By
From the * Centre National de la Recherche Scientifique-UPR420, B.P.8, F-94801 Villejuif, France; The Burnham Institute, La Jolla, California 92037; § Institute for General and Experimental
Pathology of the University of Innsbruck, A-6020 Innsbruck, Austria; and
Division Santé, Domain
Immunologie, ROUSSEL UCLAF, F-93235 Romainville, France
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive.
When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of
interleukin-1 converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (
m). In contrast, cytosolic CPP32/
Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the
m is fully disrupted. Transfection with the
cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the
m collapse and subsequent apoptosis. Cytosols from
anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified
ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their
m. In addition, ICE-treated mitochondria release an apoptosis-inducing factor
(AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA
fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be
inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling +
m collapse + release of AIF) induced by prooxidants or
cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of
apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.
It is currently assumed that the apoptotic process can be
divided into at least three functionally distinct phases (1). During the heterogeneous initiation phase, cells receive the
death-inducing stimulus via certain receptors such as the
TNF receptor or Fas/APO-1/CD95, shortage of obligatory growth factors, oxygen or metabolic supply, or subnecrotic physical and chemical damage. The biochemical
events participating in the initiation phase constitute "private" pathways in the sense that they depend on the lethal
stimulus. It is only during the subsequent phases that these
initiating events are translated into a regular common pattern
of metabolic reactions. The common pathway can be subdivided into an initial effector phase, during which the
"central executioner of apoptosis" is still subject to regulatory mechanisms, and a later degradation phase, beyond the
"point of no return", during which catabolic enzymes become activated in an irreversible fashion. During the degradation phase the morphology and characteristic biochemistry of apoptosis (e.g., step-wise DNA fragmentation, and
specific proteolysis of cytoplasmic and nuclear substrates)
become manifest (1).
Two nonexclusive mechanisms have been proposed to
intervene as central executioners of the apoptotic effector
phase. On one hand, it appears that apoptosis is associated
with the critical activation of a family of specific proteases
that include interleukin-1 Two mitochondrial proapoptotic factors have been purified: (a) the 15-kD cytochrome c protein, which acts together with cytosolic factors to induce nuclear apoptosis
(10), and (b) a ~50-kD protease that by itself suffices to
cause nuclear apoptosis (11). We have recently shown that
mitochondria release such a ~50-kD apoptogenic protein
(apoptosis-inducing factor, AIF) when they undergo PT (9,
11), a phenomenon that accounts for The hierarchical relationship between protease activation
and mitochondrial PT appears complex. The available data
suggest three levels of interaction between proteases and
PT. First, proteases may act upstream of PT. Thus, inhibitors of serine proteases such as N-tosyl-L-lysyl chloromethylketone and degenerate tripeptidic inhibitors of ICE-like
proteases such as N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk) prevent or retard the glucocorticoid-induced Prompted by these findings, we have studied the possible
impact of proteases on PT in one prototypic model of apoptosis, namely apoptosis induced via ligation of the Fas surface receptor. Fas-mediated cell death is thought to contribute to the maintenance of immune homeostasis, to immune
surveillance of mutating or virus-infected cells, as well as to
the pathological depletion of CD4+ lymphocytes in AIDS
(18, 19). It involves the activation of specific proteases,
namely an ICE-like protease associated with the Fas receptor complex (FLICE/Mach-1/Mch-5/caspase 8; references 20, 21), ICE (caspase 1; references 22), and CPP32
(caspase 3; reference 25). Fas-triggered apoptosis is unique
in the sense that it constitutes the only known apoptosis induction pathway that relies on the specific intervention of
ICE (22). In addition, Fas-induced apoptosis is not inhibited by Bcl-2, at least in some cell types (26). This
has lead to the speculation that Fas and Bcl-2 would regulate different pathways of apoptosis induction (26).
Thus, Fas could trigger an apoptotic pathway that bypasses the putative Bcl-2/PT checkpoint of the apoptotic effector
phase. Alternatively, it could induce PT in a fashion that is
not controlled by Bcl-2.
In this work, we discriminate between these possibilities
and present evidence indicating that, during Fas-induced
apoptosis, ICE functions as a direct inducer of mitochondrial PT. Although Bcl-2 efficiently inhibits mitochondrial
PT induced by a variety of different stimuli including
prooxidants, it completely fails to interfere with ICE-induced
signs of mitochondrial PT including Cell Lines and Culture Conditions.
Human CEM-C7.H2 lymphoma cells were transfected with a PHD1.2 crmA cDNA (1.46 kb) cloned in the sense orientation into a Cytofluorometric Determinations of Apoptosis-associated Alterations
in Whole Cells.
To evaluate the Preparation of Cytosols and Determination of the Activity of ICE-like
Proteases.
100 µl cytosols (107 cells/100 µl in cell-free system
[CFS] buffer [220 mM mannitol, 68 mM sucrose, 2 mM NaCl,
2.5 mM PO4H2K, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM PMSF, 1 mM dithiotreitol, 10 mM Hepes-NaOH])
and pH 7.4 buffer (supplemented with additional protease inhibitors: 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg/ml antipain,
10 µg/ml chymopapain) were prepared by five freeze/thaw cycles in liquid nitrogen, followed by centrifugation (1.5 × 105 g,
4°C, 1 h) as described (32). The protein concentration in the supernatant was determined by the Bradford assay (Bio Rad Labs.,
Hercules, CA). ICE activity was measured using a fluorogenic substrate containing the cleavage site YVAD, 4-(4 Preparation of Organelles.
Mitochondria were isolated from
BALB/c hepatocytes (6-8 wk, female) or from different CEM-C7.H2 lines. Mitochondria were purified on a Percoll® (Pharmacia, Uppsala, Sweden) gradient (34) and were stored on ice in
CFS buffer for up to 4 h. Mitochondria were washed and resuspended in CFS supplemented with 2 mM ATP. Nuclei from
HeLa cells were purified on a sucrose gradient and conserved in
50% glycerol (Sigma Chemical Co.) in HeLa nuclei buffer at
Purification of the Mitochondrial AIF.
Purified hepatocyte mitochondria were treated with atractyloside (5 mM; Atr; Sigma
Chemical Co.) to induce PT and liberation of AIF (9, 11). Supernatants (150,000 g, 1 h, 4°C) from these mitochondria were concentrated on Centricon 10 membranes ( Determination of Mitochondrial PT.
For the induction of PT,
mitochondria from different cell lines were incubated with cytosolic extracts from Cell-free System of Apoptosis.
Nuclei from mouse liver cells
were purified on a sucrose gradient (35), washed two times (1,000 g,
10 min, 4°C), and resuspended in CFS buffer. In standard conditions, nuclei (103 nuclei/µl) were cultured in the presence of mitochondrial preparations for 90 min at 37°C. Nuclei were stained
with propidium iodide (10 µg/ml; Sigma Chemical Co.) and the
lipophilic dye 5-methyl-bodipy-3-dodecanoic acid (100 nM; Molecular Probes Inc., reference 37), followed by cytofluorometric
analysis in an analyzer (EPICS Profile II Analyzer; Coulter
Corp.). Only membrane surrounded (5-methyl-bodipy-3-dodecanoic acid-labeled) particles were gated. A good correlation between the frequency of nuclei exhibiting chromatin condensation
with 4 Expression of Human CPP32 and Generation of a Specific Antiserum.
A cDNA encoding human CPP32 was generated by PCR using
the plasmid pKSII-CPP32 (gift from Guy Salvesen, Burnham Institute, La Jolla, CA) as a template and the primers 5 Western Blot Analysis.
AIF-mediated cleavage of nuclear substrates was determined by the comparative analysis of SDS-PAGE
of HeLa nuclei (5 × 106/lane) cultured in the presence or absence of supernatant from Atr-treated mitochondria (10 µg protein/ml, 90 min, 37°C) in the presence or absence of Z-VAD.fmk,
as described (11). Western blots of these nuclei were tested for
degradation of poly (ADP-ribose) polymerase (PARP) using the
monoclonal antibody C2-10 (purchased from Guy Poirier, Montreal University, Canada; reference 38). Cleavage of CPP32 in
cells (8 × 105 cells/lane) or in vitro (10 ng recombinant CPP32+
10 µg protein of mitochondrial supernatant in 50 µl CFS buffer ± 100 µM Z-VAD.fmk, 15 min at 37°C) was determined by using
a polyclonal rabbit antiserum recognizing both CPP32 and the
p17 fragment of proteolytically activated CPP32 (39). Enzymatic
activation of CPP32 (100 ng CPP32 + variable amounts of mitochondrial supernatant in 100 µl CFS buffer ± 100 µM Z-VAD.fmk,
15 min at 37°C) was detected by adding 1 µM Ac-DEVD-amino-4-methylcoumarin (30 min, 37°C), as described above. In
one control experiment, Z-VAD.fmk (100 µM) was added together with Ac-DEVD-amino-4-methylcoumarin.
Human CEM-C7.H2 lymphoma cells
can be induced to undergo apoptosis by cross-linking of
Fas. As shown in Fig. 1 A, cells manifest a rapid activation
of protease(s) capable of cleaving a fluorogenic substrate
containing the tetrapeptide YVAD. As described (22, 25),
activation of ICE-like proteases is a rapid process that peaks
15-30 min after Fas cross-linking. It thus precedes the Fas-induced
ICE-like proteases are involved in both the To determine the mechanism by which activation of ICE or ICE-like proteases causes
In accordance with the data obtained with cytosolic extracts,
recombinant purified ICE suffices to induce both large amplitude swelling and dissipation of the
Table 1.
Differential Regulation of ICE- and prooxidant-induced
Mitochondrial PT
converting enzyme (ICE1/
caspase 1), CPP32 (Yama/Apopain/caspase 3), and other
proteases homologous to the Caenorhabditis elegans protein
Ced-3 (1, 5). On the other hand, the disruption of the
mitochondrial inner transmembrane potential (
m) marks
a point of no return for the apoptotic cascade (6). Moreover, mitochondria that undergo permeability transition
(PT) or products derived from these organelles induce
chromatin condensation and DNA fragmentation in cell-free systems of apoptosis (7).
m disruption in intact cells (9, 12, 13) and that is accompanied by the release of cytochrome c (14). The oncoprotein Bcl-2 is an inhibitor of PT induced in isolated mitochondria (9, 11),
anucleate cytoplasts (15), and cells (12), underscoring the
idea that PT may indeed constitute a central checkpoint of
the apoptotic cascade. Pharmacological inhibition of PT by
mitochondrion-targeted drugs can inhibit all cytoplasmic
and nuclear manifestations of apoptosis (9, 13, 15), suggesting that PT is a rate-limiting, coordinating step of apoptosis. PT is induced by many different physiological effectors
(reactive oxygen species, blockade of the respiratory chain,
changes in the ATP/ADP concentration, pyrimidine nucleotide oxidation, thiol redox potentials, calcium, etc.), and thus may allow for the convergence of very different
inducers of apoptosis. The multiplicity of PT induction
pathways is underscored by the fact that none of the
known inhibitors of PT, including Bcl-2, can block PT induction in all circumstances (9, 11, 16).
m disruption and subsequent apoptosis in thymocytes (13). Second, PT may be regulated
directly by mitochondrial proteases. Thus, calcium- and
prooxidant-induced PT may involve the action of a mitochondrial calpain-like protease (17). Third, proteases may
also act downstream of PT. We have recently shown that
the apoptogenic protein (AIF) released from mitochondria
undergoing PT possesses a proteolytic activity that is also
inhibited by Z-VAD.fmk (11).
m disruption and
release of AIF. We show that AIF possesses unique biological properties. In addition to its direct apoptotic effect on
isolated nuclei in a cell-free, cytosol-free system, AIF is itself an inducer of PT, and thus may be involved in a positive amplification loop disrupting mitochondrial function. Moreover, AIF proteolytically activates CPP32, one of the
signature proteases of mammalian cell death. These findings
underscore the implication of mitochondria in the apoptotic effector phase, provide multiple links between proteases and mitochondrial regulation, and explain the limited apoptosis-inhibitory effect of Bcl-2. Moreover, our
data suggest a scenario according to which ICE (or ICE-like proteases), mitochondrial AIF, and CPP32 are sequentially activated and participate in the induction, effector,
and degradation phases of apoptosis, respectively.
-actin STneo B vector
(crmA cells) or a vector-only control (Neo). Three different
clones hyperexpressing crmA at the protein level yielded similar
functional results. Results are shown for the C7.H2/D1.2/2E8 clone. Alternatively, CEM-C7.H2 cells were transfected with
pEF-tTA 2A10, a doxycyclin-inhibitable transactivator (tTa) and
super-transfected with a tTa-repressed bcl-2 construct in a tk-Hyg
selection vector (pUGD10-3 Bcl-2 tkHyg; reference 29; results
are shown for one out of two clones yielding similar data). Bcl-2
expression was repressed by culture on doxycycline (10 ng/ml, 48 h),
as described (30). Apoptosis was induced by stimulation of 1-5 × 106 cells/ml with the Fas-cross-linking IgM monoclonal antibody
CH-11 (500 ng/ml; Immunotech, Marseille, France) in the presence or absence of the membrane-permeant-specific inhibitor of
ICE, Ac-YVAD.cmk (100 µM; Bachem, Basel, Switzerland), or
alternatively with C2 ceramide (50 µM; Sigma Chemical Co., St.
Louis, MO).
m, cells (106/ml) were incubated with the cationic lipophilic dye chloromethyl-X-rosamine
(CMXRos; 150 nM; Molecular Probes, Inc., Eugene, OR; reference 15). As a control, cells were simultaneously treated with the
uncoupling agent carbonyl cyanide m-chlorophenylhydrazone
(mClCCP; 50 µM; Sigma Chemical Co.). CMXRos incorporates into mitochondria driven by the
m (15) and reacts with
thiol residues to form covalent aldehyde-fixable thiol ester bonds
(31). After fixation (4% paraformaldehyde in PBS for 15 min at
room temperature), cells were washed and stained for the detection of chromatinolysis using the TUNEL method, as described
(31). In one series of experiments, cells were stained with the potential-sensitive dye DiOC6(3) (15 min, 37°C, 40 nM) together
with a biotin-Annexin V conjugate (50 × dilution; revealed by
streptavidine-phycoerythrine at 5 µg/ml, following the manufacturer's protocol; Boehringer Mannheim GmbH, Mannheim, Germany), followed by sorting of DiOC6(3)low Annexin V+,
DiOC6 (3) Annexin V
and DiOC6(3)high Annexin V+ cells on
an Elite cytofluorometer (Coulter Corp., Miami, FL), as described
(6, 15).
-deimethylaminophenylazo) benzoic-YVADAPV-5-(-2-aminoethyl-amino)
naphtalene-1-sulfonic acid (Bachem), as described (22), using a
spectrofluorometer (Kontron SFM 25; Kontron AG, Zurich,
Switzerland). The capacity of cytosols or purified recombinant
human CPP32 activity to cleave the CPP32 recognition site
DEVD was determined using Ac-DEVD-amino-4-methylcoumarin (Bachem) as fluorogenic substrate (33).
20°C for a maximum of 15 d, as described (35).
10 kD; Amicon, Beverly, MA) and then injected into a FPLC column (MonoQ
(HR5/5); Pharmacia) preequilibrated with protein-free CFS buffer (see below). Elution was performed on a linear gradient from
0 to 250 mM NaCl at 0.5 ml/min over 30 min, followed by elution at 1 M NaCl thereafter. All fractionation steps were carried
out at 4°C to avoid loss of biological AIF activity. The active
fraction (eluting at 110 mM NaCl; reference 11) was dialyzed
against protein-free CFS buffer (4°C, overnight, 5,000× excess of
CFS buffer), concentrated on Centricon 10 membranes, adjusted
to a concentration of 30 µg/ml, and aliquoted to be snap frozen
in liquid nitrogen and stored at
80°C.
Fas-treated cells (standard dose of 30 µg protein/ml), purified recombinant ICE (50 µg/ml), the prooxidant
ter-butylhydroperoxide (t-BHP; 30 µM), atractyloside (5 mM;
Sigma Chemical Co.), the protonophore mClCCP (100 µl; Sigma
Chemical Co.), bongkrekic acid (50 µM; provided by Dr. Duine,
Delf University, Delf, The Netherlands), monochlorobiman (30 µM; Sigma Chemical Co.), and/or the calpain inhibitor N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-tyrosine diazomethylketone
(100 µM; Molecular Probes Inc., reference 9). Recombinant ICE
was produced following standard procedures (36) and was allowed to partially (
5%) autoactivate by incubation at 20°C for 2 h,
followed by storage on ice for a maximum of 4 h. Two different
consequences of PT were assessed: (a) mitochondrial large amplitude swelling and (b) collapse of the
m. For determination of
swelling, mitochondria were washed and resuspended in CFS
buffer supplemented with 2 mM ATP at a concentration of 100 µg
mitochondrial protein/10 µl buffer, followed by addition of 90 µM CFS containing 2 mM ATP and recording of adsorption at
540 nm in a spectrophotometer (DU 7400; Beckman Instrs.,
Carlsbad, CA), as described (9). After stabilization of the adsorption during a minimum interval of 30 s, the indicated substance
was added in a volume of 5 µl. The loss of absorption induced by
5 mM atrectyloside within 5 min was considered 100% of the
large amplitude swelling, as described (9). The
m was measured using DiOC6 (3) (40 nM, 15 min at 37°C; Molecular
Probes Inc.), after having added the indicated PT inducer (30 min, room temperature). Mitochondria were analyzed in an Elite
cytofluorometer (Coulter Corp.). All
m determinations were
performed at least three times in each experiment.
-6-diamidino-2-phenylindole dihydrochloride (10 µM;
Molecular Probes, Leiden, The Netherlands; reference 38) and
hypoploidy with PI was obtained (Susin, S.A., and G. Kroemer,
manuscript in preparation). DNA fragmentation was determined
by horizontal agarose gel electrophoresis and ethidium bromide
staining as described (9). Electron microscopy of osmium tetroxyde-fixed nuclei was performed as described (13).
-GGAATTCCATATGGAGAACACTGAAAACTCAGTG-3
(forward)
and 5
-CCGCTCGAGGTGATAAAAATAGAGTTCTTTTG-3
(reverse). After digestion with Xho1 and Nde1, this PCR-generated cDNA was subcloned into pET21b at the Nde1/Xho1
sites to produce CPP32 protein with six histidine resides at its
COOH terminus. Recombinant purified CPP32 was purified as
described (39). New Zealand white female rabbits were injected
subcutaneously with 200 µg of purified CPP32-His6 fusion protein mixed (1:1 vol/vol) with Freund's complete adjuvant and
then boosted seven times with 200 µg of protein in Freund's incomplete adjuvant before collecting blood and obtaining immune
serum.
Fas Cross-linking Provokes Sequential Activation of ICE-like
Proteases, m Disruption plus Activation of CPP32, and Nuclear Apoptosis.
m disruption, as quantified by means of the
m-sensitive dye CMXRos. This
m collapse affects
only a minor fraction of the cells beginning at 30 min after
Fas ligation. An important fraction of cells (~ 40%) exhibits a disrupted
m about 2 h after Fas cross-linking, when
DEVDase activity is also significantly augmented. To further investigate the relationship between Fas-induced
m
disruption and activation of CPP32, CEM-C7.H2 cells
were stimulated during 2 h by Fas cross-linking, followed
by staining with the
m-sensitive dye DiOC6(3) as well
as Annexin V (which measures the aberrant phosphatidyl
serine exposure on the outer plasma membrane leaflet) and
cytofluorometric purification of cells with a still normal
m (DiOC6(3)high) as well as cells with a disrupted
m
(DiOC6(3)low) that are either in an early stage of the apoptotic process (Annexin V
) or in an advanced stage (Annexin V+) (Fig. 1 B). Only
mlow cells have cleaved the
CPP32 precursor to yield CPP32 fragments (p21 and p17)
and exhibit DEVDase activity (Fig. 1 B). This is observed
for both
mlow Annexin V
and
mlow Annexin V+
cells, indicating that CPP32/DEVDase activation occurs
concomitant with (or shortly after) the
m disruption. In
contrast,
mhigh cells behave like unstimulated control
cells and lack any detectable CPP32 cleavage or DEVDase
activation (Fig. 1 B). Thus, CPP32 is only activated in cells
whose
m is disrupted. Similar results have been obtained
in other models of apoptosis induction, including ceramide-induced cell death (not shown). As in other models of apoptosis induction (4, 6, 9, 12, 31), the Fas-induced
m collapse precedes nuclear chromatinolysis as identified with the TUNEL technique (Fig. 1 A). Thus, cells that have disrupted their
m (CMXRoslow cells) can be subdivided
into TUNEL+ and TUNEL
populations, whereas
TUNEL+ cells uniformly possess a
mlow (CMXRoslow)
phenotype (Fig. 1 C). These findings place
m disruption
upstream of nuclear apoptosis.
Fig. 1.
Chronology and
cause effect relationship between activation of ICE (or
ICE-like) protease(s) and m
disruption. (A) Chronology of
the activation of ICE,
m disruption, and nuclear DNA fragmentation in human CEM-C7.H2 lymphoma cells subjected
to Fas cross-linking. The frequency of
mlow cells and of
cells exhibiting DNA strand
breaks were determined by double staining with the potential-sensitive dye CMXRos and TdT-catalyzed FITC-dUTP incorporation (TUNEL method),
as described in Materials and
Methods. Note that the
TUNEL+ population is actually
a subset of CMXRoslow cells (see
B). Activation of ICE (-like)
protease(s) was determined by a
fluorogenic substrate containing
the ICE cleavage site YVAD (filled symbols), the maximum activity being defined as 100%. Similarly, the activation of
CPP32 (-like) protease(s) was
determined by means of a fluorogenic substrate containing the
cleavage site DEVD (open symbols). (B) Temporal relationship
between Fas-induced
m disruption and CPP32 cleavage, as
well as DEVDase activation. CEM-C7.H2 cells were cultured
during 120 min in the presence
of anti-Fas antibody, followed by
staining with the
m-sensitive dye DiOC6(3) plus Annexin V (revealed by phycoerythrin). Cells were then separated in the cytofluorometer into cells with a normal
m
(DiOC6(3)high Annexin V
) or cells with a DiOC6(3)low Annexin V
or DiOC6(3)low Annexin V+ phenotype (sorting according to Windows), followed
by determination of CPP32 cleavage using Western blots (lane 1, unstimulated control cells; lane 2, nonseparated Fas-stimulated cells; lane 3), purified DiOC6(3)high cells; lane 4, purified DiOC6(3)low Annexin V
cells; lane 5, purified DiOC6(3)low Annexin V+ cells, 8 × 105 cells/lane). Alternatively, cytosols from these cell populations were tested for DEVDase activity in vitro as in A (C) Determination of
m disruption and DNA strand breaks in different cells. CEM-C7.H2 lymphoma cell stably transfected with a Neomycin selection vector (Neo) only (fluorescence displays 1-4), with the crmA
cowpox protease inhibitor (graphs 5 and 6), or with a Bcl-2-expressing construct negatively regulated by doxycyclin (graphs 7-12). Cells were either pretreated with doxycyclin (10 ng/ml, 48 h before starting of the experiment) to repress Bcl-2 expression (Bcl-2
, graphs 7-9) or left untreated (Bcl-2+,
graphs 10-12), and then subjected to apoptosis induction with C2 ceramide (50 µM; graphs 9 and 12), anti-Fas (graphs 3, 4, 6, 8, and 11) and/or the
ICE inhibitor Ac-YVAD.cmk (50 µM, all during 4 h; graph 4), followed by double staining with CMXRos and the TUNEL method. Neo control cells
were treated during 15 min with 100 µM of the protonophore mClCCP, providing a negative control for the CMXRos staining (graph 2). Numbers indicate the percentage of cells in each quadrant. Results are representative for three independent experiments.
[View Larger Version of this Image (71K GIF file)]
m disruption and chromatinolysis, because cells treated with the
ICE-specific inhibitor Ac-YVAD.cmk or cells stably transfected with the ICE inhibitor crmA fail to demonstrate mitochondrial or nuclear manifestations of apoptosis in response to Fas cross-linking (Fig. 1 C). Thus, in Fas-induced
apoptosis, ICE (or ICE-like proteases) function upstream of
mitochondria. Transfection-enforced hyperexpression of
Bcl-2 does not interfere with Fas-triggered apoptotic
changes, although it does prevent both the
m dissipation
and DNA loss induced by the apoptosis-inducing second
messenger ceramide (Fig. 1 C). This finding confirms previous observations that Bcl-2 does not prevent Fas-induced
apoptosis, at least in certain experimental systems (26).
m disruption, we incubated isolated hepatocyte mitochondria with
cytosolic extracts from
Fas-treated cells. Cytosols from
Fas-treated cells, but not cytosols from sham-treated control cells, were found to induce large amplitude swelling of isolated mitochondria (Fig. 2 A), a sign of PT. In addition,
mitochondria treated with cytosols from
Fas-treated cells
manifest
m disruption, another sign of PT (Fig. 2 B).
This PT-inducing activity was maximal in cytosols obtained from cells subjected to Fas cross-linking for 30 min
(not shown), coinciding with the maximum activity of ICE
(-like) proteases (Fig. 1 A). As expected based on the results in intact cells (Fig. 1 C), transfection with crmA impeded
the cytosolic accumulation of such a PT-inducing activity
(Fig. 2). In addition, the ICE-specific inhibitor Ac-YVAD.
cmk and another inhibitor of ICE, Ac-YVAD.CHO (not
shown), prevented the formation of the PT-inducing activity in cytosols from
Fas-treated cells. Ac-YVAD.cmk also
prevented the action of cytosols that already contained the
PT-inducing activity on mitochondria, when added to the
cytosol derived from
Fas-treated cells in vitro (Fig. 2). In
contrast, the peptide Ac-DEVD.CHO, an inhibitor of
CPP32, was ineffective (not shown). This suggests that an
ICE-like protease participates in the induction of mitochondrial PT, both in cells (Fig. 1 C) and in a cell-free system (Fig. 2).
Fig. 2.
A cytosolic factor
neutralized by ICE-specific protease inhibitors causes mitochondrial PT in vitro. Isolated hepatocyte mitochondria were exposed
to cytosols (final concentration:
100 µg/ml protein) prepared from
CEM-C7.H2 lymphoma cells
stably transfected with a Neomycin selection vector (Neo) only
(graphs 1-4) or cells transfected with the crmA cowpox protease
inhibitor (graphs 5 and 6) that were either treated with anti-Fas
antibody during 30 min (graphs 2-4, 6) or were left untreated
(graphs 1 and 5). Cytosols were tested for their capacity to induce mitochondrial swelling,
100% of swelling being defined
as the loss of the OD540 observed
5 min after addition of 5 mM atractyloside (A). Arrows indicate addition of the cytosolic extract. Alternatively, the m was assessed cytofluorometrically on a per-mitochondrion basis of mitochondria treated with the indicated cytosol preparation and then stained with the potential-sensitive dye
DiOC6(3) (B). Treatment with the protonophore mClCCP served as a negative control for DiOC6(3) staining (dotted line, graph 1 B). The effect of the ICE-specific inhibitor Ac-YVAD.cmk was tested in two different ways. Ac-YVAD.cmk was either used with the cells exposed to
Fas (Ac-YVAD.cmk+
Fas,
graph 3) or, alternatively, was added to the cytosol prepared from Fas-treated cells (
Fas+Ac
YVAD.cmk, graph 4).
[View Larger Version of this Image (30K GIF file)]
m in isolated mitochondria in vitro (Fig. 3, A and B). This effect of ICE is
rapid (<30 s) and can be neutralized by Ac-YVAD.cmk
and Ac-YVAD.CHO, but not by AcDEVD.CHO, indicating that it relies on the enzymatic activity of ICE. In
contrast, Ac-YVAD.cmk does not interfere with t-BHP-induced PT, thus excluding that this modified tetrapeptide
might prevent PT in a nonspecific fashion (Fig. 3, A and
B). The use of additional inhibitors underscores the different mechanisms involved in ICE- and t-BHP-triggered
PT. For example, cyclosporin A, bongkrekic acid, monochlorobiman (9), and the calpain inhibitor Cbz-LLT. CHN2
(17) all inhibited the t-BHP- but not the ICE-induced PT in vitro (Table 1). Other proteases besides ICE, such as
trypsin and proteinase K, also induce PT in isolated mitochondria (not shown), in accord with previous observations
that microinjection of such proteases induces apoptosis in
cells (40).
Fig. 3.
Recombinant purified ICE is sufficient to induce
PT, as well as the release of an
apoptosis-inducing factor from
mitochondria. Purified liver mitochondria were treated with CFS
buffer only (graph 1), purified
recombinant human ICE (graphs
2-4), the prooxidant t-BHP
(graphs 5 and 6), and/or different protease inhibitors (Ac-YVAD.cmk, graphs 3 and 6 or
Ac-DEVD.CHO, graph 4). These
reagents were added together to
the mitochondria and the following parameters were assessed: large
amplitude swelling (A), m
(DiOC6(3) staining, 30 min after
addition of the reagents) (B), and release of AIF (C). Arrows in A
indicate addition of the indicated combination of reagents or
buffer only (Control). The dotted line in graph B 1 indicates
the negative control of DiOC6(3)
staining obtained in the presence of the
m-dissipating reagent mClCCP. For the determination of AIF release (C), mitochondria were centrifuged (1.5 × 10
5 g, 1 h) after 5 min of treatment, and the supernatant was incubated for 30 min with purified HeLa nuclei, followed by determination of their DNA content using the flurochrome propidium iodide, as described in Materials and Methods. Percentages detail the percentage of nuclei exhibiting an apparent subdiploidy.
[View Larger Version of this Image (46K GIF file)]
Inhibitor of PT
Inhibitory effect on
large amplitude
swelling* induced by
t-BHP
ICE
Cyclosporin A (1 µM)
+
Bongkrekic acid (50 µM)
+
Monochlorobimane (30 µM)
+
Chz-LLY.CHN2 (100 µM)
+
Ac-YVAD.CHO (100 µM)
+
Ac-DEVD.CHO (100 µM)
z-VAD.fmk (100 µM)
+
Positive symbols denote significant (>90%) inhibition of large amplitude swelling; negative symbols indicate <10% inhibition.
*
Purified mouse hepatocyte mitochondria were tested for the large amplitude swelling induced by either ICE (50 µg/ml) and t-BHP (30 µM)
as in Fig. 3 A. The indicated PT inhibitors were added 15 min before
t-BHP or ICE, and the inhibition of large amplitude swelling was determined over a period of 5 min.
Thus, ICE (or ICE-like) protease(s) is/are necessary and
sufficient to mediate the m disruption in cells subjected
to Fas ligation. The mechanism of ICE-induced PT differs
from that of prooxidant-induced PT, suggesting a direct
proteolytic effect of ICE on mitochondria.
We have previously shown that PT is accompanied by the release of an apoptogenic protein that induces isolated nuclei to undergo chromatin condensation and oligonucleosomal DNA fragmentation (9, 11). Accordingly, the ICE-induced PT is accompanied by the release of such an apoptogenic protein (AIF), which causes purified HeLa nuclei to manifest DNA condensation and loss of chromosomal DNA (subdiploidy). The ICE inhibitor Ac-YVAD.cmk prevents the ICE-induced mitochondrial release of AIF (Fig. 3 C), yet does not interfere with the activity of AIF itself in the cell-free system of nuclear apoptosis induction (9, 11, and see below), consistent with the fact that ICE by itself is insufficient to induce apoptosis in isolated nuclei (38). In conclusion, ICE-induced PT is accompanied by the release of a mitochondrial apoptogenic factor.
Bcl-2 Overexpression Prevents prooxidant-induced and Ceramide-elicited PT, and Associated Release of AIF, yet Fails to Prevent ICE-induced PT and AIF Release.As outlined in the
Introduction, Bcl-2 is incapable of suppressing the Fas-induced apoptosis in a number of different models (26).
This applies also to CEM-C7-H2 lymphoma cells (Fig. 1 C, see above). Since mitochondrial hyperexpression of Bcl-2
prevents the induction of PT by different substances, including prooxidants (9, 11; Fig. 4), we tested whether it
would also interfere with ICE-induced PT. Mitochondria
isolated from Bcl-2-transfected cells manifest large amplitude swelling when treated with recombinant ICE, exactly
as do control mitochondria from cells not hyperexpressing Bcl-2. In addition, Bcl-2 hyperexpression does not prevent
the mitochondrial release of AIF induced by ICE, although
it does suppress the t-BHP-induced PT and release of AIF
(Fig. 4). This dichotomy in the Bcl-2-mediated regulation
of PT, inhibition of prooxidant-induced PT and failure to
prevent ICE-induced PT, was observed in human CEM-C7-H2 cells transfected with tetracycline-repressable bcl-2
construct (Fig. 4), as well as in murine 2B4.11 T cell hybridoma cell lines stably transfected with the human bcl-2 gene
(not shown). Thus, Bcl-2 fails to neutralize the effects of
ICE on mitochondria in vitro, consistent with its inability
to prevent ICE-dependent apoptosis in cells. Since Bcl-2
prevents ceramide-induced apoptosis and m disruption
(Fig. 1 C), we investigated the AIF release of Bcl-2 hyperexpressing mitochondria treated with cytosolic extracts from cells that have been treated during a short interval (30 min) with either ceramide or anti-Fas. Control mitochondria readily release AIF upon incubation with such cytosols
(Fig. 5). Bcl-2-hyperexpressing mitochondria demonstrate
a selective protection against yet unidentified ceramide-elicited PT inducers, yet release AIF upon incubation with
ICE-containing cytosols from anti-Fas-treated cells (Fig. 5). These results are compatible with the hypothesis that
Bcl-2 prevents ceramide-induced apoptosis at the level of
mitochondria.
AIF Is an Apoptogenic Protease Which Itself Induces PT.
AIF is a preformed ~50-kD intermembrane protein that is
released from mitochondria undergoing PT (11). Isolated
nuclei exposed to AIF exhibit a step-wise alteration in the
morphology of the nucleus which consists in a first step of
chromatin condensation (15 min), followed by disruption
of the nuclear envelope and an associated loss of electron-dense material (60 min). At this latter stage, nuclei frequently demonstrate two homogeneous zones that differ in
their electron density and resemble nuclei from cells at an
advanced stage of apoptosis (Fig. 6 A). In addition, isolated nuclei exposed to AIF display two biochemical hallmarks
of apoptosis: (a) loss of total nuclear DNA (hypoploidy)
(11; and Fig. 6 B) and (b) oligonucleosomal DNA fragmentation (11; and Fig. 6 C). The apoptogenic effect of AIF
liberated from ICE-treated mitochondria is neutralized by
the broad spectrum inhibitor of ICE-like proteases Z-VAD.
fmk, but not by the more specific ICE inhibitor Ac-YVAD.cmk (9, 11; and Fig. 6, B and C), in accord with
the fact that ICE itself is not apoptogenic (38), whereas
ICE-like proteases do have the capacity to provoke nuclear
apoptosis in vitro (41). Since AIF apparently has an ICE-like catalytic activity (11), we tested whether AIF would
share with ICE the PT-inducing potential. Indeed, the supernatant of ICE-treated mitochondria provokes PT of
freshly isolated mitochondria (Fig. 6 D). This activity is not
neutralized by Ac-YVAD.cmk, yet is completely abolished
by Z-VAD.fmk, suggesting that it is mediated by AIF and
not by residual ICE. Accordingly, AIF enriched on an
anion exchange FPLC column (11) can induce PT in a
Z-VAD.fmk-inhibitable fashion (Fig. 6 D). These data suggest that AIF possesses a biological spectrum of activities
that overlaps, but is not identical, with that of ICE.
AIF Proteolytically Activates CPP32.
The proteolytical
activation of CPP32 has been previously shown to be inhibited by Z-VAD.fmk (42), which also inhibits the effects
of AIF (11; and Fig. 6). Stimulated by this fact, we have tested whether mitochondrial intermembrane fractions containing AIF would activate purified recombinant CPP32. As
shown in Fig. 7 A, supernatants of mitochondria induced
to undergo PT in response to Atr (9, 11) indeed activate the
cleavage of a fluorogeneic peptide substrate containing the
CPP32 cleavage site DEVD. This effect is inhibited by
Z-VAD.fmk. Moreover, the same effects were observed
with FPLC-purified AIF (Fig. 7 A). In addition, AIF
(which is by itself incapable of cleaving the "death substrate" PARP) activates CPP32 to digest PARP (Fig. 7 B).
As expected by the finding that AIF activates CPP32,
CPP32 digestion by AIF-containing preparations yields a
21 precursor and a canonical p17 fragment (Fig. 7 C) that
may associate with the p12 fragment to yield a biologically
active heterotetramer (33, 43). This activation does not require the autocatalytic processing of CPP32, since it is not
inhibited by Ac-DEVD.CHO (Fig. 7 C). In conclusion, AIF is endowed with the capacity of activating one of the
signature processes of apoptosis, CPP32.
The data presented in this work provide multiple novel connections between proteases and mitochondrial PT during the apoptotic effector phase. These interactions are bidirectional. On the one hand, ICE can provoke mitochondrial PT, and, on the other hand, PT entails the mitochondrial release of a CPP32-activating protease.
A Novel Pathway ofAs outlined in the Introduction, m
disruption constitutes an early event of apoptosis that precedes nuclear apoptosis. The apoptotic
m disruption involves opening PT pores on the inner mitochondrial membrane, based on the observation that PT pore antagonists
such as bongkrekic acid inhibit the apoptotic
m loss (4,
9, 12). Abundant literature (for review see reference 16) indicates that numerous physiological effectors regulate PT:
concentrations of divalent cations and protons, the redox status of mitochondrial thiols (in equilibrium with the redox status of glutathione), the redox status of the pyridine
nucleotide pool (NADH2/NAD + NADPH2/NADP; reference 44), concentrations of adenine nucleotides (ADP,
ATP), specific peptides, lipid acid oxidation products (16,
45), and proteases from the calpain family (17). Here we
show that ICE (or ICE-like proteases) contained in the cytosol of Fas-activated cells, as well as recombinant purified
ICE, are capable of inducing a PT-like effect in isolated mitochondria. ICE induces all three hallmarks of PT: (a)
colloidosmotic swelling (Fig. 3 A), (b) disruption of the
m (Fig. 3 B), and (c) release of AIF (Fig. 3 C), which is
self-sufficient to provoke nuclear apoptosis in a cell-free,
cytosol-free system. In contrast with other methods of PT
induction, ICE-mediated PT is not regulated by various
pharmacological inhibitors of PT (e.g., monochlorobiman, bongkrekic acid; Table 1) and it is not inhibited by overexpression of Bcl-2 in the mitochondrial membrane (Fig. 4).
Thus, it appears that the direct proteolytic effect of ICE on
unidentified mitochondrial substrates provoke PT and a
consequent
m collapse that disrupts mitochondrial functions. The ICE-induced PT is accompanied by AIF release
from mitochondria, similar to PT induced by other compounds including calcium, pro-oxidants, or the thiol-cross-linking agent diamide (9).
It thus emerges that mitochondria function as a cellular sensor of stress including changes in redox potentials, direct oxidative effects, and protease activation. These data support PT as a candidate for the "central apoptotic executioner" that has been postulated by several groups (1) and that would allow for the convergence of very different apoptosis induction pathways into one event downstream of which would follow the final common pathway of apoptosis.
A Novel Effector Protease of Mitochondrial Origin, AIF.Irrespective of the PT-triggering stimulus, PT results in the
mitochondrial release of an apoptogenic protease that we
have termed AIF (9, 11). Although the molecular cloning
of cDNA encoding AIF is still in progress, functional tests
performed on purified AIF indicate that it possesses three
unique features. First, AIF is the first protease that has been
shown to suffice in inducing apoptotic changes in isolated
nuclei (9, 11; and Fig. 6, A-C). Thus, at difference with
another mitochondrial product, cytochrome c (10), AIF
does not appear to require the presence of further cytosolic
factors to induce nuclear apoptosis. We cannot exclude the
possibility, however, that purified nuclei are associated with
factors derived from cytosol that are necessary for AIF
function. Second, AIF shares at least one biological effect of
ICE, namely the capacity to trigger PT (Fig. 6 D). Thus,
AIF liberated from mitochondria undergoing PT may engage in a self-amplifying apoptotic switch, and thus aid to
lock the cell in an irreversible stage of apoptosis, beyond
the point of no return. Since the effects of AIF are inhibited by a degenerate tripeptidic inhibitor of ICE and ICE-like proteases, Z-VAD.fmk (which acts as universal inhibitor of nuclear apoptosis in mammalian cells, perhaps with the exception of blastomeres; references 46, 47), this may explain why Z-VAD.fmk can inhibit m disruption, at
least in some systems of apoptosis induction (13). Third,
AIF induces cleavage and activation of CPP32 in vitro (Fig.
7). This is a rapid process, with detectable CPP32 cleavage
in as little as 5 min (not shown). CPP32 activation appears
to be a consistent concomitant of the apoptotic process that
may contribute to the apoptotic degradation of different
cellular and nuclear substrates (33, 41, 43), including those
that are not cleaved by AIF such as PARP (Fig. 7 B). This
finding is in accord with the fact that the apoptosis-associated activation of CPP32 is inhibited by Z-VAD-fmk in
vivo (39, 47). In conclusion, AIF has biological properties
which render it a firm candidate to act as a central molecule
of the apoptotic effector phase.
When
integrated with the current literature, the data reported in
this work suggest the following scenario for Fas-mediated apoptosis (Fig. 8). After Fas cross-linking, the Fas receptor complex rapidly (within seconds) recruits and causes the
proteolytic activation of a protease (pro-FLICE/MACH1/
Mch5/caspase 8; references 20, 21, 48), which indirectly
facilitates the activation of pro-ICE to activate ICE (peak:
~30 min). Thereafter, ICE (or possibly ICE-like proteases)
would cause mitochondrial PT (beginning at ~60 min),
which in turn would provoke the liberation of AIF from
the mitochondrial intermembrane space. AIF then acts as
an effector protease or protease activator and activates other
downstream enzymes including CPP32. In this scheme,
FLICE/MACH1 and ICE would act as "initiator" and
"amplifier" proteases (49), within the private initiation phase
of Fas-induced apoptosis. ICE and perhaps other ICE-like
proteases would then induce mitochondrial PT, a process
that causes the release of the "effector" protease/protease activator AIF from mitochondria, which in turn would
contribute to further induction of mitochondrial PT. In
cells in which Fas-induced apoptosis relies on ICE rather
than on other pathways (e.g., ceramide), Bcl-2 would fail
to impede the ICE-dependent induction of PT. Thus, PT
and associated AIF release would constitute the first event
of the common pathway of apoptosis and the central executioner of the effector phase. AIF release then would activate the "machinery" protease (49) CPP32 and perhaps
other Ced-3-like proteases, which may participate in the
degradation phase of apoptosis.
A possible critique against this sequence of events stems
from the evidence that caspases can activate each other via
direct interactions, at least in vitro (1, 5). Moreover, in
some cell-free systems, proteases, in combination with yet
unknown cytosolic factors, can provoke nuclear apoptosis
(FLICE, ICE, CPP32; references 25, 50). Nonetheless, recombinant caspases including CPP32 do not induce nuclear DNA fragmentation in vitro on their own, in the absence of additional cytoplasmic extracts (33). Moreover, a
putative direct caspase activation cascade fails to explain
important facts such as the latency between YVADase and
DEVDase activation (~2 h) or the temporal sequence between YVADase activation, mitochondrial changes plus
CPP32 activation, and late nuclear apoptosis, which is observed in intact cells and is mimicked by our cell-free system. In this context, it may be important to note that cytosolic extracts from cells which have been treated with
apoptosis inducers (Fas, ceramide) for a short period (30 min) themselves are inefficient inducers to nuclear apoptosis in vitro, unless mitochondria are added into the system
(Fig. 5). Thus, the cell-free system that we are using in this
study confirms and extends the notion that mitochondrial products have a major, and perhaps essential, apoptogenic
potential (7).
At first glance, our model may appear to be in contradiction with findings reported by Enari et al. (25) who attribute a decisive regulatory role to CPP32 in apoptosis regulation, based on the fact that preincubation of cells with the CPP32 inhibitor Ac-DEVD.CHO prevents Fas-induced apoptosis. However, addition of Ac-DEVD.CHO to cells after Fas cross-linking has no apoptosis-inhibitory effect (25), suggesting that Ac-DEVD.CHO acts on upstream proteases such as caspase 8, which cleaves the sequence motif DEVD (and thus is inhibited by Ac-DEVD. CHO; Reed, J.C., unpublished observation). Thus, the temporal and functional analysis of different proteases activated during Fas-induced apoptosis would suggest that CPP32 participates in the degradation, rather than in the execution, phase of apoptosis. Accordingly, addition of CPP32 inhibitors can suppress detectable DEVDase activation without affecting the mitochondrial phase of the apoptotic process (data not shown). As a caveat, this does not imply that CPP32 (and other closely related Ced-3 homologues) would only participate in the late phase of apoptosis. Indeed, it is conceivable that in response to other apoptosis inducers (e.g., developmentally programmed cell death), CPP32 may be involved in an earlier (private) step of the apoptotic cascade. It has been shown that CPP32 could activate unknown cytosolic factors (25) (which likely include mitochondrial products; reference 10) to become apoptogenic and thus to induce nuclear apoptosis in a cell-free system. These data suggest that CPP32 can activate other, soluble apoptogenic factors.
Irrespective of these possibilities, the results of this work suggest a unified view of protease-dependent and mitochondrial events of the apoptotic cascade. Proteases may have a major impact on mitochondrial function at the same time that mitochondria can release proteases and/or protease activators with apoptosis-inducing properties. The data reported here, therefore, provide new clues about the events that trigger the effector phase of apoptosis.
Address correspondence to Dr. Guido Kroemer, 19 rue Guy Môquet, B.P. 8, F-94801 Villejuif, France. FAX: 33-1-49-58-35-09.
Received for publication 25 November 1996 and in revised form 19 March 1997.
1Abbreviations used in this paper:We thank Anita Diu-Hercend (Roussel Uclaf, Romainville, France) for critical comments and Dr. Maurice Geuskens (Université Libre de Bruxelles, Bruxelles, Belgium) for electron microscopy.
This work was supported by Agence Nationale pour la Recherche contre le SIDA, Association pour la Recherche contre le Cancer, Centre Nationale de la Recherche Scientifique, Fondation de France, Fondation pour la Recherche Médicale/Sidaction, Ligue Française contre le Cancer, Institute National de la Santé et de la Recherche Médicale, North Atlantic Treaty Organization, the French Ministry of Science (to G. Kroemer), and National Institutes of Health grant CA72994 (to J.C. Reed). S.A. Susin receives a postdoctoral fellowship from the European Commission.
1. | Oltvai, Z.N., and S.J. Korsmeyer. 1994. Checkpoints of dueling dimers foil death wishes. Cell. 79: 189-192 [Medline]. |
2. | Thompson, C.B.. 1995. Apoptosis in the pathogenesis and treatment of disease. Science (Wash. DC). 267: 1456-1462 [Medline]. |
3. | Martin, S.J., and D.R. Green. 1995. Protease activation during apoptosis: death by a thousand cuts? Cell. 82: 349-352 [Medline]. |
4. |
Kroemer, G.,
P.X. Petit,
N. Zamzami,
J.-L. Vayssière, and
B. Mignotte.
1995.
The biochemistry of apoptosis.
FASEB J.
9:
1277-1287
|
5. | Henkart, P.A.. 1996. ICE family proteases:mediators of all apoptotic cell death? Immunity. 4: 195-201 [Medline]. |
6. | Zamzami, N., P. Marchetti, M. Castedo, C. Zanin, J.-L. Vayssière, P.X. Petit, and G. Kroemer. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181: 1661-1672 [Abstract]. |
7. | Newmeyer, D.D., D.M. Farschon, and J.C. Reed. 1994. Cell-free apoptosis in xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell. 79: 353-364 [Medline]. |
8. | Martin, S.J., D.D. Newmeyer, S. Mathisa, D.M. Farschon, H.G. Wang, J.C. Reed, R.N. Kolesnick, and D.R. Green. 1995. Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis. EMBO (Eur. Mol. Biol. Organ.) J. 14: 5191-5200 [Abstract]. |
9. | Zamzami, N., S.A. Susin, P. Marchetti, T. Hirsch, I. Gómez-Monterrey, M. Castedo, and G. Kroemer. 1996. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183: 1533-1544 [Abstract]. |
10. | Liu, X., C.N. Kim, J. Yang, R. Jemmerson, and X. Wang. 1996. Induction of apoptic program in cell-free extracts: requirement for dATP and cytochrome C. Cell. 86: 147-157 [Medline]. |
11. | Susin, S.A., N. Zamzami, M. Castedo, T. Hirsch, P. Marchetti, A. Macho, E. Daugas, M. Geuskens, and G. Kroemer. 1996. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184: 1331-1342 [Abstract]. |
12. | Zamzami, N., P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch, S.A. Susin, P.X. Petit, B. Mignotte, and G. Kroemer. 1995. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182: 367-377 [Abstract]. |
13. | Marchetti, P., M. Castedo, S.A. Susin, N. Zamzami, T. Hirsch, A. Haeffner, F. Hirsch, M. Geuskens, and G. Kroemer. 1996. Mitochondrial permeability transition is a central coordinating event of apoptosis. J. Exp. Med. 184: 1155-1160 [Abstract]. |
14. | Crofts, A.R., and J.B. Chappell. 1965. Calcium ion accumulation and volume changes in isolated liver mitochondria. Biochem. J. 95: 387-392 [Medline]. |
15. | Castedo, M., T. Hirsch, S.A. Susin, N. Zamzami, P. Marchetti, A. Macho, and G. Kroemer. 1996. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J. Immunol. 157: 512-521 [Abstract]. |
16. | Zoratti, M., and I. Szabò. 1995. The mitochondrial permeability transition. Biochim. Biophys. Acta. 1241: 139-176 [Medline]. |
17. | Aguilar, H.I., R. Botla, A.S. Arora, S.F. Bronk, and G.J. Gores. 1996. Induction of the mitochondrial permeability transition by protease activity in rats: a mechanism of hepatocyte necrosis. Gastroenterology. 110: 558-566 [Medline]. |
18. | Krammer, P.H., J. Dhein, H. Walczak, I. Behrmann, S. Mariani, B. Matiba, M. Fath, P.T. Daniel, E. Knipping, M.O. Westendorp, et al . 1994. The role of APO-1-mediated apoptosis in the immune system. Immunol. Rev. 142: 175-191 [Medline]. |
19. | Nagata, S., and P. Golstein. 1995. The Fas death factor. Science (Wash. DC). 267: 1449-1456 [Medline]. |
20. | Boldin, M.P., T.M. Goncharov, Y.V. Goltsev, and D. Wallach. 1996. Involvement of MACH, a novel MORT1/ FADD-interacting protease, in Fas/APO- and TNF receptor-induced cell death. Cell. 85: 803-815 [Medline]. |
21. | Muzio, M., A.M. Chinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Bretz, M. Zhang, R. Gentz, et al . 1996. FLICE, a novel FADD-homologous ICE/ CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 85: 817-827 [Medline]. |
22. | Los, M., M. Van de Craen, L.C. Penning, H. Schenk, M. Westendorp, P.A. Bauerle, W. Dröge, P.H. Krammer, W. Fiers, and K. Schulze-Osthoff. 1995. Requirement of an ICE/CED-3 protease for Fas/Apo-1-mediated apoptose. Nature (Lond.). 375: 81-83 [Medline]. |
23. | Enari, M., H. Hug, and S. Nagata. 1995. Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature (Lond.). 375: 78-81 [Medline]. |
24. |
Kuida, K.,
J.A. Lippke,
G. Ku,
M.W. Harding,
D.J. Livingston,
M.-S. Su, and
R.A. Flavell.
1995.
Altered cytokine export and apoptosis in mice deficient in interleukin-1![]() |
25. | Enari, M., R.V. Talanian, W.W. Wong, and S. Nagata. 1996. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature (Lond.). 380: 723-726 [Medline]. |
26. |
Chiu, V.K.,
C.M. Walsh,
L. Chau-Ching,
J.C. Reed, and
W.R. Clark.
1995.
bcl-2 blocks degranulation but not Fas-based cell-mediated cytotoxicity.
J. Immunol.
154:
2023-2029
|
27. | Memon, S.A., M.B. Moreno, D. Petrak, and C.M. Zacharchuk. 1995. Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma. J. Immunol. 155: 4644-4652 [Abstract]. |
28. | Strasser, A., A.W. Harris, D.C.S. Huang, P.H. Krammer, and S. Cory. 1995. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO (Eur. Mol. Biol. Organ.) J. 14: 6136-6147 [Abstract]. |
29. | Gossen, M., and H. Bujard. 1992. Tight control of gene expression in mammalian cells by tetracycline responsive promoters. Proc. Natl. Acad. Sci. USA. 89: 5547-5551 [Abstract]. |
30. | Yin, D.X., and R.T. Schimke. 1995. BCL-2 expression delays drug-induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells. Cancer Res. 55: 4922-4928 [Abstract]. |
31. | Macho, A., D. Decaudin, M. Castedo, T. Hirsch, S.A. Susin, N. Zamzami, and G. Kroemer. 1996. Chloromethyl-X-rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis. Cytometry. 25: 333-340 [Medline]. |
32. | Enari, M., A. Hase, and S. Nagata. 1995. Apoptosis by a cytosolic extract from Fas-activated cells. EMBO (Eur. Mol. Biol. Organ.) J. 14: 5201-5208 [Abstract]. |
33. | Nicholson, D.W., A. All, N.A. Thornberry, J.P. Vaillancourt, C.K. Ding, M. Gallant, Y. Gareau, P.R. Griffin, M. Labelle, Y.A. Lazebnik, et al . 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature (Lond.). 376: 37-43 [Medline]. |
34. | Petit, P.X., J.E. O'Connor, D. Grunwald, and S.C. Brown. 1990. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur. J. Biochem. 220: 389-397 . |
35. | Lazebnik, Y.A., S. Cole, C.A. Cooke, W.G. Nelson, and W.C. Earnshaw. 1993. Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. 123: 7-22 [Abstract]. |
36. |
Miossec, C.,
M.C. Decoen,
L. Durand,
F. Fassy, and
A. Diu-Hercend.
1996.
Use of monoclonal antibodies to study interleukin-1![]() ![]() |
37. | Macho, A., Z. Mishal, and J. Uriel. 1996. Molar quantification by flow cytometry of fatty acid binding to cells using dipyrrometheneboron difluoride derivatives. Cytometry 23: 166-173 [Medline]. |
38. | Lazebnik, Y.A., S.H. Kaufmann, S. Desnoyers, G.G. Poirier, and W.C. Earnshaw. 1994. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature (Lond.). 371: 346-347 [Medline]. |
39. |
Chinnaiyan, A.M.,
K. Orth,
K. O'Rourke,
H.J. Duan,
G.G. Poirier, and
V.M. Dixit.
1996.
Molecular ordering of the cell
death pathway: Bcl-2 and Bcl-X(L) function upstream of the
CED-3-like apoptotic proteases.
J. Biol. Chem.
271:
4573-4576
|
40. |
Williams, M.S., and
P.A. Henkart.
1994.
Apoptotic cell death
induced by intracellular proteolysis.
J. Immunol.
153:
4247-4255
|
41. | Lazebnik, Y.A., A. Takayashi, R.D. Moir, R.D. Goldman, G.G. Poirier, S.H. Kaufmann, and W.C. Earnshaw. 1995. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc. Natl. Acad. Sci. USA. 92: 9042-9046 [Abstract]. |
42. | Slee, E.A., H.J. Zhu, S.C. Chow, M. Macfarlane, D.W. Nicholson, and G.M. Cohen. 1996. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.fmk) inhibits apoptosis by blocking the processing of CPP32. Biochem. J. 315: 21-24 [Medline]. |
43. | Tewari, M., L.T. Quan, K. O'Rourke, S. Desnoyers, Z. Zeng, D.R. Beidler, G.G. Poirier, G.S. Salvesen, and V.M. Dixit. 1995. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 81: 801-809 [Medline]. |
44. |
Costantini, P.,
B.V. Chernyak,
V. Petronilli, and
P. Bernardi.
1996.
Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at
two separate sites.
J. Biol. Chem.
271:
6746-6751
|
45. |
Kirstal, B.S.,
B.K. Park, and
B.P. Yu.
1996.
4-Hydroxyhexenal is a potent inducer of the mitochondrial permeability
transition.
J. Biol. Chem.
271:
6033-6038
|
46. | Zhivotovsky, B., A. Gahm, M. Ankarcrona, P. Nicotera, and S. Orrenius. 1995. Multiple proteases are involved in thymocyte apoptosis. Exp. Cell Res. 221: 404-412 [Medline]. |
47. | Jacobson, M.D., M. Weil, and M.C. Raff. 1996. Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J Cell Biol. 133: 1041-1051 [Abstract]. |
48. |
Fernandes-Alnemri, T.,
R.C. Armstrong,
J. Krebs,
S.M. Srinivasula,
L. Wang,
F. Bullrich,
L.C. Fritz,
J.A. Trapani,
K.J. Tomaselli,
G. Litwack, and
E.S. Alnemri.
1996.
In vitro
activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.
Proc. Natl. Acad. Sci. USA.
93:
7464-7469
|
49. | Fraser, A., and G. Evan. 1996. A license to kill. Cell. 85: 781-784 [Medline]. |
50. |
Muzio, M.,
G.S. Salvesen, and
V.M. Dixit.
1997.
FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens.
J. Biol. Chem.
272:
2952-2956
|