From the Department of Oncology-Pathology, CancerCenterKarolinska, Karolinska Institute and Hospital, S-171 76 Stockholm, Sweden
Received for publication, October 8, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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DNA damage is believed to be the main cause of
the antiproliferative effect of cisplatin, a cornerstone agent in
anticancer therapy. However, cisplatin can be expected to react also
with nucleophiles other than DNA. Using enucleated cells (cytoplasts) we demonstrate here that cisplatin-induced apoptotic signaling may
occur independently of DNA damage. Cisplatin-induced
caspase-3 activation in cytoplasts required calcium and the activity of the calcium-dependent protease calpain. It is known that
calpain activation may be associated with endoplasmic reticulum (ER)
stress, suggesting that the ER is a cytosolic target of cisplatin.
Consistent with this hypothesis, cisplatin induced
calpain-dependent activation of the ER-specific caspase-12
in cytoplasts as well as in intact cells. Cisplatin also induced
increased expression of Grp78/BiP, another marker of ER stress.
By contrast, the DNA-damaging topoisomerase II inhibitor etoposide did
not induce apoptotic signaling in cytoplasts nor ER stress in intact
cells. We have thus identified a novel mechanism of action of
cisplatin. The results have implications for the understanding of
resistance mechanisms as well as the unique efficiency of this drug.
Cisplatin is a widely used chemotherapeutic agent generally
recognized as a DNA-damaging drug. The molecular mechanisms that link
the formation of DNA adducts to cell death-inducing signaling are not
well understood. We have previously reported that cisplatin induces at
least two apoptotic signaling pathways. One involves calpain activation
and calpain-mediated cleavage of the proapoptotic BH3-only protein Bid.
The other one results in
MEKK11-dependent
modulation of the proapoptotic protein Bak (1, 2). Both pathways
contribute to cytochrome c release and subsequent caspase activation.
In aqueous solutions, the chloride ligands of cisplatin are replaced by
water molecules generating a positively charged electrophile. This
electrophile reacts with nucleophilic sites on intracellular macromolecules to form DNA, RNA, and protein adducts (3). Approximately 1% of intracellular cisplatin reacts with DNA resulting in intra- and
interstrand cross-links, with an intrastrand cross-link between adjacent guanines as the most common adduct (4, 5). DNA adducts are
considered the key toxic lesions induced by cisplatin; however, some
studies have not shown a clear correlation between DNA adducts and
cisplatin cytotoxicity (6, 7). The potential contribution of
cisplatin-induced RNA or protein damage to cytotoxicity has not been
examined in this respect (8).
This prompted us to investigate the ability of cisplatin to induce
apoptosis independently of DNA damage, and we here report the ability
of cisplatin to induce apoptosis in enucleated cells. The cisplatin
response was also found to involve endoplasmic reticulum (ER) stress.
Altogether, we have here identified an apoptotic pathway induced by
cisplatin independently of its DNA-damaging activity. This novel
mechanism of action may contribute to the understanding of the causes
of sensitivity and resistance to cisplatin.
Cells--
The human melanoma cell line 224 and two variants of
the colon cancer cell lines HCT116 (wt and p53-deficient) (9) were used. The cells were maintained at 37 °C in 5% CO2 in
RPMI medium supplemented with fetal calf serum (10%),
L-glutamate, penicillin, and streptomycin.
Cytoplast Preparation--
The cells were harvested, resuspended
in 12.5% Ficoll containing complete medium supplemented with
cytochalasin B (10 µg/ml), and incubated for 30 min at 37 °C.
Three ml of this cell suspension was layered onto a density gradient
prepared in ultracentrifuge tubes with the following layers: 2 ml of
25%, 2 ml of 17%, 0.5 ml of 16%, and 0.5 ml of 15% Ficoll. The
gradient was prepared in complete medium supplemented with 10 µg/ml
of cytochalasin B and pre-equilibrated in a CO2 incubator
overnight. The cells were then centrifuged in a prewarmed (32 °C)
Beckman SW41 swing bucket rotor for 60 min at 25,000 rpm. The resulting
enucleated cytoplasts were collected at the interface between the 16 and 17% Ficoll layers and were then washed with medium and allowed to
recover for 2 h before drug treatment.
The purity of the cytoplast preparation was determined by resuspending
cytoplasts in PBS containing digitonin (50 µg/ml) and labeling with 5 µg/ml propidium iodide (PI). After 10 min of incubation at room
temperature, fluorescence was monitored using the FL2 channel on a FACS
Calibur flow cytometer.
Western Blot Analysis--
Cell extract proteins (30 µg) were
resolved by SDS-PAGE and transferred onto a polyvinylidene
difluoride membrane for Western blotting. The following
antibodies were used: anti-caspase-12 (1: 250, a kind gift from Dr. J. Yuan), anti-Grp78/BiP (1:500; BD Transduction Laboratories), and
anti-Bid (1:1,000; Molecular Probes, Inc.). Tubulin was used as an
internal standard for loading.
Assessment of Caspase-3 and DEVDase Activity--
After
treatment cells were harvested, washed with PBS, fixed in
paraformaldehyde (0.25%, 5 min), washed three times with PBS, and
incubated for 60 min with a fluorescein isothiocyanate-conjugated antibody recognizing active caspase-3 (Pharmingen). The antibody was
diluted 1:50 in PBS containing digitonin (50 µg/ml). After incubation, the cells were washed with PBS, and fluorescence was monitored using the FL1 channel of a FACS Calibur flow cytometer.
DEVDase activity was assessed using the fluorigenic Ac-DEVD-AMC
substrate (CaspACE Assay; Promega Life Science). The harvested cells
were centrifuged, washed in ice-cold PBS, and resuspended in lysis
buffer (25 mM HEPES, pH 7.5, 5 mM
MgCl2, 5 mM EDTA, 5 mM
dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin and leupeptin). The cells were lysed by three
cycles of freeze-thawing in liquid nitrogen. The lysates were
centrifuged (16,000 × g for 20 min), and supernatant
fractions were collected. The caspase activity was assayed according to the manufacturer's instructions. The fluorescence produced upon cleavage of the labeled substrate is proportional to the caspase activity in the sample.
Flow Cytometric Analysis of Bak-associated
Immunofluorescence--
Upon induction of apoptosis, the proapoptotic
Bak protein undergoes a conformational change that exposes an otherwise
inaccessible N-terminal epitope (10). In the present study, we have
used the same antibody that was shown to specifically recognize this epitope (mouse monoclonal antibody against amino acids 1-52 of Bak;
Oncogene Research Products; number AM03, clone TC100). Using a
fluorescein isothiocyanate-conjugated secondary antibody, the increases
in accessibility of the epitope were monitored by flow cytometry as
earlier described (1). The data are presented as fold increases in
immunofluorescence from control levels.
Cisplatin Induces Nucleus-independent Apoptosis--
224 human
melanoma and HCT 116 human colon cancer cells were enucleated as
described and treated with cisplatin to investigate whether cisplatin
can induce nucleus-independent apoptosis. The purity of the
cytoplast preparation was determined by staining digitonin-permeabilized cytoplasts with PI and analyzing the sample by
flow cytometry. As shown in Fig.
1A (panel I),
intact cells demonstrate a 10-fold higher PI fluorescence than
cytoplasts, allowing analysis of these two populations separately, by
way of electronic gating.
Apoptosis was assessed as activation of caspase-3 using an antibody
that specifically recognizes active caspase-3. After treatment with 15 and 20 µM cisplatin for 16 h, the cytoplast
preparations were analyzed for PI fluorescence and active caspase-3. In
untreated samples, the cut-off for activated caspase-3 was set
immediately to the right of the cell/cytoplast population (Fig.
1A). After cisplatin treatment, 43 and 48% of the 224 cytoplasts, respectively, showed activation of caspase-3 (Fig.
1B). 224 cells harbor mutated p53. To confirm that
cisplatin-induced apoptosis is independent of p53 status, we compared
the effect of cisplatin on wt and p53
Bak is a proapoptotic Bcl-2 family protein involved in cytochrome
c release from mitochondria. We have previously found that cisplatin induces a proapoptotic conformational modulation of Bak in
224 cells (1). This modulation is assessed with an antibody recognizing
a specific N-terminal epitope that is exposed only when Bak is in its
activated, proapoptotic form. To determine whether cisplatin can induce
nucleus-independent activation of Bak, we examined conformational
modulation of Bak in cisplatin-treated cytoplasts. As shown in Fig.
2A, cisplatin induces
activation of Bak in 224 cytoplasts. However, this activation is weaker
compared with Bak activation in intact 224 cells. Weak Bak activation
was also observed in HCT 116 p53 wt and p53-deficient cytoplasts (Fig. 2B).
We have previously shown that cisplatin induces cleavage of the
proapoptotic BH3-only protein Bid to its active form tBid (2). tBid is
in turn involved in cytochrome c release from mitochondria
in both the extrinsic (11, 12) and intrinsic proapoptotic pathways
(13, 14). Cisplatin-induced Bid cleavage was therefore examined also in
224 cytoplasts, and the results show that Bid is indeed cleaved
(Fig. 2C).
Caspase-3 Activation in Cytoplasts Is Blocked by a Calpain
Inhibitor and a Calcium Chelator--
As we have reported,
cisplatin-induced Bid cleavage is carried out by calpain (2). To
investigate whether calpain is involved in nucleus-independent
apoptosis, we studied the effect of the calpain inhibitor calpeptin on
caspase-3 activation in enucleated 224 cells. Cotreatment with
calpeptin was found to block cisplatin-induced caspase-3 activation by
approximately half (Fig.
3A).
An increase in intracellular calcium is required for calpain
activation. Accordingly, cisplatin-induced calpain activation in 224 cells is calcium-dependent (2). Because increased
intracellular calcium was also observed in cisplatin-treated enucleated
224 cells (not shown), we examined the effect of the calcium chelator BAPTA-AM on caspase-3 activation in cisplatin-treated
cytoplasts. As shown in Fig. 3B, cotreatment with BAPTA-AM
inhibited caspase-3 activation by ~60%.
Cisplatin Induces ER Stress in 224 Cells and Cytoplasts--
The
ER participates in regulation of cellular responses to stress and
alterations in calcium homeostasis (15, 16). Caspase-12 is an
ER-specific caspase that is activated by ER stress and that specifically participates in ER stress-induced apoptosis (17). Calpain
has been indicated as a protease responsible for activation of
caspase-12 (18).
The finding that cisplatin induces increased cytosolic calcium and
calpain activation suggested that the ER might be a non-nuclear target
of cisplatin. The ability of cisplatin to induce ER stress was assessed
as activation of caspase-12, reflected in cleavage of 60-kDa
procaspase-12. This cleavage was significant at 4 h after
cisplatin treatment and was confirmed at 8 h (Fig.
4A). Cleavage was furthermore
blocked by calpeptin and by BAPTA-AM (Fig. 4A). Cleavage of
caspase-12 was also observed in cytoplasts after 4 and 16 h (Fig.
4B).
DEVDase activity (caspase-7) has also been reported as mediator of
caspase-12 activation (19). To rule out involvement of these caspases
in the initial cleavage of caspase-12, DEVDase activity in 224 cells
and cytoplasts was assessed at 4 h after cisplatin treatment. No
increase in DEVDase activity was observed in 224 cells or cytoplasts at
this time point, whereas markedly increased activity was seen at
16 h (Fig. 4C).
Grp78 (glucose-regulated protein
78) is an ER chaperone protein that is up-regulated by ER
stress (15, 16). To confirm the ability of cisplatin to induce ER
stress, we examined expression of this protein after cisplatin
treatment. In 224 cells a 2.6-fold increase in Grp78 protein expression
was seen at 16 h after cisplatin treatment (Fig.
5A). Induction of Grp78
expression was confirmed by flow cytometry (Fig. 5B) and was
not affected by the caspase inhibitor zVAD-fmk (Fig.
5C).
Cisplatin has been shown to induce production of reactive oxygen
species (ROS) (20, 21) that have been reported as important mediators
of the stress response in many cell types (22). We have previously
observed that pretreatment of 224 cells with the ROS scavenger
N-acetyl cysteine (NAC) inhibits cisplatin-induced mitochondrial depolarization and nuclear fragmentation by ~60% (not
shown). However, NAC failed to inhibit calpain activation in
cisplatin-treated cells (not shown); thus, cisplatin-induced apoptosis
also involves events that are not ROS-dependent. To investigate the possible involvement of oxidative stress in ER stress,
we examined the expression of Grp78 in cells treated with cisplatin in
the presence or absence of NAC. As shown in Fig. 5C, the
presence of NAC did not affect up-regulation of Grp78 levels after
cisplatin treatment. This indicates that oxidative stress is not
involved in the induction of ER stress by cisplatin.
Apoptosis induced by ER stress involves downstream activation of
caspase-3. To investigate whether Grp78 induction correlates with
caspase-3 activation, both events were studied simultaneously in
cisplatin-treated 224 cells. The cells were permeabilized with digitonin, double-stained with anti-Grp78 and anti-caspase-3
antibodies, and analyzed by flow cytometry (Fig.
6). At 16 h post-treatment the
median value for Grp78-associated immunofluorescence had increased, and
activation of caspase-3 occurred in the population showing the highest
Grp78 levels (Fig. 6). We have earlier shown that caspase-3 activation
commences around 16 h post-treatment and increases sharply between
16 and 24 h (1). At 20 h, a population of cells with
activated caspase-3 has lower Grp78, indicating the presence of
ER-independent activation of caspase-3 at this later time point.
Etoposide-induced Apoptosis Requires Nuclear Events--
To
demonstrate the specificity of cisplatin in inducing
nucleus-independent apoptosis, we treated 224 cytoplasts with
etoposide. This is also a DNA-damaging drug, but as a topoisomerase
II-inhibitor it has a different mechanism of action than cisplatin.
Etoposide failed to induce caspase-3 activation in enucleated cells
(Fig. 7A), and thus, in
contrast to cisplatin, the effect of etoposide is dependent on an
effect on DNA. Furthermore, etoposide did not have any effect on ER,
because no up-regulation of Grp78 (Fig. 7B) nor caspase-12
activation (Fig. 7C) was observed in etoposide-treated
cells.
Cisplatin is a broadly active cytotoxic anticancer drug. In
aqueous solutions, the molecule is rendered highly reactive and reacts
with nucleophilic sites on intracellular macromolecules. The role of
cisplatin adducts on proteins and/or RNA in cisplatin-induced apoptosis
is at present unclear. Instead, it is generally accepted that nuclear
DNA is the critical target of cisplatin, even though only 1% of the
intracellular cisplatin reacts with DNA. Accordingly, nuclear DNA
repair-deficient cell lines may show enhanced sensitivity to cisplatin
(23-25). Cisplatin has also been found to be able to bind to
mitochondrial DNA (mtDNA) (26). However, mtDNA constitutes about
1/1,000 of the cell DNA, and, to our knowledge, no molecular sensors
have been identified that would link damaged mtDNA to the
apoptotic machinery, suggesting that mtDNA is not a major target.
Nevertheless, there are also studies reporting a lack of correlation
between cisplatin sensitivity and accumulation of intracellular cisplatin and/or DNA platination (5, 6) and conflicting reports on the
correlation of p53 status with tumor cell resistance or susceptibility
to cisplatin (7). In a panel of nine melanoma cell lines, we have found
no correlation between cisplatin sensitivity and p53
status.2
Platination of cellular proteins and/or RNA is a very likely effect of
cisplatin in treatment of cells (27), but the contribution of
platination to the cytotoxicity of cisplatin has not been investigated. This is the first report to show that cisplatin-induced apoptosis is
not exclusively dependent on DNA damage and the presence of nuclei.
We show here that cisplatin is able to induce proapoptotic signaling
and caspase-3 activation in enucleated cells (cytoplasts). Cytoplast
preparations also contain a small amount of intact cells with nuclei,
which were treated in the same way as cytoplasts, including incubation
with cytochalasin B and gradient centrifugation. This population of
intact cells was used to directly compare levels of caspase-3 and Bak
activation in cytoplasts and intact cells. Higher caspase-3 activation
induced by cisplatin was observed in cytoplasts than in intact cells in
both 224 and HCT 116 cell lines. This increased activity may be due to
the ability of nucleus-containing cells to induce expression of
protective proteins in response to cisplatin-induced stress,
e.g. Hsp70 (heat shock
protein 70) or XIAP (X-linked
inhibitor of apoptosis protein).
Hsp70 can block apoptosis by interfering with proteins involved in the
execution phase of apoptosis such as protease activating factor-1 (28) and apoptosis inducing factor (29). XIAP blocks apoptosis by inhibiting
active caspase-3 and caspase-9 (30).
Although the cisplatin-induced caspase-3 activity was higher in
cytoplasts than in intact cells, Bak activation in 224 cytoplasts was
only slightly increased compared with the 2.5-fold induction of Bak
activity in 224 cells. A possible explanation is that Bak activation is
predominantly dependent on MEKK1-mediated signaling and that this
pathway may be activated by DNA damage rather than by ER stress. The
lower Bak activation seen in cytoplasts might instead represent
activation of the calpain-Bid pathway, which we have shown to be
distinct from the MEKK1 pathway (2). tBid has been reported to
stimulate formation of Bak oligomer pores in the mitochondrial membrane
(31). Because cleavage and activation of Bid was found in 224 cytoplasts, we suggest that Bid-mediated modulation of Bak might be
responsible for the slight activation observed in 224 and HCT 116 cytoplasts.
Calpains are a family of cytosolic cysteine proteases, and their
activation is preceded by increased levels of cytosolic calcium. We
have previously found that increased cytosolic calcium and calpain
activation are early events in cisplatin-induced apoptosis and have
also presented evidence for calpain-mediated cleavage of Bid in
cisplatin-treated 224 cells (2). Here we have shown that these events
may occur independently of DNA damage.
These findings also suggested that the ER might be the non-nuclear
target of cisplatin. The ER plays an important role in maintenance of
intracellular calcium homeostasis, protein synthesis, post-translational modifications, and proper folding of proteins as
well as their sorting and trafficking. Alterations in calcium homeostasis and accumulation of unfolded proteins in the ER cause ER
stress. A variety of agents, including chemical toxicants, oxidative
stress, inhibitors of protein glycosylation, calcium ionophores, and
other agents that alter calcium homeostasis can all induce ER stress
followed by cell death (16).
To investigate the involvement of the ER in cisplatin-induced
apoptosis, we examined activation of caspase-12 and up-regulation of
Grp78. Apoptosis induced by ER stress has been shown to involve calpain-mediated activation of caspase-12 (18). Caspase-12 is localized
to the ER and may be activated by disruption of calcium homeostasis and
accumulation of excess proteins in ER but not by membrane- or
mitochondria-targeted apoptotic signals (17). Murine caspase-12 is a
member of the ICE (interleukin-1 This is the first report to show the involvement of ER stress,
specifically seen as caspase-12 activation and up-regulation of Grp78,
in cisplatin-induced apoptosis. We also present evidence for
nucleus-independent activation of caspase-12. In accordance with
calpain-mediated activation of caspase-12, both calpeptin and BAPTA
were able to block cisplatin-induced cleavage of caspase-12 in 224 cells. Although caspase-7 has also been reported to cleave procaspase-12 (19), it is probably not involved in our system, because
no DEVDase was detected at 4 h post-treatment, when caspase-12 was
already cleaved.
Cisplatin has been shown to induce oxidative stress in variety of cell
lines. As expected, cotreatment of 224 cells with the ROS scavenger NAC
resulted in decreased nuclear fragmentation and mitochondrial
depolarization. However, NAC did not have any effect on the induction
of Grp78, showing that ROS production is not required for ER stress.
This is supported by the findings that calpain is likely involved in
caspase-12 activation and that NAC did not block calpain activation.
For unknown reasons, basal expression levels of Grp78 varied widely in
the 224 cells. However, cisplatin induced a general increase in Grp78
expression, and this effect was independent of caspase-12 and caspase-3
activation, because the pan-caspase inhibitor zVAD-fmk did not affect
it. At 16 h, caspase-3 was activated in cells with high Grp78
expression. Interestingly, there was also a population with high Grp78
expression but no caspase-3 activation (Fig. 6b,
upper left quadrant). The size of this population is reduced
to below control levels only at the later time point and with the
higher dose. This observation is in accordance with the antiapoptotic
or protective effect of Grp78, which possibly has some specificity
against drugs inducing calcium depletion from the ER (16). With this
antiapoptotic action, Grp78 may well contribute to resistance against
cisplatin, which may now be counted among the ER-active anti-cancer
drugs. The basal level of Grp78 is up-regulated in many tumors (16),
but to our knowledge, the relationship between Grp78 levels and
cisplatin sensitivity/resistance has not been studied.
In contrast to cisplatin, etoposide, which is also a DNA-damaging drug
but which achieves its effect by inhibition of topoisomerase II, did
not induce caspase-3 activation in cytoplasts. Etoposide thus requires
nuclear events to induce apoptosis. Furthermore, etoposide-induced
apoptosis does not involve ER stress because it did not induce
caspase-12 activation in 224 cells or cytoplasts, nor did it lead to
increased Grp78 expression in 224 cells.
In summary, we here demonstrate a novel mechanism of action for
cisplatin, a chemotherapeutic agent hitherto regarded as a typical
DNA-damaging agent. Cisplatin is here shown to induce apoptosis in the
absence of DNA damage, and the ER is likely its non-nuclear target. We
propose that the ability to activate two, rather than a single, major
pathways to apoptosis makes cisplatin so generally efficient as an
anti-cancer agent. Lastly, we believe that our findings will lead to a
re-evaluation of resistance-determining factors for cisplatin and
possibly to new, improved treatment strategies to overcome resistance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cisplatin induces nucleus-independent
caspase-3 activation. Cells (224 human melanoma and HCT 116 human
colon cancer) and enucleated cytoplasts prepared from these cell lines
were treated with the indicated doses of cisplatin for 16 h. The
caspase-3 activity and DNA content were then assessed by flow cytometry
using an antibody that specifically recognizes active caspase-3, and
PI, respectively. A, intact 224 cells (with ~10-fold
higher PI fluorescence, in the top part of the dot
plot) represent a contamination of the cytoplast preparation
allowing analysis of both populations separately by electronic gating.
Caspase-3 activation in cisplatin-treated cytoplasts is compared.
Panel I, untreated cytoplasts; panel II,
cytoplasts treated with 20 µM cisplatin for 16 h. The
shift to the right in the dot plot
represents an increase in active-caspase-3 immunofluorescence. In
B-D, the results are shown as percentages of cells or
cytoplasts with activated caspase-3. B, 224 cells;
C, HCT 116 wt cells; D, HCT 116 p53 /
cells.
/
HCT 116 cytoplasts,
respectively. Similar levels of induction of caspase-3 activation were
observed in wt and p53
/
cytoplasts after treatment with cisplatin
(Fig. 1, C and D).
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Fig. 2.
Cisplatin induces nucleus-independent Bak
activation. 224 cells and cytoplasts as well as cytoplasts from
HCT 116 human colon cancer cells were treated with cisplatin with the
indicated doses for 16 h. Bak modulation was then monitored by
flow cytometry using an antibody that specifically recognizes the
activated conformation of Bak. The results are shown as fold increases
in Bak-associated immunofluorescence. A, 224 cells and
cytoplasts. B, HCT 116 wt and p53 /
cytoplasts.
C, cleaved Bid (14-kDa tBid) may contribute to Bak
activation. Shown here is cleavage of Bid in cisplatin-treated 224 cytoplasts (20 µM, 16 h).
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Fig. 3.
Effects of a calpain inhibitor and a calcium
chelator on caspase-3 activation in cytoplasts. 224 cytoplasts
were treated as indicated. Caspase-3 activity was assessed by flow
cytometry after 16 h of cisplatin treatment (20 µM)
in the presence or absence of a calpain inhibitor, calpeptin (10 µM), or a calcium chelator, BAPTA-AM (10 µM). The results are shown as percentages of cells with
activated caspase-3. A, calpeptin; B,
BAPTA-AM.
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Fig. 4.
Cleavage of caspase-12 in cisplatin-treated
cells and cytoplasts. 224 cells (A) or cytoplasts
(B) were treated with 20 µM cisplatin for the
indicated time periods in the presence or absence of inhibitors.
Cleavage of full-length 60-kDa caspase-12 was examined by Western
blotting of cell lysates. The indicated relative amounts were assessed
by laser densitometry and corrected for loading. A,
top blot, 4 h. lane 1, control; lane
2, cisplatin; lane 3, cisplatin + calpeptin (10 µM). Bottom blot, 8 h. lane 1,
control; lane 2, cisplatin; lane 3, cisplatin + BAPTA-AM (10 µM); lane 4, cisplatin + calpeptin (10 µM). B, top blot,
4 h. Bottom blot, 16 h. lane 1,
control; lane 2, cisplatin. C, to investigate
caspase-3 and -7 activation, DEVDase activity against Ac-DEVD-AMC was
measured fluorimetrically in extracts from cisplatin-treated 224 cells
and cytoplasts at the indicated time points. The results are shown as
inductions of activity relative to that in control samples.
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Fig. 5.
Induction of Grp78 expression in
cisplatin-treated cells. 224 cells were treated with 20 µM cisplatin for 16 h. The resulting Grp78 protein
levels were assessed by Western blotting of cell lysates (A)
or FACS analysis (B). A, the relative amounts are
indicated as assessed by laser densitometry and corrected for loading.
B, the results are shown as a histogram for Grp78-associated
immunofluorescence. Gray peak, control cells; dark
line, cisplatin-treated cells. The shift in the histogram
represents increased Grp78-associated immunofluorescence. C,
effects of ROS scavenger NAC (5 mM) and caspase inhibitor
zVAD-fmk (20 µM) on increased expression of Grp78 after
cisplatin treatment. The cells were treated with 20 µM
cisplatin for 16 h in the presence or absence of inhibitor, and
the Grp78 protein levels were assessed by FACS analysis. The results
are shown as fold increases in Grp78-associated
immunofluorescence.
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Fig. 6.
Correlation between caspase-3 activation and
Grp78 levels. 224 cells were treated with 20 or 30 µM cisplatin for 16 h and 20 h.
Digitonin-permeabilized cells were double-stained with antibodies
against active caspase-3 and Grp78, respectively, for FACS analysis.
The values in each upper left corner represent the
percentages of cells in this quadrant. Active caspase-3 was monitored
using fluorescein isothiocyanate-conjugated antibody (all x
axes), and the percentage of cells showing activation of caspase-3 is
also indicated. The levels of Grp78-associated immunofluorescence (all
y axes) are also shown as median values. Panel a,
untreated cells; panel b, 20 µM cisplatin,
16 h; panel c, 30 µM cisplatin, 16 h; panel d, 20 µM cisplatin, 20 h;
panel e, 30 µM cisplatin, 20 h.
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Fig. 7.
Etoposide-induced apoptosis requires nuclear
events. A, no caspase-3 activation was observed in
etoposide-treated cytoplasts. 224 cells and cytoplasts were treated
with the indicated doses of etoposide for 16 h. Caspase-3 activity
was assessed by flow cytometry. The results are presented as
percentages of cells with activated caspase-3. B, etoposide
failed to induce expression of Grp78. 224 cells were treated with 15 µM etoposide for 16 h. The levels of Grp78 were
examined by Western blotting. C, no cleavage of
procaspase-12 was seen in etoposide-treated cells. 224 cells were
treated with 15 µM etoposide for 4 and 8 h, and
extracts were made for Western blotting. The numbers
represent relative levels of protein assessed by laser
densitometry.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
converting enzyme) subfamily of caspases. The amino acid sequence of
caspase-12 shows high homology with human caspase-4 (48% identity) and
caspase-5 (45% identity). The monoclonal antibody used in the present
study has been shown to detect a human counterpart of caspase-12 and to
locate it to the ER in HeLa cells (17). The same antibody has also been
used for detection of caspase-12 cleavage in human epithelial cells
(32).
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ACKNOWLEDGEMENT |
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We are grateful for the kind gift of caspase-12 antibody from Dr. J. Yuan (Harvard Medical School).
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FOOTNOTES |
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* This work was supported by the Gustaf V Jubilee Foundation and by the Swedish Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
46-8-51-77-54-60; Fax: 46-8-33-90-31; E-mail:
mimmi.shoshan@onkpat.ki.se.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M210284200
2 A. Mandic, J. Hansson, S. Linder, and M. C. Shoshan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1; ER, endoplasmic reticulum; wt, wild type; PBS, phosphate-buffered saline; PI, propidium iodide; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy methyl ester); ROS, reactive oxygen species; NAC, N-acetyl cysteine; mtDNA, mitochondrial DNA; FACS, fluorescence-activated cell sorter.
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REFERENCES |
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1. |
Mandic, A.,
Viktorsson, K.,
Molin, M.,
Akusjärvi, G.,
Hansson, J.,
Linder, S.,
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