Carcinogenesis vol.21 no.3 pp.

485495, 2000
Apoptosis in cancer
Scott W.Lowe
1
and Athena W.Lin of this death was assumed to arise from necrosisa cata-
strophic (and easily discernible) type of cell deathKerr et al.
Cold Spring Harbor Laboratory, 1 Bungtown Road, PO Box 100,
raised the possibility that a large percentage of cell loss from
Cold Spring Harbor, New York, NY 11724, USA
tumors was due to apoptosis (1). Subsequent studies revealed
1
To whom correspondence should be addressed
a high frequency of apoptosis in spontaneously regressing
E-mail: lowe@cshl.org
tumors and in tumors treated with cytotoxic anticancer agents
In the last decade, basic cancer research has produced
(3). Together, these observations suggested that apoptosis
remarkable advances in our understanding of cancer bio-
contributed to the high rate of cell loss in malignant tumors
logy and cancer genetics. Among the most important of
and, moreover, could promote tumor progression. Nevertheless,
these advances is the realization that apoptosis and the
the importance of apoptosis in cancer remained under-appreci-
genes that control it have a profound effect on the malignant
ated for 15 years.
phenotype. For example, it is now clear that some oncogenic
mutations disrupt apoptosis, leading to tumor initiation,
Apoptosis and tumorigenesis
progression or metastasis. Conversely, compelling evidence
indicates that other oncogenic changes promote apoptosis, The cloning and characterization of the bcl-2 oncogene estab-
thereby producing selective pressure to override apoptosis lished the importance of apoptosis in tumor development. bcl-
during multistage carcinogenesis. Finally, it is now well 2 was rst identied at the chromosomal breakpoint of t(14;
documented that most cytotoxic anticancer agents induce 18) in a human leukemia line and was later shown to be a
apoptosis, raising the intriguing possibility that defects in common event in follicular lymphoma (5,6). At this time,
apoptotic programs contribute to treatment failure. oncogenes were classied as either transforming or immor-
Because the same mutations that suppress apoptosis during talizing oncogenes based on their properties in rodent cell
tumor development also reduce treatment sensitivity, transformation assays. However, bcl-2 did not behave like a
apoptosis provides a conceptual framework to link cancer typical oncogene: instead of disrupting normal proliferation
genetics with cancer therapy. An intense research effort is controls, Bcl-2 promoted cell survival by blocking programmed
uncovering the underlying mechanisms of apoptosis such cell death (79). Moreover, in transgenic mice, Bcl-2 overexpr-
that, in the next decade, one envisions that this information ession promoted lymphoproliferation and accelerated c-Myc-
will produce new strategies to exploit apoptosis for thera- induced lymphomagenesis (8,10). To date, at least 15 Bcl-2
peutic benet. family member proteins have been identied in mammalian
cells, including proteins that promote apoptosis and those that
prevent apoptosis (11). In addition to Bcl-2, Bcl-x
L
is a potent
Introduction death suppressor that is upregulated in some tumor types (12).
Conversely, Bax is a death promoter that is inactivated in
Apoptosis was initially described by its morphological charac-
certain types of colon cancer and in hematopoietic malignanc-
teristics, including cell shrinkage, membrane blebbing, chro-
ies (13,14).
matin condensation and nuclear fragmentation (13). The
p53 was the rst tumor suppressor gene linked to apoptosis.
realization that apoptosis is a gene-directed program has had
p53 mutations occur in the majority of human tumors and are
profound implications for our understanding of developmental
often associated with advanced tumor stage and poor patient
biology and tissue homeostasis, for it implies that cell numbers
prognosis (15). By 1992, p53 was clearly established as a
can be regulated by factors that inuence cell survival as well
checkpoint protein involved in cell-cycle arrest and maintaining
as those that control proliferation and differentiation. Moreover,
genomic integrity following DNA damage. However, p53
the genetic basis for apoptosis implies that cell death, like any
could induce apoptosis when overexpressed in a myeloid
other metabolic or developmental program, can be disrupted
leukemia cell line, suggesting that p53 might also regulate cell
by mutation. In fact, defects in apoptotic pathways are now
survival (16). Studies using p53 knockout mice demonstrated
thought to contribute to a number of human diseases, ranging
that endogenous p53 could participate in apoptosis: p53 was
from neurodegenerative disorders to malignancy (4).
required for radiation-induced cell death in the thymus, but
The notion that apoptosis might inuence the malignant
not cell death induced by glucocorticoids or other apoptotic
phenotype goes back to the early 1970s. Kinetic studies of
stimuli (17,18). Hence, the role of p53 in apoptosis was
tumor growth implied that cell loss from tumors could be
indirectly linked to DNA damage and could be stimulus-
massive; indeed, observed tumor growth rates could be 5%
(radiation) and tissue-specic (thymocytes). It is now known
of that predicted from proliferation measurements alone (1,2).
that other stimuli can activate p53 to promote apoptosis,
In principle, changes in this cell loss factor could have a
including hypoxia and mitogenic oncogenes (see below).
major impact on tumor growth or regression. Although most
Moreover, several upstream and downstream components of
the p53 pathway (e.g. Mdm-2, ARF and Bax) are mutated in
Abbreviations: IGF, insulin-like growth factor; NF-B, nuclear factor B;
human tumors (15).
PTP, permeability transition pore; TNF-, tumor necrosis factor ; TRAF-2,
TNF receptor-associated factor. Although the initial studies on Bcl-2 and p53 established
Oxford University Press 485
S.W.Lowe and A.W.Lin
the importance of apoptosis in carcinogenesis, it is now example, IGF-1 promotes cell survival through the PI-3 kinase
pathway (34), and depletion of IGF-1 or other survival factors clear that mutations in many cancer-related genes can disrupt
apoptosis. For example, the Fas/CD95 receptor normally con- can trigger death by default (35). In contrast, other stimuli
involve true pro-apoptotic factors; as an example, many forms trols cell numbers in the immune system by eliminating cells
through apoptosis, and disruption of this pathway can lead to of cellular stress can activate p53, which promotes apoptosis
through pro-apoptotic molecules like Bax (25,28,36). lymphoproliferative disorders and even cancers (19). In addi-
tion, several signal transduction pathways promote cell survival The identication of apoptotic triggers provides insight
into the forces of tumor evolution (see also next section). For in response to growth and/or survival factors, and these
pathways may be crucial in controlling cell numbers in vivo. example, in the skin, excessive exposure to UV radiation
induces apoptosis, which presumably serves to eliminate heav- One critical pathway involves signaling through PI-3 kinase
(20), which can be activated by Ras and is downregulated by ily damaged cells. UV radiation induces apoptosis, and loss
of p53 function leads to the survival of these damaged cells the PTEN tumor suppressor (21). Ras activation and PTEN
loss are common in human tumors. thereby initiating tumor development (37). Other apoptotic
triggers are important in tumor progression. As developing Studies using transgenic and knockout mice provide direct
evidence that disruption of apoptosis can promote tumor tumors outgrow their blood supply, they encounter hypoxia
(low oxygen), which can activate p53 to promote apoptosis development. In addition to the lymphomas (10), bcl-2 trans-
genes accelerate SV40 large T antigen-induced mammary (38). Cells acquiring apoptosis defects (e.g. p53 mutations)
can survive hypoxic stress, leading to their clonal expansion carcinogenesis (22). Similarly, p53 loss produced by gene
knockout accelerates tumor progression in many settings, within the tumor (38). Similarly, as developing tumor cells
undergo repeated divisions, telomeres are shortened until some including the murine retina, lens, choroid plexus and in the
lymphoid compartment (23). In many cases, p53 loss is malfunction triggers either senescence or apoptosis. Like
hypoxia, p53 is required for apoptosis induced by telomere associated with reduced apoptosis in situ. Additionally, mouse
studies reveal the crucial role of extracellular survival factors malfunction; thus p53 mutant cells survive this response and
are genomically unstable (39). Indeed, suppression of the in supporting tumor progression. For example, large T antigen-
induced pancreatic tumors require the upregulation of apoptotic response to telomere malfunction may explain why
combined loss of telomerase and p53 stimulates tumor develop- insulin-like growth factor 2 (IGF-2) for progression to
carcinomasin fact, tumors arising in IGF-2 animals remain ment (40). These studies may explain why p53 mutations are
usually late events in tumor developmenta cell acquiring a hyperplastic and display excessive apoptosis (24). Moreover,
inactivation of PTEN promotes tumorigenesis and cell survival p53 mutation might not have a selective advantage until the
developing tumor encounters hypoxic conditions or achieves when disrupted in mice (21), and disruption of the pro-apoptotic
bax gene accelerates brain and mammary tumorigenesis in sufcient telomere erosion.
Disruption of apoptosis may also contribute to tumor meta- large T antigen transgenic mice (25,26).
Because molecules that regulate apoptosis can have other stasis. To metastasize, a tumor cell must acquire the ability to
survive in the bloodstream and invade a foreign tissue. Norm- activities, it is often difcult to demonstrate that mutations in
tumors actually confer a survival advantage. For example, p53 ally, this process is prevented by the propensity of epithelial
cells to die in suspension, or in the absence of the appropriate can promote apoptosis, cell-cycle arrest and senescence such
that loss of p53 function increases viability, chromosomal tissue survival (41). Clearly, the fact that these processes
trigger apoptosis creates selective pressure to mutate apoptotic instability and cellular lifespan. However, compelling evidence
indicates its apoptotic activity is important in tumor suppres- programs during tumor development. Apoptosis in suspension
is controlled by a number of other molecules, including the sion. First, marked decreases in apoptosis correlate with the
occurrence of p53 mutations in some transgenic mice (23) and focal adhesion kinase and signal transduction pathways (42).
Some studies argue that p53 and Bcl-2 can also inuence cell in clonal progression of Wilms tumor (27). Second, disruption
of several p53 effectors in apoptosis (e.g. bax, apaf-1 and death in suspension (43), and others observe enrichment for
p53 mutations or Bcl-2 overexpression in metastases (44,45). casp-9) can promote oncogenic transformation and tumor
development in mouse model systems (25,28,29). In colon Hence, loss of apoptosis can impact tumor initiation, progres-
sion and metastasis. cancer, bax and p53 mutations appear mutually exclusive,
consistent with a pathway relationship (30). In contrast, p21,
which is essential for p53-mediated arrest, is rarely mutated
Mechanisms of apoptosis
in human tumors (31). Finally, some tumor-derived p53 mutants
remain capable of promoting cell-cycle arrest while losing Although the primary focus of this review is on the biology
of apoptosis in cancer, much is known about the biochemical their apoptotic potential (32,33). Nevertheless, these data do
not imply that the other p53 activities are dispensible for tumor action of many apoptotic players, and key components are
being assembled into pathways. At the molecular level, the suppression; rather, they simply argue that its apoptotic activity
is important. best understood cell death pathways involve those initiated by
death receptors, including Fas/CD95, TNFR1, DR3, DR4 What triggers apoptosis during tumor development? A
variety of signals appear important. Extracellular triggers and DR5 (46). Upon binding, tumor necrosis factor (TNF-
) trimerizes its ligand TNFR1 and results in the subsequent include growth/survival factor depletion, hypoxia, radiation
and loss of cell-matrix interactions. Internal imbalances can recruitment of the signal transducing molecules TRADD
through conserved protein interaction regions known as death also trigger apoptosis, including DNA damage (produced by
cell-cycle checkpoint defects or exogenous toxins), telomere domains. TRADD recruits RIP and TNF receptor-associated
factor (TRAF-2), leading to activation of nuclear factor B malfunction and inappropriate proliferative signals produced
by oncogenic mutations. In some instances the apoptotic (NF-B), which suppresses TNF--induced apoptosis (47).
While the recruitment of FADDby TRADD results in apoptosis trigger actually alleviates an anti-apoptotic signal. For
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Apoptosis in cancer
through activation of a cell death protease, caspase-8. Activated apoptosis results from a complex integration of internal and
external pro- and anti-apoptotic signals. caspase-8 initiates a protease cascade that cleaves cellular
Apoptotic programs cannot simply be described as two targets and results in apoptotic cell death (46). Hence, disrup-
parallel programs converging on a common caspase machinery. tion of FADD can prevent activation of caspase-8, thereby
First, genetic studies using caspase-decient mice demonstrate producing defects in receptor-mediated cell death (48). This
that the requirement for different death effector molecules pathway is rarely the target of oncogenic mutations, but, if
during apoptosis is highly variable, being cell-type and stimulus anything, it is enhanced during tumor development (see below).
specic (6165). Second, a large degree of cross-talk can
Growth factors, cytokines and DNA damage appear to signal
exist between pathways. For example, p53 can transactivate
cell death through the mitochondria, and this pathway is the
genes encoding death receptors (73). Also, receptor-mediated
target of many oncogenic mutations. These diverse signals
activation of caspase-8 can cleave and activate BID, a pro-
affect the function of Bcl-2 family members which, in turn, can
apoptotic Bcl-2 family member that can facilitate cytochrome
modulate the mitochondrial function though the permeability
c release from the mitochondria (74,75). Animal studies
transition pore (PTP), a proposed channel evolved in mitochon-
indicate that the caspase-8/BID pathway is highly cell-type
dria following necrotic or apoptotic signals. The PTP is thought
dependent (76). It is likely that more complexities will be
to be composed of clustered components of the mitochondrial
identied; although confounding to the experimentalist, cell
membranes, including the voltage-dependent anion channel
type and stimuli specicities provide avenues for designing
and adenine nucleotide translocator; and the opening of PTP
selective therapeutics.
results in the release of cytochrome c from mitochondria (49).
Consistent with this idea, the crystal structure of Bcl-x
L
is
reminiscent of pore-forming proteins of some bacterial toxins
Oncogenic mutations can promote apoptosis
(50). Enforced expression of the pro-apoptotic molecules Bax
Some oncogenic changes promote, rather than suppress
or Bak can result in increased mitochondria membrane potential
apoptosis. Although this nding has ramications for multistep
and release of cytochrome c, which can be blocked by
carcinogenesis and cancer therapy (see below), the initial
overexpression of Bcl-2 (51). Cytosolic cytochrome c can
insights came from studies on adenovirus. Adenovirus encodes
interact with Apaf-1 and pro-caspase-9 to initiate a protease
several oncoproteins that, when expressed together, transform
cascade similar to that described above (5254).
rodent cells (77). The E1A oncoprotein induces quiescent cells
As eluded to above, a series of enzymes known as caspases
to enter S phase (presumably to make the host cell permissive
are considered the engine of apoptotic cell death. Caspases
for virus replication) and, as a result, acts as a potent oncogene.
are cysteine proteases that are expressed as inactive pro-
The E1B 19 and 55K oncoproteins are required for efcient
enzymes, and can be broadly classied into signaling or
virus replication and cooperate with E1A in transformation.
effector caspases (55). Signaling pro-caspases associate with
Adenovirus mutants lacking the E1B 19K protein induce an
specic adapter molecules that facilitate caspase activation by
E1A-dependent cytocidal phenotype that is associated with
induced proximity (5658). For example, caspase-9 associates
degradation of both viral and host-cell DNA (78,79). As a
with Apaf-1, and oligomerization of this complex in the
result, E1B mutant adenoviruses produce poor virus yield.
presence of cytochrome c can activate the downstream caspase
Subsequent studies explained these puzzling observation: E1A
cascade. Other adapter/caspase complexes include FADD/
induces apoptosis, and the E1B 19K oncoprotein acts like Bcl-
caspase-8 and RAIDD/caspase-2 (46). In mitochondrial path-
2 to suppresses apoptosis (80). In virus-infected cells, E1B
ways, most caspases act downstream of cytochrome c release,
prevents E1A-induced apoptosis, allowing viral replication to
and some evidence suggests that disruption of caspases only
proceed. This implies that apoptosis can act to counter virus
delays cell death (59,60). However, in other circumstances,
replication and provides a biologic basis for cooperation
loss of these proteases produces pathological increases in
between E1A and E1B in adenovirus transformation.
cell numbers (29,6165). To date, little is known about the
Studies on the c-myc oncogene highlight the importance of
involvement of caspase mutations in cancers. Nevertheless,
oncogene-induced apoptosis in human cancer (81). In normal
disruption of Apaf-1 is associated with Noonans Syndrome,
cells, ectopic c-Myc expression drives proliferation and pre-
caspase-10 mutations contribute to autoimmune lymphoprolif-
vents cell-cycle arrest upon serum withdrawal. However, while
erative syndrome type II (65,66), and frameshift mutations in
c-Myc-expressing cells continue to proliferate in low serum,
caspase-5 can occur in hereditary nonpolyposis colorectal
cells do not accumulate because they die by apoptosis (82).
cancers, gastrointestinal and endometrial tumors (67).
Importantly, survival factors such as IGF-1 can suppress c-
Certain signal transduction pathways alter the probability
Myc-induced cell death without producing substantial effects
with which pro-apoptotic signals induce apoptosis. For
on c-Myc-induced proliferation (83). The observation that c-
example, cytokines such as IL-6 can suppress p53-induced
Myc actively promotes apoptosis explains the potent
apoptosis in certain cell types (16). Also, TNF--induced
cooperative effects observed between c-myc and bcl-2 in
apoptosis is modulated by TRADDs ability to bind TRAF2,
murine lymphomagenesis (10). In fact, much like E1A and
which facilitates NF-B-mediated cell survival (47,68). Finally,
E1B, c-Myc cooperates with Bcl-2 to transform rodent
PI-3 kinase pathway mediates cell survival signaling from
broblasts (84,85). The ability of c-Myc to cooperate with
extracellular cytokines receptors. These receptors activate Ras
Bcl-2 in transformation could be viewed very much like E1A
and a kinase cascade involving PI-3 kinase and Akt leading to and E1B; Bcl-2 allows c-Myc-induced proliferation to proceed
the ultimate phosphorylation and inactivation of pro-apoptotic without apoptosis.
molecules such as BAD and caspase-9 (69,70). PTEN acts as Why do some oncogenes promote apoptosis? Studies on c-
a lipid phosphatase to inactivate 3-phosphorylated phosphoino- Myc have been unable to separate its proliferative functions
sitides, thereby downregulating this pathway (71,72). Together, from those which promote apoptosis, implying that the pro-
cesses are coupled (86). Similarly, in normal broblasts, the these studies imply that the ultimate decision to initiate
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S.W.Lowe and A.W.Lin
ability of E1A to promote apoptosis depends on its ability to
inactivate the retinoblastoma tumor suppressor, and cannot be
separated from its transforming functions (87). However, Rb
loss leads to increased apoptosis in animal models (88,89),
and reintroduction of Rb into cells lacking functional protein
can suppress apoptosis (90). Both E1A and loss of Rb
deregulate the E2F transcription factors, and E2F-1 overexpres-
sion itself can induce apoptosis (9193). Hence, the
pro-apoptotic activities of E1A and c-Myc appear directly or
indirectly coupled to their hyperproliferative activity, implying
that apoptosis acts as part of a cellular fail-safe mechanism to
limit the consequences of aberrant mitogenic signaling.
The critical involvement of p53 in oncogene-induced cell
death underscores the importance of this response as a protect-
ive measure against oncogenic transformation. Although p53
is not required for normal apoptotic deaths during development,
E1A can stabilize p53 protein (94), and p53-decient cells are
resistant to E1A-induced apoptosis (95,96). p53 loss substitutes
for the E1B proteins in promoting the growth, survival and
transformation of E1A-expressing cells (95). This implies that
the primary function of the E1B proteins in adenovirus
transformation is to circumvent this program. c-Myc also
Fig. 1. Oncogene-induced apoptosis. Oncogenes such as E1A and c-myc induces apoptosis in a p53-dependent manner (97,98). In vivo,
induce apoptosis through p53-dependent and independent pathways, and
loss of p53 function can dramatically accelerate tumorigenesis
both pathways may facilitate cytochrome c release from mitochondria. In
induced by mitogenic oncogenes such as large T antigen, E7
any case, the Apaf-1/caspase-9 death effector complex appears important for
and c-Myc, which is associated with decreased apoptosis in
oncogene-induced death. Current evidence has not ruled out the possibility
that oncogenes and/or p53 inuence Apaf-1 and/or caspase-9 independent of situ (99102). Importantly, in MMTV/c-myc transgenic mice,
cytochrome c, but this remains a possibility. Components of the oncogene-
loss of p53 function accelerates thymic lymphoma but not
induced cell-death program that are mutated in human tumors are shown in
mammary cancers, suggesting the involvement of p53-indepen-
black, candidate tumor suppressors are shown in gray.
edent mechanism. Hence, oncogenes can induce apoptosis in
both p53-dependent and -independent manners, depending on
cellular context. as well as active induction of cell death in oncogene-expressing
Recent studies indicate that p19
ARF
acts as an essential cells (Figure 1).
intermediate in oncogene signaling to p53 (103). For example, Caspase-9 and its adapter Apaf-1 are essential downstream
oncogenes such as E1A or c-myc induce ARF message and components of p53 in oncogene-induced death (29). Hence,
protein in normal mouse embryo broblasts, which correlates disruption of either Apaf-1 or caspase-9 in broblasts prevents
c-Myc-induced death without affecting p53 accumulation or with their ability to activate p53 and promote apoptosis.
activation. In fact, inactivation of either caspase-9 or apaf-1 Recently, ARF was shown to be a transcriptional target of
completely substitutes for p53 loss in promoting the oncogenic c-Myc (104). In contrast, these oncogenes fail to activate p53
transformation of cells by c-Myc and Ras (29). Concordantly, in ARF-null cells, and promote proliferation without substantial
Apaf-1 has been identied in cell free systems as an oncogene- apoptosis (105,106). Together, these studies indicate that
generated activity important for apoptosis in E1A-expressing p19
ARF
acts as part of a p53-dependent fail-safe mechanism
cells (107,111). Because cytochrome c is an essential co-factor
to counter hyperproliferative signals, and predict that disruption
that is required for activation of the Apaf-1/caspase-9 protease
of ARF, or the INK4a/ARF locus, should cooperate with
complex, these studies suggest how oncogenes are linked to
mitogenic oncogenes during tumor development. Studies using
the effector phase of apoptosis (Figure 1). Of note, several
c-myc transgenic mice support this view (102).
molecules that modulate oncogene-induced apoptosis are mut-
One question is whether oncogenes directly induce apoptosis
ated in human tumors, and the data described above identify
or sensitize the cell to various apoptotic stimuli. Clearly,
Apaf-1 and caspase-9 as candidate tumor suppressors (Figure
oncogenes can induce apoptosis directly if expressed at suf-
1). However, to date, no mutations in either apaf-1 or caspase-
cient levels, and E1A-expressing cells possess an oncogene-
9 have been described. Perhaps these components act too late
generated activity capable of activating apoptosis in cell free
in the cell death program to provide a long-term survival
systems (107). However, cells stably expressing E1A or c-
advantage; alternatively, the mutational status of these genes
Myc are sensitized to diverse apoptotic stimuli, including
may not have been thoroughly examined.
serum depletion, DNA damaging agents, hypoxia, Fas, TNF-
and other stimuli (81). One target of oncogene-induced
Apoptosis and cancer therapy
sensitization is the mitochondria. Both E1A and c-Myc facilit-
ate cytochrome c release from mitochondria, and cytochrome Most anticancer agents now in use were developed using
c alone is sufcient to sensitize cells to diverse agents empirical screens designed to identify agents that selectively
(107,108). Oncogenes can facilitate cytochrome c release kill tumor cells. Until recently, most research into drug action
independently of p53 (108), although it is worth noting that focused on their intracellular targets, the nature of the cellular
p53 can induce Bax and proteins that affect mitochondrial damage produced by the drugtarget interaction, or resistance
mechanisms that prevent the drug target interaction. However, function (109,110). Hence, p53 may be involved in sensitization
488
Apoptosis in cancer
treatment sensitivity in carcinomas is less clear: whereas some
studies identify striking correlations between p53 mutations
and poor treatment response, others see no effect (116,128).
Few studies have associated Bcl-2 with drug resistance in
patients and, in fact, high Bcl-2 levels may be a good prognostic
indicator for breast cancer. Finally, in some settings loss of
Bcl-2 and p53 delays therapy-induced apoptosis but does not
enhance long-term survival in clonogenic assays (128).
What could explain these discrepancies? Though it is pos-
sible that apoptosis does not contribute to treatment sensitivity
in solid tumors, some caveats are worth mentioning. First,
clinical studies typically examine single alterations (e.g. p53
mutation) relying on detection methods that are not perfect
and cannot exclude extragenic mutations in the same pathway
Fig. 2. Anticancer agents induce apoptosis. Hematoxylin and eosin staining
(15,116). This makes it virtually impossible to determine
of lymph nodes from lymphoma-bearing mice (B cell lymphoma) left
negative results. As an example, murine lymphomas harboring
untreated (A) or isolated 5 h after treatment with cyclophosphamide (B).
INK4a/ARF mutations are chemoresistant, display defective
Apoptotic cells are identied by their reduced size and by the presence of
p53 function, but retain wild-type p53 genes (102). These highly condensed chromatin. Massive apoptosis occurs in response to
cyclophosphamide.
tumors would be mistakenly classied as p53 normal by
current technologies. Secondly, while clonogenic survival is
often considered the gold-standard of cytotoxicity assays,
in the 1970s pathologists noticed that radiation and chemother-
this readout does not always reect the in vivo response (129).
apy can induce cell death with morphological features of
It is possible that extracellular survival factorsinuenced by
apoptosis (112) (Figure 2), although the signicance of these
cell density or microenvironmentcan affect drug-induced
observations was not widely appreciated. In particular, the
death (130).
premise that anticancer agents induce apoptotic cell death
Anticancer agents induce apoptosis in normal tissues as well
implies that cellular responses occurring after the drugtarget
as in tumors. In fact, many of the pathologists who identied
interaction can have impact on drug-induced cell death (113
apoptosis in tumors realized that apoptotic cell death was
115).
induced in a subset of normal tissues (e.g. bone marrow and
It is now well-established that anticancer agents induce
intestine), and it was suggested that the process might contrib-
apoptosis, and that disruption of apoptotic programs can reduce
ute to the toxicity associated with chemotherapy (112).
treatment sensitivity (116). Since agents with distinct primary
Studies using mouse models provide strong support for this
targets can induce apoptosis through similar mechanisms,
idea. For example, moderate doses of radiation and chemother-
mutations in apoptotic programs produce multi-drug resistance
apy induce apoptosis in the murine thymus, spleen, bone
(113,117). For example, many agents activate p53, and that
marrow and intestine, the same tissues that account for the
p53 loss can attenuate drug-induced cell death (15). Moreover,
deleterious side-effects of chemotherapy. However, these tis-
p53 mutations reduce therapy-induced apoptosis and tumor
sues in p53 knockout mice display much reduced apoptosis
regression in experimentally generated and spontaneous murine
and cell loss following radiation or chemotherapy (17,18,131
tumors (102,118), whereas re-introduction of normal p53 to
134), and these animals are resistant to otherwise lethal doses
p53 mutant tumor lines and xenographs cooperates with
of ionizing radiation (135). Similarly, ectopic expression of
chemotherapy to induce apoptosis and tumor regression
Bcl-2 in bone marrow cells achieves a similar effect (136).
(15,119). p53 is not strictly required for drug-induced cell
Together, these studies strongly suggest that drug-induced
death; indeed, at sufcient doses virtually all anticancer agents
apoptosis causes loss of normal cells and contributes to the
induce apoptosis (and other types of death) independently of
side effects of cancer therapy.
p53 (15). In fact, the contribution of p53 to drug-induced
apoptosis is determined by a variety of factors, including
agent, dose, tissue and mutational background of the tumor
Apoptosis: a biological link between cancer genetics and
(15,120,121). In short-term assays, Bcl-2 can promote resist-
cancer therapy
ance to a wide range of anticancer agents (122,123) and can
The studies described above highlight the fact that disruption
even prevent p53-independent deaths (124). Because Bcl-2 is
of apoptosis can promote tumor initiation, progression and
considered as a general apoptosis inhibitor, these results argue
treatment resistance. Indeed, it is remarkable that the same
for the broad importance of apoptosis in treatment sensitivity.
genetic alterations that inuence apoptosis during tumori-
Additionally, death receptor pathways may also contribute to
genesis also modulate treatment sensitivity. For example,
therapy-induced apoptosis (125), although the relative contribu-
c-Myc enhances apoptosis in low concentrations of survival
tion of these effects is controversial (126).
factors or oxygen and following treatment with diverse
In human cancer, the most compelling links between
cytotoxic agents (38,82,137), conversely, loss of p53 and
apoptosis and treatment sensitivity occur in patients with
overexpression of Bcl-2 suppress apoptosis induced by onco- leukemia or lymphoma. In these malignancies, p53 mutations
genes, depletion of survival factors, hypoxia and cytotoxic correlate with short remissions and drug resistance following
drugs (17,118,138). As a result, anti-apoptotic mutations arising therapy (15). Also, INK4a/ARF mutations, which reduce cyclo-
during the course of tumor development can simultaneously phosphamide-induced death in murine lymphomas (102), are
select for chemoresistant cells. This pattern of co-selection associated with poor treatment outcome in acute lymphoblastic
leukemia (127). The extent to which apoptosis contributes to may explain the phenomenon of de novo drug resistance, i.e.
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S.W.Lowe and A.W.Lin
tumors that initially respond poorly to therapy despite having feasible. First, most anti-apoptotic mutations act relatively
upstream in the program (e.g. PTEN and p53 loss; Ras never been selected in the presence of drug.
Studies on c-Myc-induced lymphomagenesis support and NF-B activation), implying that tumor cells retain the
machinery and latent potential for apoptosis. Secondly, tumor- directly link disruption of apoptosis during tumor development
to de novo resistance (102). In a mouse lymphoma model, specic alterations in apoptotic programs provide opportunities
to target cell death in a selective manner. Several current INK4a/ARF or p53 mutations dramatically accelerate c-Myc-
induced lymphomagenesis, owing to a defect in oncogene- strategies are discussed below.
induced apoptosis. Moreover, these tumors respond poorly to
Targeting anti-apoptotic activities
therapy; whereas mice harboring tumors with an intact p53
In instances where apoptosis is disabled by dominant onco-
pathway are cured, the vast majority harboring tumors with
genes, agents that disrupt their anti-apoptotic function can
INK4a/ARF or p53 mutations relapse shortly after treatment.
produce remarkable increases in cell death. Overexpression of
This pattern of resistance is particularly common in advanced
anti-apoptotic Bcl-2 family members can promote tumorigen-
solid tumors. These tumor-types probably encounter many
esis and chemoresistance, suggesting that functional inhibition
triggers of apoptosis during tumor evolution (e.g. survival
of these proteins might be lethal to cancer cells. Indeed,
factor depletion, hypoxia, loss of cell-matrix interactions), and
adenoviral gene transfer of Bax results in cytotoxicity in
it seems likely that one or more apoptotic programs must be
human ovarian cancer cell lines, and administration of Ad-
lost to reach the malignant state. Perhaps this explains why
DF3-Bax following tumor inoculation eradicated 99% of
many advanced solid tumors are inherently difcult to treat.
tumor implants in nude mice (146). Adenovirus-mediated gene
transfer of Bcl-x
S
, a dominant-negative repressor of Bcl-2 and
Non-apoptotic forms of programmed cell death
Bcl-x
L
, can synergize with chemotherapy and promote tumor
Although apoptosis is a cell death program, not all programmed regression in xenographs (147,148), but produces minimal
deaths are apoptotic. In addition to cell death, other pro- apoptosis when introduced into normal epithelial cells (149).
grammed responses contribute to the deletion of potentially Some current anticancer agents may unwittingly target the
cancerous cells. Senescence is an irreversible program of cell- Bcl-2 family; for example, taxanes may induce phosphorylation
cycle arrest that is disrupted in many tumors or tumor-derived and inactivation of Bcl-2 (150). As more is learned about
lines (139). Replicative senescence was originally dened by Bcl-2 structure and function, small molecule inhibitors of Bcl-
the observation that primary cells have a genetically determined 2 action might become feasible.
limit to their proliferative potential in cell culture, after which Hyperactivation of cell survival signaling may accompany
they permanently arrest with characteristic features. Owing to tumor development, and these pathways are particularly excit-
the end-replication problem, telomeres shorten during each ing targets for small molecule inhibition. NF-B activity can
cell division unless telomerase is expressed, and it is thought be induced by oncogenes or anticancer agents to promote cell
that some aspect of excessive telomere shortening activates survival, such that inactivating NF-B enhances cell death in
cell-cycle arrest and other characteristics of senescence (139). response to tumor necrosis factor and certain chemotherapeutic
However, other stimuli can induce phenotypes suggestive agents (151). In fact, inhibiting NF-B activity using I-B or
of senescence, including mitogenic oncogenes and ionizing proteosome inhibitors synergizes with radiation and chemother-
radiation (140143). apy to induce cell death in tumor lines and xenographs
These observations imply that cellular senescence can be (152,153). The PI-3 kinase/Akt pathway involves a series of
induced by diverse stimuli leading to the engagement of a enzymes that transit the survival signals. In principle, small
common cell-cycle arrest program. In this view, senescence is molecule inhibition of any of these molecules might restore
conceptually similar to apoptosis, which is induced by diverse apoptosis to tumor cells, or synergize with more classic agents
stimuli leading to the engagement of a common cell death to induce cell death (154).
program. Consequently, the biological roles of cellular senes- Oncogenic ras mutations deregulate normal growth control
cence may go beyond the control of cellular or organismal but can also signal cell survival (34). Therefore, agents that
aging, and reect a global anti-proliferative response to a interfere with Ras function might be cytostatic or cytotoxic.
variety of cellular stresses. In this view, senescence may To be biologically active, Ras must be modied by a
represent an important form of programmed cellular death farnysltransferase, and many groups have developed
that limits tumor development. Recent evidence suggests that farnysltransferase inhibitors as anti-tumor agents. Although
anticancer agents induce cellular senescence in human tumor- these agents were predicted to be cytostatic, they can induce
derived lines treated in culture or as xenographs (144,145). massive apoptosis and tumor regression of mammary carcin-
Together, the non-apoptotic forms of programmed cell death, omas arising in ras transgenic mice (120). In addition, inhibi-
such as senescence, might also be important for therapy. tion of Ras-GAP induces apoptosis specically in tumor, but
not in normal cells, suggesting Ras-GAP as a novel target for
cancer therapy (155). Why would these agents induce apoptosis Apoptosis and new therapeutic strategies
and, moreover, why would they be selective? The answer is
Since apoptotic programs can be manipulated to produce
unknown, but it is possible that tumor cells become dependent
massive changes in cell death, the genes and proteins control-
on survival signaling through the PI-3 kinase pathway, which
ling apoptosis are potential drug targets. As indicated above,
could be suppressed by Ras inhibition.
many empirically derived cytotoxic drugs already may target
Restoring pro-apoptotic activities
apoptosis, albeit indirectly and non-exclusively. They are also
mutagenic and toxic to normal tissues. In contrast, agents that In circumstances where apoptosis is lost by a recessive
mutation, restoring the dysfunctional gene or activity can directly induce apoptosis may provide less opportunity for
acquired drug resistance, decrease mutagenesis and reduce promote massive cell death. For example, reintroduction of
p53 into p53 mutant tumor cells can directly induce apoptosis toxicity. Two observations suggest that such strategies are
490
Apoptosis in cancer
or enhance treatment sensitivity in tumor cell lines or in that simultaneous inactivation of Rb and cdk2 would be
xenographs (156,157). Indeed, strategies using this approach particularly pro-apoptotic, and produce a synthetic lethal effect
are currently in clinical trials (158). As a general rule, tumor in cells with a mutant Rb pathway (168). Consistent with this
cells are inherently more sensitive to p53 inhibition than hypothesis, cell-permeable peptides that interfere with cdk2
normal cells, perhaps because mitogenic oncogenes can activate activity readily induce apoptosis in tumor cells while having
p53 to promote apoptosis (117). Hence, there is some rationale little effect on normal cell lines (168). Interestingly, although
for selectivity of this approach. Of note, strategies to counter E2F-induced apoptosis is potentiated by p53 (91), these pep-
recessive anti-apoptotic mutations need not rely on gene or
tides can induce apoptosis in p53-decient tumor cell lines.
protein therapy. In some instances, inactivation of a pro-
Therefore, this strategy has widespread potential.
apoptotic gene might actually promote cell survival by relieving
Chemoprotection
inhibition of a downstream death suppressor (e.g. PTEN loss
As indicated above, the propensity of certain tissues (e.g. bone
de-represses Akt). In such instances, the downstream effector
marrow and intestine) to drug-induced apoptosis may limit the
may be a more suitable drug target (154).
effectiveness of many current therapies. Studies using mouse
Death ligands
models have clearly documented the importance of p53 for
Although mutations in death receptors are rare events in human
apoptosis in thymocytes, bone marrow and intestinal stem
tumors, changes accompanying tumorigenesis can alter the
cells (see above); consequently, agents which suppress p53
regulation of these pathways. Mitogenic oncogenes like c-myc
function may be effective radio- or chemoprotective agents
and E1A can increase sensitivity to Fas and TNF- in cultured
and/or allow dose intensication of current regimens. Since
cells (159,160), and many human tumors have alterations in
most advanced solid tumors have lost p53 function, these
TRAIL-mediated death pathways (19). TRAIL is a TNF-
inhibitors should not interfere with cell death of most tumor
related protein that initiates p53-independent apoptosis by
cells. Recently, a small molecule (designated pithrin-) has
binding to its receptors DR4 or DR5. Normal cells are resistant
been identied that inhibits p53-mediated transcriptional
to TRAIL-induced apoptosis, apparently because these cells
responses and p53-induced apoptosis in cultured cells (169).
express decoy receptors that compete with DR4 and DR5 for
In mice, pithrin- is a potent radioprotective agent; pithrin-
TRAIL but do not transmit a death-inducing signal (161,162).
treated mice survive otherwise lethal doses of ionizing
Through an unknown mechanism, the expression of the decoy
radiation.
receptor 1 seems to be widely lost on tumor cells, making
Several caveats exist with respect to p53 inhibition in
them exquisitely susceptible to TRAIL-mediated cell death.
patients. First, the premise is based on animal studies, and it
Because DR5 is a p53-responsive gene, combination therapy
is not yet known the extent to which p53 contributes to
with TRAIL and classic cytotoxic agents may be particularly
anticancer agent toxicity in humans. Secondly, although the
effective in treating tumors with functional p53 (73).
protective effects in mice are striking, the magnitude of the
A conceptually related strategy for selective tumor-cell
protective effect over repeated doses and time is unknown.
killing involves a viral protein known as apoptin, the VP3
Thirdly, p53 inhibition may promote mutations by allowing
product of chicken anemia virus (CAV). This protein is
the survival of mutated cells. This is clearly a concern, since
responsible for the cytopathic effects of CAV (163) and induces
p53 knockout mice are extremely sensitive to radiation-
apoptosis in tumor cells but not normal cells (164). Although
induced carcinogenesis. However, transient inhibition of p53
its mechanism of action remains unknown, apoptin-induced
may be less mutagenic (169). In any case, most current
apoptosis is independent of p53 and is enhanced by Bcl-2
anticancer agents are directly mutagenic, and it is not clear
(165,166). At the very least, the remarkable effects of apoptin
whether p53 inhibitors (which would only indirectly promote
underscore the point that selective induction of apoptosis in
mutations) would be worse. At the very least, this strategy
tumor cells is achievable.
warrants further consideration.
Enhancing the effects of pro-apoptotic mutations
Basic studies suggest that it is possible to directly harness the
Conclusion
pro-apoptotic forces produced by certain oncogenic mutations
to selectively kill tumor cells. As mentioned earlier, oncogenes
The last decade has seen an extraordinary increase in our
like c-myc and inactivation of tumor suppressors such as Rb
understanding of apoptosis, and its contribution to cancer and
force proliferation but at the same time promote apoptosis,
cancer therapy. Furthermore, the molecular mechanisms that
presumably as a cellular safe-guard against tumorigenesis. In
control and execute apoptotic cell death are coming into focus.
normal cells, E1A promotes apoptosis and enhances chemosen-
Although there is much more to learn, our current understanding
sitivity through a dual mechanism that involves inactivation
of apoptosis provides new avenues for cancer diagnostics,
of the retinoblastoma protein and binding the p300/CBP
prognosis and therapy. In the coming years, it seems likely
transcription co-activators. As a result, E1A mutants unable
that rational strategies to manipulate cell suicide programs will
to bind Rb (but retaining the p300/CBP interaction) promote
produce new therapies that are less toxic and mutagenic than
apoptosis in Rb-decient cells but not normal cells (87). In
current treatment regimens.
principle, these E1A mutants, or small molecules which mimic
their action, may provide tumor-specic anticancer agents by
exploiting the fact that the Rb pathway is disrupted in the
Acknowledgements
majority of human cancers.
The authors apologize to those investigators whose work was not cited due
Rb mutations lead to increased E2F activity, and this
to an oversight or space constraints. We thank M.McCurrach for editorial
promotes both proliferation and apoptosis (9193). Whereas
comments and support. S.W.L. is a Rita Allen Scholar; S.W.L. and A.W.L.
Rb represses E2F activity during G
0
/G
1,
cdk2 kinase can
receive support from grants CA13106 and AG16379 from the National
Institutes of Health. repress E2F activity during S phase (167). It has been suggested
491
S.W.Lowe and A.W.Lin
transgenic mice: reduction in protective apoptotic response at the
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