Apoptosis and cytotoxins
John A Hickman* and Catherine C Boyle^
*School of Biological Sciences, University of Manchester, Manchester, UK; ^Department of Clinical
Biochemistry, University of Edinburgh, Edinburgh, UK
Most xenobiotics ultimately become lethally cytotoxic, according to concentration.
Toxins with completely disparate mechanisms of action induce apoptotic cell death.
This suggests that the threshold for the onset of cell death can be determined by the
relative expression levels of genes which promote or suppress apoptosis.The
selectivity of a toxin may thus be determined not only by the selective imposition of
perturbation or the amount of damage inflicted, but also by how readily that cell
engages apoptosis. Measuring damage to cells, therefore, does not necessarily
predict outcome.The threshold for apoptosis is determined not only by the static
phenotype of the cell, which may confer a high or low survival potential, but also by
its capability when stressed to modulate the expression of genes which control
survival.The trophic environment of a cell can also influence the threshold for death.
These findings have a profound impact on concepts defining toxin selectivity and on
attempts to use mechanistic information to predict toxicity to organisms.
Toxins induce apoptosis in vivo
Many natural products and chemical compounds are cytotoxic. For
some agents this represents a hazard and is an unwelcome effect. Such
compounds define for themselves a toxicology as noxious substances.
Other cytotoxins have been harnessed as the mainstream of present day
antitumour therapy. Unfortunately, as drugs, they possess many of the
properties of the noxious toxins. Almost without exception toxins
induce death by apoptosis. The test of whether a compound induces
apoptosis has naturally rested largely on observation of the classical
morphological features of the apoptotic cell, discussed elsewhere in this
issue. Perhaps the most critical feature of the concept of a programmed,
Correspondence to apoptotic cell death is not that it has a conserved morphology but that it
John A Hickman, is regulated by the expression of certain genes1. This provides a
Schoo/ofBio/ogico/ mechanistic understanding of why some cells die readily and others do
o» c i ,Sc'"''ces' not. Morphological analysis of toxin-induced cell death may be
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©The Brihth Council 1997
British Medical Bull.hn 1996,52 (No 3).632-643
Apoptosis and cytotoxins
and rapidly move to express the appearance of a necrotic cell. In vivo,
similar kinetic considerations may influence whether one observes
necrosis or apoptosis: if membrane integrity is lost rapidly, or if cells are
not rapidly cleared by phagocytosis (see this issue), it is possible to
interpret the death as being by necrosis. Under some circumstances, if
the degree of injury to the cell is overwhelming, and/or the integrity of
membrane gradients are compromised, necrosis without apoptosis will
ensue. In the absence of secure morphological evidence, particularly in
vivo, a reasonable test of whether a toxin induces apoptosis, as defined
as a programmed cell death, is whether the amount and/or kinetics of
cell death is genetically modulatable without changing the amount of
toxin-induced damage accumulated.
In experiments performed in vivo, a wide variety of agents induce
apoptosis in different organs2. Irradiation of a mouse induces significant
amounts of apoptosis in the thymus and in splenocytes3, as well as in the
epithelial crypts of the small intestine and colon4. The intestinal tract has
been the subject of intensive studies of the induction of apoptosis by
cytotoxins in vivo5'7. Interestingly, even when high doses of whole body
radiation are administered to mice (10 Gy) apoptosis remains the mode
of cell death in the crypt epithelia, with no indications of necrosis8. In
recent experiments of ours, we have attempted to ascertain what the
relationship is between the amount of apoptosis observed acutely in the
intestinal tract after radiation or a cytotoxin and 'toxicity' as defined
more broadly by a toxicologist. Whilst the symptoms of toxicity present
themselves as a continuing morbidity (with weight loss, diarrhoea)
apoptosis is strictly a pathological snapshot, usually of early events
which may then translate to those later pathological changes observed in
histological sections. We measured morbidity, changes in animal body
weight and changes in histology (crypt integrity/cellularity, crypt height)
and compared these with acute changes in mitotic indices and apoptosis
to establish if there was any relationship. Using the gut toxin 5fluorouracil (5-FU), patterns of acute apoptosis indeed translated to
standard aspects of toxicity. As is discussed elsewhere in this issue,
toxins that induce damage to DNA, either directly or indirectly, such as
5-FU, induce apoptosis in a p53-dependent manner. The changed kinetic
patterns of apoptosis observed in mice in which are p53 null (—/—)8>9
provides a critical test of the relationship between acute induction of
apoptosis and the onset of pathological change and animal morbidity. In
these p53 null ( — / —) mice, our preliminary data strongly support the
view that reduced patterns of apoptosis relate to reductions in
pathological changes typical of 5-FU toxicity10. Similarly, important
recent experiments have sought to answer the question of whether
intervention in the apoptotic process itself, by either inhibition of the
proteases involved in cellular execution, or by inhibition of the initiation
Bnhsh Madxal Bulletm 199733 (No 3)
633
Apoptosis
of apoptosis via overexpression of bcl-2, can influence the onset of
neuronal toxicity induced by either neuronal toxins or hypoxia11-12.
Again, the evidence supports the view that toxicity does indeed reflect
the engagement of death by apoptosis11'12 and that inhibition of
apoptosis by expression of bcl-2 (a suppressor of apoptosis) permits
the maintenance of functional aspects of neurones13.
Toxicity is modulated by genes which control apoptosis
The observations that disparate toxins induce apoptosis changes a
traditional framework defining the determinants of a toxin-induced cell
death. It is generally considered that the both the quality and quantity of
damage delivered are the sole determmants of the final fate of the cell.
For example, a leading text on toxicology14 states:
The magnitude of the effect is related to dose... this assumption is
often a source of misunderstanding. It is really a composite of three
assumptions that occur frequently: (i) there is a molecular receptor
site (or sites) with which the chemical reacts to produce the response;
(ii) the production of a response and the degree of response are
related to the concentration of the agent at the receptor site; and (iii)
the concentration at the site is, in turn, related to the dose
administered.
This description of how a toxin induces a biological response assumes
that the damaged or perturbed cell is a passive recipient. This is not
always the case. The induction of a cellular response, made in an attempt
to restore homeostasis to the whole organism, may involve a number of
endpoints, only one of which is death (Fig. 1). The 'choice' between the
different end-points and the relative ease of their engagement, even at the
same level of toxin-'receptor' interaction, differs according to cell type.
There are essentially two phases to the 'response': the first is directly
related to events at the so-called 'receptor site', for example how much
inhibition of an enzyme may have occurred or the quantity and quality
of DNA damage. The second phase might be considered as a
homeostatic adjustment to that perturbation. At some point damaged
cells 'commit' to a particular pathway and the molecular events which
allow this 'decision' to be made are now becoming clear. It is possible
that a reiterative programme is initiated, perhaps involving stoichiometric balances of opposing signals — for a cell cycle check point versus
cell death in proliferating cells, for example. Thus, although two
different cell types might have received an equal quantity of enzyme
634
Bnhth Mtdical Bulletin 1997,53 (No 3)
Apoptosis and cytotoxini
Death
Cytostasis
Terminal Dlffn
Repair
Recovery
F i g . 1 Possible stages in the processing of responses to cellular damage or perturbation After
initiation, DNA damage for example, there may be a reiterative series of responses made (circular
arrows) This can be influenced by epigenetic factors, such as cell—cell, cell—matrix contacts or the
trophic environment The 'stoichiometry'or balance of these possibly opposing responses (e.g survival
versus death) ultimately leads to a point where an individual cell becomes committed to a 'response',
such as a cell cycle checkpoint (cytostasis) or to apoptosis (death) Individual cells in a population may
commit differently giving a mixed population of response (some death some cytostasis) according to the
amount of damage imposed.This type of scheme clearly implies that the amount of damage imposed to
initiate the response is not the only determinant of outcome.
inhibition or of DNA damage, the individual cellular response to that
perturbation may be very different. Consequently, the 'selectivity' of a
toxin and its dose response curve is not determined by estimations of the
concentration of the agent at the receptor site alone. Instead, selectivity
and the threshold dose for the expression of toxicity will also be
determined by differences in, for example, genomic and signalling
responses to the products of the receptor and the toxin and the
perturbation that they impose.
Whilst it is clear that the cellular response induced by a toxic insult is
concentration and context dependent, there is also evidence to suggest
that cells from the same population may respond in different ways to a
toxic insult. A recent study showed that exposure to the non-genotoxic
carcinogens CPA or nafenopin stimulated DNA synthesis and that their
withdrawal induced apoptosis15. Interestingly, the responses were
observed in different hepatocyte populations: those that remained
quiescent during exposure were preferentially deleted by apoptosis
following withdrawal of the toxins. This suggests that even within the
hepatocyte phenotype, toxic insult can establish and maintain a
Bnhsb Medical BulUhn 1997;53 (No 3)
635
Apoptosis
hierarchy for cell death which can respond to changes in the
environment (here the withdrawal of toxic insult)15.
'Sensing' toxin-induced damage
Clearly there are ways in which cells 'sense' damage. Perhaps the most
well defined of these sensors are with respect to protein damage, which
then initiates a heat shock response16, and DNA damage which initiates
repair17. How the 'sensing' of DNA damage in turn sets in train
responses such as cell cycle inhibition (a 'checkpoint') or cell death is
dealt with elsewhere in this issue, particularly with respect to the
activation of p53. In ways which are not entirely clear, individual cells
may either 'sense' damage differently or after 'sensing' they may engage
responses which differ qualitatively and/or quantitatively. A good
example of this differential is illustrated by a study of the expression
of the tumour suppressor protein p53 after whole body radiation:
whereas accumulation of the protein was observed in murine thymocytes, splenocytes and osteocytes, expression was not observed in
hepatocytes. Furthermore, whilst apoptosis was observed in the
thymocytes and splenocytes it was not observed in the osteocytes3.
Analysis of toxin-induced DNA damage to two types of tumour cell
lines, derived from testicular and bladder cancers, showed that at
equidamaging concentrations of the toxin etoposide (equal numbers of
DNA strand breaks), survival was very different, even when cell lines
were shown to both have functional p53. p53 protein was elevated after
damage in both cell types18. The testicular cancer cell lines readily
underwent apoptosis and failed to initiate a proficient Gl cell cycle
checkpoint, with only a modest expression of the checkpoint protein
waf-l/p21, whereas a bladder tumour cell line also with wild-type p53
showed a strong Gl checkpoint after the synthesis of waf-l/p21, the
cyclin-dependent kinase inhibitor. Comparison of the clonogenic
survival of these cell lines showed that at equidamaging concentrations,
the bladder cell line, which underwent a checkpoint permissive for DNA
repair, was less sensitive than the testicular lines18. Quantitation of
damage did not therefore predict outcome.
Sensors and signals
There are other 'sensors' of the damage induced by highly reactive toxins
which, directly or indirectly, damage not only the genome but other
cellular targets. Damage induced by irradiation, UV light and certain
636
British Medico/ Buffrtin 1997,53 (No 3)
Apoptosis and cytotoxins
alkylating agents appears to be 'sensed' by components of the plasma
membrane and activates signalling pathways which are normally
associated with hgation by cytokines and growth factors19. One such
pathway is the JNKs (Jun kinases, also known as the stress-activated
kinases, SAPKs) which mediate the transcnptional activation of the
immediate-early genes c-jun and c-fos. In murine fibroblasts it has been
suggested that JNK activation induced by the alkylating agent
methylmethanesulphonate may signal for survival rather than cell
death20 whereas, in other studies, with other toxins, JNK activation by
stress was associated with apoptosis21'22. Toxin-induced synthesis of the
signalling molecule ceramide, either by stimulation of cerarrude synthase
or activation of a sphingomyelinase, directly stimulates an apoptotic
signalling cascade23 and again suggests that membranes can be 'sensors'
of a variety of disparate toxins. In addition, a cytotoxins have been
shown to activate signals for apoptosis via the APO-1/FAS pathway24
(see elsewhere in this issue).
Genotoxic damage of cells in vitro initiates the transcription of a
variety of genes leading to growth arrest with associated stimulation of
repair, as is the case with GADD4525. However, in some contexts
damage can also lead to the up-regulation of the receptors for growth
factors, such as EGF26 and PDGF27, and for transcription factors such as
NPMB28, a somewhat paradoxical response at first sight. NFMB
activation has recently been shown to inhibit apoptosis when activated
through the tumour necrosis factor receptor29"31, so that in addition to a
signalling cascade which may promote cell death another cascade may
be activated to attempt to promote survival. In recent studies of ours, we
have found that treatment of Rat-1 rat fibroblasts in vitro with a nongenotoxic toxin (dimethylformamide) increased the expression of IGF-1
receptors at concentrations of toxin below that causing a 50% fall in
viability. Interestingly, the toxin-induced up-regulation of IGF-1
receptors was associated with an increase in survival, so that the cells
effectively shifted their dose response curve to the right in response to the
toxin (Fig. 2). Transfection and over expression of IGF-1 receptors or
pretreatment with PDGF, which up-regulates IF-1 receptors, also shifted
the dose response curve, making the cells less sensitive to the toxin32.
Insulin-like growth factor is a poor mitogen for fibroblasts, but provides
a strong survival signal, increasing the threshold at which apoptosis can
be engaged33. Insulin-like growth factor is thought to play a key role in
carcinogenesis34. Because it inhibits apoptosis in certain cell types,
inappropriate expression or up-regulation of expression under conditions of stress may promote the survival of cells with a damaged genome.
The up-regulation of growth/survival factors in response to toxins
illustrates the way in which a genomic response to toxicity can
consequently modulate the dose-response curve. There are analogies
British Mtdical Bulletin 1997^3 (No 3)
637
Apoptosis
toxin
Fig. 2
Cartoon to
illustrate how an adaptive
response to toxic insult
may change the doseresponse curve upon
chronic exposure to a
toxin.Up-regulation of
survival-factor receptors,
and subsequent
intracellular signalling
after ligation of trophic
factor, may change the
threshold at which cell
death is initiated (right)
either by shifting the
dose-response-curve to
the right (broken line) or
changing the threshold at
which toxicity is engaged
(full line) Other examples
of this type of adaptive
response are given in the
text.
concentration
concentration
here with the heat shock response, which induces cellular thermotolerance: an otherwise lethal heat shock is ineffective once a moderate heat
shock triggering the synthesis of protective heat shock proteins,
effectively shifting the dose response curve to the right16.
Endogenous modulators of survival and protection
against toxin-induced death
The stoichiometry of expression of members of the bcl-2 family of genes
is thought to be a major determinant of the survival potential of a cell
(see Brown, this issue). The balance between pro-and anti-apoptotic
proteins, such as bax and bcl-2, respectively, has been suggested to set
the cellular threshold for death35. Although this suggests a 'set-point' for
survival, the expression of members of the family is regulated by toxins
in some cell types. For example, genotoxic agents which activate p53
have been shown to increase the synthesis of the pro-apoptotic bax in
some cells, possibly because of a p53 transcriptional activation site on
the bax gene promoter36. Recent studies of ours have shown that ligation
of CD40 on B-cells provides cells with a clonogenic survival advantage
638
British Madical Bullttin 1997;53 |No 3)
Apoptosis and cytotoxinj
after genotoxic insult, probably because CD40 rapidly up-regulated the
expression of bcl-xL, a suppressor of apoptosis. This result is interesting
because it again suggests that the trophic environment of a cell may play
a critical role in determining fate after toxic insult, by modulating the
endogenous survival threshold. It also raises important questions about
the relevance of performing and extrapolating from toxicity experiments
in vitro where the experimenter defines a fixed trophic environment,
which is likely to be heterogenous in vivo. We consider this to be of
critical importance in the resistance of some tumours to cytotoxic
therapy, where small numbers of cells may occupy a survival 'niche'.
A wide variety of studies have shown that over-expression of bcl-2 or
bcl-xL changes the kinetics of the onset of toxin induced apoptosis35-37.
Nearly all of these reports concern the toxicity of cytotoxic antitumour
drugs with widely different mechanisms of action. They show that
overexpression of bcl-2 or bcl-xL promotes pleotropic drug resistance.
Formally, a study of ours38 showed that overexpression of bcl-2 in a Bcell lymphoma line had no effect on the damage imposed by 5fluorodeoxyuridine (inhibition of thymidylate synthase, a fall in
thymidine pools, the induction of breaks in nascent DNA) but very
significantly delayed the onset of apoptosis. This again challenges the
notion that the toxicity of an agent is defined by the amount of toxininduced damage, as discussed above. However, many of the in vitro
studies of toxins in cells overexpressing bcl-2 and bcl-xL have only
measured acute changes in survival. Usually, this has been over a 48-72 h
period using vital dyes as an indicator of viability. The long-term fate of
the bcl-2 protected cells has rarely been established, for example by a
clonogenic assay. This is critical if it is to be suggested that 'downstream'
events from toxin-induced damage are the final arbiters of survival,
translating into long-term tolerance of the toxin. In fact, there are
conflicting in vitro data, showing on the one hand that expression of bcl2 provides a true long term survival to lymphoid cells treated with
radiation or cytotoxins39 and on the other that fibroblasts are provided
with no long-term survival after treatment with aphidocolin40. However,
in vivo studies of neural toxicity imposed by hypoxia arising from
ischemia have shown convincingly that over-expression of bcl-2 driven
by neuron-specific enolase or phosphoglycerate kinase promoters
protects against brain infarction by 5 0 % n . Moreover, axotomized
neonatal neurons from transgenic animals overexpressing bcl-2 not only
survived but retained functional electrophysiological properties13. These
data show that ectopic bcl-2 expression is critical to long-term neuronal
survival after toxin challenge. Incidentally, bcl-2 knockout mice display
normal morphology and it is considered that the homologue bcl-xL may
be more critical in developed neurones, so that ectopic bcl-2 expression
can supplement its suppression of apoptosis.
Bnftth Mod/col BuJ/.f.n 1997,53 (No. 3)
639
Apoptosis
The effects of bcl-2 expression may be to delay the onset of apoptosis
rather than to promote long-term survival per se in some cells. In a clever
study, Schimke and colleagues showed that the kinetic advantage of
limited but extended survival in vitro provided by bcl-2 expression may
allow sufficient time for the toxin-treated cell to up-regulate mechanisms
of defence against particular classes of toxins which can initiate gene
amplification. Here, resistance to the toxin per se does not arise from the
expression of bcl-2 alone but as a consequence of its activity to delay
death for long enough to permit repair/resistance mechanisms to emerge
against a background of chronic toxin challenge41. One might speculate
that this effect of bcl-2 expression to promote mechanisms of
accumulating survival in the presence of chronic toxin exposure might
be quite extensive. In that respect, although an accumulating survival
threshold may allow the cell to become more and more tolerant, it may
also promote tolerance to genotoxic damage, thus allowing the
accumulation of mutations with potential for the emergence of
malignancy.
Perspectives
In this review, we have suggested that toxin induced apoptosis is
important because it implies that the toxin-induced death of the cell is, in
part, determined by the genes modulating this process. Some evidence in
favour of this hypothesis has been reviewed. Further experiments need to
be performed to establish that the end-points recognised by toxicologists
can be related to changes in the patterns of apoptosis brought about by
manipulation of these genes. The studies of bcl-2 overexpression in the
brain on the effects of hypoxia are an excellent example11. Further
studies of gene knockout animals will also be important. In the context
of multicellular organisms the concept that death by apoptosis is part of
an adaptive response, additionally suggests that adaptive responses to
toxins which are initiated in the 'downstream' aspects of toxicity (Fig. 1)
are important arbiters of toxicity. Responses to toxic insult which
modulate the amount of damage are well documented (drug metabolising enzymes, DNA repair, etc.) but genomic responses to damage, made
before, during or after limited repair, are critical in determining the
selectivity of a toxin. Measuring damage is, therefore, insufficient to
allow prediction of outcome. Furthermore, some of these responses, even
if conserved across different phenotypes, may be engaged differently
depending upon cell-cell, cell stroma and trophic environments. These
paradigms, invoking context, and adaptability according to context,
present problems when attempting to analyse causative mechanisms of
640
Bnbsh Medical Bulletin 1997,53 (No. 3)
Apoptosis and cytotoxim
toxicity in vitro. It is not surprising that, recently, attempts to model in
vivo toxicological tests in vitro have foundered42. Despite the excitement
of a growing definition of the molecular controls of toxin-induced cell
death, based on the reductionist approach of the molecular biologist,
toxicology demands a strategy of holistic, integrative biology in order to
be able to predict and modulate toxicity. Only in a whole organism can
the effect of contextual elements be determined with any certainty.
Acknowledgements
Both authors wish to thank the Wellcome Trust for support of their
work, through their Molecular and Cellular Toxicology initiative. JAH
also thanks The Cancer Research Campaign and Zeneca Pharmaceuticals for long term funding.
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