From the Unité INSERM 419, Institut de Biologie, 9 Quai
Moncousu 44035, Nantes Cedex 01, France
Calcium is involved in several steps of the
apoptotic process. In nuclei, endonucleases are presumed to be the main
targets of calcium; however, little is known about its role during the cytosolic phase of apoptosis. We used a cell-free system to address this question. Our results show that CaCl2 triggered
nuclear apoptosis (i.e. typical morphological change and
DNA fragmentation) at concentrations of 5 mM. This
concentration was lowered 10-fold by the co-incubation with cytosolic
extracts from nonapoptotic cells. Apoptotic changes induced by the
incubation of nuclei with CaCl2 in the presence of these
cytosols were strongly reduced in the presence of an inhibitor of
caspase-3 and to a lesser extent by an inhibitor of caspase-1. We also
show that calcium-induced apoptosis is affected by protease inhibitors
such as N-tosyl-L-phenylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system increased a
caspase-3 activity in nonapoptotic cytosols as shown by specific
antibodies and an enzymatic assay. No activation of a caspase-3-like
activity by the addition of cytochrome c was observed in
these extracts under similar conditions. The enhanced caspase-3
activity induced by calcium was inhibited by protease inhibitors
affecting morphological nuclear apoptosis except for those responsible
for the degradation of lamin A. These results suggest that
CaCl2 could trigger, in normal cells, an apoptotic cascade
through the activation of cytosolic caspase-3 activity.
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INTRODUCTION |
Apoptosis is a cell death program originally characterized by
specific morphological and biochemical modifications in higher eukaryotic cells (1, 2). These structural changes such as plasma and
nuclear membrane blebbings, chromatin condensation, proteases
activation, and DNA fragmentation are considered as landmarks of the
apoptotic process (3). Although apoptosis plays an important role in
the normal physiology of the cell, and in many pathological situations,
little is known about the molecular mechanisms involved in the
regulation and/or in the execution of this program (4, 5). Specific
proteases called caspases and protein members of the proto-oncogene
BCL-2 family have been shown to be the key elements of the
executive/terminal phase of apoptosis (6-8). The caspases involved in
apoptosis are generally divided into initiators and excecutioners (9). The initiators caspases (e.g. caspase-1 or -8) are
implicated in the activation of the executioners (e.g.
caspase-3 or -6), which in turn are responsible for the terminal phase
of apoptosis along with other enzymes such as proteases,
nucleases, kinases, and so forth (reviewed in Refs. 9-11). It has
recently been proposed that BCL-2-like proteins regulate the activation
of caspases through complex interactions with other proteins (7) and/or
through the control of ion fluxes across membranes of organelles (12, 13). Indeed, since BCL-2 overexpression has been shown to inhibit apoptosis-associated Ca2+ waves in the endoplasmic
reticulum or the nuclear membranes, it has been proposed that members
of the BCL-2 family could regulate the cellular calcium homeostasis
(14-18). Numerous data have shown the involvement of Ca2+
homeostasis in apoptosis and in particular the prelethal increase of
its intracellular concentration (reviewed by McConkey and Orrenius (19)). Indeed, the addition of Ca2+ to isolated nuclei can
directly promote apoptotic nuclear changes by a mechanism
apparently involving nuclear endonucleases and proteases (20). However,
despite the accumulation of data, the link between intracellular
calcium homeostasis and the activation of the apoptotic program remains
unknown. We have shown, using a cell-free system, that calcium could
play an important role in the induction of caspase-3-like activity by
regulating the release of cytochrome c from mitochondria, a
protein linked to the activation of
caspases.1 In the present
study, we investigated the direct effect of CaCl2 on
apoptotic-like modifications of purified rat liver nuclei incubated with cytosolic extracts derived from nonapoptotic or apoptotic cells.
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EXPERIMENTAL PROCEDURES |
Peptide inhibitors of caspase 1 (Ac-YVAD-CHO) or caspase 3 (Ac-DEVD-CHO), calpain inhibitor I (Ac-Leu-Leu-norleucinal) and II
(Ac-Leu-Leu-methioninal), h-APF-OH, an inhibitor of the nuclear scaffold protease (NSP)2
(20), ac-DEVD-AMC, a fluorescent caspase-3 substrate, and h-FFG-AMC, a
fluorescent chymotrypsin-like substrate were obtained from Bachem (France). Leupeptin, pepstatin, aprotinin, phenylmethylsulfonyl fluoride, E64,
N
-p-tosyl-L-lysine
chloromethyl ketone (TLCK), and
N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK) were from Sigma (St. Quentin, France). A peptide mimicking the
apoptotic cleavage site of lamin A (RLVEIDNGKQR) and thus an
inhibitor of caspase-6 (21) was synthesized by Genosys (Cambridge, UK).
The chemicals used in this study unless otherwise specified were
obtained from Sigma (St. Quentin, France). Antibodies against
cytochrome c were obtained from Pharmingen (Clinisciences, France), anti-procaspase-3 were from Transduction Laboratories (Medgene
Science, France), and anti-procaspase-8 were from Santa Cruz (TEBU,
France).
Purification of Nuclei and Obtention of Cytosolic
Extracts--
Rat liver nuclei were prepared according to Newmeyer and
Wilson (22). The rat glioblastoma cell line A15A5 and the human promyelocyte leukemic cells HL60 were grown to confluency at 37 °C and in 5% CO2. Apoptosis was then induced in these cells
by a short UV-B treatment (1 min). Most of the cells displayed, after 48 h, characteristic morphological apoptotic changes. The cytosol extracts (CE), from both control (CCE) or apoptotic cells (ACE), were
obtained as described in Cotter and Martin (23). Briefly, the cells
were washed twice in phosphate-buffered saline, pH 7.2, the resulting
pellet was resuspended in the cell extract buffer (CEB) (50 mM HEPES, pH 7.4, 50 mM KCl, 2 mM
MgCl2, 1 mM dithiothreitol, 10 µM
cytochalasin B) supplemented with 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride in order to minimize
proteolysis degradation in the extracts. Cells or apoptotic bodies were
centrifuged at 3,000 × g for 10 min at 4 °C, and
the supernatant was discarded. The pellets were transferred to a 2-ml
glass Dounce homogenizer in the remaining CEB. Cells were allowed to
swell in a 1:1 dilution in CEB for 30 min on ice. Cells were lysed
gently with 30 strokes with a B-type pestle. The cell lysate was then
transferred to a 1-ml tube and centrifuged at 4 °C for 15 min at
13,000 × g. The cytosol was removed and kept frozen at
a concentration of approximately 10 mg/ml at
80 °C.
Analysis of DNA Content and Caspase-3-like
Activity--
Cell-free reactions (50 µl) were carried out as
follows. Cytosols were diluted to the desired concentrations in the
reaction mixture which contained 2 × 105 rat liver
nuclei, 10 mM HEPES, pH 7.4, 50 mM KCl, 1 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM ATP, 10 mM
phosphocreatine, 50 µg/ml creatine kinase, 10 mM malate,
8 mM succinate, and 250 mM sucrose. Calcium and
peptides were then added, and the resulting mixture was incubated for
2 h at 37 °C. At the end of the incubation, a small aliquot of
the mix was removed and stained for 10 min with Hoechst 33342 (2.5 µM) at room temperature and examined by fluorescence
microscopy (Olympus, BX 60). The rest of the mix was analyzed for
intranucleosomal degradation of the DNA and caspase-3 activity. After
centrifugation of the samples (15 min, 400 × g at
4 °C), caspase-3 activity was measured in the supernatant and DNA
fragmentation in the pellet. DNA was purified and analyzed on 1.5%
agarose gel after staining by ethidium bromide. Caspase-3 activity was
measured in the supernatant by following the cleavage of 50 µM peptide Ac-DEVD-AMC. The fluorescence of the cleaved substrate was determined every 30 min using a spectrophotometer (Fluorolite 1000, Dynatech Laboratories) set at an excitation wavelength of 365 nm and emission wavelength of 465 nm (24).
Western Blot Analysis and Quantification of Procaspase-3 and
-8--
Western blots were performed as described in Chatel et
al. (25) using first antibodies diluted 1:100 for
anti-procaspase-8 and cytochrome c and 1:1000 for
anti-procaspase-3. The antibodies bound to Immobilon-P (Millipore,
France) were detected by enhanced chemiluminescence (Amersham, France)
and the amounts of procaspases after scanning with a Appligene-Oncor
Imager (Strasbourg, France) were quantified with IPLab Gel Program
(Signal Analytics, Vienna, VA).
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RESULTS |
Morphological Modifications and DNA Fragmentation Induced by
CaCl2 in Isolated Rat Liver Nuclei: Potentiation by
Nonapoptotic Cytosolic Extracts--
Rat liver nuclei (2 × 105) were incubated in CEB (see "Experimental
Procedures") for a fixed period of time in the presence of increasing
amounts of CaCl2 (from 0 to 5 mM), in the
absence or in the presence of nonapoptotic cytosolic extracts (100 µg of CCE). After a 2-h incubation and in the absence of CCE, the intranucleosomal degradation of DNA was observed only in nuclei incubated with 5 mM CaCl2 (Fig.
1). This is in agreement with a previous
report (20). The addition of non-CCE derived from the rat glioblastoma
cell line, A15A5, lowered considerably the amount of CaCl2
required to trigger an apoptotic-like DNA fragmentation, since the
intranucleosomal degradation was then observed with concentrations of
CaCl2 as low as 0.5 mM (Fig. 1). A similar
effect was observed with cytosols derived from different sources such as the human HL60 or K562 cells and the rat pheochromocytoma cell line
PC12 (data not shown).

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Fig. 1.
Calcium-induced DNA fragmentation is enhanced
by nonapoptotic cytosols. As described under "Experimental
Procedures," genomic DNA was prepared from 2 × 105
rat liver nuclei incubated in an isotonic buffer in the absence
( CCE) or in the presence (+CCE) of 100 µg of
A15A5 cytosols (CCE) incubated with the indicated increasing
concentrations of CaCl2. DNA was analyzed with a 1.5%
agarose gel electrophoresis. One experiment, representative of at least
three independent analyses, is shown.
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We also studied the apoptotic morphological events occurring in the
nuclei after incubation with calcium. Little or no morphological changes were observed in nuclei incubated in the absence of CCE with
concentrations of up to 1 mM CaCl2. At higher
concentrations (5 mM CaCl2), the nuclei
displayed the typical apoptotic changes in the chromatin structure
appearing highly condensed at the nuclear periphery (data not shown).
In the presence of CCE (100 µg) and in the absence of added
CaCl2, rat liver nuclei exhibited a normal pattern, and
their nuclear envelope remained intact upon co-incubation with CCE
(Fig. 2A). The addition of
CaCl2 to the CCE triggered nuclear morphological changes.
After identical periods of incubation, structural changes exhibited by
nuclei were enhanced with the addition of increasing concentrations of
CaCl2 to the cytosolic extracts. Chromatin condensed
progressively at calcium concentration as low as 0.05 mM
(Fig. 2B), to eventually either cluster against the nuclear
periphery or display the characteristic half-moon features observed at
higher concentrations (i.e. 0.5 mM
CaCl2) (Fig. 2C). At concentrations equal or
superior to 1 mM CaCl2, the nuclei were
shrinking and the chromatin started to collapse into high density
structures (Fig. 2D). These morphological changes were
highly reminiscent to those observed in apoptotic cells in vitro or in vivo (26-28).

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Fig. 2.
Influence of calcium on the morphological
aspect of rat liver nuclei incubated with nonapoptotic cytosol.
Rat liver nuclei (2 × 105) were incubated for 2 h at 37 °C in the presence of 100 µg of CCE with increasing
concentrations of CaCl2 then stained with Hoechst 33342 as
described under "Experimental Procedures" (magnification, × 300).
A, control; B, + 0.05 mM
CaCl2; C, + 0.5 mM
CaCl2; D, + 1 mM CaCl2.
The results shown are representative of at least three independent
experiments.
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These changes were observed after a 2-h incubation at 37 °C, but it
should be noted that the addition of CaCl2, even at the lower concentrations (i.e. 0.2 mM
CaCl2), resulted to the dramatic final condensation of
chromatin and degradation of DNA but after much longer periods of
incubation (data not shown). Under these conditions, the addition of
ZnCl2 (1 mM) abolished the nuclear apoptosis
even in the presence of 5 mM CaCl2 (data not
shown). The addition of CCE appeared thus to potentiate, both
morphologically and biochemically, the calcium-induced apoptosis in the
cell-free system. It should be noted that the total (free and bound)
calcium concentration in CCEs was always below 170 µM
(data not shown) and thus could not account for the enhanced apoptotic
effect observed in the presence of cytosols.
Influence of Protease Inhibitors and Free Radicals Scavengers on
the Cytosolic Potentiated and Calcium-induced Apoptosis--
We found
that either trypsin or heat pretreatment of CCEs abolished the
potentiation of the calcium-induced apoptosis by cytosolic extracts (data not shown). We thus postulated that proteinaceous components were involved in this process. Apoptosis was initiated in
the cell-free system by the incubation of rat liver nuclei with 0.5 mM CaCl2 and 100 µg of CCE in the presence of
inhibitors of caspases such as ac-YVAD-CHO (100 µM), an
inhibitor of caspase-1, or ac-DEVD-CHO (100 µM), an
inhibitor of caspase-3-like proteases. As shown in Fig.
3B, in the absence of these
inhibitors, the addition of CaCl2 resulted, after 2 h,
in an almost complete disintegration of rat liver nuclei. The addition
of 100 µM ac-YVAD-CHO also affected this process, but the
changes occurred more slowly and were not as dramatic as those observed
in calcium-treated nuclei (Fig. 3C). The addition of 100 µM ac-DEVD-CHO, on the other hand, completely prevented
the onset of apoptosis (Fig. 3D). Since calpain was shown to
be involved in the apoptotic process (11), calpain inhibitors I and II
were added to the reaction mixture with CCE and calcium. However, no
effects of these inhibitors were observed on calcium-induced apoptosis
in the cell-free system (data not shown). On the contrary, the addition
of the radical scavenger GSH, as well as the inhibitor of the NSP
(h-APF-oh) inhibited the morphological changes observed upon the
addition of CaCl2 and CCE (data not shown). Next, we
examined the effect of these inhibitors on the DNA integrity and found,
in agreement with morphological observations, that E64, a calpain
inhibitor, had no effect on the DNA fragmentation. On the other hand,
GSH protected the DNA structure in the presence of CCE and calcium.
However, the correlation between biochemical and morphological changes
was not absolute, because the fragmentation of the DNA was unaffected
by the addition of ac-YVAD-CHO (Fig. 4).
On the other hand, the addition of 100 µM ac-DEVD-CHO
inhibited the DNA intranucleosomal degradation (Fig. 4).

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Fig. 3.
Effect of different caspase inhibitors on the
calcium-induced apoptosis: morphological aspects. Rat liver nuclei
(2 × 105) were incubated for 2 h at 37 °C in
the presence of 100 µg of CCE (control) and stained with Hoechst
33342 as described under "Experimental Procedures" (magnification, × 300). A, control; B, + 0.5 mM
CaCl2; C, + 0.5 mM CaCl2 + 100 µM ac-YVAD-cmk; D, + 0.5 mM
CaCl2 + 100 µM ac-DEVD-CHO. The experiments
shown are representative of at least three independent ones.
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Fig. 4.
Effect of protease inhibitors and a free
radical scavenger on the calcium-induced apoptosis: DNA
fragmentation. Genomic DNA was prepared from 2 × 105 rat liver nuclei incubated in an isotonic buffer in the
absence or in the presence of 100 µg of CCE and 0.5 mM
CaCl2 and incubated with the following inhibitors:
lane 1, control DNA; lane 2, + 0.5 mM
CaCl2; lane 3, same as 2 + 100 µM E64; lane 4, same as 2 + 100 µM ac-YVAD-cmk; lane 5, same as 2 + 100 µM ac-DEVD-CHO; lane 6, same as
2 + 1 mM GSH. The DNA was analyzed with a 1.5%
agarose gel electrophoresis. The data shown are representative of at
least three independent experiments.
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Induction of a DEVDase Activity in Nonapoptotic Cytosols--
Both
morphological and DNA analyses suggested that caspase-like activities
were involved in the CCE plus calcium-induced apoptosis in the
cell-free system. We examined the effect of calcium on the caspase
activity present in our extracts using the cleavage of the peptide
DEVD-AMC as an index of caspase-3-like activity. CCE (100 µg)
displayed low levels of caspase-3 like activity. As shown in Fig.
5, a 3-fold increase of a DEVDase
activity was observed upon the addition of 0.5 mM
CaCl2. As this increase was observed both in the absence
and in the presence of nuclei, we ruled out the contribution of a
nuclear protease to this activity (data not shown). Under these
conditions, we found no YVADase activity both in absence and in
presence of CaCl2 (data not shown). The calcium induction
of the cytosolic DEVDase appeared to be specific of nonapopototic
cytosols as the addition of CaCl2 had no effect on the
caspase-3-like activity present in ACE at protein concentrations of 25 µg (Fig. 5) and higher (data not shown).

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Fig. 5.
Induction of a DEVDase activity by calcium in
apoptotic (ACE) and nonapoptotic (CCE)
cytosolic extracts. The effect of CaCl2 was analyzed
in control or apoptotic A15A5 cytosolic extracts exhibiting similar
DEVDase activity (either 100 µg of CCE or 25 µg of ACE). ACE and
CCE were incubated for 30 min with increasing amounts of
CaCl2, and the resulting DEVDase activity was expressed as
a proportion of the initial DEVDase activity in the absence of calcium.
The results are expressed as the mean ± S.D. of three independent
experiments.
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Several reports have shown that cytochrome c also induced a
caspase-3-like activity in normal cytosols (29-32). As illustrated in
Fig. 6A, no or little
cytochrome c was found in CCEs derived from HL60 or A15A5
cells, whereas HL60 ACE exhibited large amounts of this protein. We
compared the kinetics of induction of the DEVDase activity by calcium
and cytochrome c in 40 µg of ACE at 37 °C (Fig.
6B). Under these conditions no activation of DEVDase activity was observed in the presence 2 µM bovine
cytochrome c and 1 mM dATP (Fig. 6B).
On the other hand, the addition of 0.5 mM CaCl2
produced a rapid and important increase of the caspase-3-like activity,
which reached a plateau after 120 min (Fig. 6B). We have
verified the specificity of the DEVDase activation using a chymotrypsin
substrate (GGF-AMC). No significant difference in the chymotrypsin
activity was observed between control CCEs and cytochrome
c/dATP or CaCl2-treated CCEs (Fig.
6C).

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Fig. 6.
Involvement of cytochrome c in
the calcium-induced DEVDase activity. A, cytosolic
extracts (50 µg) from nonapoptotic HL60 or A15A5 (CCE) or
apoptotic HL60 (ACE) were analyzed by Western blot using a
anti-cytochrome c antibody. B, the DEVdase
activity was measured as described under "Experimental Procedures"
in 50 µg of control CCE ( ), in the presence of 0.5 mM
CaCl2 ( ), or in 2 µM bovine cytochrome
c plus 1 mM dATP ( ). C, the
chymotrypsin-like activity (GGFase) was measured as described under
"Experimental Procedures" in 50 µg of control CCE ( ) or after
the addition of 0.5 mM CaCl2 ( ) or of 2 µM bovine cytochrome c plus 1 mM
dATP ( ). Note that samples used in this assay are similar to those
used in B. The data shown are representative of at least
three independent experiments.
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The DEVDase Activity Induced by CaCl2 Is Likely Due to
Caspase-3--
We used several protease inhibitors to study the nature
of the caspase involved in this activation as well as the possible involvement of other proteases. The DEVDase activated in CCE in the
presence of CaCl2 was slightly inhibited by an inhibitor of caspase-1 and completely abolished by the addition of an inhibitor of
caspase-3 (Fig. 7). Since, as previously
shown by McConkey (20), lamins are cleaved during calcium-induced
apoptosis, a peptide mimicking the cleavage site of lamin A (21) was
used to test the involvement of caspase-6 in the calcium-induced
DEVDase. The caspase-3-like activity was not affected by this peptide, thus ruling out the involvement of caspase-6/Mch2 in this process. Other protease inhibitors such as TPCK and TLCK inhibited the DEVDase
activity but with different affinities. TPCK was a more powerful
inhibitor than TLCK (data not shown), suggesting the involvement of a
chymotrypsin-like protease in this process. Calpain inhibitors such as
E64 or calpain inhibitors I and II had no affect on this activity (Fig.
7). Other inhibitors of the calcium-induced morphological changes such
as GSH or NSP did not affect the onset of the caspase-3-like activity
(Fig. 7), suggesting that they acted downstream of this activation.

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Fig. 7.
Effect of antiapoptotic and/or
antiproteolytic agents on the calcium induced DEVDase activity.
The DEVDase activity was measured in 1, 100 µg of CCE with
0.5 mM CaCl2 and in the presence of
2, 100 µM ac-YVAD-cmk; 3, 100 µM ac-DEVD-al; 4, 100 µM lamin
peptide (see "Experimental Procedures"); 5, 2 µg/ml
leupeptin; 6, 100 µM TLCK; 7, 100 µM TPCK; 8, 100 µM NSP inhibitor
(see "Experimental Procedures"); and 9, 1 mM
GSH. The DEVDase activity was followed by measuring the cleavage of the
fluorogenic peptide ac-DEVD-AMC as described under "Experimental
Procedures." The results are expressed as the mean ± S.D. of
three independent experiments.
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We have used antibodies to detect procaspase-3 and procaspase-8 in CCE
incubated with increasing concentrations of CaCl2. The
Western blot in Fig. 8 reveals a
disappearance of the procaspase-3 upon incubation with
CaCl2 which parallels the activation of a DEVDase.
Conversely, procaspase-8, an initiator caspase linked to Fas or tumor
necrosis factor apoptotic pathways (33), was not affected under
these conditions. The latter result suggests that caspase-3 is
responsible at least partially for the DEVDase activity induced by
calcium in CCEs.

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Fig. 8.
Involvement of caspase-3 in the
calcium-activated DEVDase. CCEs from HL60 cells were incubated
with increasing amount of CaCl2 and the amounts of
procaspase-3 or -8 were analyzed by Western blot analysis and
quantified as described under "Experimental Procedures."
A, procaspase-3 or -8, detected by Western blot, present
after incubation with increasing concentrations of CaCl2.
B, amount of procaspase-3 or -8 quantified from the
immunoblots. These results are representative of two independent
experiments.
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DISCUSSION |
Since apoptosis does not always require protein synthesis,
elements involved in this process have to be already present in nonapoptotic cells. Our data show that calcium could induce
apoptotic-like changes in isolated nuclei and that these changes were
potentiated by the addition of nonapoptotic cytosols. Different CCEs
were analyzed derived either from human hematopoietic cell lines (HL60 and K562) or from cells derived from rat nervous tissues (A15A5 and
PC12). We have also shown that addition of calcium to these extracts
activated a DEVD cleaving protease. As caspase-3 inhibitors blocked the
calcium-induced apoptosis in the presence of CCEs, proteases belonging
to this family were likely to be involved in this potentiation.
Inhibition of the NSP or the addition of GSH blocked the
calcium-induced nuclear apoptosis in the presence of cytosol but not
the DEVDase activity. These results suggest that an antioxidant process
as well as a resident mechanism of nuclear degradation involved in
nuclear apoptosis functions downstream of cytosolic caspase activation.
Recently, Liu et al. (34) isolated a heterodimeric protein
called DFF (DNA fragmentation factor) which could provide a link
between activation of caspase-3 and DNA fragmentation. This factor,
which is not regulated by calcium, induces DNA fragmentation in HeLa
cells after cleavage by caspase-3.
The DEVDase activity appears to be rapidly induced by calcium at very
low concentrations of CCEs. Under the same conditions, cytochrome
c and dATP had little effect on DEVDase activation. However,
cytochrome c activation of DEVDase can be observed with higher concentrations of CCEs, indicating different thresholds of
activation for calcium and cytochrome c, which probably thus rely on different
mechanisms.3 Of all the
nonanticaspase agents, only TPCK appears to specifically and
efficiently inihibit the calcium induced DEVDase activity (Fig. 7).
Recently, TPCK has been shown to to block the conversion of the
inactive 32-kDa caspase-3 precursor into the mature caspase-3 (35),
thus providing an explanation of its inhibitory effect on apopotosis
reported in several studies (11). It is noteworthy that, under our
conditions, no increase in a chymotrypsin activity was observed (Fig.
6), ruling out a general proteolytic activation by calcium which in
turn would lead to an nonspecific activation of a DEVDase activity.
The calcium induced DEVDase activity was attributed to caspase-3, as no
YVADase (caspase-1-like) activity was found, the amount of procaspase-3
was specifically affected by calcium (Fig. 8) and as the inhibition of
caspase-6 did not interfere with this activity (Fig. 7). The nature of
this activation remains to be established, in particular the
intervention of other proteases (including other caspases) and/or
cytoplasmic components in this process. Even if the calcium
concentrations used in our study are elevated compared with resting
intracellular calcium concentrations, one could postulate that, under
certain circumstances, the release of calcium from intracellular
organelles could lead to high local cytosolic concentration which could
activate a caspase-3-like protease. Our observation could thus be
relevant in certain physiopathological situations. In agreement with
this hypothesis, it has recently been shown that the glutamate-induced
apoptosis of cerebellar granule neurons, which is characterized by a
sustained intracellular rise of Ca2+, is mediated by a
post-translational activation of a caspase-3-like protease (36).
We thank Dr. J. L. Orsonneau
(Laboratoire de Biochimie Générale, CHR de Nantes) for the
quantification of cytosolic calcium. We thank Dr. J. Menanteau (U419
INSERM) for fruitful discussions throughout this study.