J. Pathol.

187: 127137 (1999)

APOPTOSIS AND THERAPY

. . *
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, U.S.A.

SUMMARY
The dogma that antineoplastic treatments kill tumour cells by damaging essential biological functions has been countered by the
notion that treatment itself initiates a programmed cellular response. This response often produces the morphological features of
apoptosis and is determined by a network of proliferation and survival genes, some of which are differentially expressed in normal and
malignant cells. Correspondingly, mutations that interfere with the initiation or execution of apoptosis may produce tumour-cell drug
resistance. Remarkably, many of the genes that modulate apoptosis in response to cytotoxic drugs also affect apoptosis during tumour
development; hence, the process of apoptosis provides a conceptual framework for understanding how cancer genes can influence the
outcome of cancer therapy. Although the relative contribution of apoptosis to radiation and drug-induced cell death remains
controversial, clinical studies have associated anti-apoptotic mutations with treatment failure. While careful preclinical and clinical
studies will be necessary to resolve this point, our current understanding of apoptosis should facilitate the design of rational new
therapies. Copyright  1999 John Wiley & Sons, Ltd.
KEY WORDSanti-cancer treatment; resistance; DNA damage; clinical outcome; p53; programmed cell death

APOPTOSIS IS PROGRAMMED CELL DEATH Receptor-linked apoptosis

Apoptosis is a process of cell death that was initially
described by its morphological characteristics, including
cell shrinkage, membrane blebbing, chromatin conden-
sation, and nuclear fragmentation.1 Apoptosis acts in
embryonic development and tissue homeostasis,2 and
can also be induced by pathological insults. The fact that
apoptosis is controlled by genes reveals its clinical
relevance: as in other developmental or homeostatic
processes, cell death can be disrupted by mutations.
Hence, a variety of disease states involve either hyper-
activation or suppression of apoptosis, including
neurodegenerative diseases and cancer, respectively.3
While the terms apoptosis and programmed cell death
are often used synonymously, cell deaths can be pro-
grammed without undergoing the morphological char-
acteristics of apoptosis4,5 and can occur long after the
death-initiating event.6
A comprehensive overview of apoptosis is beyond the
scope of this discussion (for review, see refs 2 and 710).
However, as a general summary, most apoptotic path-
ways involve a sensor that detects a death-inducing
signal, a signal transduction network, and an execution
machinery that actively carries out the process of cell
death. The probability with which each signal activates
the execution machinery is controlled by a series of
regulators that, in turn, are influenced by cellular con-
text. Hence, stimuli which induce apoptosis in one
setting may not in another. In order to lay the ground-
work for therapy-induced apoptosis, two different
apoptotic pathways are outlined below.
*Correspondence to: S. W. Lowe, Cold Spring Harbor Laboratory,
1 Bungtown Road, P.O. Box 100, Cold Spring Harbor, NY 11724,
U.S.A. E-mail: lowe@cshl.org
Contract grant sponsor: Dr Mildred Scheel Cancer Foundation.
Contract grant sponsor: National Cancer Institute; Contract grant
number: CA13106.
CCC 00223417/99/01012711$17.50
Copyright  1999 John Wiley & Sons, Ltd.
At the molecular level, the best understood cell death
pathways involve those initiated by death receptors,
including Fas/CD95, tumour necrosis factor (TNF)
receptor type 1 (TNFR1), DR3, DR4 and DR5.8,11,12
For example, binding of TNF to TNFR1 results in
its trimerization and the subsequent recruitment of
the signal transducing molecules TRADD (TNFR1-
associated death domain protein) and FADD
(Fas-associating protein with death domain) through
conserved protein interaction regions known as death
domains. This complex then associates with caspase-8, a
cell death protease that becomes activated in the
receptor/adapter complex. Activated caspase-8 initiates
a protease cascade that cleaves cellular targets and
results in apoptotic cell death.10,13,14 Hence, disruption
of FADD can prevent activation of caspase-8, thereby
producing defects in receptor-mediated cell death (e.g.
ref. 15). Importantly, TNF-induced apoptosis can be
modulated by TRADDs ability to bind TRAF2 (TNF
receptor-associated factor), which facilitates nuclear
factor kappa B (NF-B)-mediated cell survival.16,17
DNA damage-induced apoptosis
DNA damage can also induce apoptosis through a
central player, p53, although p53-independent pathways
clearly exist. Current evidence suggests that DNA strand
breaks are sensed by kinases such as the DNA-
dependent protein kinase (DNA-PK) or the ataxia-
telangiectasia-mutated gene product (ATM) leading to
phosphorylation and activation of p53.18,19 p53 trans-
mits the apoptotic signal by a complicated mechanism
that involves, at least in part, its ability to transactivate
target genes such as bax and a series of p53-inducible
genes (PIGs).20,21 The next step is poorly understood,
but may involve changes in mitochondrial membrane
potential and release of cytochrome c.22 In any case,
128 C. A. SCHMITT AND S. W. LOWE
once in the cytosol, cytochrome c can interact in a
multi-protein complex with Apaf1 and pro-caspase-9,
leading to caspase-9 activation and the initiation of a
protease cascade similar to that described above.23,24
Concordantly, inactivation of caspase-9 disrupts apop-
tosis in situations where p53 is required for programmed
cell death.25,26
Apoptosis induced by DNA-damaging agents can be
dramatically affected by cellular context, including tissue
type and genetic background (see below). One of the
most important modulators is Bcl-2, owing to its ability
to affect cytochrome c release from mitochondria9,27
and/or modulate the Apaf1/caspase-9 interaction.24,28,29
Given these complexities, the scenario described above
must be considered a conceptual framework for further
research and discussion. In reality, the apoptotic net-
work is much too complicated to be accurately described
by simple linear pathways.
APOPTOSIS IN NEOPLASIA
Failure to maintain an appropriate balance of cell
numbers is a hallmark of neoplasia. Apoptosis is the
counterweight to proliferation, and disruption of
apoptosis is a key event in tumourigenesis. For
example, Bcl-2 was cloned by virtue of its high frequency
of constitutive activation following translocation in
follicular lymphomas.30 Indeed, several key players in
apoptosis were originally identified by their role
in oncogenic transformation or as genes mutated in
human tumours.31,32 Moreover, inactivation of p53
is an extremely common event in human tumours:
while p53 functions in several cellular processes, its
role in apoptosis clearly contributes to tumour
development.33,34
Paradoxically, the forces of tumour development can
actually promote apoptotic cell death, owing largely to
cellular mechanisms that couple excessive proliferation
to apoptosis. Specifically, studies using cultured cells
demonstrate that many mitogenic oncogenes (e.g. myc,
E1A, E2F-1, cdc25, ras) force uncontrolled proliferation
but at the same time can enhance apoptosis.2 Of note,
apoptosis in oncogene-expressing cells is markedly
potentiated by growth inhibitory signals prevalent in
developing tumours, including low concentrations of
survival/growth factors35,36 and low oxygen (hypoxia).37
Hence, these developing tumour cells strike a balance
between proliferation and apoptosis which is dependent
on environmental cues.
Promotion of apoptosis by oncogenes may function as
a cellular fail-safe mechanism that limits tumour devel-
opment. Consistent with this view, molecules that modu-
late oncogene-induced cell death in cultured cells are
commonly mutated in tumours. For example, the p53
tumour suppressor is activated by a variety of mitogenic
oncogenes. p53 is necessary for oncogene-induced apop-
tosis in a variety of settings,3841 and recent studies have
demonstrated that yet another tumour suppressor,
p19ARF, mediates this effect.4246 Moreover, oncogene-
induced cell death is efficiently blocked by Bcl-2.2,9
Importantly, these data predict that pro-apoptotic onco-
Copyright  1999 John Wiley & Sons, Ltd.
genes should create selective pressure for disruption of
apoptosis during tumour progression. That pro- and
anti-apoptotic mutations co-operate during tumour
development has extensive support from animal
models.33,4749
APOPTOSIS LINKS CANCER GENETICS WITH
CANCER THERAPY
The importance of programmed forms of cell death is
not restricted to tumour development, but has relevance
for cancer therapy and mechanisms of drug sensitivity
and resistance. However, until recently, the tumour-
specific toxicity of most anticancer agents was attributed
to their debilitating effects on actively proliferating cells.
The interaction of an agent with its primary intercellular
target was presumed to produce massive cellular dam-
age, thereby disrupting vital metabolic functions and
causing catastrophic cell death. Based on this view,
efforts to understand drug resistance have focused on
mutations that disrupt or prevent the drugtarget inter-
action. More recently, it has become apparent that most
anticancer agents induce apoptosis. Thus, the effective-
ness of anticancer drugs may not simply reflect their
ability to cause cellular damage, but also the cells ability
to detect and respond to this damage.1,50 Conversely,
since drugs with distinct primary targets can induce
apoptosis through similar mechanisms, mutations
in apoptotic programmes can produce multi-drug
resistance.50,51
Cytotoxic anticancer agents are effective only when
tumour cells are more readily killed than the surround-
ing normal tissue. If the efficacy of these agents is partly
determined by their ability to induce apoptosis, then
tumour cells must be more susceptible to apoptosis than
the normal tissue from which they arose. Interestingly,
pro-oncogenes such as E1A and c-myc dramatically
increase cellular radio- and chemosensitivity in cultured
cells and xenografts.5153 Since changes induced by these
oncogenes are unique to malignant cells, it has been
suggested that this effect contributes to the therapeutic
index of cytotoxic agents.51,54 Conversely, disruption of
p53 or overexpression of Bcl-2 can produce pleiotropic
resistance to anticancer agents by preventing apoptosis
(see below).
It is remarkable that the same genetic alterations that
influence apoptosis during tumour development also
modulate drug sensitivity and resistance. For example,
c-myc enhances apoptosis in low concentrations of sur-
vival factors or oxygen and following treatment with
diverse cytotoxic agents;35,37,53 conversely, loss of p53
and overexpression of Bcl-2 suppress apoptosis induced
by oncogenes and depletion of survival factors, hypoxia,
and cytotoxic drugs.35,37,39,51 As such, the concept of
apoptosis provides an attractive link between the forces
of tumour development and factors influencing drug
sensitivity and resistance. Indeed, this association may
explain the phenomenon of de novo drug resistance in
advanced solid tumours: selection against apoptosis
during tumour development can simultaneously produce
drug-resistant cells.
J. Pathol. 187: 127137 (1999)
 APOPTOSIS AND THERAPY
APOPTOSIS AND THE CLASSIC VIEW OF
DRUG TOXICITY
Although the role of apoptosis in treatment is a recent
concept, the predictions arising from this model can be
integrated with classic views of drug action to explain
previous observations. The classic view is based on the
drugtarget interaction and its relation to a distinct
cell-cycle phase. For example, pharmacological S-phase
synchronization was thought to sensitize tumour cells to
anticancer drugs most toxic to DNA synthesis or the
replication machinery.55,56 Apoptosis links the cell-cycle
phase preference of anticancer agents to a DNA
damage-checkpoint-initiated cell death programme.
Given an intact checkpoint control, drug-mediated cell
death is now understood as a checkpoint response to
DNA damage.
DNA damage-induced cell death can occur immedi-
ately or be delayed. The majority of drugs target DNA
synthesis, whereas others are more related to cell metab-
olism and division. Hence, some drugtarget interactions
mediate immediate genomic injury while others cause
delayed genotoxicity, shedding light on the phenomenon
of acute versus delayed apoptosis.6 The acuteness and
severity of the drug-induced DNA damage are not only
related to the drugtarget interaction, but also depend
on the dose and the time [the area-under-the-curve
(AUC)]. While low doses resulting in minor, repairable
damage may only delay cell-cycle progression, higher
doses leading to severe DNA damage can initiate
apoptosis or even necrosis. Furthermore, the onset
of apoptosis is dose-dependent: topoisomerase in-
hibitors like adriamycin execute cell death directly in the
affected S-phase without proceeding to the following
checkpoint; however, lower doses result in apoptosis
in subsequent cell-cycle phases following checkpoint
controls.57
Thus, the present concept of drug-mediated cyto-
toxicity, improved by new insights into programmed cell
death, dependency on checkpoint control, AUC, and the
kind of genomic injury, reflects the complexity of the
cellular response to anticancer treatment. While this
concept provides an exciting new perspective on how
anticancer drugs work, it also underlines that individual
effects are highly dependent on cell type, specific treat-
ment, and environmental conditions. In addition, it
elucidates why modified treatment conditions may cause
apoptotic cell death even in a formerly resistant setting.
This aspect may be important for the understanding
of high-dose strategies, which are able to override
resistance.58
APOPTOTIC GENES AND ANTICANCER AGENT
CYTOTOXICITY
Studies performed nearly 25 years ago noted that
apoptosis accompanies tumour regression during cancer
therapy.59 However, the view that the integrity of apop-
totic pathways can affect the outcome of cancer therapy
stems from basic and preclinical studies on the apoptotic
genes themselves. Typically, these studies use genetically
Copyright  1999 John Wiley & Sons, Ltd.
129
engineered cells to ask whether controlled alterations in
the expression of apoptotic genes can increase or
decrease cellular sensitivity to radiation or various
chemotherapeutic agents. Although these systems are
artificial, they clearly demonstrate that defects in apop-
tosis can produce resistance to anticancer agents. A
sampling of some apoptotic genes and their relationship
to drug toxicity under preclinical conditions are
discussed below.
The p53 tumour suppressor
The most well-studied gene in terms of its impact on
DNA damage-induced cell death is p53. Early studies
linking p53 loss with radiation and drug resistance came
from model systems using p53 knockout mice. For
example, loss of p53 markedly attenuates radiation
and drug-induced apoptosis in a variety of murine
tissues.6065 Importantly, oncogenically transformed
cells derived from p53-deficient fibroblasts are cross-
resistant to a variety of cytotoxic agents both in cell
culture and in nude mice.51,66 In addition, studies using
the more complicated genetic background of tumour-
derived lines also found a high dependency of most
clinically established anticancer drugs on functional
p53.67,68 Moreover, many studies document an increase
in radiation and chemosensitivity following transfer of
p53 genes into p53-deficient tumour cells in culture and
in xenografts.69,70
Bcl-2 family
Overexpression of Bcl-2 in human tumour cells pro-
duces resistance to a wide variety of toxic agents.71
Unlike classic modes of drug resistance, Bcl-2 does not
inhibit drug uptake or the ensuing cellular damage but
instead blocks apoptosis.72 Experiments using antisense
technology to interfere with Bcl-2 function have shown
an inverse correlation between chemosensitivity and
Bcl-2 levels, indicating that endogenous Bcl-2 contrib-
utes to the drug-resistant state.73 This also suggests that
agents designed to disrupt Bcl-2 function might be
effective therapeutics.
Bcl-2 is the founding member of an expanding gene
family74 that includes other anti-apoptotic molecules
such as Bcl-xL as well as pro-apoptotic members such as
bax. Preclinical studies have shown that Bcl-xL over-
expression can confer resistance to anticancer drugs and
radiotherapy.75,76 By contrast, other members of the
Bcl-2 family like bax, bcl-xS, and bak appear to promote
apoptosis.77 While bax deficiency mediates drug resist-
ance,78 Bax overexpression enhances radiation sensitiv-
ity or sensitizes to anticancer drugs.79,80 The activity of
the Bcl-2 family members involves their ability to form
homodimeric or heterodimeric complexes. Several
studies suggest that cell viability following an apoptotic
stimulus is associated with the Bcl-2/Bax ratio,81 but
exactly how these complexes regulate apoptosis is
unclear. Interestingly, antitubulin agents like paclitaxel
or vincristine induce Bcl-2 hyperphosphorylation and
inhibit its ability to heterodimerize with Bax.82
J. Pathol. 187: 127137 (1999)
130 C. A. SCHMITT AND S. W. LOWE

Fig. 1DNA damage and oncogenes initiate pathways involved in apoptosis, cell-cycle arrest,
or survival. p53 is activated by mitogenic oncogenes such as E1A and myc with p19ARF as an
intermediary, or by kinases such as the DNA-dependent protein kinase (DNA-PK) or the
product of the ataxia-telangiectasia gene (ATM) in response to DNA damage. The apoptotic
p53 pathway includes bax, cytochrome c release, activation of Apaf1, and the caspase family,
while the p53-target p21 mediates cell-cycle arrest. Oncogenes may also contribute to p53-
independent apoptosis by sensitization of the Fas/FasL death receptor pathway or by other
pro-apoptotic pathways. Induction of NF-B results in subsequent activation of survival genes

Death receptors and therapy the contribution of dysregulated caspases to anticancer

Some studies suggest that classic anticancer agents
require the FasL/Fas system to induce cell death,
although this remains highly controversial. For example,
adriamycin-induced apoptosis of human T-cell leukae-
mia cells can be blocked by anti-Fas antibodies or
up-regulation of FasL expression.83,84 In contrast, both
Fas-resistant Jurkat cells and FADD-deficient cells are
defective in Fas-induced death but not chemotherapy-
induced apoptosis.15,85 In principle, cross-resistance
against Fas- and drug-induced apoptosis may be due to
escape mechanisms of shared downstream components
of the apoptotic machinery8587 (Fig. 1). Alternatively,
the relationship may be more direct. In this regard, p53
can transcriptionally activate DR5 in some contexts,
suggesting a mechanism whereby these pathways
could facilitate drug-induced death.88,89 Still, much
work is needed to understand when and how these
death receptor pathways contribute to radio- and
chemosensitivity.
The terminal apoptotic cascade
At the final stage of apoptosis, the different death
pathways converge to activate members of the caspase
family.90 Preclinical studies on tumour cells analysing
Copyright  1999 John Wiley & Sons, Ltd.
treatment resistance have not yet been published. How-
ever, exogenous inhibition of caspases has conferred
resistance to different anticancer drugs.91 The potential
impact of caspases and their activators on resistance is
also supported by knock-out mouse models with germ-
line disruptions of the Apaf1, caspase-3 or caspase-9
gene, which are resistant to various apoptotic stimuli in
different tissues.4,25,26,92
Cell survival pathways
Extracellular survival factors play an important role
in maintaining cell viability.93 Although these cell sur-
vival pathways may not directly act downstream of
anticancer drugs, recent studies indicate that they can
synergize with anti-apoptotic mutations to reduce
chemosensitivity.94 One critical factor in cell survival is
NF-B, a transcription factor that is induced by a
variety of signals including oncogenes, cytokines, and
anticancer drugs.9599 Remarkably, inactivation of
NF-B dramatically enhances apoptosis following treat-
ment with TNF- and certain anticancer agents,100
owing, at least partially, to NF-Bs ability to modulate
the expression of components of death receptor com-
plexes.17 Another survival signal cascade, the PI3K/Akt
J. Pathol. 187: 127137 (1999)
 APOPTOSIS AND THERAPY
pathway, may link extracellular survival signals to the
apoptotic machinery by phosphorylation of specific cell
death-related molecules such as Bad.98 Recent evidence
suggests that the PI3K/Akt cell survival pathway can be
negatively regulated by the PTEN tumour suppressor, a
lipid phosphatase that itself induces apoptosis.101,102
Since PTEN is mutated in diverse tumour types, it will
be interesting to determine whether loss of PTEN can
influence the cytotoxicity of anticancer agents.
APOPTOTIC MUTATIONS AND DRUG
RESISTANCE IN PATIENTS
Although laboratory studies like those described
above have established that mutations in apoptotic
programmes produce treatment-resistant tumours, the
extent to which similar mechanisms operate in cancer
patients remains to be established. With varying degrees
of success, investigators have attempted to extend these
preclinical observations to patients. These studies can be
extremely difficult, as suitable clinical material is often
hard to obtain and the genetics of human tumours are so
variable as to preclude simple comparisons. Neverthe-
less, emerging evidence suggests that mutations that
suppress apoptosis may indeed contribute to treatment
failure in human cancer. In fact, p53 mutations were
found to be associated with poor prognosis, de novo or
acquired resistance, and relapse in a broad field of solid
and haematological malignancies.67,103110
Recent data underline the link between extragenic
alterations of the apoptotic p53 pathway and impaired
response to therapy. Mdm2 overexpression, resulting in
p53 degradation, correlates with early treatment failure
in childhood acute lymphoblastic leukaemia.111 Loss-of-
function mutations of the p53-target bax confer resist-
ance in haematopoietic malignancies.112 Expression
levels of Bax are positively correlated with response to
cytotoxic treatment and survival in breast cancer
patients.113,114 Increased Bcl-2/Bax ratios were observed
in B-cell chronic lymphocytic leukaemia cells and were
particularly pronounced in leukaemia cells from patients
non-responsive to chemotherapy.115 Overexpression of
the anti-apoptotic long form of Bcl-x correlated strongly
with decreased response rates in multiple myeloma
patient groups treated with different classes of anti-
cancer agents.116 Although fewer studies have focused
on apoptotic genes outside the p53 axis and Bcl-2 family,
overexpression of Bcr-Abl, a fusion protein arising from
the Philadelphia translocation in leukaemia, has been
shown to suppress apoptosis and reduce the therapeutic
outcome of relapsed childhood acute lymphoblastic
leukaemia.117
TO WHAT EXTENT DOES APOPTOSIS
CONTRIBUTE TO THE OUTCOME OF
CURRENT CANCER THERAPIES?
The above studies provide direct evidence that apop-
tosis can impact on therapy and correlative evidence
that anti-apoptotic mutations can contribute to drug
Copyright  1999 John Wiley & Sons, Ltd.
131
resistance in human tumours. However, not all studies
find correlations between sensitivity to apoptosis and
radio- and chemosensitivity. For example, some studies
fail to correlate p53 mutations with reduced anticancer
agent toxicity and, indeed, others see loss of p53 enhanc-
ing cell death.118,119 Moreover, in some settings, Bcl-2
does not enhance cell viability following irradiation or
drug treatment and in fact, some studies suggest that
Bcl-2 expression is indicative of a good prognosis and
outcome in breast cancer.120,121 These results confound
our understanding of the overall impact of apoptosis
on cancer therapy. Although more research will be
needed to resolve these inconsistencies, both methodo-
logical and technical problems contribute to this
confusion.
Apoptosis and clonogenic survival
In some contexts, anti-apoptotic changes such as
Bcl-2 overexpression simply delay radiation or drug-
induced apoptosis but do not enhance clonogenic
survival, raising the possibility that non-apoptotic
mechanisms are ultimately responsible for cell
death.122124 While it is likely that delayed cell death can
contribute to the toxicity of anticancer agents, several
caveats are worth mentioning. While the clonogenic
survival assays are often considered the gold standard,
recent evidence of the role of p21 in response to
irradiation indicates that clonogenic survival may not
accurately reflect in vivo cell death and regrowth of
long-term arrested cells.125 It is possible that autocrine
or paracrine survival factorsinfluenced by cell density
or micro-environmentcan impact the mechanisms of
drug toxicity. Indeed, a recent study has confirmed that
Bcl-2 has dramatically different effects on the long-term
survival of lymphoma cells depending on the culture
conditions used in the clonogenic assay.94
Despite the exceptions, a large number of studies
indicate that apoptotic genes can affect clonogenic sur-
vival and, as indicated above, can affect tumour regres-
sion in animal models or patients. However, in addition
to acute apoptosis detectable in short-term survival
assays, other forms of programmed cell death can
include delayed apoptosis and senescence-like tumour
cell arrest.6,126 Genes that affect these latter processes
are poorly understood; therefore definitive experiments
are difficult to perform.
Technical and conceptual considerations
What other factors could explain a lack of correlation
between specific apoptotic genes and chemosensitivity
and/or therapeutic outcome in patients? A major issue
relates to the technical and conceptual problems that
accompany correlative studies examining the relation-
ship between single apoptotic genes, chemosensitivity,
and/or therapeutic outcome. The problem can be illus-
trated by studying the issues associated with understand-
ing the role of one apoptotic gene, p53, in therapeutic
outcome.
In correlative studies using human cell lines or
tumours, the ability to make statistically significant
J. Pathol. 187: 127137 (1999)
132 C. A. SCHMITT AND S. W. LOWE
comparisons requires that samples are accurately
classified based on p53 status. Most clinical studies rely
on p53 immunohistochemistry as a surrogate marker for
p53 mutations, but evaluation is subjective and the
premise that detectable p53 reflects mutated p53 contrib-
utes to a potential misclassification of a subset of
tumours.127 Direct sequencing of p53 is more accurate
and can identify associations between p53 mutations and
clinical parameters where immunohistochemistry can-
not,127 but is subject to normal cell contamination. In
studies with limited material, misclassification of even a
few tumours can affect the conclusions. Even with
accurate diagnosis of p53 status, tumour classification is
confounded by the observation that not all p53 muta-
tions are functionally equivalent.128 Moreover, a large
number of extragenic mutations can abolish p53 activity
in tumours that retain a wild-type p53 gene. Many such
alterations are common in human tumours, including
HPV-E6 in cervical carcinomas, Mdm2 in a variety of
tumours, and bax in HNPCC-type colon cancer.128130
In these settings, p53 status would be misclassified by
current technology. Given these caveats, it is impossible
to interpret negative results in clinical studies.
Finally, basic studies indicate that the role of p53 in
chemosensitivity is context-dependent. Specifically, the
prediction that p53 mutations should promote drug
resistance is largely based on the observations that
DNA-damaging agents can activate p53 to promote
apoptosis. However, p53 has additional biological
activities, including the ability to promote cell-cycle
checkpoints and senescence, and little is known about
how these activities modulate chemosensitivity. Perhaps
positive correlations between p53 and chemosensitivity
will only occur where p53s apoptotic function predomi-
nates. Whether p53 functions in an apoptotic mode will
depend on several criteria, including agent, dose, tissue
type, and mutational background of the tumour cells
(see above). These are not understood in sufficient detail
to make accurate predictions as to p53s role in a given
setting.
The question is: to what extent does apoptosis con-
tribute to the clinical outcome? Although p53 can be
used as an example to address the question of drug
sensitivity and resistance, the same caveats apply to
clinical studies investigating other single genes that
control apoptosis. Besides the difficulty in assessing
pathways instead of single genes, outcome not only
depends on the therapeutic impact on cell death, but is
also the result of the tumour growth minus apoptosis
equation. Progression under treatment may reflect resist-
ance, but can occur in a fast-growing, sensitive tumour
as well. On the one hand, poor prognosis can be solely
due to an aggressive histological subtype, potentially
due to genetic alterations of apoptotic genes. On the
other hand, it can be a result of an impaired treatment
response due to non-apoptotic mechanisms attenuating
the therapeutic efficacy. Between these extremes, a sub-
stantial contribution of apoptotic dysregulation to treat-
ment failure is likely, however, in patients technically
hard to prove.
A promising approach to monitor drug-induced
apoptosis in vivo is animal models with a defined genetic
Copyright  1999 John Wiley & Sons, Ltd.
setting, leading to comparable and treatment-assessable
tumours. Since xenograft models have their own restric-
tions rooted in the grafthost collision mice harbouring
germ-line mutations in apoptotic genes may be useful.
For example, we addressed the question of treatment-
mediated apoptosis in the E-myc transgenic mouse.131
When crossed to mice heterozygous for p53, the F1
generation with wild-type p53 developed B-cell lym-
phoma within the usual latency. However, lymphoma in
the p53 +/
mice occurred much faster. While the p53
wild-type tumours responded to radiation or adria-
mycin, two thirds of the p53 +/
derived tumours were de
novo-resistant and the responsive subgroup relapsed
after very short remission periods (C. Schmitt, R. R.
Wallace-Brodeur, M. E. McCurrach, and S. W. Lowe,
unpublished observations). This is exactly what one
would predict with respect to p53s role in DNA
damage-mediated apoptosis and underlines the validity
of the model. In principle, this model or others may
become useful in genetically-oriented drug-discovery
programmes.
NEW THERAPEUTIC APPROACHES
Irrespective of the relative extent to which apoptosis
contributes to the success of present-day cancer thera-
pies, our current understanding of apoptosis leaves no
doubt that these programmes can be manipulated to
produce massive changes in cell death. Hence, the genes
and proteins that control apoptosis provide exciting new
targets for rational drug discovery. Of course, as indi-
cated above, the clinic is filled with potent inducers of
tumour-cell apoptosismolecules which are also highly
toxic due to their deleterious effects on normal cells.
Thus, strategies designed to identify novel compounds
that simply induce apoptosis are unlikely to provide
improvements over current therapies; rather, new strat-
egies must exploit apoptosis in a selective manner. A
sampling of current approaches to exploit apoptosis will
be discussed below.
Selective delivery
One strategy to achieve selectivity is through tumour-
specific delivery of apoptosis-inducing molecules. For
example, viral vectors expressing apoptotic genes can be
directly introduced into the tumour, minimizing their
uptake by normal cells (see, for example, refs 69, 132
134). This approach has proven particularly effective for
p53: intratumoural injection of p53-expressing adeno-
viruses can induce apoptosis directly and synergizes with
cytotoxic anticancer agents to promote tumour regres-
sion with minimal additional toxicity.69,135,136 Indeed,
strategies using this approach are currently undergoing
clinical trials.137 Alternatively, tumour-specific changes
in transcriptional regulation may allow selective expres-
sion of apoptosis-inducing genes. One elegant approach
takes advantage of the fact that E2F transcription
factors are often hyperactivated in tumour cells, owing
to defects in the Rb pathway. Toxic genes can be driven
from artificial promoters harbouring E2F binding sites,
J. Pathol. 187: 127137 (1999)
 APOPTOSIS AND THERAPY
leading to preferential activation of the apoptosis gene
in tumours.138 Finally, since adenovirus inefficiently
infects normal haemopoietic cells, adenoviral vectors
expressing apoptotic genes can be used to purge the
bone marrow of metastatic tumour cells.139,140
Tumour-specific alterations in apoptotic programmes
Since tumour evolution involves genetic changes that
both positively and negatively affect apoptosis, the regu-
lation of apoptosis in tumour cells may be fundamen-
tally different from normal tissues. In many instances,
apoptosis has been disabled by dominant oncogenes,
and disrupting their function can produce remarkable
increases in cell death. In chronic myelogenous leukae-
mia (CML), a chromosomal translocation leading to the
production of the Bcr-Abl fusion protein is critical for
tumour evolution and may act by suppressing apopto-
sis.141 Since this translocation is unique to these tumour
cells, the gene or protein may represent a unique target.
Indeed, inactivation of Bcr-Abl using hammerhead
ribozymes provides evidence that this approach can
enhance apoptosis.142
In many instances, tumour evolution leads to
up-regulation of one or more members of the Bcl-2
family. Although targeting these proteins with small
molecules is the ultimate goal, substantial progress has
been made using antisense inhibition;143 in fact, anti-
sense bcl-2 oligonucleotides can produce substantial
tumour regression when combined with chemotherapy
in xenograft models.144 Another gene-based approach to
target this family uses bcl-xS, a dominant-negative
repressor of Bcl-2 and Bcl-xL. Adenovirus-mediated
gene transfer of bcl-xS can synergize with chemo-
therapy145 and promote tumour regression in
xenografts,146 but produces minimal apoptosis when
introduced into normal epithelial cells.147 Why would
such therapeutic strategies be selective? The answer is
not known, but perhaps inactivation of these proteins
reveals the pro-apoptotic forces of certain oncogenic
mutations (see earlier discussion).
Indeed, preclinical studies have demonstrated that it is
possible to harness directly the pro-apoptotic forces
produced by certain oncogenic mutations. As mentioned
earlier, oncogenes like c-myc and inactivation of tumour
suppressors such as Rb force proliferation but at the
same time promote apoptosis, presumably as a cellular
safe-guard against tumourigenesis. These pro-apoptotic
activities would be even more potent if it were not for
the survival genes simultaneously induced by the family
of NF-B transcription factors, as reported for the
adenoviral oncoprotein E1A95 or for oncogenic Ras.98
Hence, inactivation of NF-B function with I-B can
enhance oncogene-induced apoptosis as well as cellular
radio- and chemosensitivity.97,100 In a similar approach,
studies on the adenovirus E1A oncogene provide proof
of concept data that tumour-specific mutations can be
exploited to selectively enhance apoptosis in tumour
cells. E1A promotes apoptosis and enhances chemo-
sensitivity through a dual mechanism that involves
inactivation of the retinoblastoma protein and binding
the p300/CBP transcription co-activators. As a result,
Copyright  1999 John Wiley & Sons, Ltd.
133
E1A mutants unable to bind Rb (but retaining the
p300/CBP interaction) promote apoptosis in Rb-
deficient cells but not in normal cells.148 In principle,
these E1A mutants, or small molecules which mimic
their action, may provide tumour-specific anticancer
agents by exploiting the fact that the Rb pathway is
disrupted in the majority of human cancers.
Although mutations in death receptors seem to be
very rare events in human tumours and, so far, no
cancer-related mutations of their ligands have been
described,149151 changes accompany tumourigenesis
have dramatic effects on the regulation of these path-
ways. Mitogenic oncogenes like myc and E1A can
increase sensitivity to Fas and TNF- in cultured
cells,152,153 and many human tumours have alterations
in TRAIL-mediated death pathways. TRAIL is a TNF-
related protein that initiates a p53-independent cell
death programme by binding to its receptors DR4 or
DR5. Normal cells are resistant to TRAIL-induced
apoptosis, apparently because normal cells express
decoy receptors that compete with DR4 and DR5 for
TRAIL but do not transmit a death-inducing signal.154
Through an unknown mechanism, the expression of the
decoy receptor 1 seems to be widely lost on tumour cells,
making them exquisitely susceptible to TRAIL-
mediated cell death.154 Interestingly, DR5 is a p53-
responsive gene; hence, combination therapy with
TRAIL and classic cytotoxics may be particularly
effective in treating tumours with functional p53.88
Consequently, the use of TRAIL or other strategies
exploiting these well-characterized apoptotic pathways
are promising avenues for drug development.
Viral proteins
In addition to E1A, a number of other viral proteins
are in preclinical studies as selective inducers of apopto-
sis. The adenovirus E4orf4 protein is responsible for
p53-independent apoptosis observed during adenovirus
infection and can produce rapid cell death when
expressed in tumour cells,155 although the specificity of
this effect remains to be determined. Another intriguing
viral protein is apoptin, the VP3 product of chicken
anaemia virus (CAV). This protein is responsible for the
cytopathic effects of CAV156 and induces apoptosis in
tumour cells but not normal cells.157 Although its mech-
anism of action remains unknown, apoptin-induced
apoptosis is independent of p53 and is enhanced by
Bcl-2.158 At the very least, the remarkable effects of
apoptin underscore the point that selective induction of
apoptosis in tumour cells is achievable.
Chemoprotection
As indicated above, there would be no truly drug-
resistant cell if cytotoxic agents could be applied in
sufficient dose. However, toxicity to normal tissues
presents a major limitation in the use of current
apoptosis-inducing cytotoxic drugs. Most tissues are
relatively insensitive to apoptosis; however, normal
haematologic cells and certain stem cells of the small
intestine are remarkably sensitive to apoptosis following
J. Pathol. 187: 127137 (1999)
134 C. A. SCHMITT AND S. W. LOWE
radiation or chemotherapy (see above). Since cell loss
from these tissues accounts for the most debilitating
side-effects of cancer therapy, exogenous interventions
conferring resistance to normal cells might allow the
administration of higher drug doses to combat the
tumour. In an ironic twist of thinking, several groups
have attempted to introduce classical drug resistance
genes into normal cells as a chemoprotective measure.
For example, the ex vivo transduction and subsequent
transplantation of haematopoietic progenitors with
MDR1 or the aldehyde dehydrogenase gene suggest that
such strategies might be effective.159,160
Based on our understanding of apoptosis in these
chemosensitive tissues, it should be possible to block this
cell death. Indeed, studies using mouse models have
clearly documented the importance of p53 for apoptosis
in thymocytes, bone marrow, and intestinal stem
ells.60,161,162 Furthermore, inactivation of p53 allows
recovery of intestinal crypts following therapy with high
dose 5-fluorouracil.163 Consequently, agents which sup-
press p53 function would make effective chemoprotec-
tive agents. Although inactivation of p53 in tumours
may be undesirable, such a strategy would likely be
suitable for advanced cancers, most of which have
already lost p53 function.
CONCLUSION
Although the precise contribution of impaired apop-
totic capability to drug resistance in tumours remains to
be determined, emerging clinical evidence correlates
mutations of apoptotic genes and treatment failure.
Understanding of the apoptotic network may help to
improve current treatment strategies. Certainly there is
still a long way to go, but the fact that apoptosis is
controlled by genes raises the exciting prospect that the
problem of drug sensitivity and resistance will be
amenable to interventions based on molecular biology.
ACKNOWLEDGEMENTS
We apologize to those investigators whose work, due
to space constraints, was not cited. We also thank M. E.
McCurrach for editorial assistance, and acknowledge
support from a Dr Mildred Scheel Cancer Foundation
Postdoctoral Fellowship (C.A.S.) and from a Sidney
Kimmel Scholar Award and Grant CA13106 from the
National Cancer Institute (S.W.L.).
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