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INTRODUCTION |
Apoptosis is a major form of cell death characterized by a series
of stereotypic morphological features. It occurs in two phases, an
initial commitment phase followed by an execution phase involving the
condensation and fragmentation of nuclear chromatin, dilation of the
endoplasmic reticulum, and alterations to the cell membrane resulting
in recognition and subsequent phagocytosis of the cell (1, 2).
Caspases, a family of cysteine proteases, play a critical role in the
execution phase of apoptosis and are responsible for many of the
biochemical and morphological changes associated with apoptosis (3, 4).
It has been proposed that "initiator" caspases with long
prodomains, such as caspase-8 (MACH/FLICE/Mch5), either directly or
indirectly activate "effector" caspases, such as caspases-3, -6, and -7 (3, 5, 6). These effector caspases then cleave intracellular
substrates, such as poly(ADP-ribose) polymerase
(PARP)1 and lamins, during
the execution phase of apoptosis. Caspase-8 is the most apical caspase
in CD95 (Fas/APO-1)-induced apoptosis (7, 8). Triggering of the CD95
receptor with its cognate ligand or agonistic antibody results in
receptor trimerization and recruitment of CD95 receptor-associated
protein with death domains (FADD/MORT1), which in turn binds to the
death effector domains in the N-terminal region of caspase-8, resulting
in its activation. As caspase-8 can activate all known caspases
in vitro (6), it is a prime candidate for an initiator
caspase in many forms of apoptosis in addition to CD95-induced
apoptosis. Procaspase-9 has also been proposed as an initiator caspase;
in the presence of dATP and cytochrome c, its long
N-terminal domain interacts with Apaf-1 resulting in the activation of
caspase-9 (9, 10). Active caspase-9 can then activate the effector
caspases-3, -6, and -7 (10, 11). Thus there are at least two major
mechanisms by which a caspase cascade resulting in the activation of
effector caspases may be initiated as follows: one involving caspase-8 and the other involving caspase-9 as the most apical caspase.
Cytochrome c, which is usually present in the mitochondrial
intermembrane space, is released into the cytosol following the induction of apoptosis by many different stimuli including CD95, tumor
necrosis factor (TNF), and chemotherapeutic and DNA-damaging agents
(12-14). Mitochondria have been proposed to act as an amplifier in
CD95-induced apoptosis when activation of caspase-8 cleaves a cytosolic
substrate leading to release of cytochrome c (15, 16).
Release of mitochondrial cytochrome c and activation of caspase-3 is blocked by anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, and promoted by proapoptotic
members, such as Bax and Bak (13, 17, 18). In chemical- or
irradiation-induced apoptosis, cytochrome c release appears
to be caspase-independent as it is not inhibited by the broad spectrum
cell-permeable caspase inhibitor, Z-VAD.FMK (13, 17, 19, 20).
Mechanisms for the release of mitochondrial cytochrome c
include opening of a mitochondrial permeability transition pore, the
presence of a specific channel for cytochrome c in the outer
membrane, or mitochondrial swelling and rupture of the outer membrane
but without loss of mitochondrial membrane potential (14). None of
these mechanisms appears generally applicable, as release of cytochrome
c occurs in cells with normal mitochondrial membrane
potential (13, 17) and by a mechanism independent of rupture of the
outer mitochondrial membrane (20). Two recent studies have highlighted
another possible mechanism of mitochondrial cytochrome c
release, involving Bid, a BH3 domain-containing proapoptotic Bcl-2
family member. Cleavage of Bid by caspase-8 results in translocation of
the cleaved Bid to the mitochondria where it induces the release of
cytochrome c, being 500-fold more potent than Bax (21, 22).
The BH3 domain of Bid is essential both for its proapoptotic activity
and its ability to induce the release of cytochrome c (22,
23).
In this study we address the order in which caspases are activated in
receptor-mediated and chemical-induced apoptosis. Our data support the
hypothesis that caspase-8 and caspase-9 are the most apical caspases in
receptor-mediated and chemical-induced apoptosis, respectively. In
chemical-induced apoptosis, cytochrome c release is
caspase-independent and is not mediated by cleavage of Bid in contrast
to receptor-mediated apoptosis. We propose that caspases act solely as
executioners of apoptosis in chemical-induced apoptosis, whereas in
receptor-mediated apoptosis they also form an integral part of the cell
death-inducing mechanism.
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EXPERIMENTAL PROCEDURES |
Materials--
Media and serum were purchased from Life
Technologies, Inc. (Paisley, UK). Z-VAD.FMK and Z-DEVD.AFC were from
Enzyme Systems Inc. (Dublin, CA), and Z-IETD.CHO was kindly provided by
Professor L. Rubin (Eisai London Research, London, UK). Anti-CD95
monoclonal antibody was obtained from Upstate Biotechnology Inc. (Lake
Placid, NY). Annexin V/FITC kit was from Bender Medsystems (Vienna,
Austria). DiOC6(3) was purchased from Molecular Probes
(Eugene, OR). All other chemicals and human recombinant TNF-
were
from Sigma (Poole, UK).
Cell Culture and Quantification of Apoptosis by Flow
Cytometry--
Jurkat T cells (clone E6-1) were obtained from ECACC
and cultured in RPMI 1640 containing 10% fetal bovine serum and 1%
Glutamax. Apoptosis in U937 cells was induced with etoposide (25 µM) or TNF-
(10 ng/ml) and cycloheximide (0.9 µM) (24). Some cells were treated for 1 h with
Z-VAD.FMK prior to exposure to the apoptotic stimulus. Apoptosis was
quantified by PS exposure or by loss of the mitochondrial membrane
potential, assessed with DiOC6(3) (20, 25).
Western Blot Analysis--
Cell samples were prepared as
described (26). Proteins were resolved on 12-15% SDS-polyacrylamide
gels and blotted onto nitrocellulose (Hybond-C extra, Amersham, Bucks,
UK). Caspases-3 and -7 were detected as described previously (26). A
rabbit polyclonal antibody to caspase-8 was raised against the large subunit of caspase-8 (amino acids 210-374). The antibody obtained was
characterized by enzyme-linked immunosorbent assay and Western blot
analysis, which verified that the antibody recognized intact procaspase-8 and the p43 and p18 subunits. An antibody to caspase-9 was
also raised, which recognized both the inactive proform and the
activated ~37- and 35-kDa processed forms. Cytochrome c
antibody was purchased from PharMingen (San Diego, CA). The Bid
antibody was kindly provided by Dr. X. Wang (22). The PARP antibody was obtained from Dr. G. Poirier (Laval University, Quebec, Canada).
Preparation of Cytosol for Measurement of Cytochrome
c--
Cells were collected at the indicated times and washed once in
ice-cold phosphate-buffered saline. Cell pellets were resuspended in
cytosol extraction buffer, and cytosolic extracts were prepared by the
method described previously (19).
Preparation of Cell Lysates for in Vitro Caspase Activation
Studies and Immunodepletion of Caspases--
Jurkat cell lysates were
prepared as described previously (26) and activated by addition of dATP
(2 mM), cytochrome c (0.25 mg/ml), and
MgCl2 (2 mM) (12). The proteolytic activity
(cleavage of Z-DEVD.AFC) of the lysate was measured as described
previously (26). Immunodepletion of capsase-8 or -9 from Jurkat cell
lysate was performed as described previously (9).
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RESULTS |
Anti-CD95 Antibody and Etoposide Induce a Similar
Time-dependent Processing of Caspases--
Both CD95
antibody and etoposide caused a time-dependent induction of
apoptosis in Jurkat T cells, as assessed both by an increase in
externalization of phosphatidylserine (PS) or by a decrease in
mitochondrial membrane potential (
m) (Figs. 1 and 2).
This was accompanied by a similar time-dependent processing of caspase-3, -7, -8, and -9 (Figs. 1 and 2). In Jurkat T cells, caspase-3 was present primarily as its intact 32-kDa proform (Fig. 1A, lane 1). Induction of both chemical- and
receptor-mediated apoptosis resulted in loss of the proform of
caspase-3 and appearance of three immunoreactive fragments of ~20 kDa
(p20), ~19 kDa (p19), and ~17 kDa (p17), following initial cleavage
at Asp-175 and then at Asp-9 and Asp-28 (27). Processing was first
detected after 2-3 h treatment with either stimulus (Figs.
1A and 2A, lanes 2-7).

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Fig. 1.
CD95-induced time-dependent
processing of caspases in Jurkat T cells. Jurkat T cells were
incubated from 0.5-6 h as indicated in the presence of anti-CD95
antibody (50 ng/ml). Cells were also treated with Z-VAD.FMK for 1 h prior to induction of apoptosis in order to facilitate its cell
permeability. Cells were then analyzed by immunoblotting for the
processing of caspase-3 (A), caspase-7 (B),
caspase-8 (C), and caspase-9 (D), as described
under "Experimental Procedures." A shorter exposure of the film
showed that the proform of caspase-8 comprised two bands of ~55 and
53 kDa. Apoptosis was assessed either by Annexin V binding to measure
the percentage of cells with externalized PS or by DiOC6(3)
to quantify the percentage of cells with decreased mitochondrial
membrane potential ( m).
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Fig. 2.
Etoposide-induced time-dependent
processing of caspases in Jurkat T cells. Jurkat T cells were
incubated for the indicated times with etoposide (50 µM)
either alone or in the presence of the indicated concentrations of
Z-VAD.FMK. Cells were then analyzed by Western blot analysis for the
processing of caspase-3 (A), caspase-7 (B),
caspase-8 (C), and caspase-9 (D), as described
under "Experimental Procedures" and the legend to Fig. 1.
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Caspase-7 was present in control Jurkat T cells primarily as a
~35-kDa protein (Fig. 1B, lane 1). Treatment with both
anti-CD95 antibody and etoposide resulted in a
time-dependent processing of caspase-7 accompanied by the
formation of two major products. These were a ~32-kDa fragment, which
probably represents the loss of the prodomain at
DSVD23
A, and a ~19-kDa (p19) fragment, which
corresponds to the catalytically active large subunit (Figs.
1B and 2B, lanes 2-7) formed following cleavage
at IQAD198
S (27). Processing of caspase-7 was first
observed 2 h after treatment with either stimulus.
In untreated Jurkat T cells, caspase-8 was present primarily as two
isoforms of ~55 kDa (Fig. 1C, lane 1), possibly
corresponding to caspase-8a and -8b (7, 28). Exposure to both anti-CD95 antibody and etoposide resulted in a time-dependent
processing of caspase-8 initially to two fragments of ~43 and 41 kDa
(p43 and p41, respectively), corresponding to cleavage of both
caspase-8a and -8b between the large and small subunits. This was
followed by the appearance of a p18 subunit resulting from removal of
the death effector domains from the 43- and 41-kDa fragments (Figs. 1C and 2C, lanes 2-7) (6, 28). An increase in
the processing of caspase-8 was first observed 2 and 2.5 h after
CD95 and etoposide treatment, respectively.
Untreated Jurkat cells contained the 46-kDa proform of caspase-9 (Fig.
1D, lane 1), which on induction of apoptosis was processed in a time-dependent manner to yield fragments of ~37 and
35 kDa (p37 and p35) (Figs. 1D and 2D, lanes
2-7), resulting from cleavage at both Asp-315 and Asp-330 (6).
The first detectable processing of caspase-9 was evident at 2 h
(Figs. 1D and 2D).
Thus, induction of apoptosis was accompanied by the activation of both
the activator caspases -8 and -9 and the effector caspases-3 and-7,
which all appeared to occur simultaneously making it extremely difficult to distinguish the order in which they were activated. In
order to further address this problem, we used the broad-spectrum caspase inhibitor Z-VAD.FMK, which inhibits apoptosis in many but not
all systems (3).
Z-VAD.FMK Inhibits Different Targets in Etoposide- and
CD-95-induced Apoptosis--
Slight inhibition of CD95-induced caspase
processing was observed at Z-VAD.FMK (0.1 µM), with
marked and almost total inhibition at 1.0 and 10 µM,
respectively (Fig. 1, A-D, lanes 8-10). Higher concentrations of Z-VAD.FMK were required to inhibit etoposide-induced processing of these caspases (Fig. 2, A
D, lanes 8-10).
Z-VAD.FMK (10 and 25 µM) largely but not completely
inhibited the processing of caspases-3, -7, and -9 (Fig. 2, A
D,
lanes 8 and 9). As caspase-9 processes caspases-3 and
-7 (10, 29), these data support the suggestion that one of the
important targets of Z-VAD.FMK in etoposide-induced apoptosis may be
either the processing or the activity of caspase-9. However Z-VAD.FMK
(10 µM) completely inhibited the processing of caspase-8,
which suggested that caspase-8 was activated downstream of caspases-3
and -7. Most importantly these data strongly suggested that the order
of caspase activation and the Z-VAD.FMK target(s) were different in
etoposide- and CD95-induced apoptosis in Jurkat cells.
The different cellular effects of Z-VAD.FMK on etoposide- and
CD95-induced apoptosis in Jurkat cells provided further support for
this hypothesis. Both CD95 and etoposide induced apoptosis, as assessed
either by an increase in externalization of PS (Fig. 3, B and D) or by a
decrease in mitochondrial membrane potential (Fig. 3, G and
I). Z-VAD.FMK (10 µM) completely inhibited
CD95-induced apoptosis assessed by both these criteria (Fig. 3,
C and H). However, Z-VAD.FMK (25 µM) did not inhibit etoposide-induced loss of
mitochondrial membrane potential (Fig. 3J) but did inhibit
PS exposure (Fig. 3E). Z-VAD.FMK (25 µM) also
did not inhibit etoposide-induced decrease in cell size as measured by
forward light scatter (Fig. 3, E and J). Taken
together, these data suggest that in CD95-induced apoptosis in Jurkat
cells the intracellular target of Z-VAD.FMK is most likely the
activator caspase-8 acting upstream of mitochondria, whereas in
etoposide-induced apoptosis the target(s) is the activation/processing of caspase-9, which is activated downstream of perturbation of mitochondria. In order to substantiate this hypothesis, we examined the
effects of Z-VAD.FMK on the release of mitochondrial cytochrome c.

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Fig. 3.
Z-VAD.FMK inhibits the decrease in both
mitochondrial membrane potential and cell size in CD95- but not in
etoposide-mediated apoptosis. Jurkat T cells were incubated for
6 h with CD95 (50 ng/ml) either alone or in the presence of
Z-VAD.FMK (10 µM). Cells were also incubated with
etoposide (50 µM) either alone or in the presence of
Z-VAD.FMK (25 µM). Apoptosis was quantified by flow
cytometric analysis as described in the legend to Fig. 1. Forward light
scatter is an indication of cell size. Cells in R2 represent
normal cells with low PS exposure and normal cell size; cells in
R3 are cells with low PS exposure but with decreased cell
size, and cells in R4 have an increase in PS exposure. Cells
in R5 have a high mitochondrial membrane potential
( m), and cells in R6 have a decreased
mitochondrial membrane potential. The numbers represent the percentage
of cells in the indicated region.
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Z-VAD.FMK Inhibits Receptor-mediated but Not Chemical-induced
Cytochrome c Release--
In agreement with other studies, CD95
induced a time-dependent increase of cytochrome
c in the cytosol, most probably due to an increased release
of mitochondrial cytochrome c (Fig.
4A). Z-VAD.FMK (10 µM) inhibited this increase (Fig. 4A), further
demonstrating that it inhibited a caspase upstream of the mitochondrial
changes. Etoposide also induced a time-dependent increase
in cytosolic cytochrome c; however, Z-VAD.FMK (25 µM) did not inhibit this increase (Fig. 4B)
further supporting the hypothesis that the target of Z-VAD.FMK in
etoposide-induced apoptosis is downstream of mitochondria.

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Fig. 4.
Z-VAD.FMK inhibits CD95- but not
etoposide-induced release of mitochondrial cytochrome c
in Jurkat cells. Jurkat T cells were incubated for the
indicated times with CD95 (50 ng/ml) (A) or etoposide (50 µM) (B). Cells were also pretreated for 1 h with the indicated concentrations of Z-VAD.FMK. Cells were then
analyzed by Western blot analysis for cytochrome c as
described under "Experimental Procedures."
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In order to determine if such differences between receptor-mediated and
chemically induced apoptosis also occurred in other cells, we examined
a human lymphoid tumor cell line, U937, which is sensitive to tumor
necrosis factor (TNF-
). Both TNF/cycloheximide and etoposide caused
an induction of apoptosis in U937 cells, assessed by PS
externalization, which was accompanied by an increase in cytosolic
cytochrome c, processing of caspase-3, and cleavage of PARP,
a commonly used measure of caspase-3-like enzymic activity (Fig.
5, A-C). Z-VAD.FMK completely
inhibited TNF/cycloheximide-induced apoptosis assessed by all these
criteria. In contrast, in etoposide-induced apoptosis Z-VAD.FMK
completely inhibited PS externalization and the cleavage of PARP but
only partially inhibited processing of caspase-3 and did not inhibit
the increase in cytosolic cytochrome c. Thus Z-VAD.FMK was
more effective at blocking the activity rather than the processing of
caspase-3. Taken together, the data from both Jurkat and U937 cells
support the hypothesis that Z-VAD.FMK inhibits a target upstream of
mitochondria in receptor (CD95 or TNF)-mediated apoptosis, whereas in
etoposide-induced apoptosis the Z-VAD.FMK target is downstream of
mitochondria.

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Fig. 5.
Z-VAD.FMK inhibits TNF- but not
etoposide-induced release of mitochondrial cytochrome c
in U937 cells. U937 cells were incubated for 3 h with
TNF- /cycloheximide (TNF/CHX) either alone or in the
presence of Z-VAD.FMK (2 µM). Cells were also incubated
for 5 h with etoposide (25 µM) either alone or in
the presence of Z-VAD.FMK (20 µM). Cells were then
analyzed by Western blot analysis as described under "Experimental
Procedures" for cytochrome c (A), processing of
caspase-3 (B), cleavage of PARP (C), and cleavage
of Bid (D). Intact PARP (116 kDa) is cleaved by a
caspase-3-like enzymic activity to an 89-kDa signature fragment. Intact
Bid (26 kDa) is cleaved into two fragments of ~15 and 14 kDa.
Con, control.
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Activation of Jurkat Lysate Results in Initial Processing of
Caspase-9--
In order to gain further insight into the order of
caspase activation in chemical-mediated apoptosis, the processing of
various caspases was studied in Jurkat cell lysates, a well established model for understanding postmitochondrial caspase cascades (12). Activation of lysates, which resulted in an increased caspase-3-like DEVDase activity, was accompanied by a time-dependent
processing of caspases (Fig. 6).
Processing of the effector caspases-3 and -7 was first observed after 5 min, and these caspases were almost completely processed after 60 min
(Fig. 6, lanes 1-7). Caspase-9 was very rapidly and
extensively processed to both its p37 and p35 fragments, initial
processing being observed by 1 min and virtually complete processing
noted at 30 min (Fig. 6, lanes 1-7). In marked contrast,
processing of caspase-8 was first observed at 30 min, when a small
amount was processed only to its ~41- and 43-kDa fragments (Fig. 6,
lanes 1-7). Co-incubation of lysates with two different
caspase inhibitors, Z-VAD.FMK or
benzyloxycarbonyl-Ile-Glu-Thr-Asp-aldehyde (IETD.CHO), resulted in
a marked inhibition of the processing of all the caspases with
caspase-9 being somewhat less sensitive to inhibition than the other
caspases (Fig. 6, lanes 8 and 9). Thus caspase
inhibitors may also block the processing of caspases activated by a
caspase cascade downstream of mitochondria.

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Fig. 6.
Caspase-9 is processed before caspases-3, -7, and -8 in activated lysates. Lysates from Jurkat cells were
prepared and activated in the presence of dATP (2 mM) and
cytochrome c (0.25 mg/ml). DEVDase activity was measured
fluorimetrically as described under "Experimental Procedures."
Processing of caspase-3 (A), caspase-7 (B),
caspase-8 (C), and caspase-9 (D) was measured
from 0-60 min by Western blot analysis as described in the legend to
Fig. 1.
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To elucidate further the role of caspases-8 and -9 in the
postmitochondrial caspase cascade, they were immunodepleted from lysates, and the subsequent ability of the lysate to be activated was
assessed. In the presence of dATP/cytochrome c, lysates from control Jurkat cells exhibited a marked DEVDase activity (Table I). Immunodepletion of caspase-8 caused
only a very slight decrease in DEVDase activity demonstrating that
caspase-8 contributed little to this activity (Table I). Western blot
analysis demonstrated that immunodepletion of caspase-8 resulted only
in loss of this caspase but not of other caspases (data not shown). In
contrast, immunodepletion of caspase-9 resulted in complete inhibition
of DEVDase activity (Table I) without loss of caspases-3, -7, and -8 (data not shown) demonstrating the key role of caspase-9 in the
postmitochondrial processing of caspases. Taken together these results
lend strong support to the hypothesis that caspase-9 is the first
caspase activated in a caspase cascade following perturbation of
mitochondria and release of cytochrome c.
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Table I
Immunodepletion of caspase-9 but not caspase-8 inhibits cytochrome
c/dATP-dependent activation
Jurkat lysate was prepared and immunodepleted with either preimmune
serum, caspase-8, or caspase-9 antibody, and the proteolytic activity
(DEVDase) of the control lysate and the immunodepleted lysates was then
measured as described under "Experimental Procedures."
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Z-VAD.FMK Inhibits Cleavage of Bid in Receptor- and
Chemical-mediated Apoptosis--
Cleavage of Bid is important for the
release of mitochondrial cytochrome c in CD95-induced
apoptosis (21, 22). We wished to investigate whether this mechanism
of cytochrome c release is important in other
receptor-mediated cell deaths or in chemical-mediated apoptosis. Bid
was present as an ~26-kDa protein in control Jurkat cells and was
cleaved initially to two major fragments of ~15 and 14 kDa (Fig.
7A), most probably following
cleavage at LQTD60
G and IEAD75
S (21, 22).
In CD95-mediated apoptosis, cleavage of Bid was first observed at 30 min (Fig. 7A). Z-VAD.FMK inhibited cleavage of Bid in a
concentration-dependent manner (Fig. 7A)
commensurate with its ability to inhibit caspase processing and release
of cytochrome c (Figs. 1 and 4A). Etoposide also
induced a time-dependent cleavage of Bid in Jurkat cells
with a small amount of fragmentation first being observed at 2 h
(Fig. 7B). Z-VAD.FMK inhibited this cleavage in a
concentrationdependent manner (Fig. 7B), but it did
not inhibit etoposide-induced release of cytochrome c (Fig. 4B). Thus, in etoposide-induced cell death in Jurkat cells,
cleavage of Bid was not required for the release of mitochondrial
cytochrome c.

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Fig. 7.
Z-VAD.FMK inhibits cleavage of Bid in both
chemical- and receptor-mediated apoptosis. Jurkat T cells were
incubated for the indicated times with anti-CD95 antibody (50 ng/ml)
(A) or etoposide (50 µM) (B). Cells
were also pretreated for 1 h with the indicated concentrations of
Z-VAD.FMK. Cells were then analyzed by Western blot analysis for Bid
and its cleaved products as indicated.
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In order to extend these studies, we examined cleavage of Bid in other
cell types. In U937 cells, induction of apoptosis with either
TNF/cycloheximide or etoposide resulted in cleavage of Bid (Fig.
5D, lanes 2 and 4). Z-VAD.FMK
prevented cleavage of Bid induced by both apoptotic stimuli (Fig.
5D, lanes 3 and 5); however, it only
inhibited cytochrome c release in TNF/cycloheximide- but not
in etoposide-induced apoptosis (Fig. 5A). Recently we have
shown that in human monocytic THP.1 cells, induction of apoptosis by
either etoposide or N-tosyl-L-phenylalanyl
chloromethyl ketone (TPCK), a chymotrypsin-like protease inhibitor, is
accompanied by the release of mitochondrial cytochrome c,
which is not blocked by Z-VAD.FMK (20). Both these chemical stimuli
also induced a time-dependent loss of intact Bid
accompanied by the formation of fragments of ~15 and 14 kDa, both of
which were prevented by Z-VAD.FMK (data not shown). Thus in three
different cellular systems, cleavage of Bid and loss of mitochondrial
cytochrome c accompany chemical induction of apoptosis. As
Z-VAD.FMK inhibited cleavage of Bid but not the loss of cytochrome
c, cleavage of Bid is not responsible for the release of
mitochondrial cytochrome c in chemical-induced apoptosis.
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DISCUSSION |
Z-VAD.FMK Inhibits Receptor-mediated Apoptosis prior to Involvement
of Mitochondria--
CD95-mediated apoptosis involves the initial
formation of a death-inducing signaling complex (DISC), which is formed
following the recruitment of FADD (Fas-associating protein with a death domain) (30). FADD binds through its death effector domain (DED) to one
of the two N-terminal DEDs of caspase-8, resulting in its activation
(7, 8). Active caspase-8 may induce apoptosis either directly following
direct activation of other caspases or indirectly following cleavage of
cytosolic factors, such as Bid, leading to involvement of mitochondria
and release of cytochrome c, which together with Apaf-1
results in activation of caspase-9 and the effector caspases (6, 16,
21, 22). Interestingly no major differences in CD95-induced apoptosis
were observed in T cells from caspase-9
/
and
Apaf-1
/
knock-out compared with wild type mice
(31-33). However, CD95-induced apoptosis was markedly reduced in
Apaf-1
/
compared with wild type embryonic fibroblasts
(34) suggesting that the same apoptotic stimulus may utilize different
caspases in different cell types. Although mitochondria may be
initially bypassed following direct activation of the effector
caspases-3 and -7 by caspase-8, these effector caspases may then
subsequently disrupt mitochondrial membrane function (Fig.
8). This will lead to a release of
intermembrane proteins, such as cytochrome c, resulting in a
further activation of caspases and the establishment of a
self-amplification loop (35). Thus receptor-mediated apoptosis may
initially either directly involve or bypass the mitochondria dependent
on the relative concentrations of caspase-8, the effector caspases and
cytosolic factors, such as Bid, in a specific cell type (16) (Fig.
8).

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Fig. 8.
Scheme for receptor- and chemical-induced
apoptosis. Triggering of death receptors, such as CD95 (Fas/Apo-1)
or TNF, results in recruitment of adapter molecules, such as FADD and
TRADD. The DED of FADD binds to the DED of caspase-8 leading to the
oligomerization and activation of caspase-8. Caspase-8 may directly
activate the effector caspases, 3 and 7, responsible for many of
the biochemical and morphological changes associated with the apoptotic
phenotype. Alternatively, caspase-8 can act indirectly by cleaving
cytosolic factors, such as Bid, leading to the release of cytochrome
c and activation of caspase-9 with subsequent activation of
the effector caspases. Active caspases-3 or -7 may then further
activate procaspase-9 or alter mitochondrial membrane permeability
setting up a self-amplification loop (- - - -) or they may cleave
DNA fragmentation factor (DFF) or inhibitor of
caspase-activated DNase (ICAD) leading to the
internucleosomal cleavage of DNA. In chemical-mediated apoptosis,
undefined signals lead to perturbation of mitochondria (Mit)
and loss of cytochrome c and then activation of
caspases.
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Anti-apoptotic members of the Bcl-2 family block mitochondrial
cytochrome c release (13, 17) and also block CD95- and TNF-induced apoptosis in some (16, 29, 36) but not all circumstances (37, 38). Thus in a variety of cell types, apoptotic signaling in
response to CD95 or TNF stimulation is regulated at least in part by a
Bcl-2 and/or Bcl-xL-inhibitable step. In this regard, CD95
has recently been shown to activate two distinct cell death pathways,
one involving FADD, which is Bcl-2-insensitive, and one involving Daxx,
which is Bcl-2 sensitive (39). Furthermore, Bcl-2 protects mice from
the lethal hepatic apoptosis induced by anti-CD95 antibody (40).
Taken together these studies demonstrate a physiological role for the
involvement of mitochondria in many instances of receptor-mediated
apoptosis. In the present study, apoptosis induced by two death
receptors, CD95 and TNF, was inhibited by low concentrations of
Z-VAD.FMK at a very early stage prior to processing of caspases-3, -7, -8, and-9, cleavage of Bid, release of mitochondrial cytochrome
c, loss of mitochondrial membrane potential, and
externalization of PS. These data strongly support the hypothesis that
the primary target of Z-VAD.FMK in receptor-mediated apoptosis is
inhibition of the activation of procaspase-8 prior to any perturbation
of mitochondria resulting in the inhibition of all downstream
biochemical effects (Fig. 8).
Z-VAD.FMK Inhibits Chemical-induced Apoptosis Downstream of
Mitochondria--
Following release from mitochondria, cytochrome
c binds to Apaf-1 resulting in the activation of caspase-9,
which in turn activates the effector caspases responsible for the
cleavage of many of the substrates associated with the characteristic
biochemical and morphological changes of apoptosis (9). In agreement
with data from chemical- and radiation-induced apoptosis in other cells (13, 17, 19, 20), Z-VAD.FMK did not inhibit release of mitochondrial
cytochrome c in etoposide-induced apoptosis in both Jurkat T
and U937 cells. Neither did it completely inhibit processing of
caspases-3, -7, and -9 (Figs. 2, 4, and 5). Taken together with the
demonstration of the critical role of caspase-9 in cytochrome c-catalyzed caspase activation in lysates (Fig. 6 and Table
I), these data support the hypothesis that the Z-VAD.FMK target in chemical-induced apoptosis is the processing or the activity of caspase-9. Recent studies on caspase-9 knock-out mice demonstrate that
caspase-9 is a critical upstream activator of a caspase cascade in vivo and in some situations is essential for the
processing of caspase-3 (31, 32).
Cleavage of Bid was observed in chemical- as well as receptor-mediated
apoptosis. Bid is most likely cleaved by low concentrations of
caspase-8 but can be cleaved by higher concentrations of caspase-3 (21). Thus in chemical-induced apoptosis, inhibition of Bid cleavage by
Z-VAD.FMK may be due to inhibition of processing/activity of either
caspase-3 or -8. In addition, effector caspases may activate caspase-8
at a later stage of the apoptotic process, resulting in cleavage of Bid
and release of mitochondrial cytochrome c, thereby further
amplifying the apoptotic program (Fig. 8). Most importantly, in
contrast to receptor-mediated apoptosis, Z-VAD.FMK blocked cleavage of
Bid but not release of cytochrome c (Figs. 4, 5 and 7)
demonstrating that cytochrome c release is caspase-independent and is not mediated by cleavage of Bid in three
different models of chemical-induced apoptosis. Whether the mechanism
of mitochondrial cytochrome c release involves another proapoptotic BH3 domain-containing member of the Bcl-2 family, such as
Bik, Hrk, Blk, or Bim, is not known. Interestingly EGL-1, a
Caenorhabditis elegans BH3-containing proapoptotic protein, has recently been isolated, and this functions upstream of CED-9, the
C. elegans homolog of Bcl-2, to induce apoptosis (41).
Caspases Are an Integral Part of the Cell Death-inducing Mechanism
in Receptor-mediated Apoptosis whereas in Chemical-induced Apoptosis
They Act Solely as Executioners--
Previous work has demonstrated an
increased clonogenic potential of cells treated with CD95 in the
presence of caspase inhibitors, such as Z-VAD.FMK (42, 43). Taken
together with the present and other studies (7, 8), it suggests that
the activator caspase-8 is an integral component of the cell
death-inducing mechanism and that in receptor-mediated apoptosis
activation of caspase-8 represents a commitment point to cell death. At
a later stage in the apoptotic program other effector caspases are
activated and are responsible for the characteristic biochemical and
morphological features of the apoptotic phenotype. We cannot exclude a
similar role for other activator caspases, such as caspase-10, which
like caspase-8 possesses a long N-terminal prodomain with two death effector domains (27).
In chemical-induced apoptosis, Z-VAD.FMK inhibited the activation of
caspases and PS externalization but not the loss of mitochondrial cytochrome c (Figs. 2, 4, and 5) or the loss of clonogenic
potential (43). Thus in chemical-induced apoptosis, Z-VAD.FMK does not inhibit the commitment to cell death but rather inhibits all the biochemical changes associated with caspase activation occurring following perturbation of mitochondria and loss of cytochrome c. We propose that Z-VAD.FMK inhibits the processing
and/or the activity of caspase-9 and thus blocks the subsequent
activation of effector caspases, resulting in the block of a
self-amplification loop (Fig. 8). Thus in chemical-induced apoptosis,
caspase activation occurs after commitment to cell death and is
primarily responsible for those stereotypic biochemical and
morphological changes commonly associated with the apoptotic phenotype.
These results have important clinical implications for the use of
caspase inhibitors as potential therapeutic agents. Cells, which are
committed to die but with caspases inhibited, may die more slowly and
with a different morphology from cells dying with characteristic
apoptotic morphology. This hypothesis is compatible with recent reports
describing a caspase-independent cell death, such as Bax- or
Bak-induced cell death (44, 45).
In summary, we have demonstrated that in receptor-mediated apoptosis,
Z-VAD.FMK inhibited apoptosis prior to commitment to cell death by
inhibiting the upstream activator caspase-8. This resulted in
inhibition of cleavage of Bid, loss of mitochondrial cytochrome
c, processing of caspases, loss of mitochondrial membrane potential, and externalization of PS. In chemical-induced apoptosis, Z-VAD.FMK inhibited apoptosis downstream of cell death commitment by
inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases, cleavage of Bid, and PS
externalization but not loss of cytochrome c or
mitochondrial membrane potential. Thus in chemical-induced apoptosis, a
mechanism other than cleavage of Bid is responsible for the release of
cytochrome c.