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Centre National de la Recherche Scientifique,
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Unité Propre de Recherche (UPR) 420, and
UPR 9044, Villejuif, France; and
Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In various tumor cells undergoing apoptosis, multiple species of caspase-3 and caspase-6 constitute the major pool of activated caspases regardless of the initial apoptotic stimulus (12). In addition to caspase-3 and caspase-6, caspase-2 and caspase-7 are activated during the execution phase of apoptosis in human monocytic tumor cells (13). In the cells (except those in the brain) of caspase-3-deficient mice, both PARP cleavage and apoptosis occur normally, demonstrating that other caspases with overlapping substrate specificity (probably caspase-7) can replace caspase-3 (14). Thus, it appears that caspases-3, -6, and -7 are the major effector caspases, with some possible contribution of caspase-2. It has been proposed that activator caspases, such as caspase-8, acting near or at the apex of the hypothetical caspase cascade, activate the effector caspases. The identity of the activator caspases is less clear, although there is evidence that caspase-8 (MACH/FLICE/Mch5) is recruited to the Fas signaling complex and initiates the caspase cascade involved in Fas-mediated apoptosis (15, 16, 17, 18). Also, activation of caspase-10 (Mch4) has been proposed to be the primary mechanism by which granzyme B initiates cell death (19). Caspases-8 and -10 can, in fact, process/activate all known procaspases as well as each other (17, 20).
Caspase inhibitors of differing specificities have also been used to implicate a critical role for caspases in the execution phase of apoptosis (10, 11). Both Z-VAD.fmk and BOC-D.fmk, two cell-permeable, broad spectrum caspase inhibitors, suppress apoptosis induced by a wide variety of stimuli (21, 22, 23, 24, 25). Additionally, BOC-D.fmk suppresses apoptosis in some Z-VAD.fmk-resistant systems, such as apoptosis triggered in activated mouse lymphoblasts by several independent stimuli (22), suggesting that each inhibitor can target distinct members of the caspase family. In intact cells, Z-VAD.fmk inhibits the processing of caspases-2, -3, -6, and -7 (13, 23) and hence may prevent the activity of an upstream caspase, possibly caspase-8 and/or caspase-10. This assumption is supported by the fact that Z-VAD.fmk prevents the activity of caspase-8 (18). Bcl-2, but not Z-VAD.fmk, blocks the release of cytochrome c from mitochondria during apoptosis (26, 27). Cytochrome c binds to apoptosis protease-activating factor-1, a human protein homologous to C. elegans CED-4 (28), and in the presence of dATP results in the activation of caspase-9 and the initiation of the caspase cascade (29).
In this study, we compared the effects of Z-VAD.fmk and
BOC-D.fmk on apoptosis induced in activated human T
lymphocytes via CD2 and via Fas, two receptors that initiate
independent death pathways (30, 31). We extended this approach to the
apoptotic pathway elicited in these cells by staurosporine, a broad
spectrum kinase inhibitor and a ubiquitous inducer of apoptosis (32)
whose lethal effect is generally blocked by Z-VAD.fmk (23). We
demonstrate that in activated T cells, CD2 and staurosporine can induce
a caspase-independent death pathway, contrasting with the
caspase-dependent pathway induced by Fas. It appears that in the
absence of caspase activation, cytoplasmic events related to apoptosis,
such as cell shrinkage, cytoplasmic condensation, loss of mitochondrial
membrane potential (
m), and exposure of phosphatidylserine, are
readily triggered. However, caspase activation is required for the
triggering of characteristic biochemical and morphologic nuclear
changes associated with apoptosis, including extreme chromatin
condensation, internucleosomal cleavage of DNA, and proteolysis of
nuclear substrates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBL were isolated from blood bank leukopheresis packs obtained from healthy volunteers of both sexes (through the courtesy of the Blood Transfusion Center from Hôpital Saint Louis, Paris, France). After Ficoll-Isopaque density (density = 1.078) gradient centrifugation, adherent cells were removed by incubation on plastic dishes for 30 min at 37°C, and the rest of the cells was fractionated by filtration over nylon wool columns.
mAbs and other reagents
Purified CD2 mAb were GT2 (IgG1), T111 (IgG1), and
D66 (IgM) from Prof. A. Bernard (INSERM, Unit 343, Nice, France).
Anti-Fas mAb CH-11 (IgM) was purchased from DIACLONE (Besançon,
France), and anti-CD3 mAb OKT3 (IgG2a) was obtained from American
Type Culture Collection (Manassas, VA). In some experiments, an
antagonistic anti-Fas mAb (provided by Dr. D. Lynch from Immunex
Research and Development, Seattle, WA) and a neutralizing rabbit
antiserum to TNF-
(provided by Dr. J. Wietzerbin, INSERM, Unit 365)
were used to block activation-induced cell death (AICD). Z-VAD.fmk,
BOC-D.fmk, and ZFA.fmk (benzyloxycarbonyl-Phe-Ala-fluoromethylketone)
were purchased from Enzyme Systems Products (Dublin, CA), dissolved at
stock solutions of 100 mM in DMSO, and stored at -20°C.
Ac-Ile-Glu-Thr-L-aspartic acid aldehyde (Ac-IETD-CHO) was
purchased from Bachem (Voisins-le-Bretonneux, France), and
Z-Ala-Ala-Asp-CH2Cl (Z-AAD-cmk) was obtained from
Calbiochem (France Biochem, Meudon, France). Etoposide (VP16) was
purchased from Sigma (St. Quentin Fallavier, France).
Culture conditions and induction of cell death
T lymphocytes (6 x 106) were cultured in the wells of six-well flat-bottom plates (Nunc, Roskilde, Denmark) in 6 ml of RPMI 1640 medium supplemented with 10% FCS and antibiotics. Stimulation of the cells was performed by using the mitogenic GT2+T111 CD2 mAb pair (2 µg/ml) plus 100 U/ml IL-2 (Roussel-UCLAF, Romainville, France) or by using 0.25 µg/ml OKT3 plus IL-2. After a 4- or 5-day culture period, the cells were washed, replaced in IL-2-containing medium, then exposed to the death signals delivered by the T111+D66 CD2 mAb pair (2 µg/ml), by anti-Fas mAb (2 µg/ml), or by staurosporine (0.1–0.5 µM).
Flow cytometric analysis of cell death
Cells were stained with 5 µg/ml propidium iodide (PI) 10 min
before examination to detect permeable dead cells (FL3 positive) using
a FACScan (Becton Dickinson, Paris, France) or a Coulter
EPICS Profile II cytofluorometer (Margency, France). To evaluate
mitochondrial m, cells were stained for 15 min at 37°C with 40
nM of the potential sensitive fluorescent dye DiOC6
(3,3'-diethyloxacarbocyanine) from Molecular Probes (Interchim,
Montluçon, France). Measurement of phosphatidylserine
externalization was performed by staining the cells with annexin V-FITC
from Boehringer Mannheim (Meylan, France), according to the
manufacturers instructions.
Hypodiploid cell assessment and microscopic detection of chromatin condensation
Cells (5 x 105) were washed twice in PBS with 5.5 mM glucose and fixed overnight in ethanol (70% in water, at 4°C). Cells were then resuspended in 0.5 ml of PBS containing 50 µg/ml PI and 100 U/ml RNase A (Sigma) and incubated for 30 min at room temperature under agitation. The DNA content of 104 cells was monitored by cytofluorometry using a Coulter EPICS profile II analyzer.
Immunoblot analysis
Cells (5 x 105) were washed and solubilized in 20 µl of Laemmli buffer. Cell lysates were then subjected to SDS-PAGE and electroblotted onto a nitrocellulose membrane. Cleavage of PARP was determined using the C2-10 mAb (purchased from G. Poirier, Montreal University, Montreal, Canada) (33); that of lamin B was determined using an anti-lamin B polyclonal rabbit antiserum provided by J. C. Courvalin (34). For detection of the cleavage products of caspase-3, we used an anti-caspase-3 rabbit antiserum (PharMingen, represented by Becton Dickinson, Le Pont de Claix, France) or an anti-caspase-3 mAb from Transduction Laboratories (Interchim, Montluçon, France) raised against the p17 subunit; both detected procaspase-3 and its large subunits. The rabbit polyclonal Abs to caspase-2, which detected procaspase-2 and its p12 small subunit, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal Abs to caspase-7, which detected procaspase-7 and the p20 large subunit, were raised as previously described (13). Blots were stained with either anti-mouse or anti-rabbit horseradish peroxidase-labeled secondary Ab (Amersham, Les Ulis, France). They were then developed using an enhanced chemiluminescence detection system (ECL kit, Amersham). Films were exposed for 1 to 15 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that the induction of apoptosis in
activated human T lymphocytes via Fas and CD2 receptors occurs by two
independent pathways (30, 31). To determine whether caspase activation
was equally involved in these pathways, we examined their sensitivities
to the caspase inhibitors, Z-VAD.fmk and BOC-D.fmk. The
irreversible cathepsin B inhibitor, Z-FA.fmk, was used as a control, as
it lacks an Asp in the P1 position and therefore does not inhibit
caspase activity (22). We also studied the effects of these inhibitors
on apoptosis induced by staurosporine, a universal inducer of apoptosis
(32). The cells were preincubated for 4 h with a range of
concentrations of the peptide inhibitors (0.01–100 µM) to allow them
to enter the cells. Z-VAD.fmk and BOC-D.fmk, but not Z-FA.fmk,
protected activated T lymphocytes from anti-Fas-mediated apoptosis
(routinely visualized by PI uptake) in a concentration-dependent manner
(Fig. 1
). Z-VAD.fmk was somewhat more
effective than BOC-D.fmk, with significant inhibition observed at 1
µM and almost complete protection afforded at 10 µM. In marked
contrast, these inhibitors, even at 100 µM, did not afford any
protection against CD2- and staurosporine-induced cell death, at odds
with the blocking effect exerted by Z-VAD.fmk in numerous cellular
models (23). Doses-response experiments further indicated that
Z-VAD.fmk and BOC-D.fmk (at 50 µM) were unable to prevent cell death
regardless of the strength of the CD2 or staurosporine apoptotic
stimulus (Fig. 2), suggesting that the
caspase inhibitors were present at concentrations adequate to fully
control their target caspases.
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Z-FA.fmk did not block the number of annexin V+
cells induced by any of the apoptotic stimuli.
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m, whose decrease represents an early and
irreversible step in the process of apoptosis (36). Anti-Fas,
anti-CD2, and staurosporine all induced a rapid increase in the
percentage of cells with low m (Fig. 4). In anti-Fas-treated lymphocytes,
the fall of m was abrogated by both Z-VAD.fmk and BOC-D.fmk, but
not by Z-FA.fmk, while it remained unaffected by all three caspase
inhibitors in anti-CD2- and staurosporine-treated lymphocytes.
Taken together, these results strongly suggested that in activated T
lymphocytes, cell death induced by anti-CD2 and staurosporine was
distinct from that induced by anti-Fas, apparently not requiring
caspase activity but retaining some apoptotic features.
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Cleavage of the 116-kDa polypeptide PARP to its characteristic
85-kDa fragment is considered a marker of apoptosis (33, 37). Although
this cleavage can be mediated by a number of caspases (10), caspase-3
and caspase-7 appear to be the most important (33, 38, 39, 40). Proteolysis
of lamins, the major structural proteins of the nuclear envelope, is
also observed in cells undergoing apoptosis and is apparently mediated
by caspase-6 rather than by caspase-3 or caspase-7 (41, 42). In
activated peripheral T lymphocytes exposed to anti-CD2,
anti-Fas, and staurosporine, both PARP and lamin B1 (the most
abundant lamin in T lymphocytes (34)) were cleaved to their
characteristic 85- and 46-kDa signature fragments, respectively.
Z-VAD.fmk and BOC-D.fmk, but not Z-FA.fmk, prevented PARP and lamin B1
cleavage irrespective of the apoptotic stimulus (Fig. 5, lanes 3–5). Inhibition of
PARP and lamin B1 proteolysis by the caspase inhibitors was long
lasting; it was still effective 24 h after apoptotic signaling
(not shown). Thus, the activities of at least caspases-3, -6, and-7
were apparently not required for the execution of anti-CD2- and
staurosporine-induced cell death in activated human T lymphocytes.
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To determine whether inhibition of caspases with PARP- and
lamin-cleaving activities altered the morphology of T lymphocytes
undergoing anti-CD2- and staurosporine-mediated cell death, we
examined the ultrastructure of these cells. In the absence of
Z-VAD.fmk and BOC-D.fmk, treatment of activated T cells with
anti-Fas, anti-CD2, or staurosporine induced varying degrees of
ultrastructural changes characteristic of apoptosis, including
condensation of the cytoplasm and the chromatin, disintegration of the
nucleolus, and, in the case of anti-CD2- and anti-Fas-mediated
apoptosis, dilatation of the endoplasmic reticulum and subsequent
formation of vesicles, some of which were fused with the cell membrane
to give a bubbling appearance (Fig. 6).
Z-VAD.fmk blocked all of these ultrastructural changes in
anti-Fas-treated cells. In marked contrast, it blocked only some,
not all, of the nuclear changes induced by anti-CD2 and
staurosporine. In these cases, the cells exhibited clumps of partially
condensed chromatin but did not possess the crescents of completely
condensed chromatin seen in cells exposed to these apoptotic stimuli in
the absence of Z-VAD.fmk (Fig. 6). It has been proposed that such
changes are consistent with the inactivation of caspase-6 and the
subsequent lack of lamin cleavage (43). Similar results were obtained
with BOC-D.fmk (data not shown). In anti-CD2- and
staurosporine-treated cells, many of the prominent features of
apoptosis, such as condensation of the cytoplasm and dilatation of the
endoplasmic reticulum (mainly seen in anti-CD2-treated cells), were
not blocked by Z-VAD.fmk, although in the latter cells the dilated
endoplasmic reticulum was not fused with the plasma membrane. Cell
shrinkage was also confirmed by forward scatter analysis of the cells
(not shown). Thus, many, but not all, of the ultrastructural changes of
apoptosis were taking in place in cells whose PARP- and lamin-cleaving
activities were inhibited by Z-VAD.fmk and BOC-D.fmk.
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The partial chromatin condensation displayed by apoptotic T
lymphocytes resisting the protection afforded by Z-VAD.fmk and
BOC-D.fmk suggested that the caspase inhibitors may have altered DNA
fragmentation. We therefore measured the effects of these inhibitors on
the appearance of hypoploid cells whose decreased DNA content has been
shown to result from internucleosomal DNA cleavage (44). The generation
of hypodiploid cells induced by anti-Fas, anti-CD2, and
staurosporine was, in fact, inhibited by Z-VAD.fmk and BOC-D.fmk in all
cases (Fig. 7). These results, together
with the lack of highly condensed chromatin observed in the
ultrastructural studies, confirm that the caspase inhibitors
preferentially affected some, but not all, nuclear manifestations of
CD2- and staurosporine-induced apoptosis.
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Given that Z-VAD.fmk and BOC-D.fmk were unable to inhibit all the
features of a classical apoptotic phenotype in anti-CD2- and
staurosporine-treated cells, we wished to examine the processing of
some of the caspases classically involved in the execution phase of
apoptosis. The cells were lysed with Laemmli buffer containing 3% SDS,
which should avoid the postlysis proteolytic processing of caspases-3
and -7 that can occur when activated T cells are lysed with RIPA
buffer (45). Granzyme B is, in fact, released from cytotoxic granules
during the lysis procedure of activated T cells, and if not
neutralized, it will activate several caspases, including caspase-3
(46) and caspase-7 (47). In some experiments (not shown), the cells
were incubated, before lysis, with PBS containing 300 µM Ac-IETD-CHO
or Z-AAD-cmk, two peptides that serve as granzyme B substrates and
should inhibit its proteolytic action. In these conditions, immunoblot
analysis revealed that control activated T cells possessed the 32-kDa
proform of caspase-3 together with the p20 subunit and small amounts of
the p19 subunit (Fig. 8A, lane 2). Both
the p20 subunit resulting from cleavage of full-length procaspase-3 at
Asp175 and the p19 subunit formed following subsequent
autocatalytic cleavage at Asp9 (20) are potentially
catalytically active, as evidenced from their capacity to interact with
biotinylated DEVD-CHO (48, 49). However, despite the presence of both
the p20 and p19 subunits in control activated cells, PARP remained
intact, as was the case for resting T cells, which only exhibited the
latent 32-kDa proform of caspase-3 (Fig. 8A, lane
1). In contrast, in activated T cells induced to undergo apoptosis
with anti-Fas, anti-CD2, or staurosporine, an additional p17
protein was observed (Fig. 8A, lanes 3,8, and 12). The formation of the p17 subunit, which is
due to further processing of the p19 subunit at Asp28 (20),
coincided with the cleavage of PARP (Fig. 5
). With all three apoptotic
stimuli, processing of caspase-3 to the p17 subunit was completely
blocked by Z-VAD.fmk (Fig. 8A, lanes
5, 10, and 14) and BOC-D.fmk (Fig. 8A, lanes 6, 11, and 15),
whereas Z-FA.fmk had little effect (Fig. 8A, lanes
4, 9, and 13).
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m that were present (at 
15–20%) in control activated T cell
preparations (see Fig. 4), would account for the partial processing of
caspases-3 and -7 observed in such cell preparations.
DiOC6high and DiOC6low cells present in control
activated T cells were separated by cell sorting. For comparison, T
cells exposed to anti-CD2 were fractionated in a similar manner. It
can be seen that >98% pure DiOC6high T cells isolated
from control populations (Fig. 8B, lane 2) and
from populations exposed to anti-CD2 (Fig. 8B,lane 5) still exhibited partial processing of caspase-3 to its p20
subunit. In marked contrast, in DiOC6low/PI-negative cells
(Fig. 8B, lane 6), the p32 proform was quite
reduced, and the large subunit was entirely processed to both the p19
and p17 subunits. In DiOC6low/PI-positive cells (Fig. 8B, lane 3), the subunits of caspase-3 were
hardly detected (they were probably degraded). These data did not
support the suggestion that the p20 observed in control activated T
cells was due to the low percentage of cells with low m. Rather,
they suggested that in live DiOC6high T cells, the p20/p12
tetramer of caspase-3 was present, but was catalytically less active
than the p17/p12 tetramer; its activity was possibly blocked by
endogenous inhibitors.
In resting T cells, caspase-7 was present primarily as its 35-kDa
proform (Fig. 8C, lane 1), whereas in activated T
cells, it was already processed to a p23 fragment (Fig. 8C, lane 2), probably following cleavage between
the large and small subunits. Induction of apoptosis by all three
apoptotic stimuli resulted in the appearance of a p20 fragment (Fig. 8C, lanes 3, 6, and 9)
following further cleavage at Asp23 (20). Formation of the
p20 subunit was inhibited by Z-VAD.fmk (Fig. 8C, lane
5), but not by Z-FA.fmk (Fig. 8C, lanes
4, 7, and 10).
Recently, we and others have shown that procaspase-2 is activated at an
early stage of the apoptotic process in some cells (13, 50). Using an
Ab that recognizes both the proform and the small p12 subunit of
caspase-2, it appeared that procaspase-2 was not processed during
anti-Fas and anti-CD2-induced apoptosis of activated human T
cells (Fig. 8D). Under similar conditions, this Ab detected
the p12 subunit of caspase-2 in human monocytic THP.1 cells induced to
undergo apoptosis by etoposide, a DNA topoisomerase II inhibitor (Fig. 8D, lane 4). Thus, apoptosis induced by several
stimuli in activated T cells leads to the selective activation of some,
but not all, caspases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In numerous cell models, Z-VAD.fmk or BOC-D.fmk block apoptosis
(21, 22, 23, 25) by irreversibly inhibiting the activity/processing of a
number of caspases (13, 18, 22). However, in a small number of recent
studies that analyzed the induction of apoptosis by Bax, Bak, GD3
ganglioside, or the CTL granule exocytosis pathway, Z-VAD.fmk and
BOC-D.fmk were ineffective in blocking cell death (19, 51, 52, 53, 54). In most
of these studies, the mode of cell death, apoptosis, necrosis, or a
combination of both was not clearly assessed. In addition, in many of
these studies, cell death was induced over a period of days, raising
the possibility that the caspase inhibitors were ineffective because
they were present intracellularly at inadequate concentrations and for
insufficient periods of time. In the present study, although this
problem was circumvented by inducing apoptosis over a short period of
time (up to 4 h), the caspase inhibitors failed to block
anti-CD2- and staurosporine-mediated cell death in conditions where
they inhibited Fas-induced apoptosis. In addition, both inhibitors were
present in the cells at concentrations that blocked the activities of
caspase-3 and caspase-7 (the caspases primarily responsible for PARP
cleavage), caspase-6 (the caspase primarily responsible for
lamin cleavage), and caspase-8 (probably by inhibiting the activity
of caspase-8 recruited to the death-inducing signaling
complex after Fas ligation) (15, 16). While this manuscript
was in preparation, we became aware of the work of Toyoshima et al.
(55), which showed that caspase-3-like peptide inhibitors (such as
Z-DEVD.fmk) were unable to block Fas-mediated cell death of
Jurkat cells. Interestingly, cell death occurring in these conditions
was characterized by cytoplasmic apoptotic responses such as cell
shrinkage and surface blebbing, but lacked the prominent features of
apoptotic nuclear responses (chromatin condensation and DNA
fragmentation), reminiscent of the changes elicited by anti-CD2 and
staurosporine in the presence of Z-VAD.fmk and BOC-D.fmk. Sarin et al.
have reported that primary murine lymphoblasts are largely protected by
BOC-D.fmk (and much less by Z-VAD.fmk) from apoptosis induced by
dexamethasone, etoposide, and irradiation (22). However, if the nuclear
events of apoptosis were almost completely prevented by this caspase
inhibitor, a substantial proportion of the murine lymphoblasts of that
study still died, in line with our own results. All these data strongly
support the presence of a caspase-independent pathway of cell death
operating in concert with a caspase-dependent one, both leading to the
complete set of changes characterizing the apoptotic phenotype.
Interestingly, it was recently shown that lymphocytes undergoing in
vivo cell death in HIV-infected lymph nodes are characterized by
reduced size, different degrees of chromatin condensation, and absence
of fragmented DNA (56). With the exception of the mitochondrial
swelling noticed in these lymphocytes, these features are similar to
those observed in the lymphocytes undergoing the caspase-independent
cell death pathway in the present study. Examination of the activation
status of caspases within the dying lymphocytes of HIV-infected
individuals would establish whether the caspase-independent pathway
described here has an in vivo relevance. It is not possible to
completely exclude a possible role of the activation of an as yet
uncharacterized caspase in anti-CD2- and staurosporine-induced
apoptosis in the presence of caspase inhibitors. However, this seems
unlikely, as Z-VAD.fmk and BOC-D.fmk are capable of inhibiting the
caspase cascades initiated by a large number of apoptotic stimuli,
including the cascade initiated by caspase-10 in the course of granzyme
B-mediated apoptosis (19). It is possible that the strength of the
apoptotic stimuli might have overcome the inhibitory effect of
Z-VAD.fmk and BOC-D.fmk against these putative uncharacterized
caspases. However, regardless of the strength of the apoptotic signal
delivered by anti-CD2 or staurosporine in our cells, the caspase
inhibitors failed to prevent cell death, arguing against this
possibility (Fig. 2).
Alterations in mitochondrial function, as evidenced by an early fall in
mitochondrial membrane potential, were proposed to be responsible for
the Bax-induced caspase-independent cell death pathway (51). Our data
also show a decrease in mitochondrial membrane potential, which, in the
case of anti-CD2- and staurosporine-treated cells, was not blocked
by the caspase inhibitors. One currently favored hypothesis for the
induction of apoptosis is that perturbations of mitochondria allow the
release of cytochrome c (26, 27), which upon binding to
apoptosis protease-activating factor-1 and in the presence of dATP
leads to the activation of a caspase cascade (28, 29). These events are
responsible for many of the biochemical changes characteristic of
apoptosis. For example, activation of caspase-3 results in the cleavage
of DNA fragmentation factor, leading to internucleosomal cleavage of
DNA (57), as well as to the cleavage of numerous other substrates,
including PARP and retinoblastoma protein (reviewed in 11 . In the
present study both Z-VAD.fmk and BOC-D.fmk inhibited internucleosomal
cleavage of DNA as well as PARP cleavage, demonstrating that events
downstream of caspase-3 and caspase-7 were effectively inhibited.
Beside the loss of mitochondrial cytochrome c, other
mitochondrial components might participate in the induction of the
characteristic biochemical and morphologic changes associated with
apoptosis. One partially characterized component is apoptosis-inducing
factor, which may also be responsible for DNA fragmentation and for
proteolytic degradation of caspase-3 (58). However, as
apoptosis-inducing factor is potently inhibited by Z-VAD.fmk (59), it
is highly unlikely that it is responsible for the caspase-independent
cell death in our study. Our data, demonstrating a decrease in m
in CD2- and staurosporine-treated activated T cells, even in the
presence of the caspase inhibitors, are compatible with the possibility
that the caspase-independent cell death pathway may be mediated in
these cells by alterations in mitochondrial function. Whether such
alterations constitute commitment for the demise of the cells or
whether they function as a potent amplificatory loop remains to be
established in anti-CD2- and staurosporine-treated cells.
Partial processing of procaspase-3 and procaspase-7, yielding their
respective prodomains with their large subunits, was observed in our
control activated peripheral T cells (displaying otherwise excellent
cell viability), whereas in unstimulated cells only the proforms of
these caspases were found. Postlysis activation of these caspases,
performed by granzyme B (released from its intracellular stores during
the lysis procedure), was unlikely to occur in our experimental
conditions. The cells were indeed lysed in Laemmli buffer
containing 3% SDS and thereafter immediately boiled, which should
avoid artifactual caspase processing (45). Moreover, adding granzyme B
inhibitors such as Ac-IETD-CHO and Z-AAD-cmk at 300 µM in the last
washing buffer just before cell lysis did not impair the formation of
the large p20 subunit of caspase-3 (not shown). AICD, occurring at
background levels during the primary stimulation period, may be
responsible for the partial processing of caspase-3 and -7 in control
activated T cells. However, adding antagonistic anti-Fas mAb,
neutralizing anti-TNF-, or BOC-D.fmk during the primary
stimulation period of the cells did not attenuate the occurrence of the
p20 subunit of caspase-3 in those cells (our unpublished data).
Interestingly, using T cells from perforin-deficient mice, Spaner et
al. have recently demonstrated that perforin may act as an internal
trigger of cell death in short term activated primary CD8+
T cells upon re-cross-linking the CD3/TCR, and that this novel form of
AICD preceded the two other forms of AICD, respectively represented by
the CD95 and the TNF receptor pathways (60). The mechanism of perforin
action is currently unknown. In our experiments, stimulation of primary
T cells with OKT3 or the GT2+T111 mAb pair in the presence
of IL-2 probably resulted in perforin synthesis. In view of the results
reported by Spaner et al. (60), it is conceivable that in control
activated live T cells, perforin was activated to a level sufficient to
trigger partial processing of caspases-3 and -7 (such a possibility
will be investigated). However, the partially processed caspases did
not appear to be catalytically active in our cells, as no cleavage of
PARP or lamins was observed. It is possible that in control activated T
cells (and in live T cells surviving anti-CD2 and staurosporine
treatments), putative endogenous inhibitors inhibited the proteolytic
activity of the processed caspases. Some support for this suggestion is
provided by a recent report showing that recombinant X-linked inhibitor
of apoptosis protein is a direct and specific inhibitor of activated
caspases-3 and 7, capable of binding to the partially processed large
subunits and of inhibiting their catalytic activity (61). On induction
of apoptosis in our model, the large subunits of both caspase-3 and
caspase-7 were further processed to their p17 and p19 subunits,
respectively. These subunits appeared to be catalytically active, as
their appearance was accompanied by PARP and lamin proteolysis. Further
support for this was provided by the studies with Z-VAD.fmk and
BOC-D.fmk, which prevented the further processing of caspase-3 and
caspase-7 and inhibited cleavage of PARP and lamins.
In summary, we have demonstrated that in activated human peripheral T lymphocytes, anti-CD2 and staurosporine induce a caspase-independent cell death that may act either independently or in concert with a caspase-dependent pathway. Alone, the caspase-independent pathway triggers major cytoplasmic events related to apoptosis, but it does not trigger the characteristic morphologic and biochemical nuclear changes associated with apoptosis, including proteolysis of lamins and PARP, internucleosomal cleavage of DNA, and intensive chromatin condensation, although partial chromatin condensation still occurs. These nuclear morphologic and biochemical changes of apoptosis require the activation of caspases.
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Acknowledgments |
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Footnotes |
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2 G.M.C. and A.S. are joint senior authors.
3 Address correspondence and reprint requests to Dr. Anna Senik, Equipe d’Immunologie Cellulaire et de Transplantation, UPR 420 du Centre National de la Recherche Scientifique, 19 rue Guy Moquet, 94801 Villejuif, France. E-mail address:
4 Abbreviations used in this paper: PARP, poly(adenosine diphosphate-ribose) polymerase; Z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone; BOC-D.fmk, BOC-Asp(OMe)-fluoromethyl ketone; m, mitochondrial membrane potential; AICD, activation-induced cell death; ZFA.fmk, benzyloxycarbonyl-Phe-Ala-fluoromethylketone; Ac-IETD-CHO, Ac-Ile-Glu-Thr-L-aspartic acid aldehyde; Z-AAD-cmk, Z-Ala-Ala-Asp-CH2Cl; DiOC6, 3,3'-diethyloxacarbocyanine; PI, propidium iodide.
Received for publication February 9, 1998. Accepted for publication June 2, 1998.
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