Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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In this study, we show that caspases 2, 3, 6, and 7 were activated during peroxynitrite-induced apoptosis in human leukemia HL-60 cells and that processing of these caspases was accompanied by cleavage of poly(ADP-ribose) polymerase and lamin B. Treatment of cells with DEVD-fluoromethyl ketone (FMK), a selective inhibitor for caspase 3-like proteases, resulted in a marked diminution of apoptotic cells. VAVAD-FMK, an inhibitor of caspase 2, partially inhibited the apoptotic response to peroxynitrite. However, selective inactivation of caspase 6 by VEID-FMK did not affect apoptosis rates. These data suggest that caspase 3-like proteases and caspase 2, but not caspase 6, are required for peroxynitrite-induced apoptosis in this cell type. Moreover, we demonstrate that peroxynitrite treatment stimulated activation of caspases 8 and 9, two initial caspases in the apoptotic signaling pathway, and preincubation of cells with their inhibitor, IETD-FMK, inhibited activation of caspase 3-like proteases and caspase 2 at the concentration that prevents the apoptosis. These observations, together, suggest that caspase 8 and/or caspase 9 mediates activation of caspase 3-like proteases and caspase 2 during the apoptosis induced by peroxynitrite in HL-60 cells.
poly(adenosine 5'-diphosphate-ribose) polymerase; lamin B; caspase cysteine protease
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INTRODUCTION |
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PEROXYNITRITE
(ONOO) is a reactive oxidant generated from nitric oxide
and superoxide anion that mediates a variety of biological processes,
including inhibition of leukocyte adhesion, blockage of platelet
aggregation, and induction of vasodilation in mammalian cells
(13, 19, 25, 45,
46). Recently, it has been shown that ONOO
can mediate apoptosis and necrosis (2) and that induction of apoptosis by ONOO
is associated with the pathogenesis
of diseases such as diabetes (35) and cardiac allograft
rejection (38). Although the mechanism by which
ONOO
causes apoptosis is not well defined, generation of
reactive oxygen species (18) and DNA strand breaks
(29) contribute to the peroxynitrite-induced
apoptosis. Caspase 3-like proteases are also involved in the
apoptosis of HL-60 cells (17, 43).
Caspases are a group of cysteine-dependent aspartate-directed proteases that mediate apoptosis induced by a variety of stimuli. To date, at least 10 mammalian caspases have been identified (44). Based on genetic and biochemical studies, caspases can be tentatively divided into three categories: group I includes caspases 1, 4, and 5; group II includes 3, 6, and 7; and group III includes caspases 2, 8, 9, and 10. Caspases are constitutively present in cells as zymogens and need to be proteolytically cleaved into the catalytic active heterodimer. Although all activated caspases consist of a 17- to 20-kDa large subunit and a small subunit of around 10 kDa, the functions of various caspases differ: group I caspases are generally considered to be required for cytokine maturation; group II caspases are effector caspases that are involved in the execution phase of apoptosis; and group III caspases function as initiator caspases that mediate the early apoptotic signaling (44). It has been shown that caspase 8 is a proximal activator in initiating activation of effector caspases in Fas-induced apoptosis (1). Binding of Fas ligand to the Fas receptor results in autoprocessing and activation of caspase 8. Active caspase 8 directly engages the caspase cascade by activating caspase 3. Alternatively, the release of cytochrome c from mitochondria can initiate a caspase cascade through binding of apoptotic protease-activating factor 1 (Apaf-1), which facilitates the activation of procaspase 9 (15). Active caspase 9 then cleaves and activates caspase 3 and other caspases (20).
Activation of effector caspases induces cleavage of a number of
targeted proteins in the progression of apoptosis, such as poly(ADP-ribose) polymerase (PARP), lamins, gelsolin, protein kinase
C, and p21-activated kinase 2 (42, 44).
Although the biological significance of these proteolytic cleavages and
their relationship with the ensuing apoptotic morphology is not well known, it has been proposed that the specific cleavage of PARP may
interfere with its key homeostatic function as a DNA repair enzyme and
could allow Ca2+/Mg2+-dependent endonucleases
to cause internucleosomal DNA fragmentation (40). The
proteolysis of lamins may also be responsible for some of the nuclear
changes in cells undergoing apoptosis, because they play a major role
in nuclear envelope integrity (12). Previous studies have
shown that PARP can be cleaved by most caspases under somewhat extreme
conditions in vitro, but in vivo PARP is mainly targeted by caspases 3 and 7 (30, 41). So far, caspase 6 is the only
caspase known to be able to cleave lamin A (27,
39), although other, as yet untested, caspases have also
been suggested to be laminases in in vitro systems (48).
Lamin B is structurally related to lamin A (26), and its
degradation occurred earlier than that of lamin A in
Fas-induced apoptosis of HeLa cells (21).
Although it has been reported that caspase 3-like caspases mediate
ONOO-induced apoptosis (17,
43), whether other caspases are activated and involved in
this apoptotic process is not clear. In the present study, we show that
ONOO
activates multiple caspases, including caspases 2, 3, 6, and 7, and induces cleavage of PARP and lamin B. Of these
activated caspases, however, only caspase 3-like caspases and caspase 2 are involved in the apoptotic response. Furthermore, we show that ONOO
activates caspases 8 and 9, and they are also
required for activation of caspase 3-like proteases and caspase 2 and
apoptosis, suggesting that caspase 8- and/or caspase 9-mediated
activation of caspase 2 and caspase 3-like caspases may contribute to
ONOO
-induced apoptosis in human leukemia HL-60 cells.
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MATERIALS AND METHODS |
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Materials.
ONOO and anti-caspases 1, 6, 8, and 10 were purchased
from Upstate Biotechnology; clone c 2-10, anti-poly (ADP-ribose)
antibody was from Biomol; anti-caspases 2 and 3 were from Transduction Laboratories; anti-caspase 9 and cytochrome c antibody were
obtained from PharMingen; the colorimetric synthetic peptide
substrates acetyl- Asp-Glu-Val-Asp-7-amino-4-p-nitroanilide
(Ac-DEVD-pNA), acetyl-Z-Val-Glu(OMe)-Lle-Asp(OMe)-4-p-nitroanilide
(Ac-VIED-pNA), acetyl-Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-4-p-nitroanilide
(Ac-VDVAD-pNA), acetyl-Lle-Glu-Thr-Asp-4-p-nitroanilide
(Ac-IETD-pNA), and
acetyl-Z-Leu-Glu(OMe)-His-Asp(OMe)-4-p-nitroanilide (Ac-LEHD-pNA) and the caspase inhibitors
Z-Asp(OMe)-Glu(OMe)Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK),
Z-Lle-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD-FMK),
Z-Val-Glu(OMe)-lle-Asp(OMe)-CH2F (Z-VEID-CH2F), and
Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VDVAD-FMK) were
supplied by Enzyme Systems Products. All chemicals and monoclonal anti-actin antibody (clone AC-40) were from Sigma Chemical.
Cell culture. Human promyelocytic leukemia HL-60 cells were grown in suspension in RPMI 1640 medium supplemented with 20% fetal bovine serum in the absence of antibiotics. Cells were passaged twice a week and used between passages 20 and 40.
ONOO treatment.
The procedure for ONOO
treatment was performed as
described by Lin et al. (17) with minor modification.
Briefly, HL-60 cells were harvested, washed once with PBS without
Ca2+ and Mg+, and diluted to 1 × 106 cells/ml. Stock ONOO
was added to the
cells suspended in PBS (pH 8.7), and they were incubated for 15 min at
37°C. Cells were incubated for 15 min in PBS (pH 8.7) without
ONOO
as control. After washing with PBS (pH 7.4), treated
or untreated cells were suspended in culture medium and then incubated
for the indicated times needed for each experiment. To make sure that the observed effects were actually caused by ONOO
rather
than its decomposing products, cells were also treated with degraded
ONOO
under conditions used for ONOO
in some
experiments before incubation in the culture medium. When required,
caspase inhibitors were added to cell culture in PBS 1 h before
ONOO
treatment.
Nuclear staining assay. After treatment and incubation, cells were harvested, fixed with methanol, and then incubated for 15 min in diamidino phenylindole (DAPI) solution (1 mg/ml). The nuclear morphology of the cells was observed under a fluorescent microscope. Cells with condensed or fragmented nuclei were considered to be apoptotic. Five hundred cells were counted for each sample, and the numbers of apoptotic cells were expressed as the percentage of the total cell population.
DNA fragmentation assay. Detection of DNA fragmentation was performed as described previously (49). Briefly, cells (2 × 106) were pelleted and then resuspended in lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0, and 1% SDS). After incubation in proteinase K (10 µg/ml) at 56°C overnight, and then in ribonuclease A (10 µg/ml) for 2 h at 37°C, DNA was extracted with 2-propanol. Pelleted DNA was resuspended in Tris-EDTA buffer, separated by horizontal electrophoresis on a 1.5% agarose gel, stained with 0.5% ethidium bromide, and visualized under ultraviolet light.
Western blot analysis. Cells were centrifuged for 5 min at 200 g, washed once with PBS, and then suspended in lysis buffer (0.25 M Tris·HCl, pH 6.8, 4% SDS, 10% glycerol, 0.1 mg/ml bromphenol blue, and 0.5% 2-mercaptoethanol). After sonication for 15 s, equal amounts of the total cellular protein lysates were separated on 10% polyacrylamide gels. The proteins were electrophoretically transferred to a nitrocellulose membrane. After treatment with 5% skim milk at 4°C overnight, the membranes were incubated with various antibodies for 1 h, followed by appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham). Bound antibodies were visualized using standard chemiluminescence on autoradiographic film.
Caspase activity assay.
Activity levels of the caspases were measured by a colorimetric assay
following the manufacturer's instructions. Briefly, untreated cells or
cells treated with ONOO were incubated in the complete
medium for the indicated times and then harvested. Cells (2 × 106) were lysed in 0.1 M HEPES buffer (pH 7.4) containing 2 mM dithiothreitol, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 1%
sucrose. Cell lysates were incubated with a colorimetric substrate,
Ac-DEVD-pNA, Ac-VDVAD-pNA, Ac-VEID-pNA, Ac-IETD-pNA, or Ac-LEHD-pNA for
30 min at 30°C. The release of chromophore p-nitroanilide
was measured with excitation at 400 nm with the use of a fluorescence
spectrophotometer (Spectra Max 340 PC).
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RESULTS |
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Induction of apoptosis by ONOO in HL-60 cells.
To test the apoptotic response after ONOO
treatment in
HL-60 cells, two procedures were performed. Nuclear staining was done using the chromatin dye, DAPI, and the change in nuclear morphology was
observed after ONOO
treatment. As described previously,
treatment of cells with ONOO
induced apoptotic morphology
characterized by fragmented or condensed nuclei (16) (data
not shown). These changes were in a dose-dependent manner, with the
maximum induction (36%) at 200 µM ONOO
(Fig.
1A). In contrast, few
cells (less than 5%) showed changes in nuclear morphology in the
degraded ONOO
-treated or the untreated samples (Fig. 1,
A and C). Time course analysis showed that
apoptotic cells began appearing in significant numbers at 2 h and
increased until 4 h after ONOO
treatment (Fig.
1C). However, the degraded ONOO
did not lead
to significant change of apoptotic rate with increasing incubation time
(Fig. 1C).
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ONOO induces cleavage of PARP and lamin B.
Although the mechanism for formation of the biochemical features of
apoptosis is not well defined, it has been proposed that proteolytic
cleavage of certain substrates by active caspases are involved in this
process. PARP and lamin B are reported to be required for fragmentation
of DNA and nuclear condensation (41). Thus we examined the
effect of ONOO
on the cleavage of PARP and lamin B. Immunoblot analysis showed that treatment of cells with
ONOO
caused the proteolytic cleavage of PARP with
accumulation of an 89-kDa fragment and the concomitant disappearance of
the original 116-kDa PARP. Cleaved PARP was detected at 2 h, and
most of the original PARP was degraded at 4 h after treatment
(Fig. 2, A and B).
Lamin B is a 69-kDa protein that was cleaved into 32-kDa fragments in
cells treated with ONOO
. Cleavage kinetics of lamin B
were similar to those of PARP, although the degree of its cleavage is
much lower than that of PARP (Fig. 2, A and B).
These results indicate that ONOO
induces cleavage of at
least two substrates in HL-60 cells, implying that caspases able to
cleave these two substrates could be activated by this oxidative
stress.
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ONOO induces activation of caspases 3 and 7.
Caspase 3-like caspases, including caspases 3 and 7, are the major
proteases responsible for the cleavage of PARP (30,
41). To assess whether these caspases were activated by
ONOO
in HL-60 cells, we first analyzed the enzymatic
activities of caspases 3 and 7 using colorimetric DEVD-pNA as a
substrate. DEVD is a synthetic peptide that mimics the PARP site
cleaved by these caspases. As shown in Fig.
3A, ONOO
treatment of HL-60 cells was accompanied by a marked increase in
cleaved DEVD-pNA. Compared with controls, there was a twofold increase
in DEVD-pNA cleavage within 2 h of incubation of
ONOO
with cells and a fourfold increase within 4 h
after ONOO
treatment, indicating that caspase 3-like
proteases are activated during apoptosis induced by ONOO
.
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ONOO induces activation of caspases 2 and 6.
Although caspase 6 may be a major laminase in cells undergoing
apoptosis, in vitro studies suggest that other caspases may also cleave
lamins (27, 39). Because lamin is a
structural protein of the nuclear envelope, the caspases with an
ability to localize in the nucleus may be the best candidates. Caspase 2 is one such protease; both its precursor and processed fragments have
been found to be distributed in the nucleus (6). With the
use of VDVAD-pNA as an indicator substrate for caspase 2 activation and VEID-pNA for caspase 6, we examined the effect of
ONOO
on the activation of these two caspases and
demonstrated that treatment of cells with ONOO
dramatically increases the levels of VDVAD-pNA cleavage activity, with
similar kinetics to DEVD-pNA cleavage (Fig.
4A). VEID-pNA cleavage
activity also increased during the course of ONOO
-induced
apoptosis, albeit at much lower levels than VDVAD cleavage (Fig.
4A). These results suggest that ONOO
also
stimulates activation of caspases 2 and 6.
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Caspase 3-like proteases and caspase 2, but not caspase 6, are
required for ONOO-induced apoptosis.
To analyze which caspases contribute to the apoptosis induced by
ONOO
, we investigated effects of several selective
caspase inhibitors on ONOO
-induced apoptosis. Previous
studies have shown that a cell-permeable inhibitor of caspase 3-like
proteases, DEVD-FMK, inhibited apoptosis induced by most stimuli in
various cell types at a concentration of 100 µM (7,
14, 36). When HL-60 cells were treated with this concentration of DEVD-FMK for 1 h and then exposed to
ONOO
, apoptotic cell death was largely inhibited (Fig.
5A), suggesting that caspase 3 and/or caspase 7 may play critical roles in mediating ONOO
-stimulated apoptosis. DEVD-FMK inhibited the
cleavage of PARP and, unexpectedly, lamin B (Fig. 5, B and
C), suggesting that this inhibitor may have an ability to
inhibit other caspases that mediate lamin B cleavage.
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DEVD-FMK inhibits processing of caspase 2.
Because both caspase 3 and caspase 2 are involved in
ONOO-stimulated apoptosis, we sought to explain the
unexpected result that caspase 3 inhibitor blocks lamin B cleavage by
examining interactions between caspases 2 and 3. As shown in Fig.
6, A and B,
treatment of cells with DEVD-FMK partially inhibited the processing of
caspase 2 as marked by an increase in the procaspase 2 form, whereas
loss of procaspase 3 was not affected by VDVAD-FMK. These data
correspond to the previous observations that DEVD-FMK blocks lamin B
cleavage, whereas PARP degradation is not affected by VDVAD-FMK (Fig.
5, B and C). In contrast, DEVD-FMK failed to
inhibit procaspase 6 cleavage. These results suggest that caspase 2 processing may be mediated, at least in part, by caspase 3-like
proteases in ONOO
-induced apoptosis, or that DEVD-FMK is
capable of inhibition of caspase 2 processing. Consistent with a
previous report that caspase 3 activation is induced partially through
self-catalytic cleavage (14), DEVD-FMK also inhibited the
cleavage of procaspases 3 and 7, caspases with structure similar to
that of caspase 3 (Fig. 6, A and B).
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ONOO induces activation of caspases 8 and 9.
To delineate the early signaling pathway that leads to activation of
effector caspases, we investigated the effect of ONOO
on
cleavage of caspases 8 and 9, two initial caspases. Caspase 8 is
synthesized as an inactive precursor of 54 kDa, is cleaved to a 44-kDa
intermediate product, and is ultimately expressed in its active form as
a p18 and p10 heterodimer (32). In untreated cells, the
antibody used can detect two protein bands that represent procaspase 8a
(55 kDa) and procaspase 8b (54 kDa). Figure
7A shows that treatment of
cells with ONOO
led to formation of a protein doublet,
the 44/41-kDa intermediate fragment, which was detectable at 2 h
and persisted until 4 h after treatment. However, the level of
procaspase 9 was kept intact, and active fragments were not detected
even after 4 h incubation, although the antibody used can detect
both 46-kDa procaspase 9 and the 37-kDa subunit of active caspase 9 (Fig. 7B). We also examined the effect of ONOO
on caspase 10 cleavage, though no small active fragment was detected, and the original form of this caspase remained intact during apoptosis (data not shown).
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IETD-FMK inhibits ONOO-induced apoptosis and cleavage
of caspases 2 and 3.
IETD-FMK, a selective inhibitor of caspases 8 and 9, was also evaluated
for its effect on ONOO
-induced apoptosis. Figure
8A shows that pretreatment of
cells with IETD-FMK led to dose-dependent inhibition of
ONOO
-stimulated apoptosis. At 100 µM, this inhibitor
reduced rates of apoptosis to near control levels. This result suggests
that these caspases 8 and/or 9 might mediate initial signaling in
ONOO
-induced apoptosis.
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DISCUSSION |
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Numerous studies have shown that caspase 3 is a major executioner
in the apoptosis induced by various stimuli in most cell types
(28). Induction of apoptosis by ONOO is also
mediated by caspase 3 in HL-60 human leukemia cells (17, 43). Recently, it has been reported that other caspases,
besides caspase 3, are also expressed in this cell line
(22). However, whether they are activated by
ONOO
and involved in apoptosis is not clear. In this
study, we show that multiple caspases are activated by
ONOO
. However, not all these caspases are required for apoptosis.
Consistent with what has been previously reported, caspase 3-like
proteases are essential for apoptosis in HL-60 cells exposed to
ONOO (17, 43). Treatment of
cells with ONOO
dramatically increases DEVD-pNA cleavage
activity (Fig. 3A). However, activation of caspase 3-like
proteases may not be the only mechanism leading to apoptosis in HL-60
cells exposed to ONOO
, because complete inhibition of
activation of these caspases with DEVD-FMK (by blockade of PARP
cleavage) only partially inhibited apoptosis (Fig. 5). In addition, it
is not clear whether both caspases 3 and 7 or just one of them are
involved in the apoptosis, although we do demonstrate that
ONOO
-stimulated apoptosis was accompanied by cleavage of
both caspases 3 and 7 (Fig. 3, B and C). Previous
studies have shown that caspase 3 and caspase 7 localize in distinct
subcellular compartments of cells (4), that caspase 7 can
be activated independently of caspase 3, and that there is no
significant functional redundancy between these two caspases
(10). These findings indicate that caspase 3 and caspase 7 may play distinct roles in the apoptotic signaling pathway. Because
analysis of caspase 3 knockout mice has shown that PARP cleavage in
apoptotic cells is seemingly unaffected by the loss of caspase 3 (23), caspase 7 activation may compensate for some of the
apoptotic biochemical events that are not mediated by caspase 3. The
development of an inhibitor specific for caspase 7 would help to
further elucidate this point.
Caspase 2 is a long prodomain caspase that is believed to play an
early acting role in the apoptosis induced by etoposide, -irradiation, or genotoxic agents (9). However, Li et
al. (14) have shown that activation of caspase 3 precedes
procaspase 2 processing in Fas-induced apoptosis and that caspase 3 has
an ability to cleave procaspase 2 to its small, active fragment. Recently, Swanton et al. (37) have provided further
evidence, in an in vitro study, that cleavage of caspase 2 is dependent on activation of caspase 3 after cytochrome c addition in
cytosolic extracts. In this study, we have demonstrated that caspase 2 mediates ONOO
-induced apoptosis (Fig. 5A).
However, we could not discern the kinetics of activation of caspase 2 and caspase 3, because the time course of activation for caspase 2 was
similar to that observed for caspase 3 (Fig. 3B and Fig.
4B). Based on the observation that inhibition of caspase
3-like caspases partially attenuated caspase 2 cleavage, whereas
caspase 3 processing was not affected by a caspase 2 inhibitor (Fig. 6,
A and B), it seems that caspase 3-like proteases
may function as upstream activators of caspase 2. However, the
possibility could not be ruled out that DEVD-FMK might directly inhibit
caspase 2 processing, because this caspase contains DXXD motif, which
is the sequence bound by DEVD-FMK (5). Further detailed
studies on kinetics of activation for caspases 2 and 3 during
ONOO
-induced apoptosis are needed before making any
conclusions on the sequence of activation of these caspases.
Although caspase 6 is also activated by ONOO, it appears
that this caspase is not required for apoptosis. This conclusion is
based on our observation that pretreatment of cells with
VEID-CH2F at a concentration of up to 100 µM did not
prevent nuclear fragmentation (Fig. 5A). We do not believe
the failure of this inhibitor to prevent apoptosis is due to the
concentrations used. VEID-CH2F is a highly selective
inhibitor that was synthesized based on the sequence within lamins
cleaved by caspase 6, but not by caspases 3 and 7 (39).
The activity of caspase 6 can be inhibited by VEID-CHO at
concentrations at least 100-fold lower than those required to inhibit
caspases 3, 4, 7, and 8. In Jurkat cells, 10 µM VEID-CHO is enough to
completely inhibit Fas-induced caspase 6 activation, as demonstrated by
the disappearance of its active fragment (10). In the
present study, 100 µM of VEID-CH2F blocked the cleavage
of lamin B but did not affect PARP degradation (Fig. 5, B
and C), revealing its effectiveness and high selectivity. These data, together with the finding that VEID-CH2F does
not affect caspase 3 processing, suggest that caspase 6 may not be involved in activation of caspase 3 in HL-60 cells. The significance of
caspase 6 activation during apoptosis induced by ONOO
is
not clear. One possibility is that caspase 6 is merely a bystander in
this mode of apoptosis. The findings that no clear role for caspase 6 was seen in Fas-induced apoptosis, and that caspase 6-deficient mice do
not display an overt phenotype (23), support this speculation.
Caspase-mediated cleavage of some specific target proteins is
associated with apoptotic changes in cellular morphology
(23). In this study, we demonstrated that PARP and lamin B
were cleaved after treatment with ONOO before significant
increases in numbers of apoptotic cells (Fig. 1, C and
D, and Fig. 2). PARP cleavage can be inhibited by DEVD-FMK, and lamin B cleavage blocked by VEID-CH2F (Fig. 5,
B and C), which is consistent with previous
reports that caspase 3-like caspases are proteases for PARP
(30, 41), and caspase 6 is a laminase (27, 39). Unexpectedly, treatment of cells
with DEVD-FMK also blocked cleavage of lamin B (Fig. 5B,
lane 3). To our knowledge, this is the first demonstration
that lamin B cleavage is regulated by DEVD-sensitive proteases.
Previous in vitro studies showed that inactivation of laminases and
PARP proteinases are distinct enzymatic activities and that caspases 3 and 7 do not cleave lamin A under conditions where caspase 6 does
cleave lamin A (27, 39). Thus
caspase 3-like caspases probably do not act as laminases. VDVAD-FMK, a
potent caspase 2 inhibitor, can block lamin B degradation and apoptosis
(Fig. 5, A-C), and DEVD-FMK partially inhibits caspase 2 processing (Fig. 6, A and B). This suggests the
possibility that caspase 2 is one of the proteases that mediates action
of caspase 3 in cleavage of lamin B. However, whether caspase 2 itself is a laminase or if it further activates other laminase(s) needs to be
further defined.
Our data show that ONOO induces activation of both
caspases 8 and 9, and their activation is required for induction of
apoptosis and activation of caspases 2 and 3 (Fig. 8). It was
previously demonstrated that caspase 8 cleavage is a rapid process in
Fas-induced apoptosis, with cleaved products detectable at as early as
5 s after receptor cross-linking in many cell lines
(24, 32). However, we did not see the cleaved
fragments of caspase 8 until 2 h after ONOO
treatment (Fig. 7A), arguing against the death
receptor-mediated mechanism in activation of this caspase by
ONOO
. Recently, Scaffidi et al. (31) have
reported that delayed cleavage of caspase 8 in some cell types occurs
downstream of mitochondrial cytochrome c release. Consistent
with this finding, we detected a marked increase in caspase 9 activity
at 2 h (Fig. 7C), when DNA fragmentation formed in
cells exposed to ONOO
. These data, together with the
observation that IETD-FMK inhibits cleavage of caspases 2 and 3, imply
that ONOO
may trigger cytochrome c release
from mitochondria, resulting in sequential activation of initiator and
effector caspases. It has been reported that ONOO
is able
to induce release of cytochrome c from heart mitochondria (3). Although caspase 8 has the same ability as caspase 9 in binding Apaf-1, a key step in caspase activation through released cytochrome c from mitochondria, no direct interaction
between caspase 8 and Apaf-1 has been demonstrated (11).
Further studies suggest that caspase 8 activation may be an event
downstream of initiator caspases in cytochrome c-mediated
apoptotic pathway, because its activation was diminished in cells from
caspase 9
/
and Apaf-1
/
mice in certain
contexts (8, 47), and because DEVD-FMK
inhibits caspase 8 activation in cytochrome c-mediated
apoptosis in HeLa cells after photodynamic therapy (PDT)
(7).
Though the activation of both effector caspases and initial caspases
induced during the course of apoptosis after ONOO
treatment is easily measured, differentiating the kinetic profiles of
their activation is more difficult. Results similar to this were
observed in PDT-induced apoptosis, a process involving cytochrome c-released mitochondria (7). It is not
clear how quickly these caspases are processed in vivo. Slee et al.
(33) have demonstrated in cell-free extracts that most
caspases, including caspases 2, 3, 6, 7, and 9, were cleaved and
activated within 30 min of addition of cytochrome c, with
the exception of caspase 8. The cleavage products of caspase 8 were
detectable at 1 h under the same conditions. This may explain our
own results, which show that all these caspases are cleaved and
activated almost simultaneously at 2 h after treatment. However,
our results also seem to indicate that caspase 8 plays a limited role
in the cascade, because its maximum processing is delayed relative to
effector caspases (Fig. 7, A and C). It is
possible that, like caspase 6, this caspase is simply a side product in
this process. If this is the case, the inhibition of apoptosis by
IETD-FMK observed in this study could be through a blockade of caspase
9 activation. This possibility is worthy of further investigation.
In summary, we have demonstrated that ONOO-induced
apoptosis is associated with activation of caspase 3-like proteases and caspase 2, although multiple caspases that cleave PARP and lamin B are
activated in HL-60 cells. The precise sequence of activation of these
caspases is not clear; cytochrome c-mediated sequential activation of caspase 9 and caspases 2 and 3 may be involved in this
process. Because ONOO
-mediated apoptosis is implicated in
the pathogenesis of many disorders, further elucidation of the
mechanism of ONOO
-induced apoptosis could provide a basis
for the design of therapeutic interventions.
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ACKNOWLEDGEMENTS |
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We thank Dr. Irene E. Kochevar for generous support of this project and Xiaochu Duan and Mary C. Lynch for assistance.
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FOOTNOTES |
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This work was supported by National Institute of General Medical Sciences Grant GM-30955.
Address for reprint requests and other correspondence: S. Zhuang, Wellman Laboratories of Photomedicine, Massachusetts General Hospital, WEL-224, 55 Fruit St., Boston, MA 02114.
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. §1734 solely to indicate this fact.
Received 4 October 1999; accepted in final form 16 February 2000.
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