(The FASEB Journal. 1999;13:2061-2070.)
© 1999 FASEB
HSP27 inhibits cytochrome c-dependent activation of procaspase-9
CARMEN GARRIDO1,
JEAN-MARIE BRUEY,
ANNIE FROMENTIN,
ARLETTE HAMMANN,
ANDRÉ PATRICK ARRIGO* and
ERIC SOLARY
INSERM U517, Groupe Biologie et Thérapie des Cancers (JE 515), Faculty of Medicine and Pharmacy, 21033 Dijon, France; and
* Stress Laboratory, CNRS UMR-5534, Claude Bernard University, Lyon, France
1Correspondence: INSERM U517, Faculty of Medicine and Pharmacy, 7 Blvd. Jeanne D'Arc, 21033 Dijon, France. E-mail: cgarrido{at}u-bourgogne.fr
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ABSTRACT
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We have previously shown that the small heat shock protein HSP27
inhibited apoptotic pathways triggered by a variety of stimuli in
mammalian cells. The present study demonstrates that HSP27
overexpression decreases U937 human leukemic cell sensitivity to
etoposide-induced cytotoxicity by preventing apoptosis. As observed for
Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage
and procaspase-3 activation. In contrast with Bcl-2, HSP27
overexpression does not prevent etoposide-induced cytochrome
c release from the mitochondria. In a cell-free system,
addition of cytochrome c and dATP to cytosolic extracts
from untreated cells induces the proteolytic activation of procaspase-3
in both control and bcl-2-transfected U937 cells but
fails to activate procaspase-3 in HSP27-overexpressing cells.
Immunodepletion of HSP27 from cytosolic extracts increases cytochrome
c/dATP-mediated activation of procaspase-3.
Overexpression of HSP27 also prevents procaspase-9 activation. In the
cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide
cleavage activity triggered by cytochrome c/dATP
treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis
by preventing cytochrome c and dATP-triggered activity
of caspase-9, downstream of cytochrome c
release.Garrido, C., Bruey, J.-M., Fromentin, A., Hammann, A.,
Arrigo, A. P., Solary, E. HSP27 inhibits cytochrome
c-dependent activation of procaspase-9.
Key Words: apoptosis cell death etoposide drug resistance leukemia
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INTRODUCTION
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EPIPODOPHYLLOTOXINS SUCH AS etoposide (VP-16) are
topoisomerase II-reactive agents that are commonly used to treat some
human tumors (1)
. These drugs produce double-strand DNA
breaks, which are thought to be critical for their cytotoxic activity
(2)
. The ability of tumor cells to undergo apoptosis in
response to this damage is a key determinant of their sensitivity to
these drugs (3)
. A balance between proapoptotic and
antiapoptotic molecules determines the fate of damaged cells. Enzymes
from the caspase family of proteases that are sensitive to the
tetrapeptide DEVD play a central role in drug-induced cell death
(4
, 5)
, whereas the antiapoptotic protein Bcl-2 delays
etoposide-induced apoptosis without modifying the formation and repair
of DNA damage provoked by the drug (6)
. Concerning the
molecular ordering of this pathway, a picture has emerged in which
Bcl-2 family proteins regulate the release of apoptogenic molecules
such as cytochrome c, which activates cytosolic procaspases,
and apoptosis-initiating factor, which triggers nuclear changes, from
the mitochondria (7
8
9
10
11
12)
.
Among other molecules that can interfere with this cell death pathway
(13
, 14)
is the small heat shock protein HSP27. Small heat
shock proteins are overexpressed in response to environmental stresses
(15)
; they vary in size from 15 to 30 kDa and share
sequence homologies and biochemical properties such as phosphorylation
and oligomerization (16)
. These proteins may act as
molecular chaperones, regulate actin cytoskeleton organization
(17
, 18)
, and modulate redox parameters (19)
.
Their overexpression efficiently protects against cell death triggered
by a variety of stimuli including hyperthermia (20)
,
oxidative stress (21
, 22)
, and several commonly used
anticancer drugs (21
, 23
24
25)
. The small stress protein
HSP27 is expressed in both normal and neoplastic human cells. We have
demonstrated the ability of this protein to protect cells against
staurosporine-, Fas/APO-1- (14)
and cytotoxic drug-induced
apoptosis (25)
. We have also shown that HSP27 protein
contributes to cancer cell tumorigenicity (26)
.
In this work, we analyzed the mechanisms by which HSP27 modulates
etoposide-induced apoptosis in U937 human leukemic cells. We show that
HSP27 overexpression prevents the activation of procaspase-9 and the
subsequent activation of procaspase-3 and cleavage of
poly(ADP-ribose)polymerase (PARP). In contrast to Bcl-2, HSP27
functions downstream of the mitochondrial release of cytochrome
c.
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MATERIALS AND METHODS
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Cells, plasmids, transfection, and drug treatment
The human leukemic cell line, U937, was grown in suspension in
RPMI 1640 medium (BioWhittaker, Fontenay-sous-bois, France)
supplemented with 10% (v/v) fetal bovine serum and 2 mM L-glutamine in
a controlled atmosphere (37°C, 5% CO2). The
plasmids used for transfections were psvhsp27 containing the
full-length human HSP27 cDNA, psvK3 as an insertless control, and
pG-hygro containing the hygromicin-B resistance gene (27)
.
Cells were transfected by electroporation (250 V, 1,500 µF) with 25
µg of psvhsp27 or psvK3 and 2.5 µg of pG-hygro, and selected
48 h later in hygromycin B-supplemented medium (Sigma-Aldrich, St.
Quentin Fallavier, France). Expression of HSP27 in isolated clones was
analyzed by Western blot. Cell growth analyses were performed by
plating 105 cells in 35 mm tissue culture dishes
on day 0, then counting the cells every 24 h by using a
hemocytometer (four independent determinations for each time point).
Etoposide (Sigma-Aldrich) stock solution was prepared in dimethyl
sulfoxide (DMSO) and stored at -20°C for less than 1 month. The
final concentration of DMSO in the culture medium never exceeded 0.1%
(v/v), which was nontoxic to the cells.
In vivo fluorescent measurement of intracellular
ROS
The fluorescent probe hydroethidine (Molecular Probe-Interchim,
Montluçon, France), which is the
NaBH4-reduced form of ethidium bromide, was used
to measure the intracellular content of ROS in living cells
(28)
. Cells were washed twice with NaCl/Pi, pH 7.4 (PBS),
and incubated for 10 min with 40 µg/ml hydroethidine. The flow
cytometry analysis was performed by using a FACScan flow cytometer
(Becton Dickinson, Le Pont de Claix, France). Mean oxidized HE
(ethidium bromide) fluorescence indices were calculated by dividing the
mean EB fluorescence of each sample by that measured in wild-type U937
cells.
Western blot analysis
Whole cell lysates were prepared by lysing the cells in 2%
sodium dodecyl sulfate (SDS), 137 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, and
NaCl/Pi (pH 7.4) at 68°C for 5 min. Protein
concentration was measured in the supernatant by the use of the micro
BCA protein assay (Pierce, Asnieres, France). Proteins were separated
in a 812% SDS-polyacrylamide gel and electroblotted to PVDF
membranes (Bio-Rad, Ivry sur Seine, France). After blocking nonspecific
binding sites with 5% nonfat milk in NaCl/Pi, pH 7.4, 0.1% Tween 20
(TPBS), blots were incubated with specific antibodies, washed in TPBS,
incubated for 30 min at room temperature with horseradish
peroxidase-conjugated goat antimouse or anti-rabbit antibodies
(Jackson ImmunoResearch Laboratories, West Grove, Pa.), and
revealed following the ECL Western blotting analysis procedure
(Amersham, Les Ullis, France). All the Western analyses were repeated
three times. Antibodies used were the mouse monoclonal anti-human
HSP27, HSP70, HSP90 (StressGen, Victoria, Canada), the rabbit
polyclonal anti-human caspase-9, the mouse monoclonal anti-human
cytochrome c, caspase-2, caspase-6, caspase-8
(PharMingen, San Diego, Calif.), the rabbit polyclonal anti human
PARP (Boehringer Mannheim SA, Meylan, France), and procaspase-3 (kindly
provided by Donald W. Nicholson, Merck-Frost Co, Toronto, Canada).
Drug cytotoxicity assay
U937 cells (104) were seeded in 96-well
microculture plates for 24 h, then treated with increasing
concentrations of etoposide. The number of surviving cells was measured
after 96 h of drug exposure by the use of an MTT assay as
described previously (29)
.
Identification of apoptosis
Exposure of phosphatidylserine on the outer membrane leaflet was
determined by the use of the annexin V-FITC kit (Bioproducts,
Boehringer Ingelheim, Heidelberg). The percentages of apoptotic cells
were calculated using a FACScan flow cytometer (Becton Dickinson).
Identification of apoptotic cells was also performed by chromatin
staining with 5 µg/ml Hoechst 33342 for 30 min at 37°C. Cells with
condensed chromatin were counted by using a Leitz microscope equipped
with an epi-illuminator and appropriate filters (Leica, Bron, France).
The percentages of apoptotic cells were determined from 300 cells
counted in triplicate.
In vivo measurement of mitochondrial
membrane potential
The cationic lipophilic fluorochrome 3,3'
dihexyloxacarbocyanine (DiOC6, Sigma Chemical
Co., St. Louis, Mo.) was used to measure the

m. Etoposide treated and untreated cells
were washed in PBS and incubated at 37°C for 30 min with 0.1 µM
DiOC6. As a positive control, cells were exposed
to 100 µM carbonyl cyanide m-chlorophenylhydrazone (mCICCP, Sigma
Chemical Co.). Results were recorded in FL1.
Cell fractionation
Nuclei-free, mitochondria-free cytosolic extracts were
prepared as described (30)
. Briefly, cells were washed in
ice-cold PBS, pH 7.2, then in hypotonic extraction buffer (HEB: 50 mM
PIPES, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2,
1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride), and
centrifuged. The pellet was resuspended in HEB, transferred to a 2 ml
Dounce homogenizer and lysed. This cell lysate was centrifuged for 30
min at 16,000 x g at 4° and the clarified
supernatant was either tested immediately or stored in aliquots at
-80°C. In some experiments, HSP27 from U937 cell-free extracts was
immunoabsorbed using 0.1 µg/ml anti-human HSP27 mAb (MedGene Science
S.A., Pantin, France) and protein A-Sepharose (Pharmacia Biotech,
Uppsala, Sweden). Mitochondrial and cytosolic (S100) fractions for
cytochrome c release studies were prepared by resuspending
the cells in ice-cold buffer A [250 mM sucrose, 20 mM HEPES, 10 mM
KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT, 17 µg/ml PMSF, 8 µg/ml aprotinin, 2 µg/ml leupeptin (pH
7.4)] before passing them through an ice-cold cylinder cell
homogenizer. Unlysed cells and nuclei were pelleted via a 10 min,
750 x g spin. The supernatant was spun at 10,000 x g for 25 min. This pellet was resuspended in buffer A and
contained the mitochondrial fraction. The supernatant was spun at
100,000 x g for 1 h. The supernatant from this
final centrifugation contained the cytosolic S100 fraction.
Measurement of caspase activity
Caspase-3 activity was assessed by cleavage of the colorimetric
substrate DEVD-para-nitroaniline (DEVD-pNA) by using the ApoAlert CPP32
Assay Kit (Clontech Laboratories Inc., Palo Alto, Calif.). One
arbitrary unit of caspase-3 activity is defined as the amount of
caspase-3 required to produce 1 pmol of pNA per minute at 25°C at
saturating substrate concentration. For caspase-9 activity, acellular
extracts were incubated with 20 µM LEHD-AFC (Calbiochem, San Diego,
Calif.) in a caspase assay buffer (100 mM HEPES, pH 7.4, 10% glycerol,
0.5 mM EDTA, 0.05% bovine serum albumin, and 1 mM dithiothreitol) for
1 h at 37°C. AFC released from the substrates were excited at
400 nm. Emission was measured at 505 nm.
 |
RESULTS
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Overexpression of HSP27 induces resistance of U937 cells to VP-16
The U937 leukemic cell line was transfected with an insertless
vector or an HSP27 containing vector. The control vector did not modify
HSP27 expression as compared with the parental U937 cells (not shown).
One control-transfected clone and two HSP27 overexpressing clones were
selected for further analysis. HSP27 expression was about fivefold
higher in the U93727-4 and U93727-7 clones than in the
control-transfected clone (Fig. 1
A). Stable transfection of hsp27 did not affect
the expression of other major heat shock proteins such as HSP70 or
HSP90 (Fig. 1A
) nor did it affect cell proliferation. The
doubling time of control-transfected and HSP27-transfected clones was
~36 h. Overexpression of a functional HSP27 was confirmed by the
1.6-fold decrease in the basal cellular content of radical oxygen
species (ROS) in hsp27-transfected compared to
control-transfected cells (19
, 25
, 28)
(Fig. 1B
). Cytotoxic and clonogenic survival assays have shown, in
different cellular models, that HSP27 increases cell resistance to
anticancer drugs (21
, 23
24
25
, 31)
. By using a 96 h
MTT assay, we consistently observed that overexpression of HSP27
protein decreased U937 cell sensitivity to etoposide-induced
cytotoxicity. Etoposide IC50 was ~0.69 µM in
the two hsp27-transfected clones as compared with 0.19 µM
in control transfected cells (Fig. 1C
). Since results
obtained with the two selected HSP27-transfected clones (U93727-7 and
U93727-4) were similar, results shown from subsequent experiments
will be those obtained in the U93727-4 clone.
HSP27 overexpression decreases etoposide-induced apoptosis in U937
cells
To determine the effect of HSP27 overexpression on apoptosis
induction by etoposide treatment, hsp27- and
control-transfected U937 cells were exposed to various concentrations
of etoposide for 4 h. The number of apoptotic cells was assessed
by counting apoptotic cells after Hoechst 33342 staining of the
condensed nuclear chromatin (Fig. 2
A). The number of apoptotic cells measured was significantly
reduced in hsp27-transfected compared with
control-transfected cells. For example, at a dose of 50 µM etoposide,
overexpression of HSP27 caused a two- to threefold decrease in the
number of apoptotic cells compared with that of control transfectants
(Fig. 2A
). These results were confirmed by monitoring the
aberrant exposure of phosphatidylserine on the outer membrane leaflet
as identified by the calcium-dependent fixation of FITC-labeled annexin
V (32)
(data not shown). Hoechst 33342 staining was also
used to assess apoptosis in hsp27-transfected U937 cells
exposed for various times to 50 µM etoposide (Fig. 2B
). In
this experiment, we tested also bcl-2-transfected U937 cells
to compare HSP27-mediated to Bcl-2-mediated antiapoptotic effects.
After a 12 h exposure to the drug, more than 80% of the
control-transfected cells were apoptotic. At this time point, the
number of hsp27- and bcl-2-transfected cells that
were apoptotic was ~40% and 25%, respectively. After 48 h of
drug exposure, ~20% of HSP27 overexpressing cells remained
nonapoptotic whereas all control cells were dead at 24 h After a
4-day drug exposure, virtually all the cells from control-,
hsp27-, and bcl-2-transfected U937 cells had
undergone apoptosis. We concluded that HSP27, like Bcl-2, delayed
apoptosis induced by continuous exposure to 50 µM etoposide in U937
cells.

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Figure 2. Effect of HSP27 on etoposide-induced apoptosis in U937
cells.A) Control-transfected (control, black bars) and
hsp27-transfected U937 (clone U93727-4) (HSP27, open
bars) cells were treated for 4 h with increasing concentrations of
etoposide (VP-16), fixed, and stained with Hoechst 33342 for chromatin
labeling. Percentages of apoptotic cells were determined from 300 cells
counted in triplicate. SDS are indicated
(n=3).B) Kinetics of apoptosis induction
by etoposide in U937 cells over 96 h. Control (black bars),
hsp27- (open bars), and bcl-2-transfected
U937 cells (shaded bars) were treated with 50 µM etoposide. At the
times indicated, cells were fixed, and stained with Hoechst 33342 for
chromatin labeling. The percentage of apoptotic cells is graphed.
SDS are indicated (n=3).
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To study the mechanism of action of HSP27 in a more physiological model
system, we tested heat-shocked U937 cells. Cells were studied after a
1 h heat shock at 42°C, followed by a 15 h incubation at
37°C. In these conditions, other main HSPs such as HSP70 and HSP90,
which had been induced early by heat shock, had returned to their basal
level whereas induction of HSP27 expression was maximal (Fig. 3A
). Apoptosis induced by a 4 h exposure to etoposide and
measured by Hoechst 33342 staining was similarly reduced in both
hsp27-transfected and heat-shocked cells compared with
control-transfected cells (Fig. 3B
).

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Figure 3. Heat-shocked U937 cells are resistant to etoposide-induced apoptosis.
A) Overexpression of HSP27 in U937 cells exposed for
1 h at 42°C, then incubated at 37°C for the indicated times.
Heat shock proteins were detected by immunoblotting with antibodies
against HSP27, HSP70, and HSP90. B) Heat-shocked cells
(1 h at 42°C and then 15 h at 37°C) (HS, shaded bars),
hsp27-transfected cells (HSP27, open bars), and U937
control cells (Co, black bars) were treated for 4 h with etoposide
(25 µM or 50 µM), fixed, and stained with Hoechst 33342 for
chromatin labeling. Percentages of apoptotic cells were determined from
300 cells counted in triplicate. SDS are indicated
(n=3).
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HSP27 functions upstream of the activation of procaspase-2L, -3,
-8, and -9.
We previously described the activation of procaspase-3 in
etoposide-treated U937 cells, as demonstrated by the appearance of p19
and p17 fragments, and the cleavage of its 116 kDa protein target PARP
into a 85 kDa fragment (5)
. These events were confirmed to
occur in control-transfected U937 cells treated for 4 h with
either 25 µM or 50 µM etoposide, whereas they were either
undetectable or significantly decreased in HSP27-overexpressing U937
cells treated under similar conditions (Fig. 4
).

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Figure 4. HSP27 overexpression prevents the activation of procaspase-3 and the
cleavage of poly(ADP-ribose)polymerase (PARP) induced by etoposide
treatment. Whole cell lysates from U937 control-transfected (control)
and hsp27-transfected cells (HSP27), either untreated
(0) or treated with etoposide (25 µM or 50 µM) for 4 h, were
subjected to Western blot analysis of procaspase-3 (Procasp.-3), its
p19 and p17 cleavage products (Casp.-3-p19 and Casp.-3-p17), and PARP
(PARP-p116) cleavage product p85 (PARP-p85). Locations of specific
products are indicated by arrowheads. As a control for protein loading,
the blot was probed with an anti-human HSP70 antibody. One
representative of three immunoblots is shown.
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To better determine the mechanism by which HSP27 interferes with
procaspase activation, we assessed the effect of etoposide on several
other procaspases in control and hsp27-transfected cells.
For this purpose, we analyzed by immunoblotting the expression of
procaspase-8, procaspase-9, and the long isoform of procaspase-2
(procaspase-2L) in hsp27- and control-transfected U937 cells
exposed to 50 µM etoposide for 4 h (Fig. 5
). In control-transfected cells, this treatment decreased the content of
the three procaspases studied, suggesting their cleavage into active
subunits. Procaspase-9 proteolysis was confirmed by the appearance of a
37 kDa fragment in these cells. Overexpression of HSP27 inhibited
procaspase-8 and procaspase-2L decrease and prevented procaspase-9
cleavage (Fig. 5)
. Altogether, these observations indicated that HSP27
interfered with the activation of at least four procaspases:
procaspase-2L, -3, -8, and 9.

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Figure 5. Immunoblot blot analysis of procaspase-8 (procasp.8), procaspase-9
(procasp.-9), and the long isoform of procaspase-2L (procasp. 2) in
U937 cells transfected with either an empty vector (control) or an
HSP27-containing vector (HSP27; clone U93727-4), either untreated (0)
or treated with 50 µM etoposide for 4 h (50). The active
fragment of caspase-9 is indicated as Casp.-9-p37. An anti-human HSP70
mAb was used as a control for protein loading. One representative of
three immunoblots is shown.
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HSP27 overexpression does not prevent cytochrome c
redistribution
Release of cytochrome c from the mitochondrial
intermembrane space has been proposed as an early central event in the
activation of procaspases by a variety of apoptotic stimuli (10
, 33
, 34)
. This cytochrome c redistribution was shown
to be prevented by the antiapoptotic protein Bcl-2 (34
, 35)
. Accordingly, in etoposide-treated U937 cells, Bcl-2
overexpression completely inhibited cytochrome c
redistribution from mitochondria to the cytosol (Fig. 6
A). By contrast, nearly all of the cytochrome c
disappeared from the mitochondrial fraction and partitioned with the
cytosolic (S100) fraction in both control- and
hsp27-transfected cells treated in the same conditions (Fig. 6A
). These results suggested that HSP27 inhibited the
activation of some procaspases downstream of the mitochondrial release
of cytochrome c. To confirm these results, we followed
cytochrome c release from the mitochondria in
etoposide-treated cells by confocal microscopy. In untreated cells,
cytochrome c demonstrated a confined localization (Fig. 6C
) similar to that observed with cytochrome oxidase (COX),
another mitochondrial marker (Fig. 6B
). After etoposide
treatment, HSP27-transfected cells demonstrated a diffuse pattern of
cytochrome c staining (Fig. 6C
), similar to that
observed when HSP27 was used as a cytosolic marker in control U937
cells (Fig. 6B
). By contrast, exposure of Bcl-2-transfected
cells to etoposide did not modify cytochrome c staining
(Fig. 6C
). Altogether, these results indicate that HSP27
interfered with etoposide-induced cell death downstream of the
mitochondrial release of cytochrome c.

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Figure 6. HSP27 overexpression does not prevent the redistribution of cytochrome
c in U937 cells undergoing apoptosis. A)
Control-transfected, (control) hsp27-transfected
(HSP27), and bcl-2-transfected (Bcl-2) U937 cells were
either left untreated (0) or treated with 50 µM etoposide (50). After
4 h, the cells were mechanically lysed and the mitochondrial and
cytosolic (S100) fractions were analyzed by Western blot with an
anti-human cytochrome c antibody. One representative of
three immunoblots is shown. B) Confocal microscopy
analysis of U937 cells labeled with an anti-cytochrome oxidase (COX Ab)
and anti-human HSP27 (HSP27 Ab) antibodies as controls for
mitochondrial and cytoplasmic localizations, respectively.
C) Confocal microscopy analysis of cytochrome
c distribution in untreated and etoposide-treated
(VP-16, 50 µM for 4 h) U937 cells stably transfected with
hsp27 (U93727-4) or bcl-2 (U937/Bcl-2).
One representative of four independent experiments is shown.
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HSP27 overexpression inhibits cytochrome c-induced
activation of procaspase-3
With the use of a cell-free system (30
, 33)
, it was
shown that in the presence of dATP, cytochrome c could
trigger the processing of procaspase-3 to active fragments in an
untreated cell extract. This system was used to test the effect of
HSP27 overexpression on cytochrome c-mediated activation of
procaspase-3 in U937 cells. Horse heart cytochrome c and
dATP (cytochrome c/dATP) were added to nuclei- and
mitochondria-free cytosolic extracts (30)
from either
hsp27-, bcl-2-, or control-transfected U937
cells. After a 2 h incubation, procaspase-3 processing was
analyzed by Western blot (Fig. 7
A) and its activity was determined spectrophotometrically
(Fig. 7B
). Cytochrome c/dATP induced procaspase-3
processing and caspase activation in cell-free extracts from control-
and bcl-2-transfected U937 cells, but not in cell-free
extracts from hsp27-transfected cells. To confirm that HSP27
was responsible for this effect, control U937 cell extracts were
immunodepleted of HSP27. Immunodepletion resulted in a significant
decrease in HSP27 content (Fig. 8
A) and an increase in cytochrome c/dATP-induced
caspase-3 activity (Fig. 8B
).

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Figure 7. Cytochrome c and dATP fail to activate procaspase-3 in
HSP27-overexpressing U937 cell-free extracts. A) Equal
amounts of cell-free extracts (500 µg of total protein) from U937
cells transfected with either an insertless (control), a
hsp27-containing (HSP27), or a bcl-2
(Bcl-2)-containing vector were untreated (0) or treated with 10 µM
cytochrome c and 1 mM dATP for 2 h at 37°C
(Cyt.c), then analyzed by Western blot. Locations of procaspase-3
(Procasp.-3) and its cleavage products (p19 and p17) are indicated by
arrowheads. As a control for protein loading, the blot was probed with
an anti-human HSP70 antibody. One representative experiment is shown
(n=3). B) Caspase-3-like activity was
measured in cell-free extracts from the indicated clones by hydrolysis
of the DEVD-pNA substrate. These extracts had been either left
untreated (0) or were treated with cytochrome c/dATP
(Cyt.c), as above. Arbitrary units of protease activity were calculated
as indicated in Materials and Methods. SDS are indicated
(n=3).
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Figure 8. Immunodepletion of HSP27 from U937 cell-free extracts increases
cytochrome c/dATP-dependent caspase-3 activity.
A) Immunoblot analysis of HSP27 and HSP70 protein levels
in U937 cell-free extracts immunodepleted with a control IgG (Co) or an
antihuman HSP27 antibody (HSP27 Ab). B) Cell-free
extracts shown in A (500 µg of total protein) were either left
untreated or treated with 10 µM cytochrome c and 1 mM
dATP (Cyt/dATP) for 2 h at 37°C. Arbitrary units of protease
activity were calculated as indicated in Materials and Methods.
SDS are indicated (n=3).
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HSP27 overexpression inhibits cytochrome c-induced
caspase-9 activity in cytochrome c/dATP-treated
cell-free extracts
According to the current picture of the apoptotic pathway
triggered by cytochrome c release from the mitochondria,
caspase-9 activity is responsible for procaspase-3 activation by
proteolytic cleavage. To determine whether caspase-9 activity was
inhibited by HSP27 overexpression, we performed in vitro
experiments using LEHD-AFC as a peptide substrate. LEHD-AFC substrate
can be cleaved by caspase-4, -5, and -9 (36)
. It has been
shown recently that procaspase-4 and -5 failed to be activated by
cytochrome c/dATP in cell-free extracts (37)
.
Therefore, measurement of LEHD-AFC cleavage in such an in
vitro system actually measured caspase-9 activity. We observed
that in contrast to Bcl-2, HSP27 prevented LEHD cleavage induced by
cytochrome c and dATP in this cell-free-system (Fig. 9
A). Immunodepletion of HSP27 from
hsp27-transfected cell extracts partially restored the
ability of cytochrome c/dATP to cleave LEHD-AFC substrate
(Fig. 9B
).

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Figure 9. HSP27 inhibits cytochrome c and dATP induced caspase-9
activity in HSP27-overexpressing U937 cell-free extracts.
A) Equal amounts of cell-free extracts (500 µg of
total protein) from U937 cells transfected with either an insertless
(control), a hsp27-containing (HSP27), or a
bcl-2-containing (Bcl-2) vector were untreated or
treated with 10 µM cytochrome c and 1 mM dATP for
2 h at 37°C. Then caspase-9 activity was analyzed in cell
lysates by measuring hydrolysis of the LEHD-AFC-specific peptide
substrate. Results are expressed as percentage of the activity measured
in control extracts. SDS are indicated
(n=3). B) Immunodepletion of HSP27
from U937 cell-free extracts increases cytochrome
c/dATP-dependent caspase-9 activity. Cell-free extracts
from HSP27-transfected cells were immunodepleted with a control
IgG [IgG(Co)] or an antihuman HSP27 antibody (HSP27Ab), then treated
with 10 µM cytochrome c and 1 mM dATP for 2 h at
37°C. Caspase-9 activity was analyzed by measuring hydrolysis of
LEHD-AFC substrate. Results are expressed as percentage of the activity
measured in control extracts. SDS are indicated
(n=3).
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DISCUSSION
|
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Small stress proteins are part of the cellular mechanism that
protects cells from the deleterious effects induced by heat or
oxidative injuries (20
21
22)
. Among these proteins, HSP27
is an endogenous pleiotropic inhibitor of apoptotic cell death. We have
previously shown HSP27's ability to block Fas/APO-1-, tumor necrosis
factor
-, and staurosporine-mediated cell death in murine L929
fibroblasts (14)
and prevent cisplatin-induced apoptosis
in human colorectal cancer cells (25)
. In the present
study, HSP27 overexpression is shown to prevent etoposide-induced
apoptosis and cytotoxicity by inhibiting cytochrome c and
dATP-triggered caspase-9 activity in the cytosol of U937 leukemic
cells.
The cleavage of intracellular proteins such as
poly(ADP-ribose)polymerase, which is associated with apoptosis, is
mediated by cysteine proteases from the caspase family
(38)
. We have previously demonstrated the central role of
a DEVD-sensitive caspase in the proteolytic cascade that leads to PARP
cleavage in etoposide-treated U937 cells (4
, 5)
. The main
caspase that is sensitive to the tetrapeptide DEVD is caspase-3
(39)
. This cytosolic protein normally exists as a 32 kDa
inactive precursor that is cleaved proteolytically at aspartic residues
in cells undergoing cell death to generate an active heterodimer of 17
kDa and 12 kDa subunits. The present study shows that HSP27
overexpression prevents the processing/activation of procaspase-3 and
the subsequent cleavage of PARP in etoposide-treated U937 cells. In
accordance with a recently described caspase cascade in which caspase-3
activates several downstream procaspases (37)
, HSP27
overexpression also prevented the activation of procaspase-2L and
procaspase-8.
Several of the biochemical perturbations common to most apoptotic
pathways result from alterations in mitochondrial functions, including
the release of cytochrome c from intermembrane space
(10
, 33
, 34
, 40
41
42
43)
. The antiapoptotic proteins Bcl-2 and
Bcl-XL have been shown to prevent mitochondrial
physiology disruption and to block cytochrome c release from
the mitochondria, whereas the proapoptotic protein Bax causes death by
direct mitochondrial effects (35
, 44
, 45)
. Here we show
that in contrast to Bcl-2 and Bcl-2-related proteins, HSP27
overexpression does not influence the mitochondrial release of
cytochrome c to the cytosol. Moreover, HSP27 overexpression
does not prevent the early fall of the mitochondrial transmembrane
potential (
m), as measured by the use of a
cationic lipophilic fluorochrome (data not shown). According to the
current picture of the common final pathway of apoptosis, these results
suggest that HSP27 overexpression interferes with etoposide-induced
death pathway between the mitochondrial release of cytochrome
c and the activation of procaspase-3.
In the presence of cytochrome c and ATP or dATP,
procaspase-9 combines with Apaf-1 to form a so-called `apoptosome'
(46)
. In this complex, procaspase-9 is processed into an
active caspase that, in turn, cleaves downstream caspases such as
procaspase-3. In a cell-free system, procaspase-3 can be activated by
the addition of cytochrome c and dATP to cytosol from
normally growing cells (30
, 33)
. Here we show that whereas
Bcl-2 overexpression fails to prevent procaspase-3 and procaspase-9
activation in this cell-free system, HSP27 overexpression blocks their
processing into an active enzyme. Addition of commercially available
recombinant HSP27 (rHSP27) to cell-free extracts failed to inhibit
procaspase-3 activation induced by cytochrome c and dATP
(data not shown). As the active forms of HSP27 with regard to
inhibition of cell death are large nonphosphorylated oligomers
(47
, 48)
, these negative results might be related to the
inability of rHSP27 to form active oligomers. We used an anti-human
HSP27 mAb and protein A-Sepharose to immunodeplete cell-free extracts
of untreated cells from HSP27 protein. These experiments confirmed that
HSP27 was responsible for the inhibition of cytochrome
c/dATP-mediated procaspase-3 activation and caspase-9
activity. In a search for HSP27 interactions with any component of the
apoptosome, we performed immunoprecipitation experiments. Although
these studies failed to identify a strong interaction between the
indicated proteins (data not shown), we cannot rule out the role of
weaker or indirect interactions.
The differential effect of Bcl-2 and HSP27 on the activation of
procaspases could be related to their subcellular distribution. Bcl-2
is localized mainly in the outer mitochondrial, outer nuclear, and
endoplasmic reticular membranes as a result of a carboxy-terminal
membrane anchor (43)
whereas small stress proteins are
dispersed in the cytosol (16)
. As the presence of
cytochrome c is required for the binding of Apaf-1 to
procaspase-9, Bcl-2 must prevent the formation of the apoptosome and
the activation of downstream caspases by inhibiting the mitochondrial
release of cytochrome c. HSP27 overexpression does not
prevent cytochrome c release from the mitochondria, but
prevents the activation of procaspase-9 in etoposide treated cells and
caspase-9 activity generated by cytochrome c and dATP in a
cell-free system. Thus, HSP27 inhibits etoposide-induced U937 cell
death between cytochrome c release from the mitochondrial
intermembrane space and the activation of procaspase-9 in the
apoptosome (Fig. 10
). It remains to be determined whether HSP27 could interact with either
cytochrome c, procaspase-9, or other molecules that
contribute to the formation of the apoptosome, such as APAF-1. Besides
its role in the formation of apoptosome, cytochrome c was
proposed to contribute to cell death by promoting free radical
production (12)
whereas small heat shock proteins were
shown to protect against oxidative stress (19)
. It can be
speculated that the antioxidative effect of HSP27 could also contribute
to the decreased activation of procaspase-9 by cytochrome
c/ATP. Whatever its mechanism, the ability of HSP27 to
prevent procaspase-9 activation might account for the role of HSP27 in
drug resistance (21
, 23
24
25)
and its poor prognostic value
in some human tumors (49
50
51)
.

View larger version (18K):
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|
Figure 10. Proposed model for the differential mechanism of inhibition of
etoposide-induced apoptosis by Bcl-2 and HSP27 in U937 human leukemic
cells. The cytotoxic drug induces cleavable complexes that are
converted into a death signal. This signal induces the release of
cytochrome c from the mitochondria. Overexpression of
Bcl-2 prevents the release of cytochrome c from the
mitochondria whereas HSP27 inhibits the activation of procaspase-9 by
cytochrome c released in the cytosol, thereby inhibiting
the activation of downstream procaspases such as procaspase-3.
|
|
 |
ACKNOWLEDGMENTS
|
|---|
We thank J. Bréard and C. Renvoizé for providing the
Bcl-2-transfected U937 cells, D. W. Nicholson for providing the
CPP32 antibody, and K. Nason-Burchenal, F. Martin, and A. Bettaieb for
helpful advice. This work was supported by grants from the Burgundy,
Saône et Loire, Nièvre and Yonne Comittees of the Ligue
Nationale Contre le Cancer, the Association pour la Recherche contre le
Cancer (ARC# 4075), and the Conseil Régional de Bourgogne.
 |
FOOTNOTES
|
|---|
Received for publication February 18, 1999. Revised for publication May 5, 1999.
 |
REFERENCES
|
|---|
-
Champlin, R., Gale, R. P. (1987) Acute myelogenous leukemia: recent advances in therapy. Blood 7,1551-1562
-
Liu, L. F. (1989) DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 58,351-375[Medline]
-
Dubrez, L., Goldwasser, F., Genne, P., Pommier, Y., Solary, E. (1995) The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemic cells to topoisomerase I and II inhibitors. Leukemia 9,1013-1024[Medline]
-
Droin, N., Dubrez, L., Eymin, B., Renvoize, C., Breard, J., Dimanche-Boitrel, M. T., Solary, E. (1998) Upregulation of CASP genes in human tumor cells undergoing etoposide-induced apoptosis. Oncogene 16,2885-2894[Medline]
-
Dubrez, L., Savoy, I., Hamman, A., Solary, E. (1996) Pivotal role of a DEVD-sensitive step in etoposide-induced and Fas-mediated apoptotic pathways. EMBO J 15,5504-5514[Medline]
-
Kamesaki, S., Kamesaki, H., Jorgensen, T. J., Tanizawa, A., Pommier, Y., Cossman, J. (1993) Bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair. Cancer Res 53,4251-4256[Abstract/Free Full Text]
-
Chinnaiyan, A. M., O'Rourke, K., Lane, B. R., Dixit, V. M. (1997) Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science 275,1122-1129[Abstract/Free Full Text]
-
Zamzami, N., Susin, S. A., Marchetti, P., Hirsch, T., Gomez-Monterey, L., Castedo, M., Kroemer, G. (1996) Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183,1533-1544[Abstract/Free Full Text]
-
Marchetti, P., Susin, S. A., Decaudin, D., Gamen, S., Castedo, M., Hirsch, T., Zamzami, N., Naval, J., Senik, A., Kroemer, G. (1996) Apoptosis associated derangement of mitochondrial function in cells lacking mitochondrial DNA. Cancer Res 56,2033-2038[Abstract/Free Full Text]
-
Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., Wang, X. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria is blocked. Science 275,1129-1132[Abstract/Free Full Text]
-
Li, P., Nijhaawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., Wang, X. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489[Medline]
-
Reed, J. C. (1997) Cytochrome c: can't live with itcan't live without it. Cell 91,559-562[Medline]
-
Dubrez, L., Eymin, B., Sordet, O., Droin, N., Turhan, A., Solary, E. (1998) BCR-ABL delays etoposide-induced apoptosis upstream of procaspase-3 activation. Blood 91,2215-2422
-
Mehlen, P., Schulze-Osthoff, K., Arrigo, A. P. (1996) Small stress proteins as novel regulators of apoptosis. J. Biol. Chem. 271,16510-16514[Abstract/Free Full Text]
-
Parsell, D. A., Lindquist, S. (1990) Heat shock proteins and stress tolerance. Morimoto, R. I. Tissières, A. Georgopoulos, C. eds. The Biology of Heat Shock Proteins and Molecular Chaperones ,457-494 Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York.
-
Arrigo, A. P., Landry, J. (1994) Expression and function of the low molecular weight heat shock proteins. Morimoto, R. I. Tissières, A. Georgopoulos, C. eds. Heat Shock Proteins: Structure, Function and Regulation ,335-73 Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York.
-
Ehrnsperger, M., Grabel, S., Gaestel, M., Buchner, J. (1997) Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J 16,221-229[Medline]
-
Hout, J., Houle, F., Spitz, D. R., Landry, J. (1996) HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 56,273-279[Abstract/Free Full Text]
-
Mehlen, P., Kretz-Remy, C., Preville, X., Arrigo, A. P. (1996) Human hsp27, Drosophila hsp27 and
B-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNF
-induced cell death. EMBO J 15,2695-2706[Medline]
-
Landry, J., Chrétien, P., Lambert, H., Hickey, E., Weber, L. A. (1989) Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J. Cell Biol. 109,7-15[Abstract/Free Full Text]
-
Huot, J., Roy, G., Lambert, H., Chrétien, P., Landry, J. (1991) Increased survival after treatments with anticancer agents of Chinese hamster cells expressing the human Mr 27,000 heat shock protein. Cancer Res 51,5245-5252[Abstract/Free Full Text]
-
Mehlen, P., Briolay, J., Smith, L., Diaz-Latoud, C., Fabre, N., Pauli, D., Arrigo, A. P. (1993) Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat shock protein. Eur. J. Biochem. 215,277-284[Medline]
-
Oesterreich, S., Weng, C. N., Qiu, M., Hilsenbeck, S. G., Osborne, C. K., Fuqua, S. W. (1993) The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines. Cancer Res 53,4443-4448[Abstract/Free Full Text]
-
Garrido, C., Mehlen, P., Fromentin, A., Hamman, A., Assem, M., Arrigo, A. P., Chauffert, B. (1996) Inconstant association between hsp27 content and doxorubicin resistance in human colon cancer cells. Eur. J. Biochem. 237,653-659[Medline]
-
Garrido, C., Ottavi, P., Fromentin, A., Hammann, A., Arrigo, A. P., Chauffert, B., Mehlen, P. (1997) Hsp27 as a mediator of confluence-dependent resistance to cell death induce by anticancer drugs. Cancer Res 57,2661-2667[Abstract/Free Full Text]
-
Garrido, C., Fromentin, A., Bonnotte, B., Favre, N., Moutet, M., Arrigo, A. P., Mehlen, P., Solary, E. (1998) Heat shock protein 27 enhances the tumorigenicity of immunogenic rat colon carcinoma cell clones. Cancer Res 58,5495-5499[Abstract/Free Full Text]
-
Mehlen, P., Preville, X., Chareyron, P., Briolay, J., Klemenz, R., Arrigo, A. P. (1995) Constitutive expression of human hsp27, Drosophila hsp27, or human
B crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J. Immunol 215,363-374
-
Mehlen, P., Kretz-Remy, C., Briolay, J., Fostan, P., Mirault, M. E., Arrigo, A. P. (1995) Intracellular reactive oxygen species as apparent modulators of heat shock protein 27 (hsp27) structural organization and phosphorylation in basal and tumor necrosis factor
-treated T47D human carcinoma cells. Biochem. J. 312,367-375
-
Solary, E., Bidan, J. M., Chauffert, B., Caillot, D., Mugneret, F., D'Athis, P., Gauville, C., Carli, P. M., Guy, H. (1991) P-glycoprotein expression and in vitro reversion of doxorubicin resistance by verapamil in clinical specimens from acute leukemia and myeloma. Leukemia 5,592-597[Medline]
-
Ellerby, H. M., Martin, S. J., Ellerby, L. M., Naiem, S. S., Rabbizadeh, S., Salvesen, G. S., Casiano, C. A., Cashman, N. R., Green, D. R., Bredesen, D. E. (1998) Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial and postmitochondrial phases. J. Neurosci. 17,6165-6178[Abstract/Free Full Text]
-
Hout, J., Roy, G., Lambert, H., Landry, J. (1992) Co-induction of HSP27 phosphorylation and drug resistance in Chinese hamster cells. Int. J. Oncol. 1,31-36
-
Koopman, G., Reutelingsperger, C. P. M., Kuijten, G. A. M., Keehnen, R. M. J., Pals, S. T., Van Oers, M. H. J. (1994) Annexin V for flow cytometry detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84,1415-1421[Abstract/Free Full Text]
-
Liu, X., Kim, C. N., Yang, J., Jemmerson, R., Wang, X. (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86,147-157[Medline]
-
Kluck, R. M., Bossy-Wetzel, E., Green, D. R., Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275,1132-1136[Abstract/Free Full Text]
-
Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumaker, P. T., Thompson, C. B. (1997) Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91,627-637[Medline]
-
Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., Nicholson, D. W. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B; Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272,17907-17911[Abstract/Free Full Text]
-
Slee, E. A., Harte, M. T., Kluck, R. M., Wolf, B. B., Casiano, C. A., Newmeyer, D. D., Wang, H-G, Reed, J. C., Nicholson, D. W., Alnemri, E. S., Green, D. R., Martin, S. J. (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of capsases-2, -3, -6, -7, -8 and -10 in a caspase-9-dependent manner. J. Cell Biol. 144,281-292[Abstract/Free Full Text]
-
Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., Yuan, J. (1993) Induction of apoptosis in fibroblasts by IL-1ß-converting enzyme, a mammalian homolog of the C. elegans cell death gene. Cell 75,653-660[Medline]
-
Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Laabelle, M., Lazebnik, Y. A., Mundaay, N. A., Raju, S. M., Smulson, M. E., Yamin, T., Yu, V. L., Miller, D. K. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature (London) 376,37-43[Medline]
-
Susin, S. A., Zamzami, N., Castedo, M., Hirsch, , Marchetti, P., Macho, A., Daugas, E., Geuskens, M., Kroemer, G. (1996) Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184,1331-1341[Abstract/Free Full Text]
-
Kharbanda, S., Panddey, P., Schofield, L., Israels, S., Roncinske, R., Yoshidda, K., Bharti, A., Yuan, Z. M., Saxena, S., Weichselbaum, R. (1997) Role for Bcl-XL as an inhibitor of cytosolic cytochrome c accumulation in DNA damage-induced apoptosis. Proc. Natl. Acad. Sci. USA 94,6939-6942[Abstract/Free Full Text]
-
Kim, C. N., Wang, X., Huang, Y., Ibrado, A. M., Liu, L., Fang, G., Bhalla, K. (1997) Overexpression of Bcl-XL inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis. Cancer Res 57,3115-3120[Abstract/Free Full Text]
-
Zhivotovsky, B., Orrenius, S., Brustugun, O. T., Doskeland, S. O. (1998) Injected cytochrome c induces apoptosis. Nature (London) 391,449-450[Medline]
-
Reed, J. C. (1997) Double identity for proteins of the Bcl-2 family. Nature (London) 387,773-776[Medline]
-
Rossé, T., Olivier, R., Monney, L., Rager, M., Connus, S., Fellay, I., Jansen, B., Borner, C. (1998) Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature (London) 391,496-499[Medline]
-
Green, D. R. (1998) Apoptotic pathways: the roads to ruin. Cell 94,695[Medline]
-
Mehlen, P., Hickey, E., Weber, L. A., Arrigo, A. P. (1997) Large unphosphorylated aggregates as the active form of hsp27 which controls intracellular reactive oxygen species and glutathione levels and generates a protection against TNF
in NIH-3T3-ras cells. Biochem. Biophys. Res. Commun. 241,187-192[Medline]
-
Preville, X., Schultz, H., Knauf, U., Gaestel, M., Arrigo, A. P. (1998) Analysis of the role of hsp25 phosphorylation reveals the importance of the oligomerization state of this small heat shock protein in its protective function against TNFalpha- and hydrogen peroxide-induced cell death. J. Biol. Chem. 69,436-452
-
Harrison, J. D., Jones, J. A., Ellis, I. O. (1991) Oestrogen receptor D5 antibody is an independent negative prognostic factor in gastric cancer. Br. J. Surg. 78,334-336[Medline]
-
Thor, A., Benz, C., Moore, D., Goldman, E., Edgerton, S., Landry, J., Schwartz, L., Mayall, B., Hickey, E., Weber, L. A. (1991) Stress response protein (hsp-27) determination in primary human breast carcinomas: clinical, histologic, and prognostic correlations. J. Natl. Cancer Inst. 83,170-178[Abstract/Free Full Text]
-
Love, S., King, R. J. (1994) A 27 kDa heat shock protein that has anomalous prognostic powers in early and advanced breast cancer. Br. J. Cancer 69,743-748[Medline]
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