From Apoptosis Technology, Inc., Cambridge, Massachusetts 02139
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ABSTRACT |
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The Bcl-2 homology 3 (BH3) domain is crucial for
the death-inducing and dimerization properties of pro-apoptotic members
of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak
bind to Bcl-xL, antagonize its anti-apoptotic
function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within
2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide
that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with
Bcl-2-related death suppressors, such as Bcl-xL, may be
critical for its activity in cells. While overexpression of
Bcl-xL can block BH3-induced apoptosis, treatment with BH3
peptides resensitized Bcl-xL-expressing cells to
Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase
inhibitors, demonstrating a dependence on caspase activation, but was
not accompanied by a dramatic early loss of mitochondrial membrane
potential or detectable translocation of cytochrome c from
mitochondria to cytosol. These findings demonstrate that the BH3 domain
itself is capable of inducing apoptosis in whole cells, possibly by
antagonizing the function of Bcl-2-related death suppressors.
The Bcl-2 protein family plays an important role in the regulation
of apoptosis in evolutionarily diverse species (1). Certain family
members, including Bcl-2 and Bcl-xL, act as potent suppressors of apoptosis, whereas other homologs (e.g. Bak
and Bax) have opposing functions and promote cell death. Although the
mechanistic details by which these proteins function to regulate apoptosis are still unclear, certain domains of homology between family
members, termed Bcl-2 homology
(BH)1 domains, are critical
for various aspects of their activities, including the induction or
suppression of cell death, and the ability to heterodimerize with other
family members (2-6).
The involvement of the BH3 domain in both the death-promoting and
protein binding functions of several of the pro-apoptotic family
members (such as Bak, Bax, Bik, and Bad) is now well established (6-9). Expression of truncated derivatives of Bak containing the BH3
domain is sufficient to induce cell death in transfected cells (6) and
synthetic peptides derived from the BH3 domains of Bak, Bax, and Bid
can induce biochemical events in a cell-free system that are
characteristic of cellular apoptosis, such as cytochrome c
release, caspase activation and DNA fragmentation (10). BH3 peptides
can bind directly to death suppressors such as Bcl-xL (3)
and block their subsequent heterodimerization with death promoters
in vitro (11, 12), suggesting BH3 domains may interfere with
the function of death suppressors in vivo. In support of
this possibility, Bad, a "BH3-only" member of the Bcl-2 family,
appears to promote apoptosis by forming inactivating dimers with death
suppressors through its BH3 domain (13).
In this study, we have examined whether synthetic BH3 peptides can
function as antagonists of Bcl-xL, and have assessed their biological activity when introduced into intact cells. A Bak BH3 peptide was found to antagonize the protective effects of microinjected Bcl-xL in Cell Culture--
HeLa cells were obtained from the American
Type Culture Collection, and maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 4 mM
L-glutamine (complete DMEM) at 37 °C with 6%
CO2. Cells were maintained in constant logarithmic growth
in plastic culture dishes (Costar) for less than 15 passages.
Peptides--
Peptides were synthesized by a commercial vendor
utilizing diisopropylcarbodiimide/1-hydroxy-benzotriazole-activated
Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acids
(Genzyme-Sygena, Cambridge, MA) on a model 396 Multiple Peptide
Synthesizer (Advanced Chemtech, Louisville, KY) and purified on Poros
R2/M 16 × 100-mm prep columns (PerSeptive Biosystems, Framingham,
MA) with a gradient of 0-80% solvent B over 40 column volumes
(solvent A = H2O/0.1% trifluoroacetic acid; solvent
B = CH3CN/0.1% trifluoroacetic acid). Confirmation of
molecular weight was determined by matrix-assisted laser desorption
ionization-time of flight mass spectrometry. Peptides were resuspended
in Me2SO at 5 or 10 mM, and stored at Microinjection--
HeLa cells were plated in complete DMEM in
60-mm dishes at 2.4 × 105 cells/dish. Mixtures
containing peptides and proteins as indicated in 25 mM
HEPES buffer, pH 7.2, containing 3.3 mM NaCl and 1 mg/ml FITC-dextran as a marker were filter-sterilized and injected into the
cytoplasm of cells using an Eppendorf micromanipulator and microinjector with femtotip capillary microtips. Following injection, the cells were returned to the incubator, and after 1 h the number of injected cells was determined by fluorescence microscopy. Cells were
then treated with Internalization of Antennapedia Peptides--
Biotinylated
control or fusion peptides were added to cells grown on glass
coverslips in serum-free DMEM supplemented with 4 mM
L-glutamine (SF-DMEM). After a 30-min incubation with the peptides, cells were washed twice with PBS and fixed with 4%
paraformaldehyde in PBS for 10 min at room temperature. Cells were then
washed twice with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. After two additional washes with PBS,
cells were incubated in blocking buffer (3% bovine serum albumin in
PBS) for 1 h at room temperature, followed by incubation with
streptavidin-FITC at 2 µg/ml in blocking buffer. Coverslips were
washed three times and mounted in aqueous mounting medium (polyvinyl
alcohol) on glass microscope slides for fluorescence microscopy using a
Nikon Microphot-FXA upright fluorescence microscope.
Induction of Apoptosis by BH3 Peptides and Assessment of Cell
Viability--
Cells were plated in complete DMEM in 96-well tissue
culture plates (Costar) at 4 × 103/well. After
24 h, cells were washed with PBS and treated with peptides (50 µM) in SF-DMEM. Cell viability was determined by staining
unfixed cells with calcein AM/ethidium homodimer (Molecular Probes),
followed by microscopic analysis of cell staining and cellular
morphology on a Nikon Diaphot 300 inverted microscope equipped with a
fluorescence module.
Transient Transfections--
HeLa cells were plated in 24-well
plates at 2.5 × 104/well 24 h prior to
transfection. Cells were transfected using the Superfect transfection
reagent (Qiagen) with a plasmid encoding the green fluorescent protein
(GFP) as a marker. Each well was transfected with 0.1 µg of
pcDNA3-GFP plasmid and 1.0 µg of either pcDNA-3 or
pcDNA3-Bcl-xL plasmids. Following transfection, cells
were incubated overnight in complete DMEM and subjected to peptide or
Analysis of Poly(ADP-ribose) Polymerase (PARP)
Cleavage--
Cells were treated with Antennapedia fusion peptides in
SF-DMEM or with Assessment of Mitochondrial Membrane Potential--
Changes in
mitochondrial membrane potential were determined by staining cells with
the indicator dye 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1). JC-1 was added directly to the cell culture medium (1 µM final
concentration) and incubated for 15 min. The medium was then replaced
with PBS, and the cells were either photographed or quantitated for
J-aggregate fluorescence intensity on a fluorescence plate reader using
excitation and emission filters of 492 and 590 nm, respectively.
Analysis of Cytochrome c Translocation--
HeLa cells (1 × 106 cells) were plated in 100-mm tissue culture dishes
and incubated overnight prior to treatment with various inducers of
apoptosis. At various time points, cells were trypsinized, washed once
with ice-cold PBS, resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 1 µM pepstatin A, 10 µg/ml leupeptin, and
2 µg/ml aprotinin, and disrupted using a Dounce homogenizer with a
tight pestle. Cytosolic fractions were isolated by centrifugation at
16,000 × g for 20 min at 4 °C in a tabletop
microcentrifuge, and membrane fractions including mitochondria were
resuspended in a volume of lysis buffer equal to the volume of cytosol
obtained to normalize the number of cells represented in each fraction. Fractions were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and analyzed for cytochrome c
content by Western blot analysis using a mouse BH3 Peptides Antagonize Bcl-xL Function--
Synthetic
BH3 peptides can bind to Bcl-2-like death suppressors and block
heterodimerization with pro-apoptotic Bcl-2 homologues in
vitro (3, 11, 14).2 To
test the ability of a BH3 peptide to antagonize the protective function
of a death suppressor, Bcl-xL was delivered into the cytosol of cells by microinjection either in the presence or absence of
a Bak BH3 peptide. Microinjection of a bacterially expressed GST-Bcl-xL fusion protein, but not GST alone, efficiently
protected HeLa cells from death induced by Fas ligation in the presence of cycloheximide (Fig. 1). Co-injection
of a 15-amino acid Bak BH3 peptide (BH3) greatly attenuated the
protective effect of Bcl-xL in this assay. A mutant Bak BH3
peptide (BH3-A78), in which an alanine has been substituted
for a leucine at position 78 (numbered according to full-length Bak),
did not block Bcl-xL-mediated protection from Fas-induced
death. Thus, the ability of these peptides to inhibit the function of
Bcl-xL correlated with their ability to bind to
Bcl-xL (3). Under these conditions, neither peptide had an
effect on cell viability in the absence of Delivery of BH3 Peptides by Fusion with the Antennapedia
Internalization Sequence--
To further investigate the activity of
this domain in cells, BH3 peptides were synthesized as fusions with the
internalization sequence from the Antennapedia (Ant) protein (Table
I). Similar Ant fusion peptides have been
shown to be internalized into cells in culture and elicit
peptide-specific biological responses (15-21). To determine whether
the Ant-BH3 peptide could be internalized, HeLa cells were treated with
biotin-tagged versions of the peptides, and peptide uptake was
monitored by staining with streptavidin-FITC. While untreated cells or
cells treated with non-biotinylated peptides did not stain with
streptavidin-FITC (data not shown), cells treated with
biotinylated Ant fusion peptides were highly positive within 30 min of addition of peptides to the culture medium (Fig.
2), indicating efficient internalization
of the peptides. A biotinylated BH3 peptide lacking the Ant
internalization sequence was not internalized.
BH3 Peptides Induce Apoptosis in Intact HeLa Cells--
In
contrast to the lack of cytotoxicity when BH3 peptides were delivered
as a single bolus dose by microinjection, treatment of HeLa cells with
the Ant-BH3 peptide, which allows for the delivery of a sustained pool
of BH3 peptide, resulted in a dramatic loss of cell viability within
6 h (Fig. 3, C and
D). Changes in cell morphology characteristic of apoptosis,
including reduction in cell volume, nuclear condensation, and membrane
blebbing, were observed as early as 2-3 h following the addition of
the fusion peptide. Treatment of cells with peptides comprising just
the BH3 domain (Fig. 3D) or the internalization sequence
alone (Ant) (Fig. 3D) had no effect on cell morphology or
viability, demonstrating that the observed cell killing activity was
BH3-dependent and required internalization. Furthermore,
the mutant Ant-BH3 peptide (Ant-BH3-A78), which is
defective in binding to Bcl-xL (3), did not exhibit
significant cell killing activity (Fig. 3, B and D). This underscores the importance of an intact BH3
sequence for the BH3 peptide's cytotoxic effects and implicates a
mechanism involving an interaction with a specific intracellular
target, presumably one (or more) of the Bcl-xL-like death
suppressors.
Bcl-xL Blocks BH3-induced Apoptosis, and BH3 Treatment
Blocks Bcl-xL-mediated Protection from Fas-induced
Apoptosis--
In consideration of the possible involvement of a
Bcl-xL-like death suppressor in BH3-induced apoptosis, we
sought to determine the effects of Bcl-xL overexpression on
the pro-apoptotic activity of BH3 peptides. HeLa cells were
cotransfected with plasmids expressing Bcl-xL and GFP as a
marker. Eighteen hours after transfection, cells were treated with the
Ant-BH3 peptide for an additional 6 h. GFP-expressing cells were
visualized by fluorescence microscopy and scored as either live or dead
based on their morphology. Overexpression of Bcl-xL blocked
the cell killing activity of the Ant-BH3 peptide in this assay (Fig.
4). Non-transfected cells in the same
well (cells not expressing GFP) were not protected and underwent
morphological changes similar to control cells when treated with
Ant-BH3 (data not shown). Bcl-xL-expressing cells were also
protected from Caspases Are Required for BH3-induced Apoptosis--
In addition
to well described morphological hallmarks, the induction of apoptosis
is often accompanied by the activation of the ICE/Ced-3 family of
cysteine proteases, or caspases (22). In order to determine the
importance of caspase activation in BH3-mediated cell killing, we first
looked for cleavage of the caspase substrate PARP (23-26) upon the
induction of cell death. Western blots of lysates prepared from HeLa
cells undergoing BH3-induced apoptosis revealed detectable PARP
cleavage within 2 h of peptide treatment, based on the appearance
of a characteristic 85-kDa breakdown product (Fig.
5A). By 4 h, a
substantial portion of PARP had been cleaved. Neither the Ant control
peptide nor the Ant-BH3-A78 mutant peptide caused
detectable PARP cleavage, even after 6 h. Interestingly, the time
course for BH3-induced caspase activation as measured by PARP cleavage
was similar to that observed upon Fas ligation in these cells,
suggesting that BH3-induced apoptosis results from rapid activation of
caspases similar to the Fas-related pathway, which involves recruitment
and activation of caspases directly at the Fas receptor. In contrast,
induction of PARP cleavage by treatment of HeLa cells with the
topoisomerase inhibitor, etoposide, does not occur until well over
6 h.
To determine whether the BH3-dependent activation of
caspases is required for induction of cell death by Ant-BH3 peptide, the effect of caspase inhibitors on BH3-induced death was tested. Treatment of cells with the broad spectrum tripeptide caspase inhibitor
z-VAD-fmk (80 µM) (25, 27) completely blocked death induced by the Ant-BH3 peptide (Fig. 5B). The inhibition of
BH3-induced death correlated with a lack of PARP cleavage in the
presence of z-VAD-fmk (Fig. 5C). The tetrapeptide inhibitor
DEVD-fmk also completely blocked BH3-induced apoptosis while YVAD-fmk
was somewhat less effective. These data indicate that the induction of
cell death by the Ant-BH3 peptide is dependent on caspase activation.
Mitochondrial Events Are Not Required for BH3-induced
Apoptosis--
It has recently become evident that the mitochondria
may play an important role in apoptotic signaling (28). Apoptosis
induced by a wide variety of stimuli is preceded by a release of
cytochrome c from the mitochondrial matrix into the cytosol,
where it has been proposed to be involved in the activation of caspases
(29-31). In addition, mitochondrial membrane permeability transition
and loss of mitochondrial membrane potential (
The mitochondrial matrix protein cytochrome c is released
from the mitochondria to the cytosol during apoptosis either prior to,
or coincident with, caspase activation, and is known to be a direct
participant in the activation of caspases in in vitro systems (29-31, 35, 36). To determine whether cytochrome c is released from the mitochondria during BH3-induced apoptosis, the
subcellular localization of cytochrome c in treated cells was determined by cell fractionation and Western blot analysis. While
staurosporine induced a rapid translocation of cytochrome c
(within 3 h) (Fig. 8) that coincided
with the cleavage of PARP (Fig. 7B), treatment of cells with
the Ant-BH3 peptide did not result in any accumulation of cytochrome
c in the cytosol by 3 h (Fig. 8), even though
substantial caspase-dependent cleavage of PARP had occurred
by this time (Fig. 7B). These data suggest that cytochrome
c release is not required for activation of cytosolic caspases by the BH3 domain.
The BH3 domain plays a critical role in mediating the cell death
and protein-binding functions of Bak and related pro-apoptotic proteins. The results of the present study demonstrate that the Bak BH3
domain alone, in the form of synthetic peptides delivered into cells,
is biologically active and able to modulate apoptosis. Bak BH3 peptides
acted as inhibitors of Bcl-xL in cells, antagonizing its
ability to suppress apoptosis induced by Fas ligation. In addition, the
Bak BH3 peptide was sufficient to rapidly induce apoptosis when
introduced into HeLa cells via fusion to the Antennapedia internalization sequence. Mutant BH3 peptides that fail to bind Bcl-xL were inactive, indicating that the biological
activity of BH3 peptides correlates with their capacity to
heterodimerize with death suppressors. Taking these findings together,
we hypothesize that the cytotoxicity of BH3 peptides is due to their
inhibitory interaction with endogenous Bcl-2-related proteins in HeLa
cells. Moreover, the ability of Bak BH3 peptides to trigger cell death, even in the absence of other apoptotic stimuli, implies that HeLa cells
require the continuous action of one or more Bcl-2 homologs for their
survival. In addition to binding Bcl-xL, Bak BH3 peptides interact directly with other Bcl-2-related death suppressors in vitro, including Bcl-2, Mcl-1, A1, and Bcl-w.2 Thus,
the inhibition of other Bcl-2 family members, either alone or in
combination, may be responsible for the cytotoxicity of Bak BH3
peptides in HeLa cells. An alternative explanation is that the
pro-apoptotic activity of Bak BH3 peptides results from an interaction
with a cellular target(s) distinct from Bcl-2-related death
suppressors; however, this as yet undefined interaction would have to
exhibit the same dependence on specific BH3 residues (e.g.
leucine 78).
How might Bak BH3 peptides antagonize the anti-apoptotic function of
Bcl-xL? The solution structure of a Bak BH3
peptide/Bcl-xL complex revealed that the BH3 peptide adopts
an Bak BH3 peptides triggered the rapid activation of caspases, and
pharmacological inhibition of caspases prevented BH3-induced apoptosis.
These observations are consistent with a mechanism whereby BH3
liberates caspase-activating factors, such as Apaf-1, from an
inhibitory interaction with Bcl-2-like proteins (as proposed above).
BH3 peptide-induced apoptosis, however, occurred in the absence of a
detectable decrease in mitochondrial The biological effects of BH3 peptides observed here appear to account
for only a part (caspase activation) of the pro-apoptotic activities
that have been previously described for the BH3-containing Bcl-2
homologs Bak or Bax. The induction of cell death by Bax has been shown
to require, at least in some cases, the loss of The importance of caspase activation in the induction of cell death by
the death-promoting members of the Bcl-2 family remains controversial.
While caspase activation is a consistent hallmark of Bax
overexpression, the ability to block Bax-induced death by caspase
inhibitors is not (45-47, 54, 55). Xiang et al. (47) have
suggested that Bax may activate two pathways, a
caspase-dependent and a caspase-independent pathway. The
latter pathway may involve crucial alterations in mitochondrial
function, such as loss of This dual pathway hypothesis may also help explain contradictory
evidence regarding the importance of the BH3 domain in the induction of
cell death by Bax and Bak (6, 56, 57). Depending on the cellular
context, death induction by Bax or Bak may require the activation of
caspases and be BH3-dependent, or proceed through caspase-independent alterations in mitochondrial function leading to
cell death that may not require BH3-mediated processes. The relative
ability of the death promoters of the Bcl-2 family, such as Bax and
Bak, to activate either pathway under specific conditions may determine
the apparent dependence on BH3 for cell death.
The apoptotic activity exhibited by a small peptide taken from the BH3
domain of the Bak protein suggests a novel pharmacological approach for
manipulating a cell's susceptibility to apoptosis. Small non-peptidyl
compounds designed to mimic the activity of the BH3 peptide may impact
the fate of the cell via mechanisms similar to those employed by the
BH3 peptide. As it has been proposed that up-regulation of
anti-apoptotic proteins such as Bcl-2 or Bcl-xL contributes
to the tumorigenesis and resistance to drug treatment in certain types
of cancers (58, 59), these results suggest that antagonizing these
death suppressors with BH3 mimics may prove to be effective in
promoting apoptosis in tumor cells that depend on such anti-apoptotic
proteins for survival.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-Fas-treated HeLa cells, whereas a mutant Bak
BH3 peptide that no longer binds Bcl-xL was inactive.
Delivery of synthetic BH3 peptides into the cytosol of intact HeLa
cells via facilitated diffusion as a fusion with the internalization
domain of the Antennapedia homeoprotein, resulted in the induction of a
caspase-dependent apoptotic program, characterized by
cleavage of poly(ADP-ribose) polymerase (PARP) and morphological
changes such as cytoplasmic contraction, membrane blebbing, and the
formation of apoptotic bodies. This BH3-mediated cell death was not,
however, accompanied by an early loss of mitochondrial membrane
potential or release of cytochrome c from the mitochondria,
suggesting the engagement of a cytochrome c-independent
pathway to caspase activation. In addition, the Antennapedia-BH3 fusion
peptide (Ant-BH3) was able to overcome the protective effects of
Bcl-xL in
-Fas-treated HeLa cells. Our results
demonstrate that the Bak BH3 domain is sufficient to antagonize the
function of Bcl-xL and to trigger the apoptotic program in
intact cells.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
-Fas mAb (7C11) and cycloheximide (10 µg/ml),
and the recovery of injected cells remaining after 18 h was determined.
-Fas mAb treatment as described above.
-Fas mAb (7C11) in complete DMEM for various times and scraped directly into 1× SDS-polyacrylamide gel electrophoresis sample buffer (with 6 M urea added). Lysates were sonicated
briefly (20 s, 40% duty cycle) and loaded onto an 8%
SDS-polyacrylamide gel. After electrophoresis, proteins were
transferred to nitrocellulose, and PARP was detected by Western
blotting using the murine
-PARP mAb (C-2-10, Biomol) at a 1:5000
dilution with ECL-based detection using a peroxidase-conjugated goat
anti-mouse IgG.
-cytochrome
c monoclonal antibody (7H8.2C12) and an enhanced
chemiluminescent-based detection system (Amersham Pharmacia Biotech).
The blots were also probed with antibodies specific for actin (AC-40)
and the mitochondrial membrane protein VDAC
(voltage-dependent anion channel) (anti-porin 31HL) as
fractionation markers.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Fas treatment (data not
shown). Similar results were obtained with MRC5 human diploid
fibroblasts where protection from Fas-induced death by microinjected
Bcl-xL was inhibited by co-injection of the wild-type, but
not mutant, Bak BH3 peptide (data not shown). These results suggest
that Bak BH3 peptides bind to Bcl-xL and interfere with its
anti-apoptotic function.
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Fig. 1.
Inhibition of Bcl-xL protection
by Bak BH3 peptides. HeLa cells (>100 cells/condition) were
microinjected with buffer, GST (0.15 mg/ml), or GST-Bcl-xL
(0.3 mg/ml) in the absence or presence of BH3 peptides (0.5 mg/ml) and
the recovery of live injected cells was determined after treatment with
-Fas and cycloheximide for 18 h. FITC-dextran was coinjected as
a marker. A 15-amino acid Bak BH3 peptide (residues 73-87;
QVGRQLAIIGDDINR) and a mutant Bak BH3 peptide with an alanine
substitution at leucine 78 (BH3-A78) were tested. Results are expressed
as mean ± S.D.
List of peptides
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Fig. 2.
Internalization of peptides. HeLa cells
were incubated with biotinylated peptides for 30 min prior to fixation
and permeablization and internalized peptide was detected with
streptavidin-FITC. A, BH3; B, Ant; C,
Ant-BH3-A78; D, Ant-BH3. Untreated cells or
cells treated with non-biotinylated peptides showed no detectable
streptavidin-FITC staining (data not shown).
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Fig. 3.
Ant-BH3, but not Ant or
Ant-BH3-A78, induces apoptotic morphology in HeLa
cells. Cells were treated with Ant (A),
Ant-BH3-A78 (B), or Ant-BH3 (C) at 50 µM for 6 h and photographed by phase contrast
microscopy. Panel D shows viability of cells as
determined by live/dead staining with calcein AM and ethidium homodimer
(expressed as mean ± S.D.). The BH3 peptide in panel
D lacks the Ant internalization sequence.
-Fas-induced death. However, treatment with the wild
type Ant-BH3 peptide, but not the loss-of-binding mutant
Ant-BH3-A78, resensitized the Bcl-xL-expressing
cells to the induction of apoptosis by
-Fas. These findings suggest
that interaction with a death suppressor such as Bcl-xL may
be an important aspect of the activity of the BH3 domain and its
ability to influence a cell's susceptibility to undergo apoptosis.
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Fig. 4.
Transient expression of Bcl-xL
protects cells from BH3-induced apoptosis. Cells were
co-transfected with a plasmid expressing GFP (pcDNA3-GFP) as a
marker and either empty vector (pcDNA3) or with a plasmid
expressing Bcl-xL (pcDNA3-Bcl-xL). 18 h after transfection, cells were left untreated or treated with
Ant-BH3, -Fas, or both for 6 h. Following treatment, cell
viability was determined by morphological analysis of transfected
(green) cells by fluorescence microscopy. Results are expressed as
mean ± S.D.
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Fig. 5.
Caspase activation is required for
Ant-BH3-induced cell death. A, cleavage of PARP
following treatment of HeLa cells with -Fas and Ant-BH3 and control
peptides (50 µM) and etoposide (0.1 mM) for
different times. B, Inhibition of Ant-BH3-induced apoptosis by
z-VAD-fmk, DEVD-fmk, and YVAD-fmk (80 µM). Cell viability
was determined as in Fig. 3. C, inhibition of PARP cleavage
in cells protected from Ant-BH3- and
-Fas-induced death by
z-VAD-fmk. Panels B and C represent
6-h time points.
m) are
events that are common to many pathways of apoptosis induction. While
several members of the Bcl-2 family, including Bax, Bcl-2, and
Bcl-xL, are located primarily in the outer
mitochondrial membrane, the specific involvement of these
proteins in the mitochondrial events associated with apoptosis remains
unclear. To analyze mitochondrial events that may be associated with
BH3-induced apoptosis, the potential-sensitive fluorescent probe JC-1
(32, 33) was used to detect loss of
m at 5 h
after exposure of cells to various apoptotic stimuli. In untreated
cells (Fig. 6A) or cells
treated with the inactive Ant-BH3-A78 peptide (Fig. 6E),
JC-1 formed the characteristic J-aggregates in the mitochondria which
emit photons at 585 nm (orange-red), demonstrating that the
mitochondrial membrane potential is intact. Treatment of cells with the
proton ionophore carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone, a potent uncoupler of
mitochondrial oxidative phosphorylation, causes a rapid loss of
membrane potential. Under these conditions, the JC-1 probe failed to
aggregate and little or no red fluorescence was observed (Fig.
6B). Etoposide treatment also resulted in a complete loss of
m at 5 h, indicated by the absence of
J-aggregates (Fig. 6D), which preceded caspase activation
(see Fig. 5A) and changes in cell morphology. In contrast,
no significant loss of
m was observed during the
induction of apoptosis by Ant-BH3 peptide (Fig. 6F) or Fas
ligation (Fig. 6C) at 5 h, even after substantial
changes in cell morphology (as in Fig. 3) and cleavage of PARP had
occurred (Fig. 5A). Fig.
7A shows a quantitative
measurement of JC-1 staining obtained on a fluorescence plate reader
3 h after induction of cell death. As expected, both etoposide and
staurosporine caused an early (3 h) loss of
m, which
preceded or coincided, respectively, with caspase activation (Fig.
7B), whereas the mitochondrial membrane potential was still
unchanged in Ant-BH3-treated cells even after caspases had been
activated. The results of these experiments suggest that depolarization
of the mitochondrial membrane is likely not required for the induction
of apoptosis by the BH3 domain in intact cells. In keeping with these
results, treatment of cells with cyclosporine A, an inhibitor of the
membrane permeability transition and subsequent loss of
m (34), did not appear to suppress BH3-induced
death.3
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Fig. 6.
Ant-BH3-induced apoptosis is not preceded by
a loss of m.
Cells were incubated with the potential-sensitive probe JC-1 (1 µM, 10 min) to visualize mitochondrial membrane potential
after treatment with various agents for 5 h. A,
untreated; B, carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (1 µM);
C,
-Fas; D, etoposide (100 µM);
E, Ant-BH3-A78 (50 µM);
F, Ant-BH3 (50 µM).
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Fig. 7.
Ant-BH3-induced PARP cleavage without loss
of m.
A, quantitation of
m by measurement of
J-aggregate (JC-1) fluorescence in cells treated without
(control) or with carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (1 µM),
staurosporine (Sts, 1 µM), etoposide
(Etop, 100 µM), or Ant-BH3 (50 µM) for 3 h. B, cleavage of PARP after
3 h treatment with the indicated agents as in A.
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Fig. 8.
Ant-BH3-induced apoptosis is not accompanied
by accumulation of cytochrome c in the cytosol.
HeLa cells (1 × 106 cells) were treated without
(control) or with Ant-BH3 (50 µM), or
staurosporine (Sts, 1 µM) for 3 h and
fractionated into membrane and cytosolic fractions as described under
"Experimental Procedures." Levels of cytochrome c in
each fraction were determined by immunoblot analysis. The same blot was
probed for VDAC (marker for mitochondria) and actin as controls for
protein loading.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical conformation and binds to a hydrophobic cleft on the
surface of Bcl-xL (3). Binding of the BH3 peptide
apparently does not induce significant conformational changes in
Bcl-xL (3). It is possible that the occupation of this
cleft by BH3 competitively displaces a cellular protein whose
interaction is critical for the anti-apoptotic function of
Bcl-xL. A candidate for such a molecule is Apaf-1, a human Ced-4 homolog implicated as an important target for suppression of
apoptosis by Bcl-xL (35, 36). Bcl-xL interacts
with Apaf-1 in transfected cells and inhibits the ability of Apaf-1 to
promote the activation of caspase-9 (37, 38). Reportedly, Bak and Bik
(a "BH3-only" death promoter) interfere with Bcl-xL
binding to Apaf-1 (38), consistent with a competition between BH3 and Apaf-1 for the BH3-binding cleft on Bcl-xL. Thus, the BH3
peptide may antagonize Bcl-xL and promote apoptosis by
preventing Bcl-xL/Apaf-1 heterodimerization, leaving Apaf-1
free to participate in the activation of caspases. In this respect, the
ability of BH3 peptides to act as antagonists of death suppressors may
serve as a model for the mode of action of a growing family of
death-promoting proteins that share homology only within their BH3
domains. Certain "BH3-only" proteins, such as Bad (9, 13), Bik/Blk
(8, 39), Bim (40), and Hrk (41), may promote apoptosis principally through BH3-mediated inhibition of Bcl-2-like death suppressors.
m or release of
cytochrome c, the latter of which is a factor required for the caspase-activating function of Apaf-1 at least in in
vitro systems (29, 42). If the proposed mechanism of BH3
peptide-induced apoptosis is correct, sufficient activation of Apaf-1
must occur either in the absence of cytochrome c release, or
in the presence of cytochrome c at levels below the limits
of detection in our assays, to initiate apoptosis in vivo.
Caspase activation in the absence of cytochrome c release
has also been observed during Fas-induced death (43, 44).
m,
and the release of cytochrome c from the mitochondrial
matrix (45-47). Cyclosporine A, a known inhibitor of the membrane
permeability transition (34), prevents both the loss of
m and cytochrome c release, as well as
Bax-induced death (45). Indeed, Bax is capable of promoting the release
of cytochrome c from isolated mitochondria, though the
ability of BH3 peptides to substitute for the full-length protein in
these assays was inconsistent (48, 49). BH3-mediated cytochrome
c release may depend on the presence of cytosolic factors as
demonstrated using extracts from Xenopus laevis oocytes
(10). The BH3 peptide in our assays with intact cells was not
sufficient (although it may be necessary) to trigger the mitochondrial
events described for Bax, suggesting that Bax delivers pro-apoptotic
signals apart from BH3-mediated effects. Additional Bax function may be
related to its membrane pore-forming activity observed in
vitro (50-53).
m and increased production
of reactive oxygen species. Based on our results using the Ant-BH3
peptide, BH3-mediated cell death may utilize a
caspase-dependent pathway. The relative contribution of
each pathway to the induction of cell death may be dependent on cell
type or context. Therefore, the requirement of caspase activation for
Bax-induced death would be dependent on the specific cellular conditions.
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FOOTNOTES |
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* This work was supported by a research collaboration agreement with Biochem Pharma, Laval, Quebec, Canada.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.
To whom correspondence should be addressed: Apoptosis Technology,
Inc., 148 Sidney St., Cambridge, MA 02139. Tel.: 617-497-1113; Fax:
617-497-5406; E-mail:
Bob_Lutz{at}Immunogen.ccmail.compuserve.com.
2 E. P. Holinger, T. Chittenden, and R. J. Lutz, unpublished results.
3 E. P. Holinger, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
BH, Bcl-2 homology;
mAb, monoclonal antibody;
PARP, poly(ADP)-ribose polymerase;
DMEM, Dulbecco's modified Eagle's medium;
SF, serum-free;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
GFP, green
fluorescent protein;
GST, glutathione S-transferase;
VDAC, voltage-dependent anion channel;
m, mitochondrial membrane potential.
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