From the Departments of Pharmacology and
¶ Radiation Oncology, University of Michigan Medical School,
Ann Arbor, Michigan 48109
Received for publication, September 7, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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We have shown previously that Bcl-XS causes acute
cell death in 3T3 cells without activating caspases (Fridman, J. S., Benedict, M. A., and Maybaum, J. (1999) Cancer
Res. 59, 5999-6004). In this study, we determined that the
explanation for lack of caspase activation is the cellular depletion of
cytochrome c. Electron microscopy revealed gross structural
changes in the mitochondria of Bcl-XS-expressing cells; however,
cytochrome c was not detected in cytosolic fractions from
these cells. Surprisingly, it was determined that cellular cytochrome
c levels decreased as Bcl-XS expression levels increased.
Experiments performed to eliminate other possible explanations for the
lack of caspase activation showed that these 3T3 cells have a
functional cytoplasmic apoptosome, a complex of proteins that form
a functional trigger capable of activating the proximal caspase in an
apoptotic pathway Chinnaiyan, A. M. (1999) Neoplasia 1, 5-15, as cytosolic extracts from these cells were capable of cleaving
pro-caspase-9. These cells were also able to release cytochrome
c from their mitochondria after appropriate stimulation,
other than Bcl-XS expression (i.e. withdrawal from serum
for 24 h), and initiate a cell death that is inhibited by a
dominant negative caspase-9. We conclude that lack of caspase activation is due to a Bcl-XS-induced depletion of active cytochrome c, a phenomenon that represents an alternative cell death
effector pathway and/or a novel mechanism for regulating caspase activation.
Bcl-XS, belongs to the Bcl-2 family of proteins, whose members can
either promote or inhibit cell death, depending on their structural
features. These features are referred to as Bcl-2 homology (BH)1 domains and are highly
conserved throughout this family of proteins). The pro-survival
proteins, Bcl-2 and Bcl-XL, contain a BH1, BH2, BH3, and a BH4 domain,
whereas the pro-death members of the family have, at least, a BH3
domain. The BH1, BH2, and BH3 domains form a hydrophobic binding pocket
into which a BH3 domain of another family member binds, forming either
a hetero- or a homodimer (3, 4). It has been proposed that the ratio of
the pro-survival to pro-death members of this family determines the
propensity of a cell to live or die (5, 6), although the specific
mechanisms by which a pro-death or a pro-survival family member act are
still poorly understood.
The most extensively studied pro-death family member, Bax, has a BH1
and a BH2 domain in addition to the obligatory BH3 domain. These
domains (BH1 and BH2) are part of the proposed membrane-spanning domain
that forms channels in synthetic membranes and lipid vesicles (7, 10).
The ability to form such channels is one of the proposed models by
which Bax may act (8, 9, 11-13).
Bcl-XS, in contrast to Bax, lacks BH1 and BH2 domains. Therefore,
Bcl-XS should not form membrane-spanning channels and thus acts through
a different mechanism. Bcl-XS does, however, have a BH3 domain as well
as a BH4 domain (thought to allow for protein-protein interactions
outside the Bcl-2 family), a unique combination among the Bcl-2 family
members. Although there are no other known pro-death Bcl-2 family
members that have only BH3 and BH4 domains, there are a number of
BH-3-only proteins including, Hrk, Bik, Bad, and EGL-1. These proteins
are thought to act either by binding to and inactivating the
pro-survival functions of Bcl-2 and Bcl-XL (or related proteins), or by
displacing pro-death Bcl-2 family members that are capable of forming
transmembrane channels, from Bcl-2 or Bcl-XL, thereby allowing them to
kill the cell. If either of these models is correct, then the outcome
will be disruption of the functionality and/or integrity of organelles
in which these proteins are concentrated, the mitochondria and
endoplasmic reticulum.
Mitochondria have been the focus of the majority of studies aimed at
explaining the role of the Bcl-2 family in cell death. Evidence for
such a role includes the following; 1) cell death has been correlated
with loss of the mitochondrial membrane potential ( We have shown recently that Bcl-XS can kill 3T3 cells and that this
cell death does not require, nor does it activate, caspases (1). In
that report we proposed four possible mechanisms of action for Bcl-XS
in 3T3 cells (see Fig. 1). The first of these models, the activation of
caspases through the release of cytochrome c from the
mitochondria, was found not to be true; however, we did find that
expression of Bcl-XS was temporally coincident with collapse of the
mitochondrial membrane potential ( Cell Lines and Tissue Culture--
3T3XS7.2 cells and stable
derivatives of these cells expressing Bcl-XL or dominant negative
caspase-9 (9DN) were derived and maintained as described previously
(1).
Electron Microscopy--
Approximately 5 × 105
3T3XS7.2 cells, or their Bcl-XL-overexpressing derivatives, were plated
in 100-mm tissue culture dishes and allowed to grow for 24-48 h in the
presence of tetracycline (TET). In the TET withdrawal time-course
experiment, plates were withdrawn from TET at different times so that
all samples were collected at the same time.
After removal of medium, cell monolayers were fixed with 2%
glutaraldehyde in 0.1 M Sorensen's buffer, pH 7.4. The
aspirated media was centrifuged to pellet floating cells that were
subsequently pooled with their respective adherent cells. Cells were
fixed in 1% osmium tetroxide and dehydrated in a graded series of
ethanol. Cell pellets were embedded in Spurr's resin. Ultrathin
sections, showing a silver interference color, were collected and
stained with uranyl acetate and lead citrate. Sections were viewed on a
Philips CM100 electron microscope.
Preparation of Cytosolic Extracts--
Cytosolic extracts were
prepared by washing cells twice in cold phosphate-buffered saline. All
additional steps were performed at 4 °C or on ice. Cells were
resuspended in cold buffer A (20 mM Hepes (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EGTA, 1 mM Na-EDTA, 1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 220 mM mannitol, 200 mM sucrose) and Complete
protease inhibitors (Roche Molecular Biochemicals) and left for 40 min
on ice. Cells were then disrupted by Dounce homogenization (45 strokes). The solution was then centrifuged twice at 500 × g to remove nuclei and unlysed cells. The supernatant was
then spun at 14,000 rpm in a microcentrifuge at 4 °C for 30 min. The
supernatant, referred to as the cytosolic extract, was removed and
stored at Immunoblot Analyses--
Protein concentrations for cytosolic
extracts were normalized and loaded onto SDS-polyacrylamide gels. For
whole cell samples, cells were counted, centrifuged at 500 × g, and resuspended in sample buffer (17) and boiled.
Equivalent numbers of cells were loaded onto SDS-polyacrylamide gels.
Following electrophoresis, proteins were transferred to polyvinylidene
difluoride membranes (equal protein loading was confirmed by Ponceau-S
staining) and blocked for at least 2 h in TBST (Tris-buffered
saline and 0.1% Tween 20) containing 5% (w/v) milk. All primary
antibodies were diluted in TBST containing 1% milk, and incubations
were carried out for either 2 h at room temperature or overnight
at 4 °C. After each incubation, membranes were washed six times in
TBST. Anti-cytochrome c antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) was used at 1:500 or 1:1000 (PharMingen, San Diego,
CA), anti-Hsp60 antibody (Stressgen Incorporated) was used at 1:2000,
and anti-VDAC (porin) (Calbiochem) was used at 1:200. Secondary
antibodies were used at 1:50,000 (Pierce).
Chemiluminescent Detection of Cytochrome c--
Chemiluminescent detection of cytochrome c was
performed as described (18, 19) with minor modifications. Briefly,
cells were lysed in 2× sample buffer (17) without In Vitro Caspase-9 Activation
Assay--
[35S]Methionine (Amersham Pharmacia Biotech)
-labeled pro-caspase-9 was made using the TNT Coupled
Reticulocyte Lysate System (Promega Corp., Madison, WI) following the
manufacturer's protocols. The translation mix was then applied to a
G-25 desalting column (Amersham Pharmacia Biotech) to remove
nucleotides and unincorporated radioactive amino acids. Aliquots were
stored at Quantification of Cellular ATP Levels by High Performance Liquid
Chromatography (HPLC)--
Cells were grown in the presence or absence
( Transient Transfection Death Assay--
3T3XS7.2 or 9DN cells
(stable derivatives of the 3T3XS7.2 cells expressing a dominant
negative caspase-9) were plated in six-well dishes and allowed to grow
for 24-48 h. Cells were maintained in the presence of TET (1 µg/ml
tetracycline) and were transfected with 0.825 µg of pK7GFP (a
constitutive GFP-expressing construct) and 2.5 µg of
pSFFVneo-Bcl-XL-FLAG, pSFFVneo-Bcl-XS-HA (a gift from Garbriel Nunez,
University of Michigan), or pCDNA3-Bax-FLAG (a gift from Claudius
Vincenz, University of Michigan) using Suprafect (Qiagen, Santa
Clarita, CA), following the manufacturer's protocol. Twenty-four hours
after transfection, cells were examined by fluorescence microscopy and
scored as live (flat cells with extensions) or dead/dying cells
(rounded, floating cells). At least 200 cells were counted whenever
possible; in most cases, however, 200 green cells did not exist in Bax
transfections of 3T3XS7.2 cells, presumably due to the toxicity of that
gene product.
Statistical analyses were performed using the analysis of variance on
the proportion of dead out of total cells counted. The major
hypothesis, that of a difference in the presence or absence of dominant
negative caspase-9 upon transfection with Bcl-XS or Bax, was tested as
an interaction contrast. Homogeneity of variance was acceptable in all
treatments except Bax, where the S.D. was 2-3 times what it was in the
other three transgenes. The untransformed proportion was used in
analyses utilizing SAS Proc GLM.
Structural Changes to 3T3 Cells upon Expression of Bcl-XS--
We
have shown previously that enforced expression of Bcl-XS causes a loss
of mitochondrial membrane potential (
The mitochondria of Bcl-XS-expressing cells appear less electron-dense
or even transparent (Fig. 2, marked
M*), indicating lack of protein and membrane content. The
structure of the cristae, in the mitochondria in which cristae were
still visible, no longer filled the interior of the mitochondria. The
cristae appeared broken and/or pushed against the outer mitochondrial
membrane. In contrast, the mitochondria of the control cells (+TET)
(Fig. 2, marked M) appeared electron-dense and, therefore,
contain a substantial amount of membranes and protein. The cristae were numerous, intact, and healthy, as they spanned all regions of the
mitochondria. The effects of Bcl-XS expression on the mitochondria were
negated by the stable expression of Bcl-XL in these cells (Fig. 2,
bottom); however, they were not prevented by pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk (up to 100 µM) or by expression of a dominant negative caspase-9
(data not shown). Expression of
In cells expressing Bcl-XS we also observed large, opaque, vesicles
that are not membrane-bound (Fig. 2, marked L). These masses
are lipid depositions often seen during mitochondrial distress (21-24)
and do not arise in control (+TET) cells or
Depletion of Cytochrome c upon Expression of Bcl-XS in 3T3
Cells--
The morphologic changes in the mitochondria upon expression
of Bcl-XS (and the depolarization of the mitochondria), led to the
assumption that cytochrome c was being released into the
cytosol of the cells. This should have initiated the formation and
activation (in the presence of sufficient ATP) of the Apaf-1-cytochrome
c-caspase-9 apoptosome (22, 23). The fact that 9DN could
inhibit cell death induced by withdrawal from serum suggested that such
an apoptosome was functional in these cells. Therefore, the previously observed lack of caspase activation upon Bcl-XS expression was unanticipated (1).
To determine whether cytochrome c was being released into
the cytosol upon expression of Bcl-XS, cytosolic extracts were made from 3T3XS7.2 cells at various times after withdrawal from TET, or
after withdrawal from serum for 24 h. Withdrawal from serum for
24 h caused approximately the same extent of cell death (as assessed morphologically and/or by trypan blue exclusion) as did withdrawal from TET for 36 h (data not shown).
Release of cytochrome c into the cytosol was not detected
upon expression of Bcl-XS (Fig.
3A). In contrast, 24 h
after serum withdrawal, cytochrome c was easily detected in
the cytosol. These results explain why serum withdrawal-induced death,
but not death induced by expression of Bcl-XS, is inhibited by 9DN
(data not shown and Ref. 1). However, the observed loss of
To investigate the possibility that cytochrome c was not
being released from the mitochondria because there was no cytochrome c to be released, we made whole cell extracts from the same
cells used to make cytosolic extracts above (Fig. 3B). Upon
expression of Bcl-XS, first detectable between 24 and 30 h after
withdrawal from TET (Fig. 3C) (1), the cellular levels of
cytochrome c decreased slightly. This decrease became more
pronounced as the time of withdrawal from TET increased. Cytochrome
c was barely detectable 36-48 h after TET withdrawal;
however, it was still present after withdrawal from serum for 24 h. To circumvent the possibility of a lack of antibody reactivity due
to cytochrome c modification(s), we used two antibodies for
immunoblot analyses, a polyclonal antibody raised against the
full-length protein and a monoclonal antibody raised against three
unique regions of the protein. Two extraction buffers, a SDS-based
buffer and a Nonidet P-40-based buffer, were used for cytochrome
c immunoblots and gave identical results (data not shown).
Additionally, we took advantage of the covalently bound iron atom in
cytochrome c and its inherent peroxidase activity to oxidize
a chemiluminescent substrate (ECL) (18, 19). Using this technique we
show that a band that comigrates with (and is the expected size of)
purified bovine cytochrome c is also depleted upon
expression of Bcl-XS, with similar kinetics as in the Western blot
(Fig. 3D).
This decrease in cytochrome c was not common to all
mitochondrial proteins as the mitochondrial matrix protein Hsp60 and
the outer membrane protein VDAC were not depleted after withdrawal from
TET (Fig. 3E). Additionally, confocal microscopy showed that the mitochondrial intermembrane space protein AIF was redistributed in
the cytoplasm, but was not degraded upon expression of Bcl-XS (data not
shown). These experiments led to the hypothesis that Bcl-XS does not
induce caspase activation because cytochrome c is not
present to a sufficient degree in the cytosol of Bcl-XS-expressing cells to complete and activate the mitochondrial apoptosome(s).
Addition of Cytochrome c Rescues the Ability of Cytosolic Extracts
to Cleave 35S-Pro-caspase-9--
To confirm that the lack
of detectable cytochrome c, in the cytosolic fractions from
Bcl-XS-expressing cells, was the explanation for absence of caspase
activation in these cells, in vitro pro-caspase-9 cleavage
assays were performed using cytosolic extracts from 3T3XS7.2 cells
(grown in the presence of TET) or from 293T cells and
35S-labeled human pro-caspase-9. 293T cells have previously
been shown to possess the very catalytically active Apaf-1XL (25) and,
therefore, served as a positive control in this assay. Reverse transcriptase-polymerase chain reaction of Apaf-1 from the
3T3XS7.2 amplified an Apaf-1 equivalent in size to the human
Apaf-1L (data not shown) and should, consequently, be slightly less
efficacious than the Apaf-1XL from 293T cells.
Cytosolic extracts from either 293T cells or 3T3XS7.2 cells were able
to process 35S-labeled human pro-caspase-9 in the presence,
but not in the absence of exogenously added dATP and bovine cytochrome
c (Fig. 4A). The
presence of two cleavage products upon apoptosome activation is the
result of both autoprocessing (caspase-9 cleavage of pro-caspase-9) and
processing by downstream effector caspases, such as caspase 3 (25).
These results confirm that a cytosolic apoptosome is functional in
these cells and that complementation of these extracts with cytochrome
c and dATP is sufficient to activate the proximal caspase in
a cell death pathway.
We also looked at the ability of cytosolic extracts from 3T3XS7.2 cells
expressing Bcl-XS ( ATP Levels in 3T3XS7.2 Cells upon Expression of Bcl-XS or Treatment
with Fas/ActD--
To strengthen the hypothesis that lack of
detectable caspase activity in the presence of Bcl-XS expression is due
to lack of cytochrome c in the cytosol, we examined another
possible explanation, insufficient ATP levels. ATP is required for the
oligomerization of Apaf-1 and activation of pro-caspase 9 (22, 23, 25). We measured ATP levels in cells that were dying by induction of Bcl-XS
expression (noncaspase mediated) or by a caspase-mediated cell death
induced by treatment with Fas/Act D. Nucleotides were extracted from
3T3XS7.2 at various times after withdrawal from TET, or after 8 h
of Fas/Act D treatment (in the presence of TET). Extracted nucleotides
were resolved and quantified by strong anion-exchange HPLC. The results
show no significant decrease in the levels of ATP from whole cells upon
expression of Bcl-XS out to 48 h (Table I). There was, however, an approximate
50% decrease in the amount of ATP extracted from cells treated
with Fas/Act D. These cells have previously been shown to have
significant caspase activity at this time point (1). As caspase
activity is an energy-consuming process, the decreased ATP levels upon
Fas/Act D treatment are not a surprise. However, it is interesting
that, after structural disruption of the mitochondria and loss of the
Bcl-XS and Bax Kill through Nonoverlapping Mechanisms--
After
determining that Bcl-XS kills 3T3 cells without caspase activation,
because of a depletion of cytochrome c, we wanted to address
another of the mechanisms proposed for Bcl-XS-induced cell death (Fig.
1). One of the described mechanisms entails the displacement, by
Bcl-XS, of a pro-death Bcl-2 family member that possesses potential
membrane-spanning domains, such as Bax. To test if Bcl-XS kills 3T3
cells by displacing such a protein, we performed the following
experiments. Stable cell lines expressing 9DN were derived from our
3T3XS7.2 cells (1). The 9DN cells were shown to express dominant
negative caspase-9 both by immunoblot analysis2 and
functionally, by resistance to serum withdrawal (1). The 3T3XS7.2 cells
and the 9DN cells were transiently transfected (in the presence of TET)
with Bcl-XS, Bcl-XL, or Bax, along
with a plasmid coding for a green fluorescent protein (pk7GFP) at a ratio of 3:1. The presence of TET throughout this experiment ensures that the TET-dependent expression of Bcl-XS is turned off
and only the transiently transfected transgenes are expressed (data not
shown). Twenty-four hours after transfection, the cells were examined
by fluorescent microscopy. Green cells were scored morphologically as
either live (flat cells with normal stellate morphology) or dead
(rounded or floating) (Fig. 5). Two
hundred cells were counted wherever possible, but, in three of four
experiments, 200 surviving green cells could not be found in the
Bax transfected 3T3XS7.2 cells. This is in comparison to
thousands of green cells in the other conditions, including the
Bax transfections of the 9DN cells (data not shown).
Analysis of variance showed that day-to-day variation was marginally
significant (p = 0.064), indicating the importance of comparing results obtained on the same day of experimentation. Therefore, all data are paired, allowing comparison of results from the
individual experiments. 9DN cells were slightly more resistant than the
3T3XS7.2 cells to base-line transfection cytotoxicity with
Bcl-XL. However, there was no difference between cell lines transfected with Bcl-XS. The hypothesis that 9DN protects
against Bax-induced, but not Bcl-XS-induced, cell death was tested
using a statistical interaction contrast (see "Experimental
Procedures" for explanation), with the cell line effect (3T3XS7.2
versus 9DN) for Bcl-XS contrasted with the cell line effect
for Bax. This contrast was significant (p = 0.031),
with the difference in the fraction of cells surviving (9DN surviving
fraction minus 3T3XS7.2 surviving fraction) being 0.006 for Bcl-XS but
0.139 for Bax. Therefore, Bcl-XS-induced cell death (since it is not
inhibited by a dominant negative caspase 9) proceeds, at least in part, through a pathway that does not utilize Bax or another Bcl-2 family member which requires a functional caspase 9. These data argue against
the proposed model for Bcl-XS-induced cell death in which Bcl-XS
displaces another pro-death Bcl-2 family member (which then kills the
cell), such as Bax.
This study presents two important findings regarding
Bcl-XS-induced cell death and the regulation of cell death as a whole. The most significant finding is the cellular depletion of cytochrome c that is temporally coincident with detectable expression
of Bcl-XS and that prevents caspase activation during Bcl-XS-induced cell death in 3T3 cells. Second, Bcl-XS was shown to kill 3T3 cells by
a pathway distinct from Bax and, therefore, does not require the
activity of a pore-forming Bcl-2 family member, such as Bax. These
findings support a model for the mechanism of action of Bcl-XS in 3T3
cells that entails inhibition of the death-suppressing effects of Bcl-2
or related proteins (i.e. a dominant negative Bcl-2) by
direct interaction with such proteins (27, 28).
The observed depletion of cytochrome c is a novel mechanism
for the prevention of caspase activation. Although various structural alterations to cytochrome c have been observed in other
systems during programmed cell death, such as Jurkat cells (29, 30) and
in Drosophila (26), in neither system was cytochrome
c depleted from the cells, nor was caspase activation
prevented. In the present study, complementation of cytosolic extracts
(from 3T3XS7.2 cells expressing Bcl-XS) with exogenous cytochrome
c restored the ability of these extracts to process
pro-caspase-9, confirming that the lack of cytosolic cytochrome
c was the explanation for the lack of caspase activation.
Of the four models to explain how Bcl-XS can kill 3T3 cells presented
in this study (Fig. 1), the data described previously (1) disproved the
first hypothesis, the activation of caspases, whereas the data
presented here describe the mechanism by which caspase activation is
prevented. Evidence supporting this mechanism, the depletion of
cytochrome c, includes the following. First, there is a
temporal coincidence between expression of Bcl-XS and the depletion of
cytochrome c from 3T3XS7.2 whole cell extracts. This was
confirmed by immunoblot analysis (using both monoclonal and polyclonal
antibodies against cytochrome c) and directly, utilizing the
peroxidase activity of the covalently bound iron atom in the heme group
of cytochrome c and a chemiluminescent substrate, which is
activated upon oxidation. The loss of detectable cytochrome
c also occurs as there are both structural and functional perturbations detected in the mitochondria. Second, complementation of
cytosolic extracts, from 3T3XS7.2 cells, with exogenous cytochrome c triggers processing of pro-caspases. Third, a mechanism
for the release of cytochrome c from the mitochondria and
the subsequent activation of caspase-9 and cell death is functional in
these cells, as they have been shown to release cytochrome c
into the cytosol upon serum withdrawal. Serum withdrawal-induced death in these cells was prevented by expression of 9DN; however, 9DN had no
effect on Bcl-XS-induced cell death. Finally, alternative mechanisms
for the inhibition of caspase activation were addressed and eliminated.
3T3XS7.2 cells can die by caspase-dependent pathways, as
activation of the Fas receptor, serum withdrawal, or transfection with
Bax have all been shown to kill in a caspase-dependent
manner. These cells have functional apoptosomes and, as we have also
demonstrated here, their ATP levels are not depleted, even after
prolonged expression of Bcl-XS. Therefore, 3T3XS7.2 cells expressing
Bcl-XS have all of the cellular machinery and the fuel to execute the apoptotic pathway, except for cytochrome c.
Interestingly, the depletion of cytochrome c appears to be a
relatively specific phenomenon as other mitochondrial proteins, each
with different localization within the mitochondria, VDAC, Hsp60, and
AIF, were not depleted upon expression of Bcl-XS. It is not
unreasonable to envision a mechanism present in either the mitochondria
or in the cytosol that rapidly degrades or grossly modifies cytochrome
c released (or when release is anticipated) in an
environment otherwise not conducive to triggering programmed cell death.
Finally, these studies were initiated with the goal of determining the
mechanism of action of Bcl-XS-induced cell death. Thus far we have
disproved the activation of caspases as a possible mechanism, and we
have also described the novel finding of depletion of cytochrome
c as a method of inhibiting caspase activation upon Bcl-XS
expression. In this study we also addressed another of the proposed
mechanisms for Bcl-XS-induced cell death (Fig. 1), that Bcl-XS acts by
displacing a pro-death Bcl-2 protein, such as Bax, that contains
membrane-spanning domains. To address this model we used the 3T3XS7.2
cells and stable derivatives of these same cells expressing 9DN to
determine whether expression of 9DN had a protective effect upon
transfection with Bcl-XS or Bax. If Bcl-XS kills by displacing Bax, the
protective effects of 9DN observed for Bax-induced death would have
also be observed upon transfection with Bcl-XS; however, this was not
the case. The lack of protection, by 9DN, from Bcl-XS implies that
Bcl-XS does not kill indirectly by the liberation and/or activation of
a Bax-like protein. These findings, combined with the fact that Bcl-XS
does not possess membrane-spanning domains and, therefore, cannot form membrane-spanning channels, supports a model in which Bcl-XS acts as a
dominant negative Bcl-2/Bcl-XL and inhibits the pro-survival function
of these proteins.
In summary, these findings suggest a novel pathway for cell death
induced by a Bcl-2 family member, Bcl-XS, that does not utilize
caspases. Additionally, there is evidence to suggest the existence of a
novel regulatory mechanism, the depletion of cytochrome c,
for the activation of caspases. Such a mechanism may have evolved in
eukaryotes to safeguard the cell from an untimely demise upon the
accidental release of cytochrome c. It will be interesting to determine what activity is responsible for the degradation of
cytochrome c in these cells and to determine whether
evidence exists in vivo for such mechanisms of regulating
cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m), and 2) release of apoptogenic factors including
cytochrome c, AIF, and caspases has been observed upon
mitochondrial disruption. The mechanism by which Bcl-2-related proteins
affect the mitochondria is not clear. However, it has been shown that
Bax can cause release of cytochrome c from mitochondria,
whereas Bcl-2 and Bcl-XL can prevent such a loss induced by Bax or
other insults (in isolated mitochondria and in whole cells) (6, 13,
14). Cytochrome c, once released into the cytosol, becomes
part of an apoptosome by binding Apaf-1 in an ATP-dependent
manner. This binding allows for the oligomerization of Apaf-1 molecules
and the recruitment of pro-caspase-9. Pro-caspase-9 is then thought to
be activated by a proximity model that utilizes the low but significant
catalytic activity of the pro-caspase (15, 16). This initiates a
caspase cascade and apoptosis.
m) and was therefore affecting the mitochondria. In this study, we sought to
determine why caspases are not activated upon expression of Bcl-XS and
to determine which, if any, of the proposed mechanisms (Fig.
1) is responsible for Bcl-XS-mediated
death in 3T3 cells.
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Fig. 1.
Possible models of Bcl-XS-induced cell
death. 1, direct release of cytochrome c
from mitochondria and subsequent activation of effector caspases;
2, inhibition of anti-apoptotic Bcl-2 family members as a
result of physical association; 3, liberation/activation of
one or more pro-apoptotic Bcl-2 family members which possess a BH1 and
BH2 domain (such as Bax), which might then act through one of the first
two models (or the last); or 4, formation of new (or
alteration of existing) membrane-spanning channels.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until used. The pellet was also saved and stored
at
80 °C and is considered to contain the membrane fraction
greatly enriched in mitochondria.
-mercaptoethanol and spun in a microcentrifuge on full speed for 5 min. Protein content
of the supernatants was determined, and 50 µg of each sample was
subjected to SDS-polyacrylamide gel electrophoresis. Proteins were
transferred to polyvinylidene difluoride membranes and incubated with
Pierce SuperSignal West Dura Extended Duration Chemiluminescent
Substrate, as indicated by the manufacturer, and signal was detected by
autoluminescence on film.
80 °C. Cleavage assays were performed with a final
volume of 50 µl with volume adjusted using Buffer A. Then 20, 40, or
80 µg of protein was incubated with 2 µl of
35S-caspase-9, in the presence or absence of bovine
cytochrome c (Sigma) and dATP. Reaction tubes were incubated
at 30 °C for 60 min, and the reaction was stopped by addition of 15 µl of 5 × sample buffer and boiling. Samples were loaded onto a
SDS-polyacrylamide gel or frozen at
80 °C. Electrophoresed gels
were fixed, dried, and exposed to BioMax MS film using a Transcreen LE
intensifying screen (Eastman Kodak Co.) at
80 °C for 2-8 days.
TET) of tetracycline for the indicated period of time. +Fas/Act D
cells are 3T3XS7.2 cells treated with activating anti-Fas antibody (500 ng/ml) and actinomycin D (4 µM) for 8 h in the
presence of TET. Medium was saved from the culture dishes to retain any
floating cells. Adherent cells were harvested and combined with their
respective floating cell populations. Nucleotides were extracted from
an equal number of cells with ice-cold 0.4 N perchloric
acid and subsequently neutralized. Nucleotides were separated and
quantified by strong anion-exchange HPLC using a Waters (Milford, MA)
gradient system controlled by Millenium 2010 software (20). Cellular ATP levels were quantified by comparison of their peak areas with that
of a known amount of standard.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m) in 3T3 cells,
and that this occurs without subsequent activation of caspases (1). To
look more closely at the mitochondrial changes, electron microscopy was
performed on 3T3XS7.2 cells (a stable derivative of 3T3 cells
expressing Bcl-XS in a tetracycline-repressible manner) as expression
of Bcl-XS was induced (
TET).
-galactosidase in the identical
expression vector also did not affect the mitochondria (data not
shown).
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Fig. 2.
Bcl-XS destroys the architecture of
mitochondria. Electron micrographs of 3T3XS7.2 cells
(upper), or a stable derivative expressing Bcl-XL
(lower), were grown in the presence (upper
left) or absence (upper right and
lower) of TET. Normal mitochondria are marked M,
and those showing structural abnormalities are marked M*.
Cells showing structural and functional alterations (1) also formed
lipid deposits, marked L. Cells overexpressing Bcl-XL do not
show disruption of their mitochondrial structure. These results are
representative of two independent experiments.
-galactosidase-expressing cells (data not shown). Pretreatment with
zVAD-fmk or expression of dominant negative caspase-9 did not prevent
the formation of these vesicles (data not shown); however, expression
of Bcl-XL did preclude their appearance (Fig. 2). These
Bcl-XL-expressing cells also did not show a loss of
m
upon expression of Bcl-XS, as opposed to the parental 3T3XS7.2
cells.2
m (1) and the apparent destruction of the
mitochondrial structure (Fig. 2) did not correlate with lack of
detectable cytochrome c release.
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Fig. 3.
Cytochrome c is not detected
in the cytosol of Bcl-XS-expressing cells because it is specifically
depleted upon expression of Bcl-XS. Immunoblots were performed on
3T3XS7.2 cells (A and B) grown in the presence or
absence of TET for the indicated times, or grown in the absence of
fetal bovine serum, marked S, as a control for
caspase-9-mediated cell death. Cytosolic (A) or whole cell
extracts (B) were assayed for the presence of cytochrome
c using a polyclonal antibody against cytochrome
c. Expression of HA-tagged Bcl-XS was examined at the
indicated time after withdrawal from TET with an anti-HA antibody. The
presence of cytochrome c in cytosolic extracts was also
examined directly by utilizing the peroxidase activity of the heme
group covalently bound to cytochrome c and chemiluminescence
(D). Positive controls (A, B, and
D) are purified bovine cytochrome c. Immunoblots
of two other mitochondrial proteins, Hsp-60 and VDAC (E),
demonstrate that depletion of cytochrome c is not a general
effect of all mitochondrial proteins. These results are representative
of at least two independent experiments.
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Fig. 4.
Addition of exogenous cytochrome c
and dATP restores the ability of cytosolic extracts from 3T3XS7.2
cells to activate pro-caspase-9. Cytosolic extracts from 3T3XS7.2
cells (7.2) grown with (A) or without
(B) TET, or from 293T cells, were incubated with
35S-pro-caspase-9 in the presence or absence of exogenous
cytochrome c and dATP and run on acrylamide gels, which were
subjected to autoradiography. The data in A show two
independent experiments, whereas B is a representative
experiment.
TET for 36 h) to cleave pro-caspase-9. These
extracts were also able to cleave pro-caspase-9 in the presence, but
not in the absence, of exogenously added cytochrome c and dATP (Fig. 4B). Finally, an alternative explanation for the
lack of caspase activation in these cells (upon expression of Bcl-XS) could be the presence of some type of inhibitor in these cells. These
experiments also serve to disprove this possibility as such an
inhibitor (e.g. an inhibitor of apoptosis) should
have also prevented the processing of pro-caspase-9 in
vitro. We do, however, recognize the possibility that an inhibitor
may exist and that it was lost during the preparation of the cytosolic
fraction. However, the fact that these cells die upon withdrawal from
serum and that this death is inhibited by 9DN argues that the
apoptosome is functional in whole cells (1).
m, Bcl-XS-expressing 3T3 cells do not show a decrease
in ATP levels. In fact, there is a reproducible increase in the
cellular ATP content. This increase may result from either an increase
in the production of ATP or from a decrease in its
utilization/degradation. Considering the observed loss of
m and the perturbations in the mitochondria upon
expression of Bcl-XS, it seems more likely that the cause is a decrease
in the utilization of ATP.
Cellular ATP levels
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Fig. 5.
Bcl-XS does not kill by displacing Bax.
3T3XS7.2 cells, or their 9DN-expressing stable derivatives, were
transiently transfected with plasmids to express Bcl-XL, Bcl-XS, or
Bax, and a GFP reporter construct at a ratio of 3:1. After 24 h,
green cells were scored as either live or dead. Four independent
experiments using two different 9DN clones are shown, with the 7.2 and
9DN samples for each experiment connected by a line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank M. Soengas, C. Schmitt, and A. Samuelson for comments on the manuscript; Dr. Gabriel Nunez (University of Michigan) for the Bax and dominant negative caspase 9 constructs; and G. Kroemer and S. A. Susin (Villejuif, France) for the anti-AIF antibody. We also thank Brenda Gillespie for help with the statistics.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA56663 and a National Institutes of Health NIGMS Pharmacological Sciences Training Grant GM07767 (to J. S. F.).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.
§ Current address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
To whom correspondence should be addressed. Tel.:
734-647-1436; E-mail: maybaum@umich.edu.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M008171200
2 J. S. Fridman, J. Parsels, A. Rehemtulla, and J. Maybaum, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: BH, Bcl-2 homology; 9DN, dominant negative caspase-9; AIF, apoptosis-inducing factor; TET, tetracycline; Act D, actinomycin D; HPLC, high performance liquid chromatography; TBST, Tris-buffered saline with Tween 20; VDAC, voltage-dependent anion channel; HA, hemagglutinin.
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