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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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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • cell death • etoposide • drug resistance • leukemia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
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 8–12% 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (U937–27-7 and
U937–27-4) were similar, results shown from subsequent experiments
will be those obtained in the U937–27-4 clone.
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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.
|
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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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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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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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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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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]
B crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J. Immunol 215,363-374
-treated T47D human carcinoma cells. Biochem. J. 312,367-375
in NIH-3T3-ras cells. Biochem. Biophys. Res. Commun. 241,187-192[Medline]
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 A. Shamaei-Tousi, J. P. Halcox, and B. Henderson Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease Cardiovasc Res, April 1, 2007; 74(1): 19 - 28. [Abstract] [Full Text] [PDF]
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 E. Schmitt, M. Gehrmann, M. Brunet, G. Multhoff, and C. Garrido Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy J. Leukoc. Biol., January 1, 2007; 81(1): 15 - 27. [Abstract] [Full Text] [PDF]
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S. Luthra, B. Fardin, J. Dong, D. Hertzog, S. Kamjoo, S. Gebremariam, V. Butani, R. Narayanan, J. K. Mungcal, B. D. Kuppermann, et al. Activation of Caspase-8 and Caspase-12 Pathways by 7-Ketocholesterol in Human Retinal Pigment Epithelial Cells Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5569 - 5575. [Abstract] [Full Text] [PDF]
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 M. Bauer-Marinovic, S. Florian, K. Muller-Schmehl, H. Glatt, and G. Jacobasch Dietary resistant starch type 3 prevents tumor induction by 1,2-dimethylhydrazine and alters proliferation, apoptosis and dedifferentiation in rat colon Carcinogenesis, September 1, 2006; 27(9): 1849 - 1859. [Abstract] [Full Text] [PDF]
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 H. Wei, W. Campbell, and R. S. Vander Heide Heat shock-induced cardioprotection activates cytoskeletal-based cell survival pathways Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H638 - H647. [Abstract] [Full Text] [PDF]
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 A. Parcellier, M. Brunet, E. Schmitt, E. Col, C. Didelot, A. Hammann, K. Nakayama, K. I. Nakayama, S. Khochbin, E. Solary, et al. HSP27 favors ubiquitination and proteasomal degradation of p27Kip1 and helps S-phase re-entry in stressed cells FASEB J, June 1, 2006; 20(8): 1179 - 1181. [Abstract] [Full Text] [PDF]
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E. Schmitt, L. Maingret, P.-E. Puig, A.-L. Rerole, F. Ghiringhelli, A. Hammann, E. Solary, G. Kroemer, and C. Garrido Heat shock protein 70 neutralization exerts potent antitumor effects in animal models of colon cancer and melanoma. Cancer Res., April 15, 2006; 66(8): 4191 - 4197. [Abstract] [Full Text] [PDF]
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B. Lelj-Garolla and A. G. Mauk Self-association and Chaperone Activity of Hsp27 Are Thermally Activated J. Biol. Chem., March 24, 2006; 281(12): 8169 - 8174. [Abstract] [Full Text] [PDF]
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Y. Chen and R. W. Currie Small interfering RNA knocks down heat shock factor-1 (HSF-1) and exacerbates pro-inflammatory activation of NF-{kappa}B and AP-1 in vascular smooth muscle cells Cardiovasc Res, January 1, 2006; 69(1): 66 - 75. [Abstract] [Full Text] [PDF]
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 D. W.-C. Li, J.-P. Liu, Y.-W. Mao, H. Xiang, J. Wang, W.-Y. Ma, Z. Dong, H. M. Pike, R. E. Brown, and J. C. Reed Calcium-activated RAF/MEK/ERK Signaling Pathway Mediates p53-dependent Apoptosis and Is Abrogated by {alpha}B-Crystallin through Inhibition of RAS Activation Mol. Biol. Cell, September 1, 2005; 16(9): 4437 - 4453. [Abstract] [Full Text] [PDF]
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Y.-J. Lee, D.-H. Lee, C.-K. Cho, S. Bae, G.-J. Jhon, S.-J. Lee, J.-W. Soh, and Y.-S. Lee HSP25 Inhibits Protein Kinase C{delta}-mediated Cell Death through Direct Interaction J. Biol. Chem., May 6, 2005; 280(18): 18108 - 18119. [Abstract] [Full Text] [PDF]
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 G.-C. Fan, X. Ren, J. Qian, Q. Yuan, P. Nicolaou, Y. Wang, W. K. Jones, G. Chu, and E. G. Kranias Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury Circulation, April 12, 2005; 111(14): 1792 - 1799. [Abstract] [Full Text] [PDF]
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 B. Xu, G.-h. Dong, H. Liu, Y.-q. Wang, H.-w. Wu, and H. Jing Recombinant Human Erythropoietin Pretreatment Attenuates Myocardial Infarct Size: A Possible Mechanism Involves Heat Shock Protein 70 and Attenuation of Nuclear Factor-kappaB Ann. Clin. Lab. Sci., April 1, 2005; 35(2): 161 - 168. [Abstract] [Full Text] [PDF]
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 S. Francois, J. El Benna, P. M. C. Dang, E. Pedruzzi, M.-A. Gougerot-Pocidalo, and C. Elbim Inhibition of Neutrophil Apoptosis by TLR Agonists in Whole Blood: Involvement of the Phosphoinositide 3-Kinase/Akt and NF-{kappa}B Signaling Pathways, Leading to Increased Levels of Mcl-1, A1, and Phosphorylated Bad J. Immunol., March 15, 2005; 174(6): 3633 - 3642. [Abstract] [Full Text] [PDF]
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N. A. Whitlock, K. Lindsey, N. Agarwal, C. E. Crosson, and J.-X. Ma Heat Shock Protein 27 Delays Ca2+-Induced Cell Death in a Caspase-Dependent and -Independent Manner in Rat Retinal Ganglion Cells Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 1085 - 1091. [Abstract] [Full Text] [PDF]
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 M. Kato, H. Fukuda, T. Nonaka, and S. Imajoh-Ohmi Cleavage of Nonmuscle Myosin Heavy Chain-A during Apoptosis in Human Jurkat T Cells J. Biochem., February 1, 2005; 137(2): 157 - 166. [Abstract] [Full Text] [PDF]
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 M. Yeo, D.-K. Kim, Y.-B. Kim, T. Y. Oh, J.-E. Lee, S. W. Cho, H. C. Kim, and K.-B. Hahm Selective Induction of Apoptosis with Proton Pump Inhibitor in Gastric Cancer Cells Clin. Cancer Res., December 15, 2004; 10(24): 8687 - 8696. [Abstract] [Full Text] [PDF]
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Y. Masuda, G. Shima, T. Aiuchi, M. Horie, K. Hori, S. Nakajo, S. Kajimoto, T. Shibayama-Imazu, and K. Nakaya Involvement of Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) in Apoptosis Induced by {beta}-Hydroxyisovalerylshikonin J. Biol. Chem., October 8, 2004; 279(41): 42503 - 42515. [Abstract] [Full Text] [PDF]
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 H.-D. Wang, P. Kazemi-Esfarjani, and S. Benzer Multiple-stress analysis for isolation of Drosophila longevity genes PNAS, August 24, 2004; 101(34): 12610 - 12615. [Abstract] [Full Text] [PDF]
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 H. M. Beere `The stress of dying': the role of heat shock proteins in the regulation of apoptosis J. Cell Sci., June 1, 2004; 117(13): 2641 - 2651. [Abstract] [Full Text] [PDF]
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E. Schmitt, A. Parcellier, F. Ghiringhelli, N. Casares, S. Gurbuxani, N. Droin, A. Hamai, M. Pequignot, A. Hammann, M. Moutet, et al. Increased Immunogenicity of Colon Cancer Cells by Selective Depletion of Cytochrome c Cancer Res., April 15, 2004; 64(8): 2705 - 2711. [Abstract] [Full Text] [PDF]
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O. Duverger, L. Paslaru, and M. Morange HSP25 Is Involved in Two Steps of the Differentiation of PAM212 Keratinocytes J. Biol. Chem., March 12, 2004; 279(11): 10252 - 10260. [Abstract] [Full Text] [PDF]
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E. Schmitt, A. Parcellier, S. Gurbuxani, C. Cande, A. Hammann, M. C. Morales, C. R. Hunt, D. J. Dix, R. T. Kroemer, F. Giordanetto, et al. Chemosensitization by a Non-apoptogenic Heat Shock Protein 70-Binding Apoptosis-Inducing Factor Mutant Cancer Res., December 1, 2003; 63(23): 8233 - 8240. [Abstract] [Full Text] [PDF]
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 A. Parcellier, E. Schmitt, S. Gurbuxani, D. Seigneurin-Berny, A. Pance, A. Chantome, S. Plenchette, S. Khochbin, E. Solary, and C. Garrido HSP27 Is a Ubiquitin-Binding Protein Involved in I-{kappa}B{alpha} Proteasomal Degradation Mol. Cell. Biol., August 15, 2003; 23(16): 5790 - 5802. [Abstract] [Full Text] [PDF]
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M. J. Rane, Y. Pan, S. Singh, D. W. Powell, R. Wu, T. Cummins, Q. Chen, K. R. McLeish, and J. B. Klein Heat Shock Protein 27 Controls Apoptosis by Regulating Akt Activation J. Biol. Chem., July 18, 2003; 278(30): 27828 - 27835. [Abstract] [Full Text] [PDF]
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 V. V. Tran, G. Chen, C. B. Newgard, and H. E. Hohmeier Discrete and Complementary Mechanisms of Protection of {beta}-Cells Against Cytokine-Induced and Oxidative Damage Achieved by bcl-2 Overexpression and a Cytokine Selection Strategy Diabetes, June 1, 2003; 52(6): 1423 - 1432. [Abstract] [Full Text] [PDF]
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C. Zhang, J. Baffi, S. W. Cousins, and K. G. Csaky Oxidant-induced cell death in retinal pigment epithelium cells mediated through the release of apoptosis-inducing factor J. Cell Sci., May 15, 2003; 116(10): 1915 - 1923. [Abstract] [Full Text] [PDF]
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M. Maccarrone, N. Battista, M. Meloni, M. Bari, G. Galleri, P. Pippia, A. Cogoli, and A. Finazzi-Agro Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release J. Leukoc. Biol., April 1, 2003; 73(4): 472 - 481. [Abstract] [Full Text] [PDF]
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Y. Li, S. Roth, M. Laser, J.-x. Ma, and C. E. Crosson Retinal Preconditioning and the Induction of Heat-Shock Protein 27 Invest. Ophthalmol. Vis. Sci., March 1, 2003; 44(3): 1299 - 1304. [Abstract] [Full Text] [PDF]
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 C. Kitamura, Y. Ogawa, T. Nishihara, T. Morotomi, and M. Terashita Transient Co-localization of c-Jun N-terminal Kinase and c-Jun with Heat Shock Protein 70 in Pulp Cells during Apoptosis Journal of Dental Research, February 1, 2003; 82(2): 91 - 95. [Abstract] [Full Text] [PDF]
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B. McLaughlin, K. A. Hartnett, J. A. Erhardt, J. J. Legos, R. F. White, F. C. Barone, and E. Aizenman Caspase 3 activation is essential for neuroprotection in preconditioning PNAS, January 21, 2003; 100(2): 715 - 720. [Abstract] [Full Text] [PDF]
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H. Sakamoto, T. Mashima, K. Yamamoto, and T. Tsuruo Modulation of Heat-shock Protein 27 (Hsp27) Anti-apoptotic Activity by Methylglyoxal Modification J. Biol. Chem., November 22, 2002; 277(48): 45770 - 45775. [Abstract] [Full Text] [PDF]
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H. S. Mchaourab, E. K. Dodson, and H. A. Koteiche Mechanism of Chaperone Function in Small Heat Shock Proteins. TWO-MODE BINDING OF THE EXCITED STATES OF T4 LYSOZYME MUTANTS BY alpha A-CRYSTALLIN J. Biol. Chem., October 18, 2002; 277(43): 40557 - 40566. [Abstract] [Full Text] [PDF]
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S. Dorion, H. Lambert, and J. Landry Activation of the p38 Signaling Pathway by Heat Shock Involves the Dissociation of Glutathione S-Transferase Mu from Ask1 J. Biol. Chem., August 16, 2002; 277(34): 30792 - 30797. [Abstract] [Full Text] [PDF]
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A. Okado-Matsumoto and I. Fridovich Amyotrophic lateral sclerosis: A proposed mechanism PNAS, June 25, 2002; 99(13): 9010 - 9014. [Abstract] [Full Text] [PDF]
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 V. L. Gabai and M. Y. Sherman Molecular Biology of Thermoregulation: Invited Review: Interplay between molecular chaperones and signaling pathways in survival of heat shock J Appl Physiol, April 1, 2002; 92(4): 1743 - 1748. [Abstract] [Full Text] [PDF]
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C. Paul, F. Manero, S. Gonin, C. Kretz-Remy, S. Virot, and A.-P. Arrigo Hsp27 as a Negative Regulator of Cytochrome c Release Mol. Cell. Biol., February 1, 2002; 22(3): 816 - 834. [Abstract] [Full Text] [PDF]
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H. Zhou, S.-H. Li, and X.-J. Li Chaperone Suppression of Cellular Toxicity of Huntingtin Is Independent of Polyglutamine Aggregation J. Biol. Chem., December 14, 2001; 276(51): 48417 - 48424. [Abstract] [Full Text] [PDF]
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M. Gao, S. Fan, I. D. Goldberg, J. Laterra, R. N. Kitsis, and E. M. Rosen Hepatocyte Growth Factor/Scatter Factor Blocks the Mitochondrial Pathway of Apoptosis Signaling in Breast Cancer Cells J. Biol. Chem., December 7, 2001; 276(50): 47257 - 47265. [Abstract] [Full Text] [PDF]
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D. S Latchman Heat shock proteins and cardiac protection Cardiovasc Res, September 1, 2001; 51(4): 637 - 646. [Abstract] [Full Text] [PDF]
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N. Strunnikova, J. Baffi, A. Gonzalez, W. Silk, S. W. Cousins, and K. G. Csaky Regulated Heat Shock Protein 27 Expression in Human Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., August 1, 2001; 42(9): 2130 - 2138. [Abstract] [Full Text] [PDF]
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S. Machida, P. Chaudhry, T. Shinohara, D. P. Singh, V. N. Reddy, L. T. Chylack Jr, P. A. Sieving, and R. A. Bush Lens Epithelium-Derived Growth Factor Promotes Photoreceptor Survival in Light-Damaged and RCS Rats Invest. Ophthalmol. Vis. Sci., April 1, 2001; 42(5): 1087 - 1095. [Abstract] [Full Text]
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 S. Sanada, M. Kitakaze, P. J. Papst, K. Hatanaka, H. Asanuma, T. Aki, Y. Shinozaki, H. Ogita, K. Node, S. Takashima, et al. Role of Phasic Dynamism of p38 Mitogen-Activated Protein Kinase Activation in Ischemic Preconditioning of the Canine Heart Circ. Res., February 2, 2001; 88(2): 175 - 180. [Abstract] [Full Text] [PDF]
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T. Jiang and S. Altman Protein-protein interactions with subunits of human nuclear RNase P PNAS, January 10, 2001; (2001) 21561498. [Abstract] [Full Text]
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S. J. Charette, J. N. Lavoie, H. Lambert, and J. Landry Inhibition of Daxx-Mediated Apoptosis by Heat Shock Protein 27 Mol. Cell. Biol., October 15, 2000; 20(20): 7602 - 7612. [Abstract] [Full Text]
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M. Maccarrone, T. Lorenzon, M. Bari, G. Melino, and A. Finazzi-Agro Anandamide Induces Apoptosis in Human Cells via Vanilloid Receptors. EVIDENCE FOR A PROTECTIVE ROLE OF CANNABINOID RECEPTORS J. Biol. Chem., October 6, 2000; 275(41): 31938 - 31945. [Abstract] [Full Text] [PDF]
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C. Gerner, U. Frohwein, J. Gotzmann, E. Bayer, D. Gelbmann, W. Bursch, and R. Schulte-Hermann The Fas-induced Apoptosis Analyzed by High Throughput Proteome Analysis J. Biol. Chem., December 8, 2000; 275(50): 39018 - 39026. [Abstract] [Full Text] [PDF]
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C.-Y. Li, J.-S. Lee, Y.-G. Ko, J.-I. Kim, and J.-S. Seo Heat Shock Protein 70 Inhibits Apoptosis Downstream of Cytochrome c Release and Upstream of Caspase-3 Activation J. Biol. Chem., August 11, 2000; 275(33): 25665 - 25671. [Abstract] [Full Text] [PDF]
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T. Jiang and S. Altman Protein-protein interactions with subunits of human nuclear RNase P PNAS, January 30, 2001; 98(3): 920 - 925. [Abstract] [Full Text] [PDF]
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) as well as control-transfected cells (
) were
treated with increasing concentrations of etoposide for 96 h. The
y axis depicts the percent cell survival and the
x axis depicts the concentration of etoposide (VP-16)
used to treat cells. Data are illustrated as the mean ±
SDS (n=4).
