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INTRODUCTION |
Genetic and biochemical data indicate that a family of cysteine
proteases with aspartate specificity, known as caspases, play a pivotal
role in the regulation and execution of apoptotic cell death (reviewed
in Refs. 1-6). Caspases are expressed as inactive proenzymes in living
cells and become activated by proteolytic processing at internal
aspartate residues when cells receive an apoptosis-inducing signal (3).
Proteolytic cleavage results in the removal of an amino-terminal
prodomain and the generation of a small and a large active subunit,
which forms a heterodimer. At present, 14 mammalian caspase family
members have been described. Some, including caspase-8, -9, and -10, contain large prodomains and are initiators of cell death. Other family
members, such as caspase-3, -6, and -7, carry small prodomains and are
mostly involved in the execution of cell death. When caspases are
activated, they cleave a number of key substrates, resulting in their
activation or inactivation, which orchestrate the morphological and
biochemical features of apoptosis (7-11).
Two pathways of caspase activation during apoptosis have been
described. The first one involves apoptosis mediated by death receptors, such as Fas or tumor necrosis factor receptors (reviewed in
Ref. 12). When Fas ligand binds to the Fas receptor, the adaptor
molecule FADD/Mort-1 becomes recruited to the receptor (13-15),
allowing binding and autoactivation of procaspase-8 (16-22). When
caspase-8 is activated, it can process effector caspases (caspase-3,
-6, and -7), inducing a cascade of caspases (23-29).
In the second pathway, diverse proapoptotic signals converge at the
mitochondrial level, provoking the translocation of cytochrome c from mitochondria to cytoplasm (30-37). Once cytochrome
c is in the cytoplasm, it binds to
Apaf-1,1 a mammalian CED-4
homologue, which then permits recruitment of procaspase-9 (38, 39).
Oligomerization results in autoactivation of procaspase-9 (40). Active
caspase-9 then cleaves and activates procaspase-3. In the mitochondrial
pathway, the complex of cytochrome c, Apaf-1, and caspase-9,
called the "apoptosome," is a critical activator of the effector
caspases. Both pathways induce a cascade of caspases, which amplifies
the apoptotic signal to ensure fast and irreversible cell death.
Cytochrome c release has been documented for apoptosis
induced by chemotherapeutic drugs, UV irradiation, growth factor
withdrawal, and ligation of Fas and tumor necrosis factor receptors
(31-37). Cell survival-promoting molecules Bcl-2 and Bcl-xL, localized
at outer mitochondrial membranes, prevent translocation of cytochrome
c from mitochondria (30, 35, 36, 41, 42).
Caspase-8 can directly cleave and activate caspase-3, serving as a
major substrate (17, 18, 21, 26, 29, 38). Thus, mitochondria may not be
required for receptor-mediated cell death. Nevertheless, electron
transport is inactivated in Fas-induced cell death, and cytochrome
c is released from mitochondria (30, 32, 41, 42). In
addition, Bcl-2 and Bcl-xL, localized to the outer mitochondrial
membrane (43), are able to block these events in some cases, arguing
for a role of mitochondria in Fas-mediated cell death (41, 42, 44-46).
To search for signals regulating cytochrome c release,
caspase-8 and other caspases were tested for their ability to provoke
cytochrome c release from mitochondria.
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EXPERIMENTAL PROCEDURES |
Reagents--
Anti-cytochrome c antibody (7H8.2 C12)
was from Pharmingen (San Diego, CA); anti-Fas monoclonal antibody CH-11
was from Kamiya Biomedical Company (Seattle, WA). Anti-actin antibody
was from ICN Biomedicals, Inc. (clone 14) (Aurora, OH). Anti-caspase-3 antibodies were from Transduction Laboratories (Lexington, KY). Anti-Fodrin antibodies were from ICN Biomedicals, Inc. Anti-protein kinase C-
antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-Bcl-2 antibodies (clone 124) were from DAKO (Missisauga, Canada). Complete mixture of protease inhibitors was purchased from
Roche Molecular Biochemicals. Protein concentrations were determined
with the Bio-Rad assay kit (Hercules, CA). zVAD-fmk (zVal-Ala-Asp-CH2F) was from Kamiya Biomedical Company
(Seattle, WA). Colorimetric substrate DEVD-pNA was from Biomol
(Plymouth, PA). Anti-mouse HRP linked antibodies were from Amersham
Pharmacia Biotech.
Immunoblotting--
Jurkat cells were collected by
centrifugation at 200 × g for 5 min at 4 °C and
washed twice with ice-cold PBS, pH 7.4. The cell pellet was resuspended
in 500 µl of extraction buffer containing 250 mM sucrose,
20 mM Hepes-KOH, pH 7.0, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1.5 mM
MgCl2, 1 mM dithiothreitol, and a mixture of protease inhibitors (Roche Molecular Biochemicals). After 20 min on
ice, cells were homogenized with a glass Dounce homogenizer (B
pestle/50 strokes). Cell homogenates were spun at 14,000 × g for 15 min at 4 °C in a microcentrifuge, and
supernatants were stored at
70 °C until gel electrophoresis.
20-50 µg of cytosolic extracts were loaded onto each lane of an 8, 12, or 15% SDS-polyacrylamide gel, separated and transferred to
Hybond-ECL nitrocellulose membrane (0.45 µm) (Amersham Pharmacia
Biotech) at 150 mA overnight in transfer buffer (20 mM
Tris-base, 150 mM glycine, 20% methanol). Nonspecific
binding was blocked by incubation with 3% bovine serum albumin, 3%
nonfat milk powder, and 0.1% Tween-20 in PBS for 3 h at room
temperature. Anti-cytochrome c antibodies were diluted at
1:1000, anti-caspase-3 at 1:2000, anti-Fodrin at 1:2000, anti-protein kinase C-
at 1 2000, anti-caspase-8 at 1:5, anti-Actin at 1:4000, and anti-Bid at 1:1000 in PBS, 3% nonfat milk powder, and 0.1% Tween-20. The rest of the procedure was done as described previously (37).
Isolation of Mouse Liver Mitochondria--
The liver of a
6-week-old Balb/c mouse was minced on ice, resuspended in 10 ml of
ice-cold Buffer A (200 mM mannitol, 50 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM Hepes-KOH, pH 7.4, 0.1% bovine serum albumin, mixture
of protease inhibitors), and homogenized with a glass Dounce
homogenizer and a tight Teflon pestle. Homogenates were centrifuged at
600 × g for 5 min at 4 °C. Supernatants were then
recentrifuged at 3500 × g for 15 min at 4 °C. After
centrifugation, supernatants and floating lipid layers were aspirated,
and the mitochondrial pellet was resuspended in Buffer A and
centrifuged at 1500 × g for 5 min at 4 °C.
Supernatants were recentrifuged at 5500 × g for 10 min. The last two steps were repeated twice. Mitochondrial pellets were
resuspended in 1 ml of Buffer B (200 mM mannitol, 50 mM sucrose, 10 mM succinate, 5 mM
potassium phosphate, pH 7.4, 10 mM Hepes-KOH, pH 7.4, 0.1%
bovine serum albumin). Mitochondria were prepared fresh for each
experiment and used within 4 h.
Preparation of Cytosolic Extracts--
Jurkat cells were grown
for 3 days in culture in RPMI medium containing 10% fetal bovine
serum. The cells were harvested by centrifugation at 200 × g for 10 min at 4 °C, washed twice with PBS, and
resuspended in 700 µl of ice-cold buffer (20 mM
Hepes-KOH, pH 7.0, 10 mM KCl, 1.5 mM
MgCl2, 1 mM sodium EDTA, 1 mM
sodium EGTA, 1 mM dithiothreitol, 250 mM
sucrose, and protease inhibitors). After incubation on ice for 20 min,
cells were disrupted by Dounce homogenization (B pestle/50 strokes).
Nuclei were removed by centrifugation at 1000 × g for
10 min at 4 °C in a microcentrifuge. Supernatants were then further
centrifuged at 100,000 × g for 1 h.
Alternatively, supernatants were centrifuged for 15 min at 14,000 × g in a microcentrifuge. The resulting supernatants were
stored at
70 °C and until used for in vitro apoptosis assays.
Isolation of Recombinant Bcl-xL Protein and
Caspases--
Bl21(DE3) cells were transformed with the
pET29b-BclxL
C plasmid. Bacteria were grown in 2× TY medium
containing 10 µg/ml kanamycin and induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside overnight at
30 °C. Bacterial pellets were lysed in 150 ml of buffer (PBS, pH
7.4, 100 mM NaCl, 1% Triton × 100, 0.5 mM EDTA, containing protease inhibitors). After being
frozen and thawed once, the bacterial lysate was sonicated on highest
microtip limit for 5 min on ice. Bacterial lysates were cleared by
centrifugation at 10,000 × rpm for 75 min and then filtered using
a Sterivex-GS filter unit (0.22 µm) (Millipore). Bcl-xL protein was
affinity purified by binding to a 2-ml nickel-chelating column and
eluted using an imidazole gradient (0-200 mM). Bcl-xL
containing fractions were pooled and dialyzed overnight at 4 °C
against buffer containing 20 mM Hepes-KOH, pH 7.0, 10 mM KCl, 1.5 mM MgCl2, 1.0 mM dithiothreitol, 5 mM EDTA and stored at
70 °C. To purify caspases, transformed BL21 bacteria were grown in
2× TY medium containing ampicillin (50 µg/ml) and chloramphenicol
(34 µg/ml) and induced with 0.02 mM
isopropyl-1-thio-
-D-galactopyranoside for 4 h at
30 °C. Bacterial pellets were lysed in 150 ml of Buffer C (50 mM Tris-Cl, pH 8.0, 100 mM NaCl). Cleared
bacterial lysates were loaded on a 2-ml nickel-chelating column. The
column was washed with 70-90 ml of Buffer D (50 mM
Tris-Cl, pH 8.0, 500 mM NaCl) and then with 10 ml of Buffer
C. Protein was eluted by an imidazole gradient (0-200 mM)
in Buffer C, pH 8.0.
Cell-free Apoptosis System--
Standard reactions were carried
out in a 50-µl reaction volume with reaction buffer (20 mM Hepes-KOH, pH 7.2, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, 10 mM succinate, 2 mM
ATP, 1 mM dATP, 10 mM phosphocreatine, 50 µg/ml creatine kinase plus complete mixture of protease inhibitors), 20-50 µg of cytosolic extract, and 5 µg of isolated liver
mitochondria. The reaction mix was incubated at 37 °C for various
time periods, and mitochondria were removed by centrifugation at
14,000 × g in a microcentrifuge for 10 min.
Supernatants were further used for SDS-PAGE and immunoblotting or for
DEVD cleavage assays.
Measurement of Caspase Activity--
Cytosolic protein (20-100
µg) was diluted in extraction buffer at a final volume of 200 µl in
a microtiter plate 100 µM DEVD-pNA substrate (Biomol).
Samples were incubated at 37 °C, and absorbance at 405 nm was
measured using a SpectraMax microtiter plate reader at 30 min, 1 h, and 2 h.
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RESULTS |
Cytochrome c Release from Mitochondria Is Prevented by Caspase
Inhibition in Fas-induced Apoptosis--
To examine whether or not Fas
signaling may go through a mitochondrial pathway, Jurkat T-lymphocytes
were treated with agonistic anti-Fas antibodies (CH-11), and cytosolic
extracts, lacking mitochondria, were prepared at various times and
analyzed by immunoblotting. Cytochrome c accumulated in
cytosolic extracts at 2 h after exposure to anti-Fas antibodies, a
time when caspase-8 was activated (detected by the 18-kDa active
caspase-8 fragment) (Fig. 1, a
and b). Following caspase-8 activation and cytochrome
c release, procaspase-3 became processed at 4 h
(recognized by the loss of its pro-form) (Fig. 1c). Cleavage
of the caspase substrates fodrin and protein kinase C-
was
detectable at 2 h (Fig. 1, d and e).
zVAD-fmk treatment prevented cytochrome c release,
procaspase-8, and procaspase-3 proteolytic processing, substrate
cleavage, and cell death (Fig. 1), suggesting that caspase-8 may
initiate cytochrome c release in this system.

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Fig. 1.
Cytochrome c is released
from mitochondria in Fas-induced apoptosis, which is inhibited by
zVAD-fmk. Jurkat T-lymphocytes were treated with anti-Fas
antibodies (CH 11, 250 ng/ml) for the indicated times, and cytosolic
extracts were prepared. When caspase inhibitors were used, cells were
preincubated with 100 µM zVAD-fmk for 1 h and then
treated with anti-Fas antibodies. Cytosolic extracts (50 µg) were
separated by SDS-PAGE and immunoblotted as described under
"Experimental Procedures." Actin was used as a protein loading
control. The percentage of cell death was determined by light
microscopy of eosin & heamatoxylin-stained cytospins. Cells were scored
as apoptotic when the cytoplasm was shrunken and chromatin was
condensed. Cyt. c, cytochrome c;
casp., caspase; PKC, protein kinase C.
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Mitochondria Amplify Caspase-8-induced DEVD-specific Cleavage
Activity--
We had previously observed that the activation of
caspase-3 by caspase-8 in vitro proceeds identically in the
presence or absence of cytosol (29). This raised the question of
whether the release of cytochrome c following Fas ligation
can contribute to caspase activation. To test the possible role of
mitochondria in cell death mediated by Fas or tumor necrosis factor
receptors, purified caspase-8 and cytosol were incubated with
increasing amounts of mitochondria for various times, and the resulting
caspase activity was measured by conversion of the colorimetric
substrate, DEVD-pNA. As shown in Fig. 2,
caspase-8-induced cleavage activity was low and reached an early
plateau after 60 min, when either no or a low amount (1%; v/v) of
mitochondria were present. In contrast, the caspase-8-mediated
DEVD-specific cleavage activity was significantly elevated in the
presence of 5, 10, and 15% (v/v) mitochondrial fraction. Thus,
mitochondria enhance caspase-8-induced DEVD-specific cleavage activity,
most likely by release of cytochrome c and activation of the
Apaf-1-procaspase-9 complex.

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Fig. 2.
Caspase-8-induced DEVD-specific cleavage
activity is enhanced by mitochondria. Cytosolic extracts (50 µg)
and caspase-8 (100 ng/ml) were incubated either with or without the
indicated amounts of a mitochondrial fraction (MF) at
37 °C; at the indicated times, samples were spun in a
microcentrifuge, and supernatants were tested for DEVD-specific
cleavage activity as described under "Experimental Procedures."
, without MF; , 1% (v/v) MF; , 5% (v/v) MF; , 10% (v/v)
MF; , 15% (v/v) MF.
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Caspase-8-induced Cytochrome c Release from Mitochondria Requires
Cytosol--
To examine whether caspase-8 would induce cytochrome
c release from mitochondria, cytosolic Jurkat T-lymphocyte
cell extracts, purified caspase-8 and mitochondria were incubated
in vitro. When cytosol was incubated with mitochondria
alone, no cytochrome c release was observed, indicating that
the mitochondrial preparation was not releasing cytochrome c
nonspecifically (Fig. 3A).
Similarly, when caspase-8 was incubated with the mitochondrial fraction
alone, no cytochrome c release was detectable by
immunoblotting, indicating that caspase-8 had no direct effect on
mitochondria (Fig. 3B). However, when caspase-8, cytosol,
and mitochondria were incubated together, cytochrome c was
released from mitochondria within 20 min (Fig. 3C, lane 4),
and reached maximum release at 90 min (Fig. 3C, lane 7).
These results indicate that caspase-8 is capable of mediating
cytochrome c release in the presence of cytosolic extract,
presumably by proteolytically activating one or several cytosolic
cytochrome c releasing factors.

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Fig. 3.
Caspase-8-induced cytochrome c
release from isolated mitochondria requires cytosol.
A, Jurkat cytosol (20 µg) was incubated with mitochondrial
fraction (10 µg) in reaction buffer. At the indicated times,
mitochondria were removed by centrifugation, supernatants were
subjected to SDS-PAGE (15%), and immunoblotting with anti-cytochrome
c antibodies was performed as described under
"Experimental Procedures." B, purified caspase-8 (50 ng/ml) was incubated with mitochondria (10 µg) for various times at
37 °C and cytochrome c release from mitochondria was
analyzed as in A. C, caspase-8 (50 ng/ml) was
incubated together with cytosolic extract (20 µg) and mitochondria
(10 µg) for the indicated time periods, and samples were treated as
described in A. Lane 9 shows cytochrome
c (Cyt.c) content of the amount of lysed
mitochondrial fraction present in each sample .
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Ultrafiltration of Cytosol Removes the Caspase-8-induced Cytochrome
c Releasing Activity--
To further characterize the
caspase-8-induced cytochrome c releasing activity, cytosolic
extract from Jurkat cells was filtered to remove proteins that were
larger than 10 kDa, prior to incubation with caspase-8, and
mitochondria. Fig. 4A shows a
control experiment, in which unfiltered cytosol and mitochondria
without caspase-8 were incubated. As shown above (Fig. 3A),
no cytochrome c release was observed under these conditions.
On the other hand, incubation of caspase-8 with unfiltered cytosolic
extract and mitochondria induced rapid cytochrome c release,
detectable after 10 min at 37 °C (Fig. 4B). However, when
cytosol was first filtered, caspase-8-induced cytochrome c
release from mitochondria was abolished (Fig. 4C). These
data indicate that the capsase-8-induced cytochrome c
releasing activity resides in the cytosol and is likely a protein
larger then 10 kDa.

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Fig. 4.
Ultrafiltration of cytsolic extracts removes
the caspase-8 induced cytochrome c releasing
activity. A, cytosolic extract (30 µg) was mixed with
mitochondria (14 µg), and at the indicated times and at 37 °C,
mitochondria were removed by centrifugation. Supernatants were
separated by SDS-PAGE (15%) and immunoblotted using anti-cytochrome
c (Cyt.c) antibodies as described under
"Experimental Procedures." B, caspase-8 (100 ng/ml) was
mixed with Jurkat cytosolic extract (30 µg) and mitochondria (14 µg) and further analyzed as described in A). C,
cytosol was filtered using an Ultra-free MC (10,000 NMWL) Millipore
filter unit at 4 °C to remove proteins >10 kDa. After filtration,
caspase-8 and mitochondria were added and further analyzed as described
in A. Right lane (MF) shows cytochrome
c content of the lysed mitochondria.
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Caspase-8-induced Cytochrome c Releasing Factor Is Not Inhibited by
zVAD-fmk--
Other caspase family members are known caspase-8
substrates (18, 21, 23-29). Therefore, it is conceivable that
caspase-8 activates another caspase, which then acts directly on
mitochondria to release cytochrome c. To test this
possibility, cytosol was treated either simultaneously with caspase-8
and caspase inhibitor, zVAD-fmk, or first with caspase-8, allowing
proteolytic processing of the putative substrates, and then with
zVAD-fmk (to inhibit possible downstream caspases), and mitochondria
were added. Fig. 5a, lanes
1-4, shows cytochrome c release in response to cytosol and caspase-8, when no caspase inhibitor was added. When caspase-8, cytosol and zVAD-fmk were incubated together from the beginning, no
cytochrome c release from mitochondria was observed,
confirming that the inhibitor was effective in blocking caspase
activity (Fig. 5b, lanes 1-4). Interestingly, when cytosol
was pretreated with caspase-8, and then zVAD-fmk and mitochondria were
added, cytochrome c release proceeded with similar kinetics
as in control experiments, without zVAD-fmk (Fig. 5b, lanes
5-8). Thus, the cytosolic target of caspase-8, which triggers the
release of cytochrome c from mitochondria, is unlikely to be
a caspase.

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Fig. 5.
Caspase-8-induced cytochrome c
releasing activity is not inhibited by zVAD-fmk. Jurkat
cytosolic extract was treated with caspase-8 alone (A) or
with zVAD-fmk (10 µM) (B) for 2 h at
37 °C followed by addition of mitochondria (15 µg) (lanes
1-4). Alternatively, cytosol was pretreated with caspase-8 (100 ng/ml) for 2 h, and then zVAD-fmk and mitochondria were added
(lanes 5-8). Lane 9 shows cytochrome
c (Cyt. c) content of the lysed mitochondrial
fraction added to each sample.
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Bcl-2/Bcl-xL Inhibit the Caspase-8-induced Cytochrome c Releasing
Activity--
Bcl-2 and Bcl-xL prevent cytochrome c release
from mitochondria during apoptosis induced by different stimuli (30,
34-36, 41, 42). To test the effect of Bcl-2 on caspase-8-induced cytochrome c release, mitochondria were isolated from
CEM-neo or CEM-Bcl-2 cells, which over-express Bcl-2. Fig.
6A, a, shows the amount of
Bcl-2 overexpression in CEM-Bcl-2 cells. Mitochondria from both cell
lines were incubated with caspase-8 and cytosolic extracts, and
cytochrome c release was measured by immunoblotting. As
shown in Fig. 6A, b, cytochrome c was rapidly
released from the mitochondria of CEM-neo cells, starting at 10 min of
incubation with active caspase-8. In contrast, cytochrome c
release from CEM-Bcl-2 mitochondria was considerably delayed, occurring
only at 40 min (Fig. 6A, b). Similarly, when increasing
amounts of purified, recombinant Bcl-xL protein were added to cytosol
containing caspase-8 and mitochondria, cytochrome c release
was inhibited (Fig. 6C). In contrast, increasing amounts of
proapoptotic Bax protein had a moderate additive effect on the
caspase-8-induced cytochrome c releasing activity. Taken
together, these results indicate that the cytochrome c
releasing activity can be inhibited by cell death inhibitors Bcl-2 or
Bcl-xL.

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Fig. 6.
Caspase-8-induced cytochrome c
release is delayed by Bcl-2/Bcl-xL. A, a,
immunoblot of CEM-neo and CEM-Bcl-2 cells, using anti-Bcl-2 antibodies,
after hypotonic lysis. b, equal amounts of mitochondria
isolated from CEM-neo and CEM-Bcl-2 cells were incubated with caspase-8
(100 ng/ml) and cytosolic extract (30 µg). Mitochondria were removed
by centrifugation at the indicated times, and supernatants were
immunoblotted with anti-cytochrome c antibodies.
B, the indicated amounts of recombinant Bcl-xL or Bax
protein were added to isolated liver mitochondria, cytosol (50 µg),
and caspase-8 (200 ng/ml). After incubation for 60 min at 37 °C,
mitochondria were removed by centrifugation, and cytochrome
c content in supernatants was analyzed by immunoblotting
using anti-cytochrome c antibodies.
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Bid Cleavage by Caspase-3 and Caspase-8--
Recent reports
identified Bid, a proapoptotic Bcl-2 family member, as a cytosolic
protein that triggers cytochrome c release from mitochondria
after proteolytic processing by caspase-8 (61-64). Therefore, we
tested the ability of caspase-3, caspase-6, caspase-7, and caspase-8 to
cleave Bid protein. Recombinant, active site-titrated caspases were
used. Cytosolic extracts from Jurkat cells were incubated with the
caspases for various time periods, and the cleavage of Bid was
determined by immunoblotting (Fig. 7).
Caspase-8 cleaved Bid most effectively, which was detectable at 5 min
and was completed at 15 min. Similarly, caspase-3 caused Bid cleavage, but with slower kinetics than caspase-8. In contrast, no cleavage of
Bid was detectable by caspase-6 and caspase-7.

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Fig. 7.
Bid cleavage by caspases in vitro.
Cytosolic extract from Jurkat T-lymphocytes (25 µg) was incubated
with the indicated recombinant caspase (50 nM) for the
indicated times at 37 °C. Protein extracts were separated by 15%
SDS-PAGE and immunoblotted using anti-Bid polyclonal antibody.
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Because caspase-8 can cleave and activate caspase-3, we also examined
how effectively Bid would be cleaved in comparison to caspase-3.
Interestingly, Bid and caspase-3 were processed by caspase-8 with
similar rates (Fig. 7).
Cytochrome c Release by Effector Caspases Requires Cytosol--
We
then tested whether effector caspases (caspase-3, caspase-6, and
caspase-7) would also promote cytochrome c translocation from mitochondria in vitro. Three different concentrations
of purified active caspases (20, 50, and 100 nM), normally
found in cells, were tested for their ability to initiate release of cytochrome c in the presence or absence of cytosol. Fig.
8A shows that all caspases
provoked cytochrome c release from isolated mitochondria
in vitro. However, this effect was dependent on the presence
of cytosol, indicating that none of the caspases had a direct effect on
mitochondria at physiological concentrations. Caspase-3 and caspase-8
were most able among the tested caspases to induce cytochrome
c release, with a maximum release at 30 min and at a
concentration of 20 nM. Similarly, caspase-6 was able to
initiate cytochrome c release. Caspase-7 was the least
efficient among the tested caspases in promoting cytochrome
c release, which only was detectable at 60 min and
concentrations of 50 and 100 nM.

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Fig. 8.
Effector caspases provoke cytochrome
c release in the presence of cytosol.
A, purified recombinant caspases were incubated at three
different concentrations (20, 50, and 100 nM) without
( cytosol) or with (+ cytosol) cytosolic
extract (30 µg) and mouse liver mitochondria (3 µg). After
incubation at 37 °C for the indicated times, mitochondria were
removed by centrifugation and supernatants were separated by 15%
SDS-PAGE. Cytochrome c (Cyt.c) release from
mitochondria was determined by immunoblotting using anti-cytochrome
c antibody. B, Bid cleavage was determined in the
same samples and analyzed as in A, by immunoblotting with
anti-Bid antibody.
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In the same experiment, we tested whether cytosolic Bid was cleaved.
Fig. 8B shows that although all caspases induced cytochrome c release from isolated mitochondria, Bid was only cleaved
by caspase-3 and caspase-8. In addition, Bid was less effectively processed by caspase-3 than by caspase-8, although both caspases released cytochrome c with similar, rapid kinetics. Neither
caspase-6 nor caspase-7 cleaved Bid (Figs. 7 and 8B),
despite their ability to induce cytochrome c release (Fig.
8A). Thus, cytosol may contain a caspase substrate, which
differs from Bid and which, in response to cleavage by effector
caspases, releases cytochrome c.
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DISCUSSION |
Binding of Fas ligand to the Fas receptor results in
autoprocessing and activation of caspase-8 (13-22). Active caspase-8
directly engages the caspase cascade by activating and cleaving
caspase-3 (29). Alternatively, caspase-8 can activate other caspases
indirectly, by inducing release of cytochrome c from
mitochondria. Once in the cytoplasm, cytochrome c binds and
activates Apaf-1 and procaspase-9 (38, 39). Active caspase-9 then
cleaves and activates procaspase-3. In this study, we examined the role
of mitochondria in Fas-mediated cell death. We have found that
cytochrome c is released from mitochondria in Jurkat cells
undergoing cell death by Fas activation (Fig. 1). Unlike cell death
induced by other stimuli (37), the cytochrome c release by
Fas signaling was effectively prevented by caspase inhibitors,
rendering caspase-8 and other caspase family members likely candidates
for this process. This is in agreement with other reports, describing
inactivation of electron transport and subsequent cytochrome
c loss from mitochondria in Fas-mediated cell death (30, 32,
41, 42). At 12 h, some cytochrome c accumulated in
zVAD-fmk plus anti-Fas treated cells (Fig. 1a). An
explanation might be that long-term Fas-receptor ligation may activate
other signaling molecules with cytochrome c releasing activity and that are not blocked by caspase inhibitors. A candidate for such a signaling molecule might be Daxx, which is implicated in
Fas-mediated apoptosis (47).
Several cell-free systems of apoptosis revealed that mitochondria play
a pivotal role in the regulation of cell death (48, 49). We show here
that purified caspase-8 induces rapid cytochrome c release
from mitochondria in vitro. (Fig. 2). However, the effect was indirect, because a cytosolic extract was required (Fig.
3C), suggesting that caspase-8 cleaves and activates one or
several cytosolic targets, provoking cytochrome c release.
Similar results were obtained in a Xenopus cell-free system,
in which caspase-8 induced cytochrome c release indirectly
by cleaving at least one cytosolic factor (50). Recent reports
identified Bid as cytosolic protein that induces cytochrome
c release in response to cleavage by caspase-8. Bid is a
proapoptotic member of the Bcl-2 family that interacts with Bcl-2 and
Bax (61). Bid is normally present in the cytosol as inactive protein
and undergoes caspase cleavage in response to Fas or tumor necrosis
factor receptor signaling (62-64). The carboxyl-terminal fragment of
Bid translocates then to mitochondrial membranes and triggers
cytochrome c release. Here, we show that Bid is cleaved
rapidly by caspase-8 (Fig. 7). In addition, caspase-3 processed Bid,
but with slower kinetics than caspase-8. These data are in agreement
with recent reports, describing Bid as an excellent substrate for
caspase-8, but a less good substrate for caspase-3 (63, 64).
The induction of cytochrome c release from mitochondria was
not restricted to caspase-8, as effector caspases, including caspase-3, caspase-6, and caspase-7, also triggered rapid cytochrome c
release when cytosol was present (Fig. 8A). Although
caspase-6 and caspase-7 promoted cytochrome c translocation,
they were unable to process Bid (Figs. 7 and 8B). Thus,
cytochrome c release initiated by effector caspases may
involve another cytosolic factor, which when cleaved by caspases
releases cytochrome c and which is different from Bid.
Bax, another proapoptotic member of the Bcl-2 family, can directly
induce cytochrome c release from mitochondria (51, 52). Bax
protein resides in the cytoplasm in living cells and translocates to
mitochondria when cells receive an apoptotic signal (53, 54). Thus, one
can speculate that another cytosolic caspase target could be either a
Bax-binding protein, which keeps it in an inactive state in the
cytoplasm, or Bax itself. There is precedent for Bcl-2 family members
being caspase substrates. Caspase-3 can cleave Bcl-xL, converting it
into a Bax-like, proapoptotic molecule (55-57). Bcl-2 or Bcl-xL in our
cell-free system inhibited the caspase-8-induced cytochrome
c activity (Fig. 6).
What role does the mitochondrial/cytochrome c pathway play
in Fas-mediated cell death? When active caspase-8 was incubated either
with cytosol alone or low amounts of a mitochondrial fraction, moderate
DEVD-specific cleavage activity was observed, suggesting that
relatively little caspase-3 was cleaved and activated directly by
caspase-8. However, with increasing amounts of mitochondria, the
DEVD-specific cleavage activity was increased (Fig. 2). This is
consistent with an observation by Kuwana et al. (50) in
which mitochondria amplified caspase-8 initiated DEVDase activity in a
Xenopus cell-free apoptosis system. As proposed by Scaffidi et al. (42), the contribution of mitochondria may depend on the amount of active caspase-8 formed in the death-inducing signaling complex, which can vary with cell type. Here we report that Bid and
caspase-3 are cleaved by caspase-8 with similar and rapid kinetics
(Fig. 7), suggesting that in intact cells both mitochondrial and
mitochondrial-independent pathways may be activated at the same time.
Mitochondria might ensure rapid and irreversible cell death by loss of
cytochrome c from mitochondria in situations in which small
amounts of caspase-8 are activated and downstream caspases are not
present or inhibited (42, 58-60). They may also permit an additional
degree of regulation, as Fas-induced cell death can be inhibited by
Bcl-2 or Bcl-xL in some cases (41, 42, 44, 46). In summary, cytochrome
c release initiated by caspases may serve as a positive feed
forward mechanism to amplify the caspase cascade.