From the Trescowthick Research Laboratories, Peter MacCallum Cancer
Institute, St. Andrews Place, East Melbourne, Victoria, Australia, 3002 and the Department of Radiation Oncology, Stanford
University, Stanford, California 94305
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ABSTRACT |
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The resistance to stress-induced apoptosis
conferred by the thermotolerant state or by exogenous expression of
HSP72 was measured in mouse embryo fibroblasts. The induction of
thermotolerance protects cells from heat, tumor necrosis factor (TNF
), and ceramide-induced apoptosis but not from ionizing
radiation. Because the development of thermotolerance is associated
with increased levels of heat shock proteins, we determined whether
constitutive expression of one of the major inducible heat shock
proteins, HSP72, could also protect cells from stress-induced
apoptosis. Cells expressing constitutive HSP72 were shown to have
significantly reduced levels of apoptosis after heat, TNF
, and
ceramide but not after ionizing radiation. Activation of
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was
found to be strongly inhibited in thermotolerant cells after heat shock
but not after other stresses. Cells that constitutively express HSP72
did not demonstrate decreased SAPK/JNK activation after any of these
stresses. Thus, factors other than HSP72 that are induced in the
thermotolerant state are able to reduce activation of SAPK/JNK after
heat stress. Notably, the level of activation of SAPK/JNK did not
correlate with the amount of apoptosis detected after different
stresses. Constitutive HSP72 expression inhibited poly(ADP-ribose)
polymerase cleavage in cells after heat shock and TNF
but not after
ceramide or ionizing radiation. The results suggest either that
SAPK/JNK activation is not required for apoptosis in mouse embryo
fibroblasts or that HSP72 acts downstream of SAPK/JNK. Furthermore, the
data support the concept that caspase activity, which can be
down-regulated by HSP72, is a crucial step in stress-induced apoptosis.
Based on data presented here and elsewhere, we propose that the heat shock protein family can be classified as a class of anti-apoptotic genes, in addition to the Bcl-2 and inhibitor of apoptosis protein families of genes.
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INTRODUCTION |
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The regulation of normal development involves a combination of cell division, differentiation, and death. Although much emphasis was previously placed on mechanisms of cell cycle progression, mechanisms of cell death have only recently begun to be understood. Programmed cell death, or apoptosis, is an active process resulting in characteristic morphological changes to the cell including condensed regions of nuclear material, internucleosomal DNA cleavage, and membrane blebbing (1). The fragmented chromatin, still membrane-bound, is phagocytosed by neighboring cells, thus avoiding an inflammatory response. Apoptosis is responsible for the removal of unwanted cells of many lineages and is thought to be an important safeguard against hyperplasia, which can be an early event in neoplasia (2).
Environmental stresses such as heat, radiation, and hypoxia; growth
factors and ligands for surface receptors; and many drugs or chemical
agents can induce apoptosis. Nevertheless, cells undergoing apoptosis
exhibit a similar morphology, suggesting that these divergent apoptotic
stimuli converge to trigger a common pathway of cell death. The common
pathway involves a family of proteases known as the
interleukin-1-converting enzyme
(ICE)-like1 proteases or
caspases, which are activated in a proteolytic cascade to cleave
specific substrates (3, 4). More recently, it has become evident that
the transmission of signals from external stresses is accompanied by
the activation of two families of kinases, the stress-activated protein
kinases (SAPKs), also known as the c-Jun N-terminal kinases (JNKs), and
the p38/HOG-1 kinases (5). Another possible signaling molecule in
apoptotic pathways is the second messenger, ceramide. Increases in
ceramide levels mediated by the activation of sphingomyelinase and
consequent hydrolysis of sphingomyelin have been observed after
exposure of cells to heat, radiation, TNF
, and peroxide (6). In
addition, exogenously added ceramide induces apoptosis (7). Thus,
whereas the early signals appear to be specific to a particular stress
or group of stresses, the subsequent events, such as the activation of the caspases and possibly the SAPKs and ceramide, are common
events.
Heat shock proteins (HSPs) are a group of inducible proteins, some of which are constitutively expressed and increase in response to stress, whereas others are expressed only after stress (8). The constitutively expressed proteins act as chaperones for other cellular proteins, binding to nascent polypeptides to prevent premature folding and to translocate proteins into organelles (9). The induction of increased levels of the stress proteins is associated with the development of thermotolerance, a transient resistance to heat induced by prior exposure to mild heat or other stress agents (10, 11). It is apparent that induced stress proteins can act to protect cells from stress-induced damage by preventing protein denaturation and/or by repairing such damage (12).
One major group of HSP is the HSP70 family that comprises a multi-gene
family with at least 11 genes in humans (13) and 8 identified so far in
the mouse (14). We and others have shown previously that induction of
thermotolerance with a parallel induction of HSP inhibits heat-induced
apoptosis in several cell lines (15, 16). The consequences of exogenous
expression of HSP72 in cells have been investigated by several groups.
Expression of human HSP72 protects cells from heat stress (17-19),
from some drugs (20), from the cytotoxic effects of TNF (21), from
monocyte killing (22), and from nitric oxide (23). In some of these studies, however, it was not demonstrated whether the cells died by
apoptosis after these stresses.
As part of our investigation of pathways of stress-induced apoptosis, we have measured the effects of either induction of thermotolerance or constitutive expression of HSP72 in blocking apoptosis induced by external stresses. The protection provided by HSP72 against diverse apoptosis-inducing stresses that we have reported here and other data in the literature lead us to propose that the heat shock gene family represents another class of apoptosis-inhibiting genes.
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EXPERIMENTAL PROCEDURES |
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Cell Cultures-- Mouse embryo fibroblasts (MEF) derived from Balb/c mice were minimally transformed with E1A and Ras as described previously (24). To generate cells with constitutive HSP72 expression, clones were infected with the pMVH/human HSP72 proviral vector containing the 2.3-kilobase human HSP72 cDNA under the control of the cytomegalovirus promoter (25), a kind gift from Dr. G. Li. Cultures were single cell cloned, and HSP72 levels were determined by Western blotting. Except where specified, the high HSP72 expressing clone 2 cell line was used in experiments.
MEF were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C in a humidified 5% CO2 atmosphere. Immediately prior to each experiment, the medium was replaced with fresh pH equilibrated medium. All experiments were performed on 50% confluent cell cultures. For heat shock treatments, cells were immersed in a circulating water bath calibrated to 43 ± 0.1 °C for 10-60 min. Lyophilized mouse TNFApoptosis Assays-- Nuclear morphology was assessed by fluorescence microscopy after propidium iodide staining. Briefly, flasks containing approximately 3 × 106 cells were trypsinized, centrifuged at 1000 rpm, and resuspended in 200 µl of phosphate-buffered saline containing 10 µg/ml propidium iodide, 0.1% Triton X-100, and 0.1% sodium acetate. Samples were incubated on ice for 30 min in the dark before being scored for apoptotic morphology using a fluorescence microscope. A cell was scored as apoptotic if it displayed one or more of the following: nuclear margination, chromatin condensation, or formation of apoptotic bodies. At least 200 cells were counted in each experiment, and the data shown are the means and standard deviation of at least three independent experiments.
Western Analysis--
HSP72 expression in cells was detected
using a mouse monoclonal antibody specific for human HSP72 (N15, a kind
gift from Dr. W. Welch) or with an antibody that detects both human and
mouse HSP72 (SPA-810, StressGen Biotechnologies). The cleavage of
poly(ADP-ribose) polymerase (PARP) from a 116-kDa protein to an 85-kDa
fragment was detected using a mouse monoclonal antibody, C-2-10 (a
kind gift from Dr. S. Kaufmann), which recognizes both the full-length and the 85-kDa fragment. Samples containing equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose. The membranes were incubated with
C-2-10, SPA-810, or N15 antibody, followed by horseradish
peroxidase-conjugated rabbit anti-mouse IgG and ECL chemiluminescence
for PARP or alkaline phosphatase-conjugated goat anti-mouse IgG and
color staining for HSP72. As a loading control, membranes were
immunostained with an anti--tubulin antibody (clone B-5-1-2, Sigma)
and detected by a color reaction using an alkaline
phosphatase-conjugated secondary antibody.
SAPK/JNK Assay--
SAPK/JNK activity was assayed in
vitro using a bacterially expressed glutathione
S-transferase (GST)-c-Jun (1-141) fusion protein as a
substrate (26). GST-c-Jun was bound to glutathione-Sepharose 4B beads
and maintained as a 10% slurry in phosphate-buffered saline/sodium
azide. Cells were washed in cold phosphate-buffered saline and lysed in
PLC buffer (50 mM Hepes, pH 7.5, 10% glycerol, 1% Triton
X-100, 150 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 5 mM dithiothreitol, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 15 mM p-NPP, 1 mM phenylmethylsulfonyl fluoride) and
incubated on ice for 10 min. The lysate was cleared by centrifugation
(13,000 rpm, 4 °C, 10 min). The supernatant was collected, and
SAPK/JNK was immunoprecipitated with a polyclonal rabbit anti-mouse
JNK1 antibody (Santa Cruz Biotechnology). Samples were resuspended in
PLC buffer and incubated with GST-c-Jun-Sepharose beads for 15 min at
4 °C. The beads were washed twice in kinase buffer (25 mM Hepes, pH 7.5, 10% glycerol, 10 mM NaCl, 5 mM MnCl2, 1 mM dithiothreitol, 15 mM p-NPP) and resuspended in kinase reaction
buffer (kinase buffer containing 60 µM ATP, 1 mM dithiothreitol, 30 mM p-NPP, 1 µCi of [-32P]ATP). The kinase reaction was performed
at 30 °C for 30 min. The reaction was stopped by washing the beads
in PLC buffer supplemented with 20 mM EDTA and 15 mM p-NPP. The beads were resuspended in 2×
Laemmli gel sample buffer and boiled for 5 min. Samples were subjected
to 10% SDS-polyacrylamide gel electrophoresis, stained with Coomassie
Blue, destained, dried, and phosphorylation of c-Jun measured by
PhosphorImager analysis (Molecular Dynamics).
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RESULTS |
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Thermotolerance Protects Cells from Apoptosis after Heat Shock,
Ceramide, and TNF but Not after Ionizing Radiation--
MEF were
treated with a mild heat shock (44 °C for 10 min) to induce
thermotolerance and allowed to recover at 37 °C for 6 h before
being subjected to more severe heat shock, ceramide, TNF
, or
ionizing radiation. Cells were scored for apoptotic morphology under a
fluorescence microscope following permeabilization and staining with
propidium iodide. As shown in Fig.
1A, cells in a thermotolerant
state showed significant decreases in apoptosis after heat shock,
ceramide, and TNF
but not after ionizing radiation. The
thermotolerant state protected cells from apoptosis after a range of
heat doses (Fig. 1B) or TNF
doses (Fig. 1C).
The induction of thermotolerance induces high levels of HSP72 (Fig.
2).
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Constitutive Expression of HSP72 Protects Cells from Apoptosis
after Heat Shock, Ceramide, and TNF but Not after Ionizing
Radiation--
Because the development of thermotolerance closely
correlates with the induction of HSP72, it was decided to investigate
whether constitutive expression of HSP72 alone could protect MEF from stress-induced apoptosis. MEF were infected with HSP72 and single cell
cloned, and a number of clones expressing human HSP72 were isolated.
The highest HSP72 expressing clone, clone 2, has less HSP72 than is
measured in thermotolerant MEF but more than is expressed by clone 3 (Fig. 2). Control and HSP72 expressing cells were treated either with
heat shock, ceramide, TNF
, or ionizing radiation as described above,
stained with propidium iodide, and scored for apoptotic morphology. As
shown in Fig. 3, cells expressing constitutive HSP72 are protected from heat shock-, ceramide-, and
TNF
-induced apoptosis but not from ionizing radiation. Although we
found that HSP72 does protect the cells from heat stress, we found
greater protection from TNF
- and ceramide-induced apoptosis. Also
shown in Fig. 3 is the response of clone 2 cells to heat or TNF
after the induction of thermotolerance. The level of HSP72 increases
further after induction of thermotolerance in clone 2 but is similar to
the level induced in the parental MEF (Fig. 2). Cells expressing both
high levels of HSP72 and thermotolerance are more resistant to heat
stress than those expressing HSP72 alone (Fig. 3A), whereas
the response to TNF
(Fig. 3B) is similar to that of HSP72
expressing cells. Thus the protection from heat-induced apoptosis
afforded by the thermotolerant state cannot be attributed solely to the
induction of HSP72, whereas protection from TNF
-induced apoptosis in
thermotolerant cells can be fully accounted for by the expression of
HSP72.
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Thermotolerance Reduces SAPK/JNK Activity after Heat Shock but Not
after Ceramide, TNF, or Ionizing Radiation--
The activation of
the SAPK/JNK cascade detected after a number of cytotoxic insults has
been proposed to be required for apoptosis (6). To determine whether
the thermotolerant state was protecting MEF from stress-induced
apoptosis through the inhibition of this pathway, SAPK/JNK activity was
measured by an in vitro kinase assay using a GST-c-Jun
(1-141) fusion protein as a substrate. Time courses were performed
after each stress to determine optimum times for detecting SAPK/JNK
activity. Maximal activity was detected at 30 min after heat shock and
TNF
. MEF show a 10-15-fold activation of SAPK/JNK after heat shock
and 2-3-fold activation after TNF
. Data shown for ceramide and
ionizing radiation were obtained 1 h after stress; however, little
SAPK/JNK activation was detected in MEF after ceramide and ionizing
radiation over a 24-h period. Using these conditions, SAPK/JNK activity
was measured in control and thermotolerant cells. Significant reduction
of SAPK/JNK activity in thermotolerant cells after heat shock was
observed, but no changes in activity were detected after other stresses
(Fig. 4A).
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Constitutive Expression of HSP72 Does Not Reduce SAPK/JNK
Activation after Stress--
Because the thermotolerant state was
shown to reduce SAPK/JNK activation after heat shock but not after
other stresses, the effect of HSP72 expression on SAPK/JNK
activity was measured. Control and HSP72 expressing cells were
subjected to heat shock, ceramide, TNF, or ionizing radiation and
SAPK/JNK activity measured. As shown in Fig. 4B, HSP72
did not reduce SAPK/JNK activation after any of these stresses, despite
protecting the cells from apoptosis.
Thermotolerance Inhibits PARP Cleavage in Cells after Heat Shock,
TNF, and Ceramide but Not after Ionizing Radiation--
A common
event in the apoptotic pathway is the activation of a caspase cascade.
PARP, a DNA repair enzyme, has been identified as a substrate for
caspase-3. Caspase-3 activity was measured by following the cleavage of
PARP from a 116-kDa protein to an 85-kDa fragment. PARP cleavage is
first detected in MEF cells 6 h after stress but is more extensive
at 16 h after heat, ceramide, and TNF
or 24 h after
ionizing radiation (data not shown). We examined the possibility that
the thermotolerant state may inhibit PARP cleavage either directly or
by regulating some upstream event in the caspase cascade. MEF were
treated with heat shock, ceramide, TNF
, or ionizing radiation and
allowed to recover for 16 or 24 h. Cell extracts were subjected to
immunoblotting with a PARP-specific antibody that recognizes both the
116- and 85-kDa fragments. As shown in Fig.
5A, the induction of
thermotolerance can reduce PARP cleavage after heat shock, TNF
, and
ceramide but not after ionizing radiation. Measurement of
-tubulin
levels demonstrates equal protein loading in each lane.
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Constitutive HSP72 Expression Inhibits PARP Cleavage in Cells after
Heat Shock and TNF but Not after Ceramide or Ionizing
Radiation--
To determine whether HSP72 could mimic the effect of
thermotolerance on PARP cleavage, MEF were treated with heat
shock, ceramide, TNF
, or ionizing radiation, and immunoblotting was
performed on cell extracts. As found for the thermotolerant state,
constitutive HSP72 expression reduced PARP cleavage in cells after heat
shock and TNF
but not after exposure to ceramide or ionizing
radiation (Fig. 5B).
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DISCUSSION |
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Many forms of cellular stress lead to the activation of two
related signaling pathways mediated by the stress-activated protein kinases, SAPK/JNK and p38/HOG-1 (5). The SAPK/JNKs, which are activated
by a kinase known as Sek1, comprise a family of eight or more isoforms
that phosphorylate the c-Jun and JunD components of the AP1
transcription factor as well as Elk-1 and ATF-2. ATF-2 is also a
substrate for p38/HOG-1, as is MAPKAP kinase-2, which in turn
phosphorylates the heat shock protein, HSP27, at the same sites at
which it is phosphorylated in response to stress (5). Many stresses
capable of activating SAPK/JNK, including heat shock, TNF, ceramide,
UV light, ionizing radiation, osmotic stress, and anti-cancer agents,
also result in apoptosis (reviewed in Ref. 5). Despite the observation
that SAPK/JNK activation and apoptosis are often co-incident, there is
strong debate about the requirement of SAPK/JNK activation for
apoptosis. Stable transfection of dominant negative mutants of either
Sek1 or c-Jun (TAM-67) has been shown to inhibit apoptosis after
ionizing radiation, ceramide, UVC light, heat shock, and hydrogen
peroxide (6). In contrast, studies performed in Sek1 (
/
) ES cells
demonstrated a normal apoptotic response after ionizing radiation, UV
light, osmotic stress, serum deprivation, anisomycin, heat shock,
dexamethasone, and anti-cancer agents, although thymocytes from Sek1
(
/
) mice showed impaired Fas-induced apoptosis (27). Controversy
surrounds the involvement of SAPK/JNK activation in TNF
-induced
apoptosis, with studies claiming that a catalytically inactive MAPKKK
(ASK1), which no longer activates SAPK/JNK and p38/HOG-1 (28), can
inhibit TNF
-induced apoptosis, as can either Sek1 or TAM67 dominant
negative mutants (6). In contrast, other studies have shown that
SAPK/JNK activation occurs through a pathway that is not required for
TNF
-induced apoptosis (29, 30).
In our study, we have shown that the thermotolerant state or
constitutive expression of HSP72 can protect cells from apoptosis induced by heat, TNF, and ceramide but not from ionizing radiation, thus demonstrating the existence of multiple pathways of stress-induced apoptosis. It is interesting to note that HSP72, which is widely believed to protect cells from heat stress, prevents heat-induced apoptosis to a lesser extent than TNF
- or ceramide-induced
apoptosis. Further, the protection seen in thermotolerant cells after
TNF
and ceramide can be accounted for by the induction of HSP72,
whereas the induction of thermotolerance provides a significantly
higher level of protection against heat-induced apoptosis than can be accounted for by HSP72 alone. An explanation for this may lie in the
fact that heat is a nonspecific stress that causes damage to many
proteins and organelles in the cell, and thus multiple targets need to
be protected from heat stress. HSP72 may be able to protect some of
these targets, but the induction of other stress proteins may be
required for more complete protection. In contrast, TNF
initiates
apoptosis through a specific pathway that begins with the activation of
TNF receptors. HSP72 could act at one stage of this pathway to minimize
the extent to which apoptosis is triggered by TNF
.
The possibility that HSP72 may regulate stress-induced apoptosis through inhibition of the SAPK/JNK pathway was investigated, but expression of HSP72 was found to not alter the extent of activation of SAPK/JNK. In addition, we found that the extent of SAPK/JNK activation after the different stresses did not correlate with the extent of apoptosis observed, possibly indicating that SAPK/JNK activation is not required for the induction of apoptosis in MEF. Alternatively, SAPK/JNK activation may be necessary but is not sufficient for apoptosis to occur. We therefore conclude either that MEF can undergo stress-induced apoptosis using a SAPK/JNK-independent pathway that can be regulated by HSP72 or that HSP72 acts downstream of SAPK/JNK activation. Interestingly, SAPK/JNK was inhibited in thermotolerant cells after heat shock but not in cells expressing constitutive HSP72. This supports the finding that thermotolerance provides a substantially higher level of protection against heat-induced apoptosis than HSP72 alone, suggesting that factors other than HSP72 that are induced in the thermotolerant state are capable of suppressing SAPK/JNK activation.
An expanding family of cysteine proteases (caspases), of which ICE is
the prototype, has been shown to play a key role in mammalian cell
apoptosis. The caspases can be divided into three main groups based on
sequence similarity: ICE, ICErel 11/Tx/ICH-2 and ICErel 111/Ty;
CPP32/Yama/apopain/prICE, ICE-LAP3/MCH3 and MCH2; and finally,
Nedd2/ICH-1 (4). Overexpression of any one of these enzymes induces
apoptosis in transfected cells (31, 32). Evidence is emerging that
multiple caspases may function sequentially after induction of
apoptosis in a cell (33). A number of substrates have now been
identified, including other caspases, proteins involved in DNA repair
(PARP and DNA-protein kinase) and some structural proteins (lamin,
actin, and vimentin) (4), but the importance of some of these
substrates in apoptosis is not clear. We investigated the possibility
that HSP72 could inhibit stress-induced apoptosis through the
regulation of the caspase pathway. Caspase-3 activity can be measured
by following the cleavage of PARP from a 116-kDa protein to an 85-kDa
fragment. We have shown that constitutive HSP72 expression can prevent
PARP cleavage after heat shock or TNF but not after ceramide or
ionizing radiation. The lack of protection of PARP by HSP72 after
ceramide exposure is surprising because HSP72 does protect against
ceramide-induced apoptosis. The data suggest that HSP72 acts to block
the activation or the activity of one of the caspases and/or to protect
the substrates of these caspases from proteolytic degradation. The
possibility that HSP72 acts as a decoy substrate for the caspases was
investigated by Mosser et al. (19), but they found that
HSP72 was unable to inhibit the caspase-3-mediated cleavage of PARP in
an in vitro assay.
Of interest in this study was the marked protection by HSP72 from
TNF-induced apoptosis, mediated through the p55 TNF receptor. While
triggering apoptosis in many cells, TNF
also activates the
transcription factor NF-
B that blocks apoptosis (30). How HSP72 may
interact with this pathway is unknown, but in WEHI-S cells, the
expression of HSP72 does not alter the TNF-induced activation of
NF-
B, nor does it alter TNF receptor levels (34). Instead, HSP72
inhibits TNF-induced activation of phospholipase A2, which
releases arachidonic acid from membrane phospholipids (34). It has been
shown previously that arachidonic acid is released in response to TNF
in sensitive cells but not in TNF-resistant cells (35).
Two other families of genes that block apoptosis have been identified. The inhibitor of apoptosis protein family, first recognized as the candidate gene for spinal muscular atrophy, can block apoptosis induced by a number of stresses (36-38). The best characterized inhibitors of apoptosis are Bcl-2 and several of its homologues, which can block apoptosis in response to a large number of, but not all, stimuli (39). The mechanism by which Bcl-2 and its homologues block apoptosis is not clear; however it has been suggested that Bcl-XL prevents disruption of the mitochondrial membrane potential that otherwise permits the release of apoptosis-inducing proteins into the cytosol (40). Bcl-2 blocks the release of cytochrome c from the mitochondria that occurs following apoptotic stimuli but prior to membrane depolarization. This in turn prevents the interaction between cytochrome c and Apaf-1 and subsequent caspase activation and apoptosis (41, 42). It is feasible that HSP72 may protect cells from apoptosis through a similar mechanism; either through the inhibition of cytochrome c release from the mitochondria, or, more likely, by binding directly to cytosolic cytochrome c and preventing activation of the caspases. HSP72 is known to bind to peptides derived from cytochrome c (43).
Heat shock proteins have been recognized for some years to protect cells from heat damage and more recently from some other cytotoxic insults. As well as the protective effects of HSP72 shown here and in other studies (17-23) the small heat shock protein, HSP27, has been shown to block apoptosis induced by Fas/Apo-1 and staurosporine (44) and by some anti-cancer drugs (20, 45). Heat shock proteins are often expressed at high levels in tumor cells, leading to suggestions that they can protect cells either from immune attack or from the type of therapy being administered. For example, several recent studies have revealed that tumor specimens from patients whose cancer therapy has resulted in a short disease-free survival contain high levels of one or more of the stress proteins (46-49). Emerging clones of tumor cells would be subjected to host initiated stresses such as TNF and may show prolonged survival if they express high levels of HSP72. Thus, we believe there is evidence to suggest that high levels of heat shock proteins may confer a survival advantage on an emerging or metastasizing clone of neoplastic cells by protecting it from immune surveillance and other host-derived stresses. We therefore propose that the heat shock gene family represents a third class of anti-apoptosis genes.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Kaufmann for the anti-PARP
antibody, C-2-10; Dr. W. Welch for the anti-human HSP72 antibody, N15;
Dr. G. Li for the pMVH human HSP72 proviral vector; and Dr. A. Strasser for human TNF and Trail ligand. We also acknowledge Dr. D. Vaux, Dr.
A Strasser, Dr. D. Bowtell, and Dr. R. Pearson for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Peter MacCallum Cancer Inst., Locked Bag #1, A'Beckett St., Melbourne, Australia, 3000. Tel.: 61-3-9656-1284; Fax: 61-3-9656-1411; E-mail: r.anderson{at}pmci.unimelb.edu.au.
1
The abbreviations used are: ICE,
interleukin-1-converting enzyme; SAPK, stress-activated protein
kinase; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; HSP,
heat shock protein; MEF, mouse embryo fibroblast(s); Gy, gray; PARP,
poly(ADP-ribose) polymerase; GST, glutathione
S-transferase; p-NPP, p-nitrophenyl
phosphate.
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REFERENCES |
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