Download cell cycle regulation and apoptosis1

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Annu. Rev. Physiol. 1998. 60:601–17
CELL CYCLE REGULATION
AND APOPTOSIS1
K. L. King∗ and J. A. Cidlowski#
∗ Department
of Molecular and Cellular Physiology, University of Cincinnati Medical
Center, P. O. Box 670576, Cincinnati, Ohio 45267-0576; #National Institutes of
Health, National Institute of Environmental Health Sciences, P.O. Box 12233 MD
E2-02, Research Triangle Park, North Carolina 27709;
e-mail: [email protected]
KEY WORDS:
p53, pRb, E2F, cell death, cell cycle, lymphocytes
ABSTRACT
Tissue homeostasis requires a balance between cell proliferation and death. Apoptosis and proliferation are linked by cell cycle regulators, and apoptotic stimuli
affect both cell proliferation and death. Glucocorticoids induce G1 arrest and
apoptosis in transformed lymphoid cells. Decreased expression of the cell cycle
components c-myc and cyclin D3 is essential for glucocorticoid-induced growth
arrest and death in dividing cells. Other G1 regulators, such as p53, pRb, and E2F,
have also been implicated in apoptosis. Mice lacking either p53 or E2F display
aberrant cell proliferation and tumor formation, suggesting that these proteins
are involved in the elimination of abnormal cells through apoptosis. In contrast,
pRb induces G1 arrest and suppresses apoptosis in cultured cells. Mice that lack
pRb are nonviable and show ectopic mitosis and massive cell death, suggesting
that pRb is an apoptotic suppressor. Further analysis of common components
of apoptotic and cell cycle machinery may provide insight into the coordinated
regulation of these antagonistic processes.
INTRODUCTION
Tissue homeostasis is dependent on the proper relationships among cell proliferation, differentiation, and cell death. As somatic cells proliferate, the cell
1 The US Government has the right to retain a nonexclusive, royalty-free license in and to any
copyright covering this paper.
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mitotic cycle progression is tightly regulated by an intricate network of positive and negative signals, and much is known about the molecules involved in
cell cycle control. Programmed cell death, or apoptosis, is also a highly regulated process by which an organism eliminates unwanted cells without eliciting
an inflammatory response. Apoptosis is involved in many physiological processes including tissue homeostasis, embryonic development, and the immune
response (1). Mitosis and apoptosis display several similar morphological features. Both mitotic and apoptotic cells lose substrate attachment and become
rounded. During both processes, cells shrink, condense their chromatin, and
display rapid membrane blebbing. Although a number of similarities exist between mitotic and apoptotic cells, several distinct differences are also apparent.
For example, only apoptotic cells display fragmentation of their DNA into approximately 200 base-pair fragments. At the end of apoptosis, the cell is broken
into multiple apoptotic bodies that are phagocytosed by neighboring cells. Because cellular contents are not released, this occurs with little inflammation.
During mitosis, DNA is segregated and the nucleus is divided into two distinct
but equal parts. The mitotic process ends with cytokinesis and the production
of a new daughter cell. Furthermore, cell cycle components such as p53, pRb,
and E2F, have been shown to participate in both cell cycle progression and
apoptosis. Hence, comparison of apoptosis and cell proliferation may provide
insight into the regulation and molecular mechanisms of these two antagonistic
processes.
Multiple inducers of apoptosis have been identified. These signals are often
cell-type specific, and a partial list includes growth factor withdrawal, ionizing
radiation, Ca2+ influx, tumor necrosis factor, viral infection, and glucocorticoids. In response to these signals, the cell induces a programmed cascade of
events that results in the destruction of the cell. A number of catalytic pathways
are induced during apoptosis, including protease activation, which leads to the
destruction of cellular proteins, and nuclease activation, which results in DNA
fragmentation and RNA degradation. Caspases, also called ICE proteases, are
a family of cysteine proteases that act as effectors of the mammalian cell death
pathway (2). Their activation leads to morphological changes characteristic of
the apoptotic process.
CELL PROLIFERATION AND APOPTOSIS
The balance between proliferation and apoptosis must be strictly maintained
to sustain tissue homeostasis. An imbalance between these two processes can
result in either unwanted tissue atrophy or tissue growth. For example, when
adrenalectomized rats are treated with the synthetic glucocorticoid dexamethasone, a steroid that induces thymocyte apoptosis, the wet weight of the rat
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thymus decreases by 50% within 24 h owing to an increase in the rate of apoptosis of cortical thymocytes that is not offset by an increase in mitosis (3).
Tumor formation can result from a decrease in cell death, as well as an increase in cell proliferation. For example, when bcl-2, an anti-apoptotic gene,
is overexpressed, neoplasia results because of a reduction of apoptosis that is
not offset by a decrease in cell proliferation (4, 4a,b).
Cell Cycle Checkpoints
Because it is essential to identify and eliminate cells proliferating inappropriately, apoptosis and proliferation are tightly coupled, and cell cycle regulators
can influence both cell division and cell death (5, 6). The timing and order
of cell cycle events are monitored during cell cycle checkpoints that occur at
the G1/S phase boundary, in S phase, and during the G2/M phases (7). These
checkpoints ensure that critical events in a particular phase of the cell cycle
are completed before a new phase is initiated, thereby preventing the formation
of genetically abnormal cells. Cell cycle progression can be blocked at these
checkpoints in response to the status of both the intracellular and extracellular
environment. For example, the expression of certain genes required for DNA
synthesis occurs only in the presence of growth factors, thus linking the extracellular environment to cell proliferation. Additionally, growth arrest can be
induced when DNA damage is detected or when chromosomes are misaligned
on the mitotic spindle (8). Upon repair of the damage, progression through
the cell cycle resumes. An alternative to repairing damaged cells is simply
to eliminate them through the process of apoptosis. Thus as a cell progresses
through the cell cycle, it must determine whether to complete cell division,
arrest growth to repair cellular damage, or undergo apoptosis if the damage is
too severe to be repaired or if the cell is incapable of repairing the DNA. It is
at the checkpoints that the cell determines which of these options is suitable.
The cell cycle control system is based on two protein families: the cyclindependent protein kinases (Cdks) and the cyclins. Cdks allow progression
through the different phases of the cell cycle by phosphorylating substrates.
Their kinase activity is dependent on the presence of activating subunits known
as cyclins. The abundance of specific cyclins increases during the phase of
the cell cycle wherein they are required and decreases during phases in which
they are not needed. For example, in most circumstances cyclin D associates
with Cdk4 and Cdk6 during early G1, whereas cyclin E activates Cdk2 during
G1 to S phase transition. Cyclin A binds to Cdk2 or Cdc2 during S phase
and the G2 to M phase transition, and the cyclin B/Cdc2 complex functions
during the G2 to M phase transition. Thus specific cyclin/Cdk complexes are
activated, and their phosphorylation of particular proteins permits the cell cycle
processes to continue. The transcription of genes necessary for S phase, for
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example, is regulated by cyclinD/Cdk4-dependent phosphorylation of the cell
cycle regulator pRb. For more extensive reviews of cyclins, Cdks, and cell
cycle progression, see References 8a,b,c.
Whereas cyclin binding is required for Cdk kinase activity, other proteins
have been identified whose association leads to the inhibition of Cdk activity.
Cyclin/Cdk complexes can be bound by Cdk inhibitor (CKI) proteins, which
inhibit kinase activity and prevent cell cycle progression. Two separate families
of CKI proteins have been identified (8b,d,e). The p21 family, composed of p21,
p27, and p57, predominantly inhibits the Cdks of the G1 to S phase transition.
The INK4 (inhibitors of Cdk4) family includes p15, p16, p18, and p19, several
of which are mutated or deleted in certain types of human cancers.
Numerous genes, including c-myc, are known to inhibit or activate cell proliferation by affecting the formation and activity of Cdk complexes. The protooncogene c-myc is an immediate early gene encoding a protein that functions
as a transcription factor. Its activity is dependent on its association with another
factor, Max, and this association is required for mitogenesis (9, 10). Expression
of c-myc, which is exquisitely sensitive to growth factors, is required for quiescent cells to enter the cell cycle (11). The c-Myc protein has been shown to
activate transcription of Cdc25, a phosphatase that activates Cdks, and expression of Cdc25 appears necessary for c-myc-induced cell cycle activation (12).
The G1- to S-phase transition also requires c-Myc, and inhibition of c-myc
expression leads to growth arrest (13). Conversely, if c-myc is expressed in
G1-arrested cells in the absence of growth factors, the cells exit G1 and divide,
a process that eventually leads to death (14–17). Deregulated expression of
this proto-oncogene has been implicated in a number of human malignancies
(18, 19). A more detailed discussion of the roles of c-myc in apoptosis may be
found in the chapter by EB Thompson (this volume).
GLUCOCORTICOIDS, THE CELL CYCLE,
AND APOPTOSIS
Glucocorticoids induce death through at least two separate pathways. When
proliferating thymocytes are treated with glucocorticoids, the signal to activate
apoptosis involves changes in cell cycle components (Figure 1). Glucocorticoids also induce apoptosis in nonproliferating thymocytes, and while not
identical, this signaling pathway shares some features in common with that in
proliferating cells. In both systems, glucocorticoid binds to its receptor and
induces changes in gene expression. However, in nonproliferating cells, the
signaling pathway leading to apoptosis does not appear to involve changes in
cell cycle proteins. Thus upstream signals induced by glucocorticoids in the
circumstances differ depending on whether the cell is progressing through the
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Figure 1 Possible pathways for glucocorticoid-induced apoptosis of lymphoid cells (21–33).
Although G1 arrest and cell death appear linked, the mechanism by which these two processes are
coordinated remains unknown.
cell cycle. The downstream components of both signaling pathways ultimately
appear to activate apoptotic effector molecules that include the caspases and
nucleases.
Many apoptotic stimuli induce cell cycle arrest before cell death, thereby
affecting both cell cycle and apoptotic machinery. Growth suppressive and
lytic effects of glucocorticoids on thymocytes and transformed lymphoid cells
have been studied (20, 21). Glucocorticoid treatment of many lymphoid cell
lines expressing markers of immature thymocytes induces G1 arrest and, in
most cases, this growth suppression is followed by cell death. The precise
mechanism by which glucocorticoids induce G1 arrest and apoptosis is currently
being investigated but is largely unknown.
Glucocorticoids Regulate Cell Cycle Genes
Glucocorticoids elicit their effects by binding to their cognate receptors, which
function as ligand-dependent transcription factors. Glucocorticoids can inhibit
gene expression and alter mRNA stability of genes that are critical for the
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G1- to S-phase transition. One of the cell cycle genes regulated by this steroid is
c-myc (22–26). Although an inappropriate increase in c-myc expression usually
leads to cell death, downregulation of c-myc by glucocorticoids causes apoptosis
in a lymphoma cell line and in a leukemic cell line (22, 27, 28). Thus at least two
pathways involving c-myc result in apoptosis. Although the precise molecular
mechanism by which glucocorticoids decrease c-myc mRNA has not yet been
elucidated, this decrease appears to be critical for induction of death. For
example, sustained expression of c-myc blocks glucocorticoid-induced death
in the human leukemic cell line CEM-C7, and antisense c-myc oligomers trigger
apoptosis in these cells (27). Furthermore, the glucocorticoid antagonist RU
486 blocks steroid-induced cell death, and this inhibition is correlated with an
increase in c-myc mRNA levels (28). Thus changes in this cell cycle regulator
play a prominent role in glucocorticoid-induced apoptosis.
Other cell cycle regulators are also sensitive to glucocorticoid treatment including cyclin D3, the primary isoform of cyclin D in thymocytes, and one
of its catalytic subunits Cdk4 (23, 29, 30). In the murine lymphoma cell line
P1798, glucocorticoids inhibit the transcription of Cdk4, a cyclin-dependent
kinase thought to be involved in progression through G1. Additionally, cyclin
D3 mRNA is rapidly downregulated in P1798 cells treated with the synthetic
glucocorticoid dexamethasone (23, 29). Destabilization of this mRNA appears
to be the main mechanism by which glucocorticoids decrease cyclin D3. In
the absence of steroid, the half life of cyclin D3 mRNA is 8 h, whereas in
the presence of glucocorticoid, half life is reduced to 1 h (29). The increase
in turnover of mRNA is independent of cell cycle progression and dependent
on protein synthesis. Thus glucocorticoids induce expression of proteins that
accelerate the degradation of cyclin D3 mRNA.
When P1798 lymphoma cells with normal cyclin D3 and c-myc are cultured
in the absence of serum and treated with dexamethasone, they rapidly undergo
apoptosis (23, 31, 32). In the presence of serum, these cells undergo G1 arrest.
Cells that overexpress both cyclin D3 and c-myc genes override glucocorticoidinduced G1 arrest (23). Additionally, cells that overexpress both proteins in the
absence of serum are resistant to glucocorticoid-induced death. Thus, cyclin D3
and c-Myc convey resistance to both G1 arrest and cell death, and glucocorticoid
inhibition of both these genes appears to be critical for steroid-mediated growth
suppression and apoptosis in P1798 cells. It is unclear whether G1 arrest and
apoptosis are parallel, independent functions or sequential, dependent events
induced by decreases in c-Myc and cyclin D.
Glucocorticoid treatment of cultured lymphoid cells affects other G1 regulators including pRb and E2F (23, 33). For example, treatment of P1798 cells
induced a 75–90% reduction in pRb phosphorylation by a cyclin D3-associated
kinase (23). Hypophosphorylation of pRb, G1 arrest, and apoptosis have been
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observed in other cell types, including Burkitt lymphoma cells treated with an
anti-immunoglobulin antibody (34) and in the myeloid leukemic cell lines HL
60 and U937 treated with the DNA-damaging agent cytosine arabinoside (35).
Glucocorticoid treatment of P1798 cells induces G1 arrest and causes changes
in the proteins associated with the transcription factor E2F (33). Although the
role that these changes play in induction of G1 arrest and apoptosis is unclear,
it is apparent that G1 checkpoint regulators play a role in both cell cycle regulation and glucocorticoid-induced apoptosis in those cells that are progressing
through the cell cycle.
THE G1 CHECKPOINT AND APOPTOSIS
p53
Insight into the roles of cell cycle checkpoints in apoptosis and proliferation has
come from in vitro studies of cells and in vivo studies of mice lacking various
cell cycle components. The G1 checkpoint regulators p53, pRb, and E2F have
been extensively analyzed by both methods (36, 37). The well-characterized
tumor suppressor p53 is either inactivated by mutation or sequestered by viral
proteins in numerous tumors (37–40). In response to cell damage, p53 has been
implicated in controlling the G1- to S-phase transition, blocking cell cycle progression at G1 in response to DNA damage (41). Protein p53 mediates these
effects through its transcriptional activation functions. A number of genes controlling cell cycle progression, including the cyclin-dependent kinase inhibitor
p21, are transcribed in a p53-dependent manner (42, 43). It has been postulated
that p53 induces the expression of p21 in response to ionizing radiation, resulting in G1 arrest. p53 also plays an important role in the induction of apoptosis.
When wild-type p53 is transfected into some cell lines lacking p53, apoptosis
is induced (44, 45). Within 15 min of exposure to the apoptotic signal delivered to oligodendrocytes by IL-2, p53 translocates from the cytoplasm, and a
mutant p53 protects these cells from death (46). Consequently, in cell culture
systems, p53 not only regulates progression through the cell cycle but under
some circumstances can also induce apoptosis in damaged cells.
p53 KNOCKOUT MICE To further study the role of p53 in mammalian physiology, p53 knockout mice have been generated (47). These mice are viable
but show a high incidence of cancer and typically die from T-cell lymphomas.
Furthermore, thymocytes derived from animals lacking p53 are resistant to
apoptosis induced by DNA damaging agents such as ionizing radiation and
topoisomerase II inhibitors, but not to cell death induced by glucocorticoids
or calcium ionophore/phorbol ester treatment (48, 49). Therefore, p53 participates in the induction of some, but not all, forms of apoptosis. Obviously p53
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is not an essential component of the machinery that carries out apoptosis but
rather is an activator of its function. Both p53-dependent and -independent
pathways may be necessary to transduce the large diversity of death signals,
or these multiple signaling pathways may provide a fail-safe mechanism if one
pathway becomes inactivated.
The mechanism by which p53 induces apoptosis remains unclear. Because
p53-dependent cell death may occur in the presence of transcriptional and
translational inhibitors, p53 transcriptional activation apparently is not essential
(50–53). Additionally, p53 mutants that are unable to transactivate have been
shown to induce apoptosis (51). However, others have found that p53 doublepoint mutants that cannot activate or repress transcription are compromised in
their ability to induce apoptosis (52). Using loss- and gain-of-function p53 mutants, Attardi et al (53) have shown that transcriptional activation is necessary
for both G1 arrest and apoptosis. However, p53 targets different genes for each
process. Rowan et al (54) isolated a mutant that retains transcriptional activation abilities and can induce G1 arrest but does not induce apoptosis, suggesting
that these two functions are separable. Discrepancies among results may be
due to the existence of multiple transactivation-dependent and transactivationindependent pathways that lead to apoptosis. Additionally, interpretation of
mutant studies may be complicated by the possibility that some mutants have
lost the ability to activate genes required for cell death but retain the ability to
induce genes required for other p53 functions. Thereby, mutants that are transactivation positive would be unable to induce apoptosis even though gene transcription would still be necessary for apoptotic induction. Finally, differences
in model systems make comparisons of results difficult. For example, some
cell lines employed contain endogenous wild-type p53 in addition to mutant
p53 protein, and others have viral proteins that target p53. Further studies will
be needed to resolve inconsistencies in results before the role of p53-mediated
transcriptional activation and repression in apoptosis is understood.
p53 REGULATES APOPTOSIS GENES p53 regulates the expression of several proteins known to influence the apoptotic process. In the murine leukemia cell
line M1, a temperature-sensitive p53 mutant decreases bcl-2 expression and increases bax expression (55). Bcl-2 and Bax have opposing effects on cell death:
Bcl-2 inhibits or delays cell death, and Bax accelerates apoptosis (56). Furthermore, the ratio of Bcl-2 to Bax has been proposed to influence the propensity of
a cell to undergo apoptosis. Additionally, Fas, a cell surface protein that triggers
apoptosis upon ligand binding, is encoded by a target gene for transcriptional
activation by p53 (57). Both wild-type p53 and a temperature-sensitive p53
mutant (at a permissive temperature) induced a three- to sixfold increase in
fas expression in several human cell lines. Accordingly, if p53 transcriptional
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activation and repression functions are required to induce apoptosis, these effects may be mediated through changes in the expression of known apoptotic
regulators.
p53, APOPTOSIS, AND CANCER THERAPY Because p53 is altered in a large number of human cancers, defects in the p53-dependent apoptotic pathway may be
a significant obstacle to overcome for successful cancer therapy. Indeed, p53
status in cancer cells does affect the efficacy of cancer treatment (58). Both
ionizing radiation and the chemotherapeutic agent adriamycin were more effective in inducing apoptosis in tumor cells that were homozygous for wild-type
p53. This information is currently being used to develop new cancer therapy
strategies. Reintroduction of normal p53 into tumor cells lacking p53 function
has been employed for human cancer treatment. Roth et al (59) injected a retroviral vector containing the wild-type p53 driven by the B actin promoter into
lung tumors of human cancer victims. They found p53 DNA in tumor cells,
and posttreatment biopsies showed that apoptosis occurred more frequently.
Tumor regression or growth stabilization were observed in approximately 60%
of the patients treated. This suggests that introducing components necessary
for a functional apoptotic pathway into tumor cells can aid in cancer treatment.
pRb
The retinoblastoma protein (pRb), similar to p53, functions as a negative regulator of cell growth and is a tumor suppressor (60). pRb inactivation or deletion is
found in many cancers, including retinoblastomas and carcinomas of the lung,
breast, bladder, and prostate. Similar to the case with p53, viral-transforming
proteins have been shown to inactivate pRb, thus leading to inappropriate cell
proliferation and tumor formation (61–63). By binding to and inhibiting transcription factors such as E2F, which are necessary for S-phase entry, pRb is
believed to inhibit cell cycle progression. In mid-to-late G1, cyclin-dependent
kinases along with their respective cyclins, phosphorylate pRb and the pRb-like
proteins p107 and p130. Once phosphorylated, pRb and p130 release the bound
transcription factors, including the E2F family. These transcription factors activate expression of genes necessary for the cell to traverse from G1 to S phase.
In addition to playing a role in growth arrest, pRb has been shown to suppress apoptosis. In a pRb-defective bladder carcinoma cell line, IFN γ induces
apoptosis, but in pRb-positive cells, IFN γ does not induce cell death (64). In
the human osteosarcoma cell line SOAS, cells transiently or stably transfected
with pRb are protected from radiation-induced apoptosis (65). These cells undergo G1 arrest after irradiation, and this block in cell cycle progression may
be responsible for the decrease in susceptibility to apoptosis. Also, pRb inhibits TGF-β1 induced apoptosis in hepatoma cells (66). TGF-β1 treatment of
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hepatoma cells is associated with inhibition of both pRb expression and phosphorylation and with induction of cell death. Overexpression of pRb in these
cells overrides the TGF-β1 death-stimulating pathway and blocks apoptosis.
Recently, pRb was shown to be proteolytically cleaved during apoptosis
(67, 68). A member of the caspase family of apoptotic proteases specifically
cleaves pRb in tumor necrosis factor, staurosporine, and cytosine arabinosideinduced apoptosis (67, 68). This proteolysis is blocked by tetrapeptide inhibitors of ICE-like enzymes. The destruction of pRb, which acts as an apoptosis suppressor, may be required to induce death.
Deletion of pRb in transgenic mice is an embryoniclethal mutation that causes death in utero at 14 to 15 days (69–71). The pRb
null embryos are defective in erythropoiesis and also show massive cell death
in the central and peripheral nervous systems, and in liver, lens, and skeletal
muscle precursors. This cell death is associated with inappropriate S-phase
entry and increased expression of cyclin E (72). Transgenic mice that express
low levels of pRb have also been generated (73). These mice die at birth due
to skeletal muscle defects. There is a large increase in myoblast apoptosis, and
surviving myoblasts do not differentiate properly. These results suggest a role
for pRb in both cell survival and terminal differentiation (74).
pRB KNOCKOUT MICE
p53 and pRB Interaction
There may be complementary roles for p53 and pRb as tumor suppressors, and
each may be able to compensate for the loss of the other’s tumor suppressor
activity. The basis for pRb and p53 cooperativity is being analyzed in several
model systems. For example, the interaction between p53 and pRb has been
examined in HeLa cells that lack both p53 and pRb (51). When p53 is transiently overexpressed in these cells, apoptosis is induced. However, cell death
can be inhibited when pRb is coexpressed with p53. In this case, cell cycle
arrest occurs instead of apoptosis. Thus pRb protects the cells from apoptosis,
possibly by inducing cell cycle arrest. This lack of aberrant cell proliferation
may prevent the induction of p53-dependent apoptosis. In addition to inducing
G1 arrest, p53 can induce cell death in response to DNA damage and inappropriate proliferation signals. There appears to be a direct link between p53 and
pRb in cell proliferation and apoptosis.
Interactions between p53 and pRb have also been examined in transgenic
animals. Mice that are pRb+/− are viable but develop thyroid and pituitary
tumors (69–71). Because pRb null mice are nonviable, pRb heterozygotes have
been used to analyze pRb and p53 regulation of apoptosis. pRb+/−/p53−/−
mice show tumors common to each knockout alone, plus additional tumors
such as pinealoblastomas and islet tumors (74). Loss of the wild-type Rb
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allele was associated with tumorigenesis in some tissues. For example, Rb
inactivation was frequently seen in pineal blastomas but not in lymphomas.
Thus it appears that in some tissues each tumor suppressor can compensate
for the loss of the other. Apoptosis is dependent on p53 because embryos that
are null for p53 and heterozygous for pRb do not show apoptotic effects seen
in mice deficient in pRb alone. Thus pRb deficiency leads to induction of
apoptosis in a p53-dependent manner. In lens tissue, p53 and pRb functions
have been extremely well characterized. In pRb null mice, lens fiber cells fail
to differentiate and undergo apoptosis (75). In a p53 null background, lens fiber
cells still do not differentiate properly, but they are not removed by apoptosis;
consequently, tumors form in the lens. These results have been confirmed using
DNA tumor viruses expressing proteins that inactivate p53 or pRb. When the
human papilloma virus oncoprotein E7 is expressed in the developing lens, pRb
is inactivated and ectopic mitosis and apoptosis is evident (76). Conversely,
if E7 and E6, oncoproteins that inactivate p53, are coexpressed, apoptosis is
inhibited and tumor formation occurs. E7 has also been expressed in lens cells in
a p53 null background with similar results (77). Similar interactions between
p53 and pRb function are also seen in the choroid plexus. A simian virus 40
(SV40) T antigen fragment that inactivates pRb was targeted to epithelial cells in
the choroid plexus (78). Inactivation of pRb led to slow growing tumors and an
increase in p53-dependent apoptosis. In contrast, when pRb was inactivated in a
p53 null background, rapidly growing tumors formed, and there was a decrease
in apoptosis. Symonds et al concluded that p53-dependent apoptosis occurring
in response to tumorigenic events is a key regulator of tumor formation (78).
Therefore, deregulation of the cell cycle by pRb mutation or inactivation and
inhibition of apoptosis by p53 disruption result in malignant transformation
in numerous tissues. These properties have been exploited by DNA tumor
viruses that can simultaneously inactivate both tumor suppressors and cause
deregulation of cell cycle progression and apoptosis.
E2F
The pRb protein is a transcriptional repressor that regulates gene expression by
physically associating with transcription factors such as those of the E2F family
(79). The function of E2Fs in cell proliferation and apoptosis has been characterized in cell culture systems and in knockout mice. The E2F family consists of
five closely related transcription factors (36). Some differences in biochemical
properties of the E2F members have been noted. E2F-1, -2, and -3 prefer to
bind to pRB, whereas E2F-4 and -5 predominantly associate with pRB-related
proteins p107 and p130. It is likely that the different isoforms of E2F have different functions or are used to differing extents in specific tissues. E2F-1,
the most well characterized member of this family, activates genes whose
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products are important in the G1- to S-phase transition (60, 79, 80). E2F binds
to the DP family of transcription factors, and heterodimers of E2F and DP
regulate gene expression necessary for G1/S-phase transition. E2F-1 plays a
prominent role in G1/S-phase transition: The ectopic expression of E2F-1 can
drive serum-starved, growth-arrested cells through the G1 to S phase, and expression of dominant-negative mutants of E2F-1 inhibits cell cycle progression
during this phase of the cell cycle (81–84). Moreover, viral oncoproteins such
as SV40 large T antigen and human papilloma virus E7 can transform cells
when they bind to pRb and release activated E2F-1 (79). Furthermore, E2F expression alone, and in conjunction with the oncogene ras, can transform cells
that are then tumorigenic in nude mice (81, 85, 86). E2F-1 has also been shown
to play a role in apoptosis in cell culture. Deregulated E2F-1 expression in
rat 2 fibroblasts causes early S-phase entry and subsequent apoptosis (82–84),
which is dependent on p53 (82, 87). When E2F-1 and p53 are coexpressed in a
mouse fibroblast cell line, apoptotic cell death is induced and p53-mediated G1
arrest is overridden. Overexpression of E2F-1 may induce apoptosis by altering
transcription of genes necessary for cell survival or by inducing inappropriate
progression through the cell cycle.
E2F-1 KNOCKOUT MICE Mice that are null for E2F-1 have been generated
(88, 89). Because E2F is required for S-phase progression, it is perhaps surprising that E2F knockout mice do not show hypoproliferation. Instead, they show
enlarged thymus glands and lymph nodes owing to the excess of single positive
(CD4+/CD8− or CD4−/CD8+) thymocytes. Additionally, lymphomas, lung
tumors, and reproductive tract tumors are detected. Thus, in addition to its
role during S phase, E2F-1 appears to suppress inappropriate cell proliferation,
perhaps because E2F null mice are defective in normal apoptotic pathways.
Depending on the cellular context, E2F-1 may function as an oncogene to promote cell growth or may function as a tumor suppressor to induce apoptosis.
For example, when E2F-1 is bound to pRb, cell cycle progression is repressed.
However, when pRb is absent, E2F-1 stimulates proliferation. The cellular
concentration of E2F or the presence or absence of other cell cycle regulators
may influence whether E2F-1 functions to promote cell growth or death.
CONCLUSIONS
Apoptotic stimuli often arrest growth before inducing cell death. Glucocorticoid treatment of transformed lymphoid cells, for example, causes cells to
arrest in G1 before entering apoptosis. Glucocorticoids have been shown to inhibit the expression of G1-phase components such as cdk4, cycD3, and c-myc.
Downregulation of c-myc is necessary for G1 arrest and the induction of
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613
Figure 2 Effects of p53, pRb, and E2F mutations in cell culture systems and transgenic mice. All
these G1 regulators are capable of altering cell cycle progression and affecting the propensity of a
cell to undergo apoptosis (45, 47–49, 51, 64–66, 69–77, 81–85, 88, 89).
apoptosis in CEM cells, and P1798 cells require decreased expression of both
cyclin D3 and c-myc, which suggests that these cell cycle regulators influence
cell death as well as proliferation. Glucocorticoids appear to act through two
separate pathways to induce apoptosis—one in proliferating cells and another
in nonproliferating cells. These pathways share common upstream and downstream components that include glucocorticoid binding to its receptor, changes
in gene expression, and activation of apoptotic effector molecules.
During mouse development, p53, pRb, and E2F function as cell cycle regulators. This is readily apparent in the phenotypes of knockout mice (Figure 2).
In all three mouse models, abnormal cell proliferation is visible in some tissues; however, the response to this uncontrolled cell division differs among the
three different knockout mice. In p53 and E2F knockout models, proliferation
is allowed to continue unchecked, resulting in the formation of tumors. In
contrast, pRb knockout mice are nonviable and display massive cell death in
response to inappropriate mitosis. Thus it appears that p53 and E2F provide
positive signals for apoptosis in response to inappropriate cell proliferation, and
removal of these regulators results in lack of cell death and tumor formation.
It appears that pRb inhibits inappropriate apoptosis from occurring during embryogenesis by inducing alternative, non-apoptotic pathways to prevent undue
growth. When this protein is deleted from cells, growth arrest cannot occur and
apoptosis is induced. Animals that are heterozygous for pRb display a different
phenotype. These mice show unregulated proliferation and tumor formation,
suggesting that pRb has other effects that may be masked by the early death of
pRb null embryos. Thus although these three proteins serve as both cell cycle
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and apoptotic regulators, their functions differ greatly. Whereas pRb induces
growth arrest to suppress apoptosis, p53 and E2F are necessary for induction
of apoptosis in response to unregulated cell growth. The mechanisms by which
these cell cycle regulators influence the apoptotic process remain unknown.
In summary, the cell has integrated control of two antagonistic processes—
cell proliferation and cell death—at cell cycle checkpoints. This coordinated
regulation provides an effective means to control inappropriate cell cycle progression.
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