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Research Communications |
* Division of Rheumatology, Department of Internal Medicine III,
Department of Immunodermatology,
Division of Infectiology, Department of Internal Medicine I, University of Vienna;
§ Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck; and
|| Ludwig Boltzmann-Institute for Rheumatology and Balneology, Vienna, Austria
1Correspondence: Division of Rheumatology, Department of Internal Medicine III, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Fas activation • antibody • heat shock element • apoptosis • hsp
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2)
or to the activation of
a cascade of molecular events leading to apoptosis (3)
. At a certain
time, a cell exposed to stress has to either activate molecular defense
strategies in order to survive or start self-destruction by apoptosis.
Stress or heat shock proteins (hsp)2
constitute a
phylogenetically old cellular system providing increased resistance to
a variety of cellular stress factors, such as elevated temperatures,
heavy metal ions, oxidants, and amino acid analogs (1,
4)
. Inducible
hsp70 (also called hsp72), one of the most widely and best studied
forms of hsp in mammalian cells, is characterized by its highly
inducible expression in response to stress and by its function as a
chaperone, facilitating the folding, unfolding, and refolding of
proteins under both normal and stressed conditions (5,
6)
. Induced
expression of hsp70 has been shown to promote cytoprotection (7,
8)
.
Stress-induced synthesis of hsp70 is regulated at the transcriptional
level via the activation of heat shock transcription factors (HSF) (9)
.
HSFs are constitutively present in the cytoplasm in a non-DNA binding
state and, upon activation, they hyperphosphorylate, trimerize,
translocate to the nucleus, and bind to DNA at a specific site of the
hsp gene promotor region called the heat shock element (HSE) (10,
11)
.
Two functionally different HSFs are known—HSF1 and HSF2—of which HSF1
has been shown to mediate signaling of stress factors, including
elevated temperatures (12)
.
The Fas protein (CD95) belongs to a signal transduction system inducing
apoptotic cell death in the presence of pro-apoptotic factors (1)
.
Activation of Fas-mediated apoptosis is induced by binding of its
physiological ligand, Fas ligand (13,
14)
or by anti-Fas antibodies
(15,
16)
. A cytoplasmic `death' domain of the transmembrane Fas
protein induces a cascade of intracellular signals (17,
18)
, finally
leading to the activation of interleukin 1-converting enzymes (ICE)
(19,
20)
. As cysteine proteases, ICE cleave a number of substrates,
such as poly(ADP) ribose polymerase, lamin, and actin, leading to the
typical morphological changes of the cells and nuclei during apoptosis.
We investigated the interplay of Fas-mediated apoptosis with the (HSF1-activated) hsp70 stress response and, in light of their contradictory actions, analyzed whether 1) Fas apoptosis, once activated, may inhibit the HSF1/hsp70 stress response, and 2) an activated hsp70 response could offer protection from Fas-induced apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heat stress and induction of Fas apoptosis
To induce both HSF1 activation and hsp70 expression, cultured
U937 cells were heat stressed for 30 min at 42°C, whereas control
cells remained at 37°C. HSF1 activation was analyzed 30 min after the
beginning of heat stress; hsp70 expression was assessed 6 h after
heat stress. Fas-mediated apoptosis was induced by the addition of an
anti-human Fas (anti-APO1; Clone CH-11, Coulter-Immunotech, Marseille,
France) monoclonal antibody at 1:200 dilution (2.5 µg/ml final),
known to activate apoptosis through binding to Fas molecules on the
cell membrane. For blocking Fas-mediated apoptosis, ICE inhibitor
(caspase-1 inhibitor I: YVAD-CHO; Boehringer Ingelheim Bioproducts
Partnership, Heidelberg, Germany; ref 21
) in various concentrations (1
µM–1 mM) was added to the cultures.
Protein extraction and Western blot analysis
Cells (5 x 106) were washed twice in
phosphate-buffered saline (PBS), pelleted by centrifugation at 1200
g for 10 min, and resuspended in 200 µl of lysis buffer
[0.15 M NaCl, 50 mM Tris, 1 mM EDTA, 0.05% sodium dodecyl sulfate
(SDS), 0,5% Triton-X100, and 1 µM phenylmethylsulfonylfluoride, pH
7.4] for 30 min at 4°C. The cell lysate was then centrifuged at
13,000 rpm for 10 min, and the supernatants were harvested and analyzed
for their protein content using a protein assay kit (Bio-Rad, Hercules,
Calif.). For electrophoresis, total cell proteins were dissolved (1:2
v/v) in sample buffer containing 5% ß-mercaptoethanol, 15%
glycerol, 3% SDS, and 0.1 M Tris (pH 6.8) and separated on a 12% SDS
gel under reducing conditions. Proteins were electrophoretically
blotted onto nitrocellulose membranes (BA85, Schleicher & Schuell,
Dassel, Germany). Hsp70 expression was detected by probing the
membranes with a monoclonal antibody specific for the inducible form of
human hsp70 (also called hsp72) (clone W28, StressGen Biotechnologies
Corp., Victoria, Canada, ref 22
), as described (23)
. HRP-conjugated
rabbit anti-mouse Ig (Dako, Glostrup, Denmark) was used as detection
antibody and the reaction visualized by 4-chloro-1-naphtol/hydrogen
peroxide (Sigma, St. Louis, Mo.). HSF1 expression was investigated
using antibodies against mammalian HSF1 (gift from Dr. R. I.
Morimoto, Northwestern University, Evanston, Ill.).
Gel mobility shift assays
Nuclear protein preparations from U937 cells were used for
analysis of HSF1 transcription factor activity. Nuclear protein
isolation was similar to that described by Schreiber et al. (24)
with
slight modifications (25)
. After washing 2 x
106 U937 cells in cold TBS (10 mM Tris, 150 mM
NaCl, 1 mM EDTA, pH 7.4), the cells were pelleted and 400 µl of cold
buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EGTA, 1 mM DTT, 1 mM Pefablock
SC, 0.5 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, pH 7.9) was
added. The suspension was incubated on ice for 15 min. Then, 25 µl
(10%) Nonidet P-40 was added, the suspension vortexed for 10 s,
and centrifuged for 30 s at 13,000 rpm. The supernatant was
discarded and the nuclear pellet incubated with 50 µl of cold buffer
B (20 mM HEPES, 0.4 M NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM
Pefablock SC, 0.5 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, pH
7.9) at 4°C for 15 min on a gyratory shaker platform with vigorous
rocking. The suspension was then centrifuged at 4°C for 5 min at
13,000 rpm, the supernatant was collected, and protein concentration
was determined.
Gel mobility shift assays were performed as described previously
(25)
. Briefly, DNA binding was determined after incubation of 5 µg of
nuclear protein with 10 fmol of an oligonucleotide containing the HSE
sequence from the Drosophila hsp70 promotor
(5'-GCCTCGAATGTTCGCGAAGTTT-3') labeled with
32P-dCTP. Reaction buffer contained 10 mM HEPES,
pH 7.9, 1 mM DTT, 1 mM EDTA, 80 mM KCl, 4% Ficoll, and 1 µg
poly(dIdC) (Pharmacia Biotech., Uppsala, Sweden) as a nonspecific
competitor. Samples were electrophoresed on 4% polyacrylamide gels in
0.5 x TBE (1xTBE: 89 mM Tris, 89 mM boric acid, 20 mM EDTA, pH
8.3). Then the gel was dried and exposed to autoradiographic film.
FACS analysis
U937 cells were seeded on 96-well, flat-bottom microtiter plates
at a density of 5 x 105 cells per 200 µl 10%
FCS/RPMI in triplicates. Anti-Fas antibody was added to all triplicate
sets except two, which were intended to serve as non-Fas controls. Heat
stress was applied before (6 h or 0.5 h) or after (0 h, 0.5 h, 1 h, 3 h, or 6 h) the addition of anti-Fas antibody.
Detection of early-stage apoptosis by annexin V staining
Cells were analyzed for their capacity to bind recombinant human
annexin V, directly conjugated to FITC (Bender MedSystems, Boehringer
Ingelheim Bioproducts Partnership) 1, 3, and 6 h after
administration of anti-Fas.
Cells were pelleted, washed in annexin V binding buffer (ABB)
containing 1.8 mM CaCl2 according to the
manufacturer's guidelines, resuspended in 200 µl ABB with 5 µl
annexin V-FITC, and incubated for 15 min at room temperature. After
washing in ABB, propidium iodide (PI) was added to a final
concentration of 1 µg/ml and samples were analyzed on a FACScan
(Becton Dickinson Immunocytometry Systems, San Jose, Calif.) by dual
color cytofluorometry. Cells that stained brightly with annexin V but
still excluded PI were considered to be early-phase apoptotic (26)
.
Simultaneous analyses of hsp70 expression and DNA fragmentation
Twelve hours after the addition of anti-CD95 antibody, terminal
deoxynucleotidyl transferase (TdT) -mediated dUTP nick end labeling
(TUNEL) was performed using an In Situ Cell Death Detection Kit
(Boehringer Mannheim, Mannheim, Germany) according to the
manufacturer's recommendations (276). Negative controls treated
exactly the same way except for the addition of TdT were included for
later determination of the autofluorescence baseline. After final TUNEL
washes, pelleted cells were resuspended in 50 µl of a 1:10 dilution
(10 µg/ml final) of unconjugated mouse anti-human HSP-70 antibody in
PBS and incubated for 1 h at 4°C. In parallel, negative control
samples were incubated with an equivalent amount of isotype-matched,
unconjugated mouse IgG2a-negative control antibody (Dako). Subsequently
all samples were washed once in PBS, resuspended in 50 µl of 1:10
diluted F(ab')2 fragments of rabbit anti-mouse Ig
antibody conjugated to R-phycoerythrin (Dako) in PBS, and incubated for
30 min at 4°C. After a final wash, samples were analyzed by dual
color immunocytofluorometry.
Immunofluorescence
Immunofluorescence studies were performed as described (28)
,
with slight modifications. Cells (1 x 105) in 8-well
chamber slides (Nunc, Naperville, Ill.) were treated with anti-Fas
antibody (Ab) (2.5 µg/ml) alone or were heat stressed (42°C, 30
min) simultaneously or 1 h after the addition of the antibody. In
addition, untreated cells (negative control) and heat-stressed cells
(positive control) were analyzed. To assess HSF1 nuclear translocation,
cells were permeabilized and fixed 30 min after the start of heat
stress; for hsp70 detection, cells were analyzed 6 h after heat
stress. Cells were permeabilized with 4% paraformaldehyde for 30 min
and fixed with absolute methanol for 5 min. Staining with antibodies to
HSF1, hsp70, or CD3 (Dako), as a control, and FITC conjugates (Dako)
was then performed. Slides were finally embedded in
n-propylgalate/glycerol (Sigma) and analyzed by confocal laser scanning
microscopy (Carl Zeiss Inc., Thornwood, N.Y.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). When heat stress and Fas stimulation were applied
simultaneously, unhampered activation of HSF1 was observed. However, if
heat stress was applied only 30 min after Fas activation, HSF1
activation was markedly diminished. As early as 1 h after addition
of anti-Fas Ab, HSF1-DNA binding was highly suppressed; after 3 h,
no significant HSF1 binding could be detected (Fig. 1A
). To
clarify whether the suppression of HSF1 activation was mediated via the
ICE pathway, serial dilutions of ICE inhibitor were added to the
system. In the presence of 10 µM to 1 mM of ICE inhibitor, Fas
activation failed to suppress HSF1-DNA binding, suggesting an
ICE-dependent mechanism (Fig. 1B
).
|
Intracellular localization of HSF1 was assessed by immunofluorescence
studies: heat stress led to a nuclear translocation of HSF1 (Fig. 2
A). In contrast, when cells were preincubated with anti-Fas
Ab 1 h before heat stress, no nuclear translocation was observed
(Fig. 2B
). Similarly, cytoplasmic staining was observed in
cells treated with anti-Fas Ab alone (Fig. 2C
) or untreated
cells (Fig. 2D
).
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Effects of Fas activation on HSF1 phosphorylation
Hyperphosphorylation of the cytoplasmic inactive form of HSF1 is
considered an initial step in HSF1 activation. To investigate whether
Fas-induced inhibition of HSF1-DNA binding was based on inhibition of
HSF1 phosphorylation, Western blot analysis of HSF1 from unstressed and
heat-stressed cells treated with anti-Fas Ab was performed. Unstressed
(Fig. 3
A, lane a) as well as Fas-treated cells (lane b) only
expressed the lower molecular weight, constitutively-phosphorylated
isoform of HSF1. In contrast, heat stress entailed a shift to the
slower migrating hyperphosphorylated isoform of HSF1 (lane c), whereas
pretreatment with anti-Fas antibody 1 h before heat stress
completely blocked this shift (lane d). Pretreatment with ICE
inhibitors (10 µM) reversed this effect (lane e). Thus, these data
demonstrated that Fas activation leads to an ICE-dependent inhibition
of phosphorylation and activation of cytoplasmic HSF1. In addition, no
signs of proteolytic cleavage of HSF1 proteins during Fas apoptosis
were found, since assessment of HSF1 proteins by highly polymerized
(16%) SDS-polyacrylamide gel electrophoresis did not detect any
proteolytic cleavage products in the presence of activated Fas
signaling (Fig. 3B
).
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Effects of Fas activation on inducible hsp70 expression
When analyzing the expression of hsp70 during activated Fas
signaling, inhibition hsp70 expression was similar to that observed
with HSF1. U937 cells receiving heat shock 1 h after Fas
activation showed highly decreased expression of hsp70, and suppression
was even stronger when heat shock was performed at later time points.
Fas activation and heat shock applied at the same time resulted in
up-regulation of hsp70 comparable to that observed after heat shock
alone (Fig. 4
A). Fas activation itself had no influence on hsp70
expression.
|
As observed with HSF1, addition of ICE inhibitors blocked Fas-mediated
suppression of hsp70 synthesis, again suggesting that the ICE cascade
was involved in Fas-dependent inhibition of the HSF1/hsp70 system (Fig. 4B
).
Furthermore, immunofluorescence studies confirmed a decreased hsp70 expression when cells were heat stressed 1 h after Fas activation compared with cells that were either heat stressed alone or simultaneously with Fas activation (not shown).
Effects of Fas activation on resistance to heat stress
Next, the effect of heat stress on a suppressed HSF1/hsp70 system
was investigated (Fig. 5
). U937 cells were treated with anti-Fas Ab, heat stressed after various
periods of time, and the rate of apoptotic cells was determined by FACS
analysis. The proportions of viable (annexin-negative, PI-negative),
apoptotic (annexin-positive, PI-negative), and dead (annexin-positive,
PI-positive) cells were compared. Fas activation in the presence of a
functionally active HSF1/hsp70 response resulted in apoptosis rates of
around 20% after 8 h of culture. However, when the HSF1/hsp70
system was suppressed due to Fas activation, apoptosis rates increased
strongly. When heat shock was applied as early as 30 min after Fas
activation, apoptosis rates increased up to 30%, 1 h after Fas
activation up to 40%, with a maximum of 50% of apoptotic cells after
3 h and 6 h, respectively. In the absence of Fas activation
and heat stress, apoptosis rates were very low (2%). The number of
dead cells was below 10% (Fig. 5)
.
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Effects of HSF1/hsp70 activation on Fas-mediated apoptosis
To investigate a possible protective effect of HSF1/hsp70
induction to Fas-mediated apoptosis, U937 cells were heat stressed
6 h before the addition of anti-Fas Ab to induce hsp70 protein
expression. Alternatively, cells were heat shocked 30 min before Fas
activation to only allow activation of HSF1-DNA binding. Control cells
were left unstressed before addition of anti-Fas Ab. A comparative
analysis of the apoptosis rates of viable (Fig. 6
A), apoptotic (Fig. 6B
), and dead cells (Fig. 6C
) between these three groups was performed. Unstressed
cells showed a 40% increase in apoptosis rates (Fig. 6B
)
3 h after addition of anti-Fas Ab. Similar rates were observed
with cells heat stressed 30 min before Fas activation (40% apoptotic
cells after 3 h), showing that activated DNA binding of HSF1 was
not sufficient to protect from Fas-mediated apoptosis. However, when
heat stress was applied 6 h before Fas activation, apoptosis rates
were significantly lower after 3 and 6 h of incubation with
anti-Fas Ab (15% and 12% apoptotic cells, respectively), suggesting
hsp70 to act as an inhibitor of Fas-mediated apoptotic cell death. In
contrast, the rates of dead (nonapoptotic) cells remained stable and
did not show significant differences among the three groups.
|
As a second technique for detection of apoptosis, FITC-TUNEL labeling
was performed. At the same time, cells were counterstained by
anti-hsp70 Ab to enable a selective assessment of the four following
staining patterns: double negative cells, hsp70-positive cells,
TUNEL-positive (apoptotic) cells, and double positive cells (Fig. 7
). Whereas the majority of untreated cells (Fig. 7A
) were
hsp70 negative (92.7% hsp70 negative), heat stress was followed by a
strong increase in hsp70 expression (Fig. 7B
; 93.0% hsp70
positive). The rate of apoptotic cells, however, was low in both
groups, 7.3% and 7.0%, respectively. Incubation with anti-Fas Ab
(Fig. 7C
) led to increased rates of apoptotic cells (33.4%
apoptotic), the majority of which were in the hsp70-negative subgroup
(23.6%; =71% of apoptotic cells). In contrast, pretreatment with heat
shock 6 h before induction of apoptosis by anti-Fas Ab displayed a
protective effect, as demonstrated by almost normal rates of apoptotic
cells (10.4% apoptotic).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The quantity of inducible hsp70 (also called hsp72) molecules in a cell
is one of the pivotal conditions determining cellular resistance to a
variety of different stress factors and is strictly dependent on
stress-regulated activation of its transcription factor HSF1. This
mechanism has repeatedly been shown to enable cytoprotection and cell
survival (29
30
31)
. However, if cellular signals leading to apoptosis
are already activated, the presence of a protective HSF1/hsp70 response
may be counterproductive, since it may impede the progression to
apoptotic cell death. Herein, we have demonstrated that activation of
the Fas pathway blocks heat shock-induced activation of HSF1 and
expression of hsp70 within 1 h after the addition of anti-Fas
antibody. Thus, Fas-mediated apoptosis, once induced, rapidly blocks
the heat shock-induced HSF1/hsp70 response rather than activates it.
This observation indicates that activation of the apoptotic pathway
leads to the suppression of anti-apoptotic mechanisms. In addition, we
have shown that Fas apoptosis does not activate HSF1 or hsp70,
suggesting that Fas-mediated apoptosis itself does not represent a
stress factor (32)
. It is therefore conceivable that the high hsp70
levels previously observed in apoptotic cells (33,
34)
are not induced
by apoptotic mechanisms, but are based on the underlying stress factors
that, despite induction of stress proteins, entail cellular apoptosis.
This assumption is supported by observations of a blocked HSF1/hsp70
pathway during Fas-mediated apoptosis: heat stress applied after
induction of apoptosis led to amplification of apoptotic cell death.
Because of an impaired HSF1/hsp70 response, these cells cannot adapt to
the presence of stress factors and rapidly die by apoptosis. Studies
performed with quercetin as an inducer of apoptosis have also shown
that the additional presence of heat stress leads to enhanced rates of
apoptosis compared with quercetin alone (35)
.
Signal transduction of Fas-mediated apoptosis from ligand receptor
binding to final apoptotic death has been partly elucidated. Activation
of the ICE-protein cascade is of central importance (36)
, since
inhibitors of caspase-1 were shown to block Fas-induced apoptosis (20)
.
The suppressive effect of Fas-mediated apoptosis on the HSF1/hsp70
response could either be mediated upstream or downstream of ICE. Since
in the presence of ICE inhibitors the HSF1/hsp70 response was still
heat-inducible despite an activated Fas pathway, activation of the ICE
cascade (or signals downstream of ICE) seems to be required for
HSF1/hsp70 suppression. HSF1 molecules, however, are unlikely to be a
substrate of ICE-mediated cleavage, since the amount of cellular HSF1
was shown to be stable during Fas activation and no cleavage products
were observed on Western blots. In contrast, hyperphosphorylation of
HSF1 as the first step of HSF1 activation seemed to be defective in the
presence of Fas signaling. The serine-kinase responsible for HSF1
hyperphosphorylation and activation is still unknown; however, these
data suggest that Fas-mediated apoptosis may inhibit this kinase via
activation of ICE. Alternatively, at least in cells transfected with
hsp70 (ref 37
) or HSF1 (ref 38
), activation of serine/threonine
phosphatase as well as inhibition of protein kinase C is associated
with dephosphorylation and subsequent inhibition of HSF1. Additional
studies elucidating the regulation of the heat shock response upstream
from HSF1, especially its regulating kinase(s), will shed further light
on these interactions.
To investigate whether the effect of Fas-mediated apoptosis on the
HSF1/hsp70 stress response was unidirectional or, in addition,
stress-induced up-regulation of hsp70 could also affect Fas-mediated
apoptosis, we studied a potential protective role of an activated
HSF1/hsp70 axis on Fas-mediated apoptosis. In fact, apoptotic cell
death induced by Fas was significantly inhibited in the presence of
heat-induced overexpression of hsp70. However, activation of HSF1
transcription factor alone was not sufficient to influence Fas-mediated
apoptosis in a protective way. These findings indicate that
up-regulation of hsp70 protein synthesis is necessary to protect from
Fas-mediated apoptosis. Hsp70, as a chaperonin, ensures protection of
proteins during folding and unfolding from proteases, and it is likely
that destructive enzymes involved in apoptosis are inhibited by hsp70.
Hsp70-transfected Jurkat cells permanently overexpressing hsp70 have
been shown to be more sensitive to Fas-induced apoptotic cell death,
whereas heat shock-induced hsp70 up-regulation in the wild-type cell
protects from Fas-mediated apoptosis (39)
. This contradictory reaction
pattern could be based on the different regulation of the hsp70
response, with non-HSF1-regulated constitutive overexpression of hsp70
in transfectants, but inducible HSF1-regulated overexpression in
wild-type cells. Taken together, these data suggest that an enhanced
hsp70 synthesis, mediated by appropriate activation (via HSF1),
protects from apoptotic cell death.
Hsp70-mediated protection from apoptosis may be based on interference
with several pro-apoptotic signal transduction pathways.
Stress-activated protein kinases (SAPK), for example, mediate signaling
of extracellular stress factors and enable stress-induced apoptosis.
Down-regulation of SAPK has been demonstrated in cells overexpressing
hsp70 (40)
. Only inducible but not constitutive up-regulation of hsp70
could block SAPKs, indicating a possible role of HSF transcription
factors and subsequent hsp70 up-regulation in this anti-apoptotic
mechanism (41,
42)
. In addition, hsp70 also inhibits the ICE cascade by
blocking caspase 3 (40)
, which might serve as an explanation for
decreased Fas-mediated apoptosis in the presence of hsp70.
The protective and anti-apoptotic effect of hsp70 seems to be
independent from the stress factor entailing hsp70 up-regulation.
Besides heat stress, oxidative stress leading to induced synthesis of
hsp70 has been shown to protect from apoptotic cell death (43)
. Ras
protein has also been demonstrated to increase hsp70 levels and lead to
an increased resistance to apoptosis. It remains to be clarified
whether all of these agents activate HSF-1 and whether activation of
HSF1 is pivotal for hsp70-dependent protection from apoptosis (44)
. The
present experiments demonstrated rapid blocking of the HSF1/hsp70
pathway in the course of Fas-mediated apoptosis, indicating the
important anti-apoptotic role of the HSF1/hsp70 system. In addition,
protection from Fas-mediated apoptosis by an induced hsp70 expression
was demonstrated. This latter effect of the HSF1/hsp70 system may
represent a double-edged sword, on the one hand enabling normal cells
to survive in hostile environments, such as heat, oxidative,
mechanical, and cytokine stresses, but on the other hand protecting
tumor cells and/or autoreactive cells from apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 18, 1998.
Revision received January 4, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, annexin- and
PI-negative), apoptotic ([hatched, annexin-positive, PI-negative), and
dead (
, annexin- and PI-positive) cells was performed. Note the
increased rates of apoptosis in response to heat stress during Fas
activation. Values are means of three independent experiments.
) before
addition of anti-Fas Ab (0 h). Heat stress 6 h before Fas
activation was to induce maximal expression of hsp70, heat stress 30
min before Fas activation to induce maximal HSF1-DNA binding in the
cells. Control cells left unstressed before addition of anti-Fas Ab
(
). Percentages of viable, apoptotic, and dead cells were assessed
1, 3, and 6 h after Fas activation. A) Viable
cells: annexin- and PI-negative; B) apoptotic cells:
annexin-positive, PI-negative, C) dead cells: annexin-
and PI-positive. Note the decreased apoptosis rate with an induced
hsp70 expression. Values are means of three independent experiments.