curr Opin Oncol. 1994;6:106–113. [PubMed]

62. Mintz B, Fleischman R A. Teratocarcinomas and other neoplasms as developmental defects in

gene expression. Adv Cancer Res. 1981;34:211–278. [PubMed]

63. Dolle P, Ruberte E, Kastner P. et al. Differential expression of genes encoding alpha, beta and
gamma retinoic acid receptors and CRABP in the developing limbs of the mouse. Nature.
1989;342:702–705. [PubMed]

64. Bjornson C R, Rietze R L, Reynolds B A, Magli M C, Vescovi A L. Turning brain into blood: a
hematopoietic fate adopted by adult neural stem cells in vivo [see comments] Science.
1999;283:534–537. [PubMed]

65. Pittenger M F, Mackay A M, Beck S C. et al. Multilineage potential of adult human mesenchymal
stem cells. Science. 1999;284:143–147. [PubMed]

66. Jackson K A, Mi T, Goodell M A. Hematopoietic potential of stem cells isolated from murine
skeletal muscle [in process citation] Proc Natl Acad Sci U S A. 1999;96:14482–14486. [PubMed]
[Free Full text in PMC]

67. Rheinwald J G, Beckett M A. Defective terminal differentiation in culture as a consistent and

selectable character of malignant human keratinocytes. Cell. 1980;22(2 Pt 2):629–632. [PubMed]

68. Wille J J, Jr, Maercklein P B, Scott R E. Neoplastic transformation and defective control of cell
proliferation and differentiation. Cancer Res. 1982;42:5139–5146. [PubMed]

69. Yuspa SH, Lichti U, Strickland J, et al. Aberrant regulation of differentiation in epidermal
carcinogenesis. In Growth Factors, Tumor Promoters and Cancer Genes. Edited by NH Colburn,
HL Moses, EJ Stanbridge. New York: Liss, 1988: pp 183–189.

70. Ekblom P, Vestweber D, Kemler R. Cell-matrix interactions and cell adhesion during development.
Annu Rev Cell Biol. 1986;2:27–47. [PubMed]

71. Petersen O W, Ronnov-Jessen L, Howlett A R, Bissell M J. Interaction with basement membrane

serves to rapidly distinguish growth and differentiation pattern of normal and malignant human
 breast epithelial cells Proc Natl Acad Sci U S A 1992. 899064–9068. [PubMed] [published erratum
appears in Proc Natl Acad Sci U S A 1993;90:2556]. [Free Full text in PMC]

72. Varticovski L, Druker B, Morrison D, Cantley L, Roberts T. The colony stimulating factor-1 receptor
associates with and activates phosphatidylinositol-3 kinase. Nature. 1989;342:699–702. [PubMed]

73. Lemmon S K, Riley M C, Thomas K A. et al. Bovine fibroblast growth factor: comparison of brain
and pituitary preparations. J Cell Biol. 1982;95:162–169. [PubMed]

74. Rifkin D B, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor.
J Cell Biol. 1989;109:1–6. [PubMed]

75. Rozengurt E. Early signals in the mitogenic response. Science. 1986;234:161–166. [PubMed]

76. Todaro G J, De Larco J E, Fryling C, Johnson P A, Sporn M B. Transforming growth factors

(TGFs): properties and possible mechanisms of action. J Supramol Struct Cell Biochem.
1981;15:287–301. [PubMed]

77. Moses H L, Yang E Y, Pietenpol J A. TGF-beta stimulation and inhibition of cell proliferation: new
mechanistic insights. Cell. 1990;63:245–247. [PubMed]

78. Peter M, Herskowitz I. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell
cycle [comment] Cell. 1994;79:181–184. [PubMed]

79. Sherr C J. G1 phase progression: cycling on cue [see comments] Cell. 1994;79:551–555.
[PubMed]

80. Twardzik D R, Ranchalis J E, McPherson J M. et al. Inhibition and promotion of differentiated-like

phenotype of a human lung carcinoma in athymic mice by natural and recombinant forms of
transforming growth factor-beta. J Natl Cancer Inst. 1989;81:1182–1185. [PubMed]

81. Sporn MB, Roberts AB, Goodman DS. The Retinoids. New York: Academic, 1984: p 56.

82. Glass C K, Devary O V, Rosenfeld M G. Multiple cell type-specific proteins differentially regulate
target sequence recognition by the alpha retinoic acid receptor. Cell. 1990;63:729–738. [PubMed]

83. Bennington J L. Cellular kinetics of invasive squamous carcinoma of the human cervix. Cancer
Res. 1969;29:1082–1088. [PubMed]

84. Vasios G W, Gold J D, Petkovich M, Chambon P, Gudas L J. A retinoic acid-responsive element is

present in the 5’ flanking region of the laminin B1 gene. Proc Natl Acad Sci U S A. 1989;86:9099–
9103. [PubMed]

85. Gehring W J. Homeo boxes in the study of development. Science. 1987;236:1245–1252.

[PubMed]

86. Holland P W, Hogan B L. Expression of homeo box genes during mouse development: a review.
Genes Dev. 1988;2:773–782. [PubMed]

87. Castronovo V, Kusaka M, Chariot A, Gielen J, Sobel M. Homeobox genes: potential candidates for
the transcriptional control of the transformed and invasive phenotype. Biochem Pharmacol.
1994;47:137–143. [PubMed]

88. Deschamps J, Meijlink F. Mammalian homeobox genes in normal development and neoplasia. Crit
Rev Oncogenesis. 1992;3(1–2):117–173. [PubMed]
 89. McCormick A, Campisi J. Cellular aging and senescence. Curr Opin Cell Biol. 1991;3:230–234.
[PubMed]

90. Singal R, Ginder G D. DNA methylation. Blood. 1999;93:4059–4070. [PubMed]

91. Feinberg A P, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from
their normal counterparts. Nature. 1983;301:89–92. [PubMed]
i. 92.
92. Goelz S E, Vogelstein B, Hamilton S R, Feinberg A P. Hypomethylation of DNA from benign and
malignant human colon neoplasms. Science. 1985;228:187–190. [PubMed]
i. 93.
93. Carlsson M, Totterman T H, Matsson P, Nilsson K. Cell cycle progression of B-chronic lymphocytic
leukemia cells induced to differentiate by TPA. Blood. 1988;71:415–421. [PubMed]
i. 94.
94. Gorstein F, Thor A. Tumor markers in diagnostic pathology. In Clinics in Laboratory Medicine.
Edited by F Gorstein, A Thor. Philadelphia: WB Saunders, 1990.
i. 95.
95. Hainsworth J D, Johnson D H, Greco F A. Poorly differentiated neuroendocrine carcinoma of
unknown primary site. A newly recognized clinicopathologic entity. Ann Intern Med. 1988;109:364–
371. [PubMed]
i. 96.
96. Reiss M, Gamba-Vitalo C, Sartorelli A C. Induction of tumor cell differentiation as a therapeutic
approach: preclinical models for hematopoietic and solid neoplasms. Cancer Treat Rep.
1986;70:201–218. [PubMed]
i. 97.
97. Waxman S, Rossi GB, Takaku F. The Status of Differentiation Therapy of Cancer. New York:
Raven, 1988.
i. 98.
98. Andreeff M, Stone R, Michaeli J. et al. Hexamethylene bisacetamide in myelodyplastic syndrome
and acute myelogenous leukemia: A Phase II clinical trial with a differentiation-inducing agent.
Blood. 1992;80:2604–2609. [PubMed]
i. 99.
99. Breitman T R, Collins S J, Keene B R. Terminal differentiation of human promyelocytic leukemic
cells in primary culture in response to retinoic acid. Blood. 1981;57:1000–1004. [PubMed]
i. 100.
100. Castaigne S, Chomienne C, Daniel M T. et al. All-trans retinoic acid as a differentiation
therapy for acute promyelocytic leukemia. I. Clinical results [see comments] Blood. 1990;76:1704–
1709. [PubMed]
i. 101.
101. Warrell R P, Jr, Frankel S R, Miller W H., Jr et al. Differentiation therapy of acute
promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med. 1991;324:1385–1393.
[PubMed]
i. 102.
102. Chabner B A. Biological basis for cancer treatment [comment] Ann Intern Med.
1993;118:633–637. [PubMed]
i. 103.
103. Cheson B D, Jasperse D M, Chun H G, Friedman M A. Differentiating agents in the
treatment of human malignancies. Cancer Treat Rev. 1986;13:129–145. [PubMed]
i. 104.
104. Andreeff M, Jiang S, Zhang X. et al. Expression of bcl-2-related genes in normal and AML
progenitors: changes induced by chemotherapy and retionic acid. Leukemia. 1999;13:1881–1892.
[PubMed]
i. 105.
105. Hong W K, Lippman S M, Itri L M. et al. Prevention of second primary tumors with
isotretinoin in squamous-cell carcinoma of the head and neck [see comments] N Engl J Med.
1990;323:795–801. [PubMed]
i. 106.
 106. Meyskens F L., Jr Coming of age — the chemoprevention of cancer [editorial; comment]
[see comments] N Engl J Med. 1990;323:825–827. [PubMed]
i. 107.
107. Rowley J D, Aster J C, Sklar J. The clinical applications of new DNA diagnostic technology
on the management of cancer patients. JAMA. 1993;270:2331–2337. [PubMed]
i. 108.
108. Reed J C. Dysregulation of apoptosis in cancer. J Clin Oncol. 1999;17:2941–2953.
[PubMed]
i. 109.
109. Alnemri E S, Livingston D J, Nicholson D W. et al. Humanf ICE?CED-3 protease
nomenclature. Cell. 1996;87:171. [PubMed]
i. 110.
110. Yuan J, Shaham S, Ledoux S. et al. The c. elegans cell death gene ced-3 encodes a
protein similar to mammalian interleukin-1 beta-convecting enzyme. Cell. 1993;75:641–652.
[PubMed]
i. 111.
111. Hengartner M O, Horvitz H R. Programmed cell death in Caenorhabditis elegans. Curr
Opin Genet Dev. 1994;4:581–586. [PubMed]
i. 112.
112. Vaux D L, Weissman I L, Kim S K. Prevention of programmed cell death in Caenorhabditis
elegans by human bcl-2. Science. 1992;258:1955–1957. [PubMed]
i. 113.
113. Hofmann K, Bucher P, Tschopp J. The CARD domain: a new apoptotic signalling motif.
Trends Biochem Sci. 1997;22:155–156. [PubMed]
i. 114.
114. Li P, Nijhawan D, Budihardjo I. et al. Cytochrome c and dATP-dependent formation of
Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997;91:479–489.
[PubMed]
i. 115.
115. Chou J J, Matsuo H, Duan H, Wagner G. Solution structure of the RAIDD CARD and
model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell. 1998;94:171–
180. [PubMed]
i. 116.
116. Kuida K, Haydar T F, Kuan C Y. et al. Reduced apoptosis and cytochrome c-mediated
caspase activation in mice lacking Caspase 9. Cell. 1998;94:325–337. [PubMed]
i. 117.
117. Nicholson D W, Thornberry N A. Caspases: killer proteases. Trends Biochem Sci.
1997;22:299–306. [PubMed]
i. 118.
118. Talanian R V, Quinlan C, Trautz S. et al. Substrate specificities of caspase family
proteases. J Biol Chem. 1997;272:9677–9682. [PubMed]
i. 119.
119. Thornberry N A, Rano T A, Peterson E P. et al. A combinatorial approach defines
specificities of members of the caspase family and granzyme B. Functional relationships
established for key mediators of apoptosis. J Biol Chem. 1997;272:17907–17911. [PubMed]
i. 120.
120. Fernandes-Alnemri T, Armstrong R C, Krebs J. et al. In vitro activation of CPP32 and
Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.
Proc Natl Acad Sci. 1996;93:7464–7469. [PubMed] [Free Full text in PMC]
i. 121.
121. Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. Involvement of MACH, a novel
MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell.
1996;85:803–815. [PubMed]
i. 122.
122. Muzio M, Chinnaiyan A M, Kischkel F C. et al. FLICE, a novel FADDl-homologous
ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex.
Cell. 1996;85:817–827. [PubMed]
i. 123.
123. Faleiro L, Kobayashi R, Fearnhead H, Lazebnik Y. Multiple species of CPP32 and Mch2
are the major active caspases present in apoptotic cells. EMBO J. 1997;16:2271–2281. [PubMed]
i. 124.
124. MacFarlane M, Cain K, Sun X M, Alnemri E S, Cohen G M. Processing/activation of at
least four interleukin-1 beta converting enzyme-like proteases occurs during the execution phase
of apoptosis in human monocytic tumor cells. J Cell Biol. 1997;137:469–479. [PubMed]
i. 125.
125. Takahashi A, Hirata H, Yonehara S. et al. Affinity labeling displays the stepwise activation
of ICE-related proteases by Fas, staurosporine, and Crm-A-sensitive caspase 8. Oncogene.
1997;14:2741–2752. [PubMed]
i. 126.
126. Srinivasula S M, Fernandes-Alnemri T, Zangrilli J. et al. The CED-3/interleukin-1 beta
converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2 alpha are substrates
for the apoptotic mediator CPP32. J Biol Chem. 1996;271:27099–27106. [PubMed]
i. 127.
127. Fernandes-Alnemri T, Takahashi A, Armstrong R. et al. Mch3, a novel human apoptotic
cysteine protease highly related to CPP32. Cancer Res. 1995;55:6045–6052. [PubMed]
i. 128.
128. Liu X, Kim C N, Yang J, Jemmerson R, Wang X. Induction of apoptotic programin cell-free
extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157. [PubMed]
i. 129.
129. Yang J, Liu X, Bhalla K. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c
from mitochondria blocked [see comments] Science. 1997;275:1129–1132. [PubMed]
i. 130.
130. Zou H, Henzel W J, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C.
elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 [see comments]
Cell. 1997;90:405–413. [PubMed]
i. 131.
131. Humke E W, Ni J, Dixit V M. ERICE, a novel FLICE-activatable caspase. J Biol Chem.
1998;273:15702–15707. [PubMed]
i. 132.
132. Green D R, Reed J C. Mitochondria and apoptosis. Science. 1998;281:1309–1312.
[PubMed]
i. 133.
133. Green D R. Apoptotic pathways: the roads to ruin. Cell. 1998;94:695–698. [PubMed]
i. 134.
134. Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA
degradation during apoptosis [see comments] Nature. 1998;391:96–99. [PubMed]
i. 135.
135. Enari M, Sakahira H, Yokoyama H. et al. A caspase-activated DNase that degrades DNA
during apoptosis, and its inhibitor ICAD [see comments] Nature 1998. 39143–50. [PubMed]
[published erratum appears in Nature 1998;393:396].
i. 136.
136. Fujita N, Nagahashi A, Nagashima K, Rokudai S, Tsuruo T. Acceleration of apoptotic cell
death after the cleavage of Bcl-XL protein by caspase-3-like proteases. Oncogene. 1998;17:1295–
1304. [PubMed]
i. 137.
137. Zhang Y, Fujita N, Tsuruo T. Caspase-mediated cleavage of p21Waf1/Cip1 converts
cancer cells from growth arrest to undergoing apoptosis. Oncogene. 1999;18:1131–1138.
[PubMed]
138. 138.
139. Cheng E H, Kirsch D G, Clem R J. et al. Conversion of bcl-2 to a Bax-like death effector by
caspases. Science. 1997;278:1966–1968. [PubMed]
140. 139.
 141. Grandgirard D, Studer E, Monney L. et al. Alphaviruses induce apoptosis in Bcl-2-
overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2. EMBO J.
1998;17:1268–1278. [PubMed]
142. 140.
143. Fattman C L, An B, Dou Q P. Characterization of interior cleavage of retinoblastoma
protein in apoptosis. J Cell Biochem. 1997;67:399–408. [PubMed]
144. 141.
145. MacCorkle R A, Freeman K W, Spencer D M. Synthetic activation of caspases: artificial
death switches. Proc Natl Acad Sci U S A. 1998;95:3655–3660. [PubMed] [Free Full text in PMC]
146. 142.
147. Clem R J, Miller L K. Control of programmed cell death by the baculovirus genes p35 and
iap. Mol Cell Biol. 1994;14:5212–5222. [PubMed]
148. 143.
149. Duckett C S, Nava V E, Gedrich R W. et al. A conserved family of cellular genes related to
the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 1996;15:2685–2694.
[PubMed]
150. 144.
151. Roy N, Mahadevan M S, McLean M. et al. The gene for neuronal apoptosis inhibitory
protein is partially deleted in individuals with spinal muscular atrophy. Cell. 1995;80:167–178.
[PubMed]
152. 145.
153. Rothe M, Pan M G, Henzel W J, Ayres T M, Goeddel D V. The TNFR2-TRAF signaling
complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell.
1995;83:1243–1252. [PubMed]
154. 146.
155. Uren A G, Pakusch M, Hawkins C J, Puls K L, Vaux D L. Cloning and expression of
apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis
factor receptor-associated factors. PNAS. 1996;93:4974–4978. [PubMed] [Free Full text in PMC]
156. 147.
157. Hay B A, Wassarman D A, Rubin G M. Drosophila homologs of baculovirus inhibitor of
apoptosis proteins function to block cell death. Cell. 1995;83:1253–1262. [PubMed]
158. 148.
159. Stennicke H R, Deveraux Q L, Humke E W. et al. Caspase-9 can be activated without
proteolytic processing. J Biol Chem. 1999;274:8359–8362. [PubMed]
160. 149.
161. Deveraux Q L, Roy N, Stennicke H R. et al. IAPs block apoptotic events induced by
caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 1998;17:2215–
2223. [PubMed]
162. 150.
163. Dierlamm J, Baens M, Wlodarska I. et al. The apoptosis inhibitor gene API2 and a novel
18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21)p6ssociated with mucosa-
associated lymphoid tissue lymphomas. Blood. 1999;93:3601–3609. [PubMed]
164. 151.
165. Greiner A, Seeberger H, Knorr C, Starostik P, Muller-Hermelinck H K. MALT-type B-cell
lymphomas escape the sensoring FAS-mediated apoptosis. Blood. 1998;92:484a.
166. 152.
167. Ambrosini G, Adida C, Altieri D C. A novel anti-apoptosis gene, survivin, expressed in
cancer and lymphoma. Nat Med. 1997;3:917–921. [PubMed]
168. 153.
169. Li F, Ambrosini G, Chu E Y. et al. Control of apoptosis and mitotic spindle checkpoint by
survivin. Nature. 1998;396:580–584. [PubMed]
170. 154.
171. Tamm I, Wang Y, Sausville E, Scudiero D A, Vigna N, Oltersdorf T, Reed J C. IAP-family
protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases,
and anticancer drugs. Cancer Res. 1998;58:5315–5320. [PubMed]
172. 155.
 173. Adida C, Berrebi D, Peuchmaur M. et al. Anti-apoptosis gene, survivin, and prognosis of
neuroblastoma [letter] Lancet. 1998;351:882–883. [PubMed]
174. 156.
175. Lu C D, Altieri D C, Tanigawa N. Expression of a novel antiapoptosis gene, survivin,
correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res.
1998;58:1808–1812. [PubMed]
176. 157.
177. Kawasaki H, Altieri D C, Lu C D. et al. Inhibition of apoptosis by survivin predicts shorter
survival rates in colorectal cancer. Cancer Res. 1998;58:5071–5074. [PubMed]
178. 158.
179. Altieri D C. Xa receptor EPR-1. FASEB J. 1995;9:860–865. [PubMed]
180. 159.
181. Ambrosini G, Adida C, Sirugo G, Altieri D C. Induction of apoptosis and inhibition of cell
proliferation by survivin gene targeting. J Biol Chem. 1998;273:11177–11182. [PubMed]
182. 160.
183. Adida C, Crotty P L, McGrath J. et al. Developmentally regulated expression of the novel
cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol.
1998;152:43–49. [PubMed]
184. 161.
185. Smith C A, Farrah T, Goodwin R G. The TNF receptor superfamily of cellular and viral
proteins: activation, costimulation, and death. Cell. 1994;76:959–962. [PubMed]
186. 162.
187. Nagata S. Apoptosis by death factor. Cell. 1997;88:355–365. [PubMed]
188. 163.
189. Consoli U, El-Tounsi I, Sandoval A. et al. Differential induction of apoptosis by fludarabine
monophosphate in leukemic B and normal T cells in chronic lymphocytic leukemia. Blood.
1998;91:1742–1748. [PubMed]
190. 164.
191. Tanaka M, Itai T, Adachi M, Nagata S. Downregulation of Fas ligand by shedding [see
comments] Nat Med. 1998;4:31–36. [PubMed]
192. 165.
193. Strand S, Hofmann W J, Hug H. et al. Lymphocyte apoptosis induced by CD95 (APO-
1/Fas) ligand-expressing tumor cells — a mechanism of immune evasion? Nature Med.
1996;2:1361–1366. [PubMed]
194. 166.
195. Hahne M, Rimoldi D, Schroter M. et al. Melanoma cell expression of Fas(Apo-1/CD95)
ligand: implications for tumor immune escape. Science. 1996;274:1363–1366. [PubMed]
196. 167.
197. O’Connell J, O’Sullivan G C, Collins J K, Shanahan F. The Fas counterattack: Fas-
mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med. 1996;184:1075–
1082. [PubMed]
198. 168.
199. Nagata S, Golstein P. The Fas death factor. Science. 1995;267:1449–1456. [PubMed]
200. 169.
201. Boldin M P, Varfolomeev E E, Pancer Z. et al. A novel protein that interacts with the death
domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem.
1995;270:7795–7798. [PubMed]
202. 170.
203. Chinnaiyan A M, O’Rourke K, Tewari M, Dixit V M. FADD, a novel death domain-
containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell.
1995;81:505–512. [PubMed]
204. 171.
205. Chinnaiyan A M, Tepper C G, Seldin M F. et al. FADD/MORT1 is a common mediator of
CD95 (Fas/APO1) and tumor necrosis factor recreptor-induced apoptosis. J Biol Chem.
1996;271:4961–4965. [PubMed]
206. 172.
 207. Duan H, Dixit V M. RAIDD is a new ‘death’ adaptor molecule. Nature. 1997;385:86–89.
[PubMed]
208. 173.
209. Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. Involvement of MACH, a novel
MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell.
1996;85:803–815. [PubMed]
210. 174.
211. Muzio M, Chinnaiyan A M, Kischkel F C. et al. FLICE, a novel FADD-homologous
ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex.
Cell. 1996;85:817–827. [PubMed]
212. 175.
213. Yang X, Khosravi-Far R, Chang H Y, Baltimore D. Daxx, a novel Fas-binding protein that
activates JNK and apoptosis. Cell. 1997;89:1067–1076. [PubMed]
214. 176.
215. Muller M, Wilder S, Bannasch D. et al. p53 activates the CD95 (APO-1/Fas) gene in
response to DNA damage by anticancer drugs. J Exp Med. 1998;188:2033–2045. [PubMed]
216. 177.
217. Fukazawa T, Fujiwara T, Morimoto Y. et al. Differential involvement of the CD95
(Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in
human cancer cells. Oncogene. 1999;18:2189–2199. [PubMed]
218. 178.
219. Owen-Schaub L B, Zhang W, Cusack J C. et al. Wild-type human p53 and a temperature-
sensitive mutant induce Fas/APO-1 expression. Mol Cell Biol. 1995;15:3032–3040. [PubMed]
220. 179.
221. Owen-Schaub L B, Radinsky R, Kruzel E, Berry K, Yonehara S. Anti-Fas on
nonhematopoietic tumors: levels of Fas/APO-1 and bcl- 2 are not predictive of biological
responsiveness. Cancer Res. 1994;54:1580–1586. [PubMed]
222. 180.
223. Maeda T, Yamada Y, Moriuchi R. et al. Fas gene mutation in the progression of adult T
cell leukemia. J Exp Med. 1999;189:1063–1071. [PubMed]
224. 181.
225. Ogasawara J, Watanabe-Fukunaga R, Adachi M. et al. Lethal effect of the anti-fas
antibody in mice. Nature. 1993;364:806–809. [PubMed]
226. 182.
227. Andreeff M, Hansen H, Cirrincione C. et al. Cellular RNA content: a major prognostic factor
in adult acute leukemia and non-Hodgkin lymphoma. Proc Intl Conf Analyt Cytol. 1984;10:25.
228. 183.
229. Schneider P, Holler N, Bodmer J L. et al. Conversion of membrane-bound Fas(CD95)
ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of
liver toxicity. J Exp Med. 1998;187:1205–1213. [PubMed]
230. 184.
231. Senkevich T G, Bugert J J, Sisler J R. et al. Genome sequence of a human tumorigenic
poxvirus: predication of specific host response-evasion genes. Science. 1996;273:813–816.
[PubMed]
232. 185.
233. Bodmer J L, Burns K, Schneider P. et al. TRAMP, a novel apoptosis-mediating receptor
with sequence homology to tumor necrosis factor receptor 1 and Fas (Apo-1/CD95). Immunity.
1997;6:79–88. [PubMed]
234. 186.
235. Kitson J, Raven T, Jiang Y P. et al. A death-domain-containing receptor that mediates
apoptosis. Nature. 1996;384:372–375. [PubMed]
236. 187.
237. Chinnaiyan A M, O’Rourke K, Yu G L. et al. Signal transduction by DR3, a death domain-
containing receptor related to TNFR-1 and CD95. Science. 1996;274:990–992. [PubMed]
238. 188.
239. Irmler M, Thome M, Hahne M. et al. Inhibition of death receptor signals by cellular FLIP.
Nature. 1997;388:190–195. [PubMed]
240. 189.
241. Griffith T S, Chin W A, Jackson G C, Lynch D H, Kubin M Z. Intracellular regulation of
TRAIL-induced apoptosis in human melanoma cells [in process citation] J Immunol.
1998;161:2833–2840. [PubMed]
242. 190.
243. Hitoshi Y, Lorens J, Kitada S I. et al. Toso, a cell surface, specific regulator of Fas-induced
apoptosis in T cells. Immunity. 1998;8:461–471. [PubMed]
244. 191.
245. Schneider T J, Fischer G M, Donohoe T J, Colarusso T P, Rothstein T L. A novel gene
coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B
lymphocytes. J Exp Med. 1999;189:949–956. [PubMed]
246. 192.
247. Johnstone R W, Cretney E, Smyth M J. P-glycoprotein protects leukemia cells against
caspase-dependent, but not caspase-independent, cell death. Blood. 1999;93:1075–1085.
[PubMed]
248. 193.
249. Thomas W D, Hersey P. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis
in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells. J Immunol.
1998;161:2195–2200. [PubMed]
250. 194.
251. Griffith T S, Chin W A, Jackson G C, Lynch D H, Kubin M Z. Intracellular regulation of
TRAIL-induced apoptosis in human melanoma cells. J Immunol. 1998;161:2833–2840. [PubMed]
252. 195.
253. Snell V, Clodi K, Zhao S. et al. Activity of TNF-related apoptosis-inducing ligand (TRAIL) in
haematological malignancies. Br J Haematol. 1997;99:618–624. [PubMed]
254. 196.
255. Andreeff M, Wong G, Koziner B, Espiritu E, Clarkson B. Non B-Non T acute lymphoblastic
leukemia (ALL): evidence for complete b cell differentiation of a quiescent subpopulation and their
response to induction therapy. Proc Am Assoc Cancer Res. 1985;26:28.
256. 197.
257. Keane M M, Ettenberg S A, Nau M M, Russell E K, Lipkowitz S. Chemotherapy augments
TRAIL-induced apoptosis in breast cell lines. Cancer Res. 1999;59:734–741. [PubMed]
258. 198.
259. Golstein P. Cell death: trail and its receptors. Curr Biol. 1997;7:R750–R753. [PubMed]
260. 199.
261. Ribeiro P, Renard N, Warzocha K. et al. CD40 regulation of death domains containing
receptors and their ligands on lymphoma B cells. Br J Haematol. 1998;103:684–689. [PubMed]
262. 200.
263. Walczak H, Miller R E, Ariail K. et al. Tumoricidal activity of tumor necrosis factor-related
apoptosis- inducing ligand in vivo [see comments] Nat Med. 1999;5:157–163. [PubMed]
264. 201.
265. Yin X M, Oltvai Z N, Korsmeyer S J. BH1 and BH2 domains of Bcl-2 are required for
inhibition of apoptosis and heterodimerization with Bax. Nature. 1994;369:321–323. [PubMed]
266. 202.
267. Chittenden T, Flemington C, Houghton A B. et al. A conserved domain in Bak, distinct from
BH1 and BH2, mediates cell death and protein binding functions. EMBO J. 1995;14:5589–5596.
[PubMed]
268. 203.
269. Zha H, Aime-Sempe C, Sato T, Reed J C. Proapoptotic protein Bax heterodimerizes with
Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol
Chem. 1996;271:7440–7444. [PubMed]
270. 204.
271. Gibson L, Holmgreen S P, Huang D C. et al. Bcl-w, a novel member of the bcl-2 family,
promotes cell survival. Oncogene. 1996;13:665–675. [PubMed]
272. 205.
273. Sato T, Irie S, Krajewski S, Reed J C. Cloning and sequencing of a cDNA encoding the rat
Bcl-2 protein. Gene. 1994;140:291–292. [PubMed]
274. 206.
275. Sedlak T W, Oltvai Z N, Yang E. et al. Multiple Bcl-2 family members demonstrate
selective dimerizations with Bax. PNAS. 1995;92:7834–7838. [PubMed] [Free Full text in PMC]
276. 207.
277. Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved
homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–619. [PubMed]
278. 208.
279. Yang E, Zha J, Jockel J. et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces
Bax and promotes cell death. Cell. 1995;80:285–291. [PubMed]
280. 209.
281. Hsu Y T, Wolter K G, Youle R J. Cytosol-to-membrane redistribution of Bax and Bcl-X(L)
during apoptosis. Proc Natl Acad Sci U S A. 1997;94:3668–3672. [PubMed] [Free Full text in PMC]
282. 210.
283. Hsu Y T, Youle R J. Bax in murine thymus is a soluble monomeric protein that displays
differential detergent-induced conformations. J Biol Chem. 1998;273:10777–10783. [PubMed]
284. 211.
285. Wang K, Gross A, Waksman G, Korsmeyer S J. Mutagenesis of the BH3 domain of BAX
identifies residues critical for dimerization and killing [in process citation] Mol Cell Biol.
1998;18:6083–6089. [PubMed] [Free Full text in PMC]
286. 212.
287. Otter I, Conus S, Ravn U. et al. The binding properties and biological activities of Bcl-2 and
Bax in cells exposed to apoptotic stimuli. J Biol Chem. 1998;273:6110–6120. [PubMed]
288. 213.
289. Hanada M, Aime-Sempe C, Sato T, Reed J C. Structure-function analysis of Bcl-2 protein.
Identification of conserved domains important for homodimerization with Bcl-2 and
heterodimerization with Bax. J Biol Chem. 1995;270:11962–11969. [PubMed]
290. 214.
291. Hunter J J, Bond B L, Parslow T G. Functional dissection of the human Bcl2 protein:
sequence requirements for inhibition of apoptosis. Mol Cell Biol. 1996;16:877–883. [PubMed]
292. 215.
293. Borner C, Martinou I, Mattmann C. et al. The protein bcl-2 alpha does not require
membrane attachment, but two conserved domains to suppress apoptosis. J Cell Biol.
1994;126:1059–1068. [PubMed]
294. 216.
295. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced
phosphorylation of BAD through the protein kinase Akt. Science. 1997;278:687–689. [PubMed]
296. 217.
297. Zha J, Harada H, Yang E, Jockel J, Korsmeyer S J. Serine phosphorylation of death
agonist BAD in response to survival factor results in binding to 14-3-3 not Bcl-X. Cell.
1996;87:619–628. [PubMed]
298. 218.
299. Alnemri E S, Robertson N M, Fernandes T F, Croce C M, Litwack G. Overexpressed full-
length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells. Proc Natl Acad
Sci U S A. 1992;89:7295–7299. [PubMed] [Free Full text in PMC]
300. 219.
301. Ito T, Deng X, Carr B, May W S. Bcl-2 phosphorylation required for anti-apoptosis function.
J Biol Chem. 1997;272:11671–11673. [PubMed]
302. 220.
303. May W S, Tyler P G, Ito T. et al. Interleukin-3 and bryostatin-1 mediate
hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis. J Biol Chem.
1994;269:26865–26870. [PubMed]
304. 221.
305. Ruvolo P P, Deng X, Carr B K, May W S. A functional role for mitochondrial protein kinase
Calpha in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem. 1998;273:25436–
25442. [PubMed]
306. 222.
 307. Poommipanit P B, Chen B, Oltvai Z N. Interleukin-3 induces the phosphorylation of a
distinct fraction of bcl-2. J Biol Chem. 1999;274:1033–1039. [PubMed]
308. 223.
309. Haldar S, Jena N, Croce C M. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci
U S A. 1995;92:4507–4511. [PubMed] [Free Full text in PMC]
310. 224.
311. Haldar S, Chintapalli J, Croce C M. Taxol induces bcl-2 phosphorylation and death of
prostate cancer cells. Cancer Res. 1996;56:1253–1255. [PubMed]
312. 225.
313. Haldar S, Basu A, Croce C M. Bcl2 is the guardian of microtubule integrity. Cancer Res.
1997;57:229–233. [PubMed]
314. 226.
315. Blagosklonny M V, Schulte T, Nguyen P, Trepel J, Neckers L M. Taxol-induced apoptosis
and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal
transduction pathway. Cancer Res. 1996;56:1851–1854. [PubMed]
316. 227.
317. Maundrell K, Antonsson B, Magnenat E. et al. Bcl-2 undergoes phosphorylation by c-Jun
N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-
binding protein Rac1. J Biol Chem. 1997;272:25238–25242. [PubMed]
318. 228.
319. Chen Y R, Wang X, Templeton D, Davis R J, Tan T H. The role of c-Jun N-terminal kinase
(JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may
determine cell death and proliferation. J Biol Chem. 1996;271:31929–31936. [PubMed]
320. 229.
321. Graves J D, Draves K E, Craxton A. et al. Involvement of stress-activated protein kinase
and p38 mitogen- activated protein kinase in mIgM-induced apoptosis of human B lymphocytes.
Proc Natl Acad Sci U S A. 1996;93:13814–13818. [PubMed] [Free Full text in PMC]
322. 230.
323. Ichijo H, Nishida E, Irie K. et al. Induction of apoptosis by ASK1, a mammalian MAPKKK
that activates SAPK/JNK and p38 signaling pathways. Science. 1997;275:90–94. [PubMed]
324. 231.
325. May W S, Tyler P G, Ito T, Armstrong D K, Qatsha K A, Davidson N E. Interleukin-3 and
bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of
apoptosis. J Biol Chem. 1994;269:26865–26870. [PubMed]
326. 232.
327. Haldar S, Jena N, Croce C M. Inactivation of Bcl-2 by phosphorylation. PNAS.
1995;92:4507–4511. [PubMed] [Free Full text in PMC]
328. 233.
329. Chang B S, Minn A J, Muchmore S W, Fesik S W, Thompson C B. Identification of a novel
regulatory domain in Bcl-XL and Bcl-2. EMBO J. 1997;16:968–977. [PubMed]
330. 234.
331. Fadeel B, Hassan Z, Hellstrom-Lindberg E. et al. Cleavage of Bcl-2 is an early event in
chemotherapy-induced apoptosis of human myeloid leukemia cells. Leukemia. 1999;13:719–728.
[PubMed]
332. 235.
333. Wesselborg S, Engels I H, Rossmann E, Los M, Schulze-Osthoff K. Anticancer drugs
induce caspase-8/FLICE activation and apoptosis in the absence of CD95 receptor/ligand
interaction. Blood. 1999;93:3053–3063. [PubMed]
334. 236.
335. Landowski T H, Shain K H, Oshiro M M. et al. Myeloma cells selected for resistance to
CD95-mediated apoptosis are not cross-resistant to cytotoxic drugs: evidence for independent
mechanisms of caspase activation. Blood. 1999;94:265–274. [PubMed]
336. 237.
337. Eischen C M, Kottke T J, Martins L M. et al. Comparison of apoptosis in wild-type and Fas-
resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions.
Blood. 1997;90:935–943. [PubMed]
338. 238.
 339. Clem R J, Cheng E H, Karp C L. et al. Modulation of cell death by Bcl-XL through caspase
interaction. PNAS. 1998;95:554–559. [PubMed] [Free Full text in PMC]
340. 239.
341. Dimmeler S, Breitschopf K, Haendeler J, Zeiher A M. Dephosphorylation targets Bcl-2 for
ubiquitin-dependent degradation: a link between the apoptosome and the proteasome pathway. J
Exp Med. 1999;189:1815–1822. [PubMed]
342. 240.
343. Zamzami N, Susin S A, Marchetti P. et al. Mitochondrial control of nuclear apoptosis (see
comments). J Exp Med. 1996;183:1533–1544. [PubMed]
344. 241.
345. Liu X, Kim C N, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free
extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157. [PubMed]
346. 242.
347. Susin S A, Zamzami N, Castedo M. et al. Bcl-2 inhibits the mitochondrial relapse of an
apoptogenic protease. J Exp Med. 1996;184:1331–1341. [PubMed]
348. 243.
349. Kluck R M, Bossy-Wetzel E, Green D R, Newmeyer D D. The release of cytochrome c
from mitochondria: a primary site for bcl-2 regulation of apoptosis. Science. 1997;275:1132–1136.
[PubMed]
350. 244.
351. Yang J, Liu X, Bhalla K. et al. Prevention of apoptosis by bcl-2: release of cytochrome c
from mitochondria blocked. Science. 1997;275:1129–1132. [PubMed]
352. 245.
353. Marzo I, Brenner C, Zamzami N. et al. The permeability transition pore complex: a target
for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med. 1998;187:1261–1271.
[PubMed]
354. 246.
355. Marzo I, Susin S A, Petit P X. et al. Caspases disrupt mitochondrial membrane barrier
function. FEBS Lett. 1998;427:198–202. [PubMed]
356. 247.
357. Pan G, O’Rourke K, Dixit V M. Caspase-9, Bcl-XL, and Apaf-1 form a ternary complex. J
Biol Chem. 1998;273:5841–5845. [PubMed]
358. 248.
359. Yang J C, Cortopassi G A. dATP causes specific release of cytochrome C from
mitochondria [in process citation] Biochem Biophys Res Commun. 1998;250:454–457. [PubMed]
360. 249.
361. Xiang J, Chao D T, Korsmeyer S J. BAX-induced cell death may not require interleukin 1
beta-converting enzyme-like proteases. Proc Natl Acad Sci U S A. 1996;93:14559–14563.
[PubMed] [Free Full text in PMC]
362. 250.
363. Li H, Zhu H, Xu C J, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial
damage in the Fas pathway of apoptosis. Cell. 1998;94:491–501. [PubMed]
364. 251.
365. Hofmann K, Bucher P, Tschopp J. The CARD domain: a new apoptotic signaling motif.
Trends Biochem Sci. 1997;22:155–156. [PubMed]
366. 252.
367. Chinnaiyan A M, O’Rourke K, Lane B R, Dixit V M. Interaction of CED-4 with CED-3 and
CED-9: a molecular framework for cell death. Science. 1997;275:1122–1126. [PubMed]
368. 253.
369. Shaham S, Horvitz H R. Developing Caenorhabditis elegans neurons may contain both
cell-death protective and killer activities. Genes Dev. 1996;10:578–591. [PubMed]
370. 254.
371. Shaham S, Horvitz H R. An alternatively spliced C. elegans ced-4 RNA encodes a novel
cell death inhibitor. Cell. 1996;86:201–208. [PubMed]
372. 255.
 373. Irmler M, Hofmann K, Vaux D, Tschopp J. Direct physical interaction between the
Caenorhabditis elegans ‘death proteins’ CED-3 and CED-4. FEBS Lett. 1997;406(1–2):189–190.
[PubMed]
374. 256.
375. Spector M S, Desnoyers S, Hoeppner D J, Hengartner M O. Interaction between the C.
elegans cell-death regulators CED-9 and CED-4. Nature. 1997;385:653–656. [PubMed]
376. 257.
377. Wu D, Wallen H D, Nunez G. Interaction and regulation of subcellular localization of CED-
4 by CED-9. Science. 1997;275:1126–1129. [PubMed]
378. 258.
379. Hu Y, Benedict M A, Wu D, Inohara N, Nunez G. Bcl-XL interacts with Apaf-1 and inhibits
Apaf-1-dependent caspase-9 activation. PNAS. 1998;95:4386–4391. [PubMed] [Free Full text in
PMC]
380. 259.
381. Zou H, Henzel W J, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C.
elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell.
1997;90:405–413. [PubMed]
382. 260.
383. Yoshida H, Kong Y Y, Yoshida R. et al. Apaf1 is required for mitochondrial pathways of
apoptosis and brain development. Cell. 1998;94:739–750. [PubMed]
384. 261.
385. Cecconi F, Alvarez-Bolado G, Meyer B I, Roth K A, Gruss P. Apaf1 (CED-4 homolog)
regulates programmed cell death in mammalian development. Cell. 1998;94:727–737. [PubMed]