From the Division of Cellular Immunology, La Jolla
Institute for Allergy and Immunology, San Diego, California 92121 and
Tumor Biology Laboratory, Biochemistry Department, University
College, Cork, Ireland
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ABSTRACT |
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A growing body of evidence supports a role for
mitochondria and mitochondria-derived factors in the cell death
process. In particular, much attention has focused on cytochrome
c, a key component of the electron transport chain, that
has been reported to translocate from the mitochondria to the cytosol
in cells undergoing apoptosis. The mechanism for this release is, as
yet, unknown. Here we report that ectopic expression of Bax induces
apoptosis with an early release of cytochrome c preceding
many apoptosis-associated morphological alterations as well as caspase
activation and subsequent substrate proteolysis. A loss of
mitochondrial transmembrane potential was detected in vivo,
although no mitochondrial swelling or loss of transmembrane potential
was observed in isolated mitochondria treated with Bax in
vitro. Caspase inhibitors, such as endogenous XIAP and synthetic
peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk),
although capable of altering the kinetics and perhaps mode of cell
death, had no influence on this release, suggesting that if cytochrome
c plays a role in caspase activation it must precede this
step in the apoptotic process. Mitochondrial permeability transition
was also shown to be significantly prevented by caspase inhibition,
indicating that the translocation of cytochrome c from
mitochondria to cytosol is not a consequence of events requiring
mitochondrial membrane depolarization. In contrast, Bcl-xL was capable
of preventing cytochrome c release while also significantly
inhibiting cell death. It would therefore appear that the mitochondrial
release of factors such as cytochrome c represents a
critical step in committing a cell to death, and this release is
independent of permeability transition and caspase activation but is
inhibited by Bcl-xL.
The stereotypical death throes of a cell undergoing apoptosis
include DNA fragmentation, nuclear condensation, cell shrinkage, blebbing, and phosphatidylserine externalization (1-3), all features that promote the physiologically silent removal of the cell by its
phagocytic neighbors. A large body of evidence supports the idea that
these events are mediated by the activation of several cytosolic
proteases, the caspases, which then orchestrate apoptosis via the
cleavage of key substrates (reviewed in Refs. 4-7). For example,
specific cleavage of two such substrates, PAK2 and DNA fragmentation
factor, activate these proteins, mediating membrane blebbing and DNA
fragmentation, respectively, without further requirements for the
proteases (for these events) (8, 9).
But how are the caspases activated during apoptosis? Recent studies
have delineated one key mechanism responsible for initiating the
executioner phase of apoptosis. Early in the process, mitochondria release cytochrome c (10), which upon entry into the cytosol forms a complex with another molecule, Apaf-1 (11, 12), and the
unprocessed (and inactive) proform of a caspase, caspase-9 (13). In the
presence of dATP or ATP, this complex processes and activates the
caspase, which in turn can now trigger a cascade by processing and
activating other caspases (in particular, caspases-3, -6, and -7) (13,
14). These then cleave key substrates and coordinate the process of
apoptotic cell death.
Bax is a pro-apoptotic Bcl-2-family protein (15, 16) that resides in
the cytosol and translocates to mitochondria upon induction of
apoptosis (17, 18). Recently, Bax has been shown to induce cytochrome
c release and caspase activation in vivo (19) and
in vitro (20). This release was reportedly dependent upon
induction of the mitochondrial permeability transition, an event that
is associated with disruption of the mitochondrial inner transmembrane
potential ( In contrast, Bcl-2 has been shown to be capable of blocking spontaneous
cytochrome c release in cell-free extracts and in cells
treated with apoptosis-inducing agents (26, 27). In the former,
cytochrome c was able to completely bypass the
anti-apoptotic effects of Bcl-2 (27). Furthermore, in both cell-free
systems and in cells undergoing apoptosis, the release of cytochrome
c can occur independently of changes in Cell Culture and Reagents--
Human embryonic kidney cells
(293T cells) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine under standard conditions. CEM
cells were grown in RPMI medium supplemented with 10% fetal calf
serum, penicillin, and streptomycin. Plasmid constructs pcDNA3, pcDNA3.Bax, and pcDNA3-myc-XIAP were generously provided by Dr. John Reed. Green fluorescent protein was purchased from
CLONTECH (Palo Alto, CA). cDNA encoding Bcl-xL
was generously provided by Dr. Craig Thompson and cloned into the
EcoRI site of pEF.neo. 3,3'-Dihexyloxacarbocyanine iodide
(DiOC61
(3)), carbamoyl cyanide n-chlorophenylhydrazone (mCICCP), MitoTracker Orange, and rhodamine green (Rh123) were obtained from
Molecular Probes, Inc. (Eugene, OR); DAPI and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) were from Sigma,
and Phiphilux-G6D2 was from OncoImmunin, Inc.
(Kensington, MD).
Anti-Bax antibody was generously provided by Dr. John Reed (Burnham
Institute, La Jolla, CA). Anti-poly(ADP-ribose) polymerase monoclonal
antibody and anti-cytochrome c were purchased from Pharmingen (San Diego, CA). Anti-fodrin (nonerythroid spectrin) and
anti- Transient Transfection of 293T Cells--
293T cells were plated
at 1 × 106/100-mm dish on day 0. On day 2, medium was
changed, and cells were incubated for an additional 3 h. Cells
were then transfected for 6 h using calcium phosphate, after which
cells were washed with PBS, and fresh medium was added. Cells were
harvested at various time points post-transfection. The amount of DNA
in all transfection experiments was made equal by including respective
amounts of vector.
Preparation of Cytosolic Extracts and Immunoblotting--
293T
cells were collected by centrifugation at 200 × g for
10 min at 4 °C. The cells were washed twice with ice-cold PBS, pH
7.2, followed by centrifugation at 200 × g for 5 min.
The cell pellet was then resuspended in 500 µl of extraction buffer
containing 220 mM mannitol, 68 mM sucrose, 50 mM Pipes-KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease
inhibitors. After a 30-min incubation on ice, cells were homogenized
using a glass dounce and a B pestle (80 strokes). Cell homogenates were
spun at 14 000 × g for 15 min, and supernatants were
removed and stored at Assessment of Apoptotic Events--
Nuclei from apoptotic cells
undergoing DNA fragmentation contain subdiploid amounts of DNA and were
therefore quantified by cell cycle analysis as described previously
(28).
Morphological changes such as cell shrinkage, rounding, and membrane
blebbing were evaluated by microscopic inspection of cells under phase
contrast. Nuclear changes such as chromatin condensation and
fragmentation and DEVD-like caspase activity were analyzed by staining
with DAPI and Phiphilux-G6D2, respectively. Phiphilux-G6D2 is a fluorogenic substrate that
is cleaved in a DEVD-dependent manner to produce rhodamine
molecules, which fluoresce red under G2A filter, whereas DAPI stains
nuclei (apoptotic or viable) blue under DAPI filter. Briefly, cells
were plated on poly(D-lysine)-coated coverslips at 4 × 105/well in a 6-well plate 24 h before
transfection. Cells were transfected with 0.5 µg of green fluorescent
protein and 2 µg of pcDNA3.Bax or empty vector as described
above. Cells were analyzed at 18 h post-transfection. Cells were
washed twice in PBS and then incubated in
Phiphilux-G6D2 (10 µM) at
37 °C for 1 h in the dark. Cells were then washed twice in PBS
and stained for 3 min with DAPI (5 µM) at room
temperature in 3.7% paraformaldehyde. Cells were then rinsed twice in
PBS, mounted in PBS, and viewied by fluorescence microscopy.
Assessment of Mitochondrial Purification of Bax Protein--
DH-5 Cell-free Apoptosis with Isolated Mouse Liver
Mitochondria--
Mitochondria were isolated from liver tissue of
6-week-old Balb/c mice. Briefly, the livers were taken and homogenized
with a Teflon glass potter in Buffer A (0.2 M mannitol,
0.05 M sucrose, 1 mM EDTA, 10 mM
KCl, 5 mM succinate, 10 mM Hepes-KOH, pH 7.4, and 0.1% bovine serum albumin). All steps were then carried out at
4 °C. Samples were centrifuged at 1,030 × g for 15 min. The supernatant was transferred to another tube and centrifuged at 3,300 × g for an additional 10 min. Pellets were
resuspended in Buffer B (0.3 M mannitol, 5 mM
potassium phosphate, 10 mM Hepes-KOH, pH 7.4, and 0.1%
bovine serum albumin) and centrifuged at 1,030 × g for
10 min. The supernatant was collected and centrifuged at 3 300 × g for 10 min. Finally mitochondrial pellets were resuspended in MSH buffer containing an ATP regenerating system (210 mM
mannitol, 70 mM sucrose, 10 mM Hepes-KOH, pH
7.4, 0.2 mM EGTA, 5 mM succinate, pH 7.0, 0.15% bovine serum albumin, 2 mM ATP, 1 mM
dATP, 10 µM phosphocreatine, 50 µg/ml creatine kinase,
10 µg/ml leupeptin, 10 µg/ml aprotinin). The freshly isolated
mitochondria were then incubated with recombinant Bax protein in the
presence or absence of S-100 cytosolic extract. After 5 to 60 min at
37 °C, mitochondria were removed by centrifugation at 20 000 × g, and supernatants were analyzed by immunoblotting as
described above.
Preparation of S-100 Extracts--
Jurkat cells were grown for 3 days. Cell pellets were then resuspended in 20 mM
extraction buffer (Hepes-KOH, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium-EDTA, 1 mM sodium-EGTA, 1 mM dithiothreitol, 250 mM sucrose, 10 mM succinate, 10 µg/ml
leupeptin, 10 µg/ml aprotinin), incubated for 30 min on ice, and
lysed by homogenization using a glass dounce (40 strokes/B-pestle).
Cell debris were removed by centrifugation at 20,000 × g in an Eppendorf centrifuge for 15 min at 4 °C.
Supernatants were re-centrifuged at 100 000 × g for
1 h in an Ultracentrifuge, and the resulting S-100 extracts were
stored at Mitochondrial Swelling Assay--
100 µg of freshly isolated
mitochondria protein was incubated with various amounts of Bax protein
in 500 µl of MSH buffer containing an ATP regenerating system, and
A520 was measured over time (20, 30). A decrease
in light scattering is consistent with an increase in mitochondrial
volume. As controls for mitochondrial swelling atractyloside (5 mM in Me2SO) and CaCl2 (100 µM) were used.
Bax promotes apoptosis induced by removal of growth factors and
other stimuli (15, 18, 31-33), and in some cases, ectopic expression
can itself induce apoptosis (34-36). To confirm this, we transfected
293T cells with a construct for expression of Bax together with one for
expression of green fluorescent protein (37). We monitored caspase
activation in individual cells using a cell-permeable, fluorescent
substrate (as described under "Materials and Methods"). As shown in
Fig. 1A, Bax-transfected cells
exhibited both morphological and biochemical characteristics of
apoptosis. Cells induced to die upon Bax expression appeared rounded
and blebbed while displaying condensed chromatin, fragmented nuclei, and active DEVD-cleaving caspases. We then examined whether expression of Bax could induce the release of mitochondrial cytochrome
c. Fig. 1B shows that as the levels of Bax
protein increased, cytochrome c could be detected in the
cytosol. In other experiments including earlier time points before
9 h (not shown), negligible Bax expression was observed with
neither cytochrome c nor caspase activation detectable.
INTRODUCTION
Top
Abstract
Introduction
References
m) (21) and has been implicated in a variety of
apoptotic phenomena (22-25). Bcl-2 was found to be capable of
inhibiting Bax-induced apoptosis but not Bax-induced cytochrome
c release in cells (19).
m. We
therefore examined the ability of Bax to induce the release of
cytochrome c and apoptosis and evaluated the relationships
between caspase activation,
m, and the effects of
anti-apoptotic Bcl-2-family proteins.
MATERIALS AND METHODS
-actin monoclonal antibody were purchased from Chemicon (Temecula, CA) and ICN (Irvine, CA), respectively. Anti-Bcl-xL was
purchased from Santa Cruz Inc. (CA). A mixture of protease inhibitors
(CompleteTM) was obtained from Boehringer Mannheim (Indianapolis, IN),
and benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) was
obtained from Kamiya Biomedical Co. (Seattle, WA). Thrombin protease
and glutathione-Sepharose-4B columns were purchased from Amersham
Pharmacia Biotech. All other chemicals were obtained from Sigma.
80 °C until analysis by SDS-polyacrylamide gel electrophoresis. Between 15-35 µg of cytosolic protein extract was boiled for 5 min and loaded. Samples were resolved under reducing conditions for 2 h at 80 V on SDS-polyacrylamide gels as described previously. Separated proteins were then blotted onto polyvinylidene difluoride and nitrocellulose membranes at 120 mA overnight. The membranes were blocked for 2 h in PBST (10 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween) containing 5%
nonfat dried milk and then probed overnight with an appropriate
dilution of the primary antibody. Reactions were detected with suitable
secondary antibody conjugated to horseradish peroxidase (The Jackson
Laboratory, Bar Harbor, ME and Amersham Pharmacia Biotech) using
enhanced chemiluminescence (Pierce).
m--
Changes in the inner
mitochondrial transmembrane potential (
m) were determined by
incubating 1 × 105 cells in 40 nM of
DiOC6 (3) or 150 nM MitoTracker Orange for 20 min at 37 °C. These two fluorochromes incorporate into cells dependent upon their mitochondrial transmembrane potential (29). The
cells were then scored using FACScan flow cytometery (Becton-Dickinson, Mountain View, CA). Controls were performed in the presence of 50 µM mitochondrial uncoupling agent mCICCP. In all cases,
cells were gated to exclude cellular debris associated with necrosis. Assessment of mitochondrial transmembrane potential in isolated mitochondria was carried out by incubating 0.2 µg of mitochondria (prepared as described below) in 80 nM Rh123 and scoring
immediately by FACScan flow cytometery. Controls were performed in the
presence of FCCP (1 µM).
bacterial cells
containing a pGEX-KG expression vector with the murine Bax protein
lacking the C-terminal hydrophobic region (Bax
C19, amino acids
1-173) were treated with 0.1 mM
isopropyl-1-thio-b-D-galactopyranoside for 4-6 h at
30 °C. Bacterial cell pellets were lysed in 0.5 mM EDTA,
1 mM dithiothreitol, 1% Triton X-100, 0.1 mg/ml
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, 1 µg/ml pepstatin in PBS and sonicated for 4 min on ice
(output 6.5; duty 90%). Cell lysates were then centrifuged at 20 000 × g for 20 min at 4 °C. The supernatant was
loaded onto a glutathione-Sepharose-4B column and the column washed
with PBS. Bound GST-Bax protein was eluted from the column by thrombin
protease treatment (10 units/liter). The eluate was incubated with 80 µg/ml
N
-p-tosyl-L-lysine
chloromethyl ketone protease inhibitor and dialyzed overnight against
20 mM Hepes-KOH pH 7.4, 10 mM KCl, 1.5 MgCl2, 1 mM dithiothreitol, 5 mM EDTA.
70 °C.
RESULTS
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Fig. 1.
Overexpression of Bax induces apoptosis.
A, 293T cells were transiently transfected with 2 µg of
empty vector or pcDNA3.Bax and 0.5 µg of green fluorescent
protein (GFP) reporter plasmid. Biochemical and
morphological changes were examined after 18 h by fluorescence
microscopy. Transfected cells were detectable under B2A filter by green
autofluorescing protein. Apoptotic cells appeared as rounded and
blebbed under phase. Caspase activation was detected using the
fluorogenic substrate Phiphilux-G6D2, which
stained cells red under G2A filter. With DAPI staining, viable cells
show homogenous staining of their nuclei. In contrast, apoptotic cells
show irregular staining as a result of chromatin condensation and
nuclear fragmentation. Magnification 60×. B, Western blot
analysis of the time-course release of cytochrome c after
Bax expression. Actin was used as loading control.
We previously observed that the release of cytochrome c
induced by staurosporine or UVB irradiation occurs before and
independently of caspase activation and subsequent apoptosis (38). To
examine this with respect to Bax-induced cytochrome c
release, we treated Bax-transfected cells with the pan-caspase
inhibitor zVAD-fmk. As described by others (39-43), zVAD-fmk
significantly inhibited apoptosis during the period studied (24 h)
(Fig. 2A). As shown in Fig.
2B, this was not because of any effect on Bax expression levels. Bax-induced apoptosis corresponded to the activation of caspases and the subsequent cleavage of fodrin and poly(ADP-ribose) polymerase, two caspase substrates previously shown to be cleaved during apoptosis (44), whereas the caspase inhibitor zVAD-fmk efficiently blocked this. Nevertheless, Bax-induced cytochrome c release proceeded with the same kinetics with or without
caspase inhibition (Fig. 2B) as measured by cell
fractionation and immunoblot analysis. This was confirmed by
densitometric analysis of the cytochrome c immunoblots. At
all time points, the cytosolic cytochrome c in the presence
of Bax plus zVAD-fmk was that of Bax alone (data not
shown).
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As another approach to inhibiting caspase activation, we co-transfected Bax and a construct for expression of XIAP. Recent studies have shown that this molecule is a potent inhibitor of caspase function, including caspase activation by cytochrome c (45). As shown in Fig. 2, XIAP coexpression completely blocked apoptotic cell death (Fig. 2C) and caspase substrate cleavage (Fig. 2D) triggered by Bax. Nevertheless, XIAP expression had no effect on Bax-induced cytochrome c release (Fig. 2D). Once again zVAD-fmk was demonstrated to be capable of blocking caspase activation but had no effect on cytochrome c release.
Cell-free systems have been extremely valuable for the analysis of apoptosis mechanisms, including cytochrome c release from mitochondria (9, 10, 13, 26, 27, 46, 47). We therefore asked whether recombinant Bax protein can induce cytochrome c release in vitro and whether this is a direct or indirect effect of the protein. As shown in Fig. 3, the addition of Bax to cytosolic extracts containing mitochondria induced a rapid release of cytochrome c. Similarly, the addition of Bax to isolated mitochondria rapidly induced cytochrome c release, which was even more pronounced than that seen in the presence of cytosol. It appears, therefore, that this release is a direct effect of Bax on the mitochondria, as observed by others (19, 20). Interestingly, the presence of cytosol appeared to delay the Bax-induced release of cytochrome c from mitochondria in vitro. It is possible that this can be simply explained by levels of inhibitors in the cytosol, such as Bcl-2 or other Bax-binding proteins, which may sequester Bax and thereby interfere with its activity.
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In many systems, apoptosis is associated with a loss of mitochondrial
inner membrane potential (m), which may correspond to the opening
of an outer membrane pore (permeability transition pore). It has been
suggested that this event is responsible for cytochrome c
release (48), although we and others (26, 27, 38) have shown that such
release can occur in the absence of a decrease in
m. Furthermore,
we have shown that inhibition of caspase activation by zVAD-fmk can
block early changes in
m without affecting cytochrome
c release. In contrast, Xiang et al. (35)
observed that the decrease in
m induced by Bax is unaffected by
caspase inhibition with zVAD-fmk.
Therefore, we examined m in our system using two fluorochromes,
DiOC6(3) and MitoTracker Orange. As shown in Fig.
4A, Bax expression induced a
loss of
m, regardless of the fluorochrome employed. The addition
of zVAD-fmk substantially decreased the number of cells displaying this
loss, although some cells continued to show a loss of
m in the
presence of zVAD-fmk, consistent with the observations of Xiang
et al. (35). Similar results were obtained when caspases
were inhibited by co-transfection with XIAP (Fig. 4C).
Despite the persistence of this phenotype in some cells, these data
suggest that the loss of
m is not required for Bax-induced
cytochrome c release, because no change in this release was
observed upon caspase inhibition (Fig. 2). The mitochondrial uncoupler,
mCICCP, was used as a positive control for
m disruption (Fig. 4,
B and D). To confirm this observation, we
examined changes in
m using Rh123 and cytochrome c
release by immunoblot over time in isolated mitochondria in the
presence or absence of recombinant Bax. As seen in Fig.
5A, Bax alone cannot induce
changes in
m. Despite this, a rapid release of cytochrome c could be detected in the presence of Bax (Fig.
5B). The uncoupler FCCP, although inducing a dramatic change
in
m, had no effect on this cytochrome c release.
Similar results were seen when
m was monitored using
DiOC6(3) (data not shown).
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Recently, Vander Heiden et al. (49) provided evidence that during apoptosis there is a disruption of the mitochondrial outer membrane, which may be responsible for the release of cytochrome c. One way this could happen would be through induction of swelling of the outer membrane until it breaks the outer membrane, as was suggested (49). We examined mitochondrial swelling as a function of light scattering, as described (20, 30). Although both CaCl2 and atractyloside induced rapid swelling, we were unable to detect Bax-induced swelling of mitochondria in vitro (Fig. 5C), suggesting either that other mechanisms are involved or that any swelling is too transient to be detected by this technique.
Altogether this suggests that Bax can induce alterations in m
in vivo, but it does so indirectly and requires cytosolic participation. Caspases are proposed as one likely candidate for this
phenomenon. One of the best indicators of the mitochondrial permeability transition is swelling of the organelle, which can be
readily observed in isolated mitochondria. Our results (discussed above), which failed to show such swelling, provide further evidence against an irreversible permeability transition induced by Bax in
vitro.
Recently two groups reported that Bcl-2 proteins can block apoptosis by acting downstream of cytochrome c (19, 50). In light of this we therefore examined whether Bcl-xL, a member of the Bcl-2 family, could inhibit Bax-induced death and cytochrome c release. Bcl-xL inhibited cell death (Fig. 6A) when co-transfected with Bax, and this was accompanied by a prevention or delay in cytochrome c release (Fig. 6B). Caspase activation as determined by poly(ADP-ribose) polymerase cleavage was also inhibited by Bcl-xL.
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The ability of Bcl-2-family members to interfere with Bax-induced apoptosis was also observed in vitro. Mitochondria were prepared from CEM cells or CEM with ectopic expression of Bcl-2. Although Bax readily induced cytochrome c release from mitochondria isolated from the parental line, the mitochondria isolated from the Bcl-2-expressing cells were relatively resistant to this effect of Bax (Fig. 6C).
Therefore, the induction of cytochrome c release from
mitochondria by Bax is inhibited by Bcl-2 and Bcl-xL.
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DISCUSSION |
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In this paper we have shown that ectopic expression of Bax induces mitochondria to release cytochrome c, caspase activation, and apoptosis. Although apoptosis depends upon caspase function, cytochrome c release does not, suggesting that if cytochrome c plays a role in caspase activation, it must precede this step in the apoptotic process. Several studies have shown that cytochrome c can trigger caspase activation in cell-free extracts (47), supporting this possibility.
In some systems, signals leading to apoptosis result in the transcription of key genes, which in turn lead to the death of the cell. Bax can function as one such gene. For example, DNA damage in some cells induces p53, which can activate the Bax promoter such that DNA damage results in elevation of Bax levels (51-54). Expression of Bax might then promote apoptosis via targeting mitochondria and inducing cytochrome c release.
In many cases, however, transcription is not required for apoptosis (55, 56). Nevertheless, Bax may play a crucial role in the apoptotic process via a number of different mechanisms. For example, Bcl-xL counteracts the effects of Bax (18, 57), and this molecule can be sequestered in the cytosol by another protein, BAD (58). Phosphorylation of BAD via growth factor receptor signaling and the Akt kinase releases Bcl-xL to target mitochondria (59). Thus, upon growth factor withdrawal, Bcl-xL becomes sequestered, and Bax may then be free to induce cytochrome c release and apoptosis. Not surprisingly, then, elevated levels of Bax exacerbate the effects of growth factor deprivation in cells (15, 31).
Some studies have shown that Bax translocates from its predominantly cytoplasmic location to the mitochondria (18) upon apoptosis induction. In cells overexpressing Bax, we similarly observed that this molecule remained mostly cytoplasmic for several h and then localized to the mitochondria around the time of cytochrome c release (approximately 12 h, data not shown). The signals and mechanisms responsible for this change in Bax distribution are not known, although our results would suggest that they can be caspase-independent, because caspase inhibitors did not block Bax-induced cytochrome c release (Fig. 2). Because Bax can clearly promote apoptosis, the nature of the translocation signal leading to induction of mitochondrial release of cytochrome c potentially takes on significance as a major apoptotic signaling pathway.
Once localized to the mitochondria, how does Bax induce the release of
cytochrome c? Our studies support the idea that this can
occur independently of a decrease in m. Although Bax induced a
dramatic decrease in mitochondrial transmembrane potential, inhibition
of caspases by XIAP or zVAD-fmk significantly reduced the number of
cells displaying such a loss while not affecting the extent of
cytochrome c release. It is possible, however, that loss of
m occurs in two stages, a minor caspase-independent loss followed
by a more dramatic caspase-dependent loss. Although this
remains a possibility, it is noteworthy that we failed to detect any
decrease in mitochondrial transmembrane potential in isolated
mitochondria treated with Bax, despite the release of cytochrome
c (Fig. 5).
Recent studies by Vander Heiden et al. (49) have suggested
that during apoptosis, a hyperpolarization of the mitochondrial inner
membrane causes a swelling that might act to puncture the outer
membrane without necessarily disrupting m in the short term. In
this model, Bcl-xL acts as an ion channel (60, 61) to offset this
hyperpolarization by allowing an efflux of protons. Bax can also act as
an ion channel (62, 63), and thus, our results are consistent with the
possibility that Bax promotes inner membrane swelling and outer
membrane puncture. However, we were unable to detect the expected
mitochondrial swelling (Fig. 4B). Either this is a transient
effect that we were simply unable to capture or else Bax promotes a
loss of mitochondrial outer membrane integrity via a different
mechanism. A similar failure to detect Bax-induced mitochondrial
swelling in vitro was recently described by
Jürgensmeier et al. (20).
Studies by Xiang et al. (35) have shown that inducible Bax expression triggers a rapid caspase-dependent apoptosis, but if caspase activity is inhibited, a slower nonapoptotic death proceeds that has been associated with generation of reactive oxygen species (whether or not these are responsible for the subsequent death still remains unknown). Bax-induced cytochrome c release helps to explain these observations. First, cytochrome c can trigger caspase activation and apoptosis (10, 13, 14, 47). In addition, however, and independently of caspase activation, the release of cytochrome c might be expected to result in disruption of electron transport, as has been observed in Fas-induced apoptosis. The resulting loss of ATP and generation of reactive oxygen species may ultimately cause cell death even in the absence of caspase function. This, of course, does not exclude the possibility that Bax has additional death-promoting activities. Nevertheless, the ability of Bax to induce the disruption of the mitochondrial outer membrane and the release of cytochrome c represents an important step in Bax-induced cell death.
Controversy over the mechanism of action of Bcl-2 and its homologue Bcl-xL has arisen recently with reports that these two anti-apoptotic oncogenes can block apoptosis by acting downstream of cytochrome c in the cell death pathway (19, 50). Bcl-2 has long been known to block cell death. Despite the vast literature dealing with this family of oncogenes, little is known about how the mechanisms used by these molecules prevent apoptosis. Some clues were provided by the observations that the structure of Bcl-xL resembles diphtheria toxin, which is able to form channels in cellular membranes (60). Furthermore, it was shown that Bcl-xL and Bcl-2 can indeed form ion channels in vitro (61). Previously it had been demonstrated that high levels of Bcl-2 can prevent the release of cytochrome c and, thus, caspase activation in response to a number of apoptosis-inducing stimuli, such as UVB, staurosporine, and etoposide (26, 27). Furthermore, in vitro, exogenous cytochrome c bypassed this inhibitory effect of Bcl-2 (27). Similarly Duckett et al. (64) showed that redistribution of cytochrome c is an early event in apoptosis that is inhibitable by Bcl-xL, but microinjection of cytochrome c overcomes this apoptotic inhibition. Together these studies proposed that a possible anti-apoptotic mechanism of Bcl-2 and its anti-apoptotic members was to inhibit cytochrome c translocation from mitochondria to the cytosol, thereby preventing caspase activation and subsequent apoptosis.
However Bcl-2 is not restricted exclusively to the mitochondrial membrane (65), and therefore, the possibility that this protein may have multiple anti-apoptotic mechanisms must be considered. To complicate matters, the Bcl-2 family members have been shown to bind to several proteins (66), not including other members of the Bcl-2 family, which may determine their localization and, therefore, activity. Reports that Bcl-xL can itself bind cytochrome c (67) and Apaf-1 (68) may possibly explain how apoptosis induced by cytochrome c microinjection could be inhibited in cells overexpressing Bcl-2. It remains possible that cells with high levels of Bcl-2 may have a large cytoplasmic fraction that is available to bind and thereby sequester exogenously added cytochrome c and, in doing so, quench its pro-apoptotic activity.
In contrast, we observed that Bcl-xL can significantly delay the release of cytochrome c from mitochondria (and subsequent apoptosis) in response to ectopic Bax. These results parallel similar observations reported in yeast studies (57). The anti-apoptotic mechanism, however, has not yet been determined. Proposed mechanisms include the formation of heterodimers between Bcl-2/Bcl-xL and Bax (15), which would interfere with the availability and translocation of the Bax protein from the cytoplasm to the mitochondria. Alternatively, counteracting ion channels at the level of the mitochondria may help to explain the antagonistic nature of these proteins (60, 61, 63, 69, 70).
In conclusion, we have shown that Bax induces the release of cytochrome
c in conjunction with apoptosis and that caspase inhibition, although altering the kinetics and perhaps mode of cell death, has no
effect on this release. In contrast, members of the anti-apoptotic oncogene family, Bcl-2 and Bcl-xL, are capable of inhibiting or delaying this release while significantly preventing cell death. This
suggests that Bcl-2 family members may play a modulating role in
blocking the mammalian cell death machinery by acting upstream of
caspase function and upstream or at the mitochondrial level.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Gross and S. Korsmeyer for
their pGEX-KG-BaxC19 construct and advice in the isolating Bax
protein. We also thank Dr. Ruth Kluck for helpful discussions.
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FOOTNOTES |
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* This is manuscript 261 from La Jolla Institute for Allergy and Immunology. This research was supported by National Institutes of Health Grants AI40646 and CA69381.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by The Children Leukemia Research Project fellowship.
¶ Supported by the Swiss National Science Foundation (Fellowship 823A-046638).
** To whom correspondence should be addressed: Div. of Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 619-558-3515; Fax: 619-558-3525; E-mail: dgreen5240{at}aol.com.
The abbreviations used are: DiOC6, dihexyloxacarbocyanine iodide; mCICCP, carbamoyl cyanide n-chlorophenylhydrazone; DAPI, 4'6-diamino-2-phenylindole dihydrochloride; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid.
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