 |
INTRODUCTION |
Programmed cell death or apoptosis is critical for both the
development and maintenance of tissues. The BCL-2 family members are
major regulators of the apoptotic process (1). The cell death
regulatory mechanism of these proteins is unknown, although it is
thought that their function depends mostly on their ability to modulate
mitochondrial function. This family is comprised of both pro-, as well
as anti-apoptotic proteins. A subset of the proapoptotics are the
BH3-only proteins. Another major component of the death machinery is a
proteolytic system involving a family of cysteine proteases named
caspases (2).
Two major apoptotic pathways have been identified: the extrinsic and
intrinsic pathways. The cell-intrinsic apoptotic pathway is initiated
when the outer mitochondrial membrane becomes permeable, resulting in
the release of cytochrome c (Cyt
c)1 and other
intermembrane space proteins (3). In the cytosol, Cyt c
induces the oligomerization of Apaf-1, which forms a multimeric complex
that recruits and leads to the activation of caspase-9. Activated
caspase-9 leads to downstream caspase-3 activation resulting in
apoptotic cell death. Strong support for the necessity and linearity of
this pathway has come from animal knockout studies. These studies have
demonstrated that Apaf-1-, caspase-9-, and caspase-3-deficient
embryonic stem cells (ES) or fibroblasts (MEFs) are resistant to
intrinsic damage signals (4). Cyt c-deficient ES cells
showed a similar resistant to various apoptotic stimuli (5).
In the extrinsic pathway, apoptosis is initiated through activation of
certain cell surface receptors. The best characterized are members of
the TNF/Fas receptor family. Once engaged by ligand, these receptors
initiate the formation of the DISC, which leads to activation of
caspase-8 (6). Active caspase-8 can initiate both the activation of a
cascade of caspases and the cleavage of the proapoptotic BID protein.
Cleavage of cytosolic BID at Asp-59 yields a p15 C-terminal truncated
fragment (tBID) that translocates to the mitochondria to induce the
release of Cyt c (7-9). Targeting of tBID to mitochondria
induces its own oligomerization (10) and the oligomerization of BAX and
BAK (11, 12). MEFs deficient in either Bak or Bax are sensitive to
tBID-induced cell death; however, Bax, Bak double deficient MEFs are
completely resistant to tBID-induced cell death as well as to multiple
apoptotic stimuli (13). The requirement for BID in the extrinsic death pathway was demonstrated in Bid-deficient mice, which are resistant to
Fas-induced hepatocellular apoptosis (14).
The fact that BID is cleaved by caspase-8 and tBID translocates to the
mitochondria to initiate an apoptotic program suggests that the
proapoptotic activity of BID depends upon its cleavage. In addition,
in vitro studies as well as studies using permeabilized cells have demonstrated that full-length BID is much less effective than tBID in targeting to mitochondria and in inducing the release of
Cyt c (8, 9, 15, 16).
Based on the studies mentioned above, full-length BID seems to be the
inactive form of this protein. However, several studies have suggested
differently. One of these studies has demonstrated that addition of
staurosporine (STS) to HeLa cells induced the translocation of BID to
the mitochondria (17). Another study using purified mitochondria has
demonstrated that BID was capable of inducing the oligomerization and
insertion of BAX into the outer mitochondrial membrane (11).
Translocation of BID to mitochondria, resulting in Cyt c
release and apoptotic cell death was recently demonstrated in Jurkat T
cells treated with either ceramide or anti-Fas antibodies in
combination with inhibitors to both caspase-8 and PARP (18). The
possible involvement of phospholipids in the translocation of BID was
demonstrated in a study showing that physiological concentrations of
certain phospholipids induced the translocation of BID to the
mitochondria (19). Taken together, these studies suggest that uncleaved
BID may play an active role in mitochondria during apoptosis.
To further define the ability of full-length BID to induce apoptosis
and to explore its mechanism of action, we mutated the caspase-8
cleavage site in BID and analyzed the effects of this mutant in Bid
/
and wild type primary MEFs. We demonstrate that the caspase-8
non-cleavable (nc) BID mutant is a potent inducer of apoptosis in both
types of MEFs. Both ncBID and wild type (wt) BID were much less
effective than tBID in inducing Cyt c release, but only
slightly less effective in inducing apoptosis. Apaf-1, caspase-9, and
both BAX and BAK, but not caspase-3 or caspase-8 were essential for
both nc/wtBID and tBID-induced apoptosis in primary MEFs. Finally,
expression of non-apoptotic levels of either ncBID or wtBID in Bid
/
MEFs induced a similar and significant enhancement in apoptosis
in response to a variety of death signals. Thus, these results
implicate full-length BID as an active player in the mitochondrial
apoptotic pathway.
 |
EXPERIMENTAL PROCEDURES |
Preparation of BID Recombinant Adenoviruses and Infection of
MEFs--
For the expression of BID in MEFs we have produced
adenovirus vectors expressing BID under the control of tetracycline
(tet)-regulatable promoters. In these constructs the E1 region of the
virus was replaced with either a D59A mutant of BID (ncBID), wild-type
(wt) BID, or tBID. Two types of adenovectors were produced: 1) a
"tet-on" inducible vector that relies on the reverse tet
transactivator (rtTA) (20) for the expression of either wtBID or ncBID
tagged with a C-terminal FLAG epitope; 2) a tet operator containing a full CMV promoter-driven vector system, which is inhibitable with the
tet repressor (tetR) (T-REx, Invitrogen), for the constitutive expression of either wtBID or tBID tagged with a C-terminal HA epitope
or for the expression of GFP. We have used this system for the
expression of tBID since tBID under control of the
tetracycline-inducible promoter was too toxic to the 293 packaging
cells and therefore viruses could not be prepared.
To produce the adenovectors, the genes to be expressed were cloned into
pCA14 (a CMV promoter-containing variant of p
E1sp1A) or variants in
which we had replaced the CMV promoter to tet-regulatable promoters.
The pCA14 construct is a shuttle vector for making adenovectors with
inserts in the E1 region of the viral genome. For the tet-on vectors,
the promoter from pTRE (Clontech) containing 2 tet operator sites and a
minimal CMV was used to replace the full CMV promoter in pCA14 to
produce pCA14Tind. For the T-REx-driven vectors, the promoter from
pcDNA4/TO (Invitrogen) containing a full CMV promoter with two
upstream tet operator sites was subcloned in place of the CMV promoter
in pCA14 to produce pCA14T-Rex. This T-REx system functions as a
constitutive promoter in all cell types lacking the tet repressor.
The ncBid gene was cloned into pCA14Tind; wtBid
was cloned into pCA14Tind and pCA14T-REx; tBid and
GFP were cloned into pCA14T-Rex, and the
rtTA gene was from pUHD17-1 (20) was cloned into pCA14. To
produce adenovirus vectors, individual pCA14 and pCA14Tind-derived shuttle vectors were introduced into 293T cells using LipofectAMINE Plus (Invitrogen) together with pJM17 (an adenovirus genomic construct lacking the E1 region of the virus) to produce recombinant viruses by
the standard procedure. At each stage of viral plaque purification, PCR
screening was used to confirm that the correct inserts were present.
The only difference with pCA14T-REx-based adenovirus vectors was that
the lipofections were done in 293T-TR cells, which are a variant of
293T cells that express the tetR from a stable plasmid integration with
pcDNA6/TR (Invitrogen). The tetR keeps the inserted gene repressed
during virus production.
Viruses were grown using 293T or 293T-TR cells depending on the
promoter they contained. Virus preparations were made from freeze-thaw
lysis of the cells, and virus titers were done on 293T-TR cells. In
experiments, cells were generally seeded at 70-80% confluence. With
the tet-inducible system, cells were infected with an MOI (multiplicity
of infection) of 100 with both the BID containing virus and the
rtTA-containing virus. 1 µg/ml doxycycline (a synthetic analog of
tetracycline; Sigma) was added to the medium 12-15 h postinfection to
activate gene expression from the tet-inducible promoter. With the
T-REx BID viruses, cells were infected at an MOI of 100. Efficiency of
infection was determined using the recombinant adenovirus carrying the
constitutive expressing vector of GFP (pCA14T-Rex-GFP) and was in the
range of 70-90%.
MEFs--
Wild type and knockout primary MEFs were prepared from
11-13 days embryos, and maintained in ISCOVE's medium containing 20% fetal bovine serum. Caspase-9
/
and caspase-3
/
primary MEFs were obtained from Richard A. Flavell and Apaf-1
/
primary MEFs were obtained from Tak W. Mak. Caspase-8
/
primary MEFs were a
generous gift from David Wallach (Weizmann Institute). Bax,Bak double-deficient primary MEFs were a generous gift from Stanley J. Korsmeyer (Dana-Farber Cancer Institute). SV-40 transformation of
primary MEFs was performed by transfecting cells with the SV-40 whole
genome using LipofectAMINE 2000 (Invitrogen). Stable clones were
collected 14-18 days post-transfection.
Cell Viability Assays--
TNF
, ActD, and staurosporine were
purchased from Sigma. Etoposide, cisplatin, and thapsigargin were
purchased from Alexis. Cell viability was determined at the designated
time points by propidium iodide (PI)/dye exclusion. PI (2.5 µg/ml)
was added to the cells immediately prior to FACScan (BD Biosciences) analysis.
Caspase Activity Assay--
Cells were lysed in buffer A
containing 5 mM EGTA, 5 mM EDTA, 10 µM digitonin, 2 mM dithiothreitol, and 25 mM HEPES, pH 7.4. The lysates were clarified by
centrifugation, and the supernatants were used for the assays.
Enzymatic reactions were carried out in buffer A containing 20 µg
of protein and 50 µM
acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC) to measure
caspase-3 activity, and acetyl-Leu-Glu-His-Asp-aminomethylcoumarin (LEHD-AMC) to measure caspase-9 activity. The reaction mixtures were
incubated at 37 °C for 30 min, and fluorescent AMC formation was
measured at excitation 380 nm and emission 460 nm using a microplate
spectrofluorometer (SPECTRAmax, Molecular Devices).
Western Blot Analysis--
Proteins were size-fractionated by
SDS-PAGE and then transferred to polyvinylidene difluoride membranes
(Immobilon-P, BioRad). Antibodies included anti-mBID (21), anti-FLAG
(M2; Sigma), and anti-active caspase-3 (CM-1; a generous gift from Idun
Pharmaceuticals). Western blots were developed by use of the enhanced
chemiluminescence reagent (Amersham Biosciences).
Immunocytochemistry--
For immunocytochemistry, primary MEFs
were grown on glass coverslips. At the designated time points, the
cells were fixed with 3% paraformaldehyde in phosphate-buffered saline
for 10 min and permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline for 5 min. For blocking, the cells were
incubated in phosphate-buffered saline containing 0.1% Triton and 3%
bovine serum albumin for 1 h at room temperature. For
double-immunostaining, cells were incubated overnight at 4 °C with
anti-cytochrome c 6H2.B4 monoclonal antibodies (BD
PharMingen, diluted 1:200) together with either anti-BID antibodies
(diluted 1:200) or CM-1 antibodies (diluted 1:50) in blocking solution.
After three washes with phosphate-buffered saline containing 0.1%
Triton, the cells were stained for 30 min at room temperature with
Cy3-labeled goat anti-mouse (dilution 1:100, Jackson ImmunoResearch)
and Alexa 488-labeled goat anti-rabbit Abs (dilution 1:150, Molecular
Probes), followed by 5 min of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (10 µg/ml). For staining of
mitochondria, cells were incubated with 100 nM Mitotracker
red (MTR; Molecular Probes) for 30 min at 37 °C prior to fixation.
The coverslips were mounted with elvanol, and the cells were viewed
under a Nikon fluorescence microscope at a magnification of
×200/×400. Pictures were taken with a 1310 digital camera (DVC).
Confocal microscopy was performed using a Zeiss Axiovert 100 TV
microscope (Oberkochen, Germany), attached to the Bio-Rad Radiance 2000 laser scanning system, operated by LaserSharp software.
 |
RESULTS |
ncBID and wtBID Induce Cell Death in Bid
/
and Caspase-8
/
Primary MEFs--
In order to define whether cleavage of BID by
caspase-8 is essential for triggering its apoptotic activity, we have
constructed recombinant adenoviruses carrying tetracycline-inducible
vectors of either wtBID or a caspase-8 ncBID-D59A mutant tagged with a C-terminal FLAG epitope. Bid
/
primary MEFs were infected with each
of the viruses and 12-15 h later doxycycline was added to the culture
to induce BID expression. Expression of BID was analyzed by Western
blot using anti-BID antibodies. Low levels of expression of both wtBID
and ncBID were detected in the absence of doxycycline because of
leakage of the tet promoter (Fig.
1A, top). A
significant increase in the expression of both forms was detected by
4.5 h post- doxycycline treatment. Importantly, wtBID was cleaved
to generate p15 tBID, whereas ncBID was not (Fig. 1A,
top). Next, we measured the levels of cell death 8- and 24-h
postinduction of BID expression. Strikingly, expression of either wtBID
or ncBID induced comparable levels of cell death (Fig. 1A,
bottom). To assure that cell death was indeed due to
expression of nc/wtBID, we constructed a recombinant adenovirus
carrying a constitutive expressing vector of GFP, and found that
infection of Bid
/
MEFs with this adenovirus had little effect on
cell viability (Fig. 1A, bottom).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
ncBID and wtBID induce similar levels of cell
death in Bid / and caspase-8 / primary MEFs. A,
BID-induced cell death in Bid / primary MEFs. Top, Bid
/ primary MEFs were infected with recombinant adenoviruses carrying
wtBID-FLAG, ncBID-FLAG, GFP, or not infected (N/T).
Expression of both BID forms was induced by doxycycline
(Dox). Cell lysates were prepared at the indicated time
points post BID induction and equal amounts of protein (20 µg per
lane) were subjected to SDS-PAGE followed by Western blot analysis using anti-BID Abs. The blot was stripped and reprobed
with anti- -actin Abs to control for the level of BID expression
(lower panel). Bottom, Bid / primary MEFs
were infected as described above. The cells were collected 8- and 24-h
postinduction, and the level of cell death was assessed by PI dye
exclusion. B, BID-induced cell death in caspase-8 /
primary MEFs. Top, Caspase-8 / primary MEFs were
infected as in A and cell death was assessed by PI dye
exclusion. Bottom, caspase-8 / primary MEFs were
infected with FLAG-tagged nc/wtBID. Cell lysates were prepared 4.5-h
post-BID induction and equal amounts of protein (20 µg per lane) were
subjected to SDS-PAGE followed by Western blot analysis using anti-BID
Abs. FLAG-BID and FLAG-tBID denote exogenous expressed proteins, and
BID and tBID denote endogenous expressed proteins. Asterisk
denotes an unidentified band.
|
|
To assess whether full-length BID can induce apoptosis in the absence
of caspase-8, caspase-8
/
primary MEFs were infected with either of
the recombinant viruses, and the levels of death were measured
following doxycycline treatment. Expression of either wtBID or ncBID
induced a similar and significant increase in the levels of cell death
(Fig. 1B, top). Thus, cleavage of BID by caspase-8 is not essential for triggering its apoptotic activity in
primary MEFs. Interestingly, Western blot analysis indicated that in
caspase-8
/
MEFs, exogenous FLAG-tagged wtBID and endogenous BID
were cleaved to generate p15 tBID but FLAG-tagged ncBID was not (Fig.
1B, bottom). Since the cleavage of wtBID probably
occurs only after the death process has been initiated, these results suggest that ncBID and wtBID (nc/wtBID) are capable of inducing the
activation of a downstream caspase (other than caspase-8) that feeds
back to cleave wtBID at Asp59. In this respect, caspase-3 was shown to
cleave BID at Asp-59 downstream of mitochondria (22, 23).
ncBID Induces the Activation of Caspase-9 and Caspase-3 in a
Similar Time Course as wtBID--
The translocation of tBID to the
mitochondria leads to Cyt c release, apoptosome formation
and caspase-9 and -3 activation. To determine whether these caspases
were activated in Bid
/
MEFs expressing nc/wtBID, the
cleavage of specific fluorogenic peptide substrates, LEHD-AMC for the
caspase-9-like subset and DEVD-AMC for the caspase-3-like subset, were
measured. In wtBID-expressing cells, caspase-9 and -3 activities
appeared 4.5-h postinduction of BID expression, peaked at 7 h, and
decreased by 10 h (Fig. 2A). In ncBID-expressing
cells, there was lower activation of caspase-9, but the overall time
course of caspase-9 and -3 activation was similar to the time course
observed in the wtBID-expressing cells (Fig. 2A). A similar
set of experiments using caspase-9- and caspase-3-deficient primary
MEFs clearly indicated that the activities measured with the LEHD-AMC
and DEVD-AMC peptide substrates were specific for caspase-9 and
caspase-3, respectively (Fig. 2A).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
Infection of Bid / primary MEFs with
either wtBID or ncBID induces a similar time course of caspase-9 and
caspase-3 activation. A, BID-induced activation of caspase-9
and -3. Bid / , caspase-9 / and caspase-3 / primary MEFs
were infected as in Fig. 1, and cells were collected at the indicated
time points post-BID induction to determine caspase activity. Caspase-9
(left) and caspase-3 (right) activities were
assessed by using the LEHD-AMC and DEVD-AMC fluorogenic tetrapeptides,
respectively. B, BID-induced cleavage of effector caspases.
Bid / primary MEFs were infected as above. Cell lysates were
prepared at the indicated time points post-BID induction, and equal
amounts of protein (40 µg per lane) were subjected to SDS-PAGE
followed by Western blot analysis using the CM-1 Ab. p17 represents the
large subunit of caspase-3. Asterisk denotes a CM-1 reactive
band that probably represents another effector caspase cleavage
product.
|
|
To further confirm that these caspases were activated and that there
was no significant difference in the time course of caspase activation
between the two BID forms, the cleavage of caspase-3 was analyzed by
Western blot. Using the CM-1 antibody, which principally recognizes the
p17 cleaved form of caspase-3 (24), we detected a weak signal by 3-h
postinduction of either wtBID or ncBID, and a much stronger signal by
5- and 8-h postinduction (Fig. 2B). At 5-h postinduction,
the level of cleaved caspase-3 in ncBID-expressing cells was lower than
the level in wtBID-expressing cells but eventually reached similar
levels by 8-h postinduction.
nc/wtBID Are Much Less Effective Than tBID in Inducing
Cyt c Release but Only Slightly Less Effective in Inducing
Apoptosis--
The results presented above suggest that ncBID acts by
inducing the release of Cyt c from mitochondria, leading to
apoptosome formation and caspase activation. Next, we analyzed the
ability of ncBID to induce Cyt c release. Since tBID is
100-fold more effective than full-length BID in inducing Cyt
c release from mitochondria in vitro (16), we
used tBID as a positive control in these experiments. For this purpose
we constructed two additional recombinant adenoviruses carrying
constitutive expressing vectors of either wtBID or tBID tagged with a
C-terminal HA epitope. The time course of wt/tBID expression using the
constitutive system was similar to the time course of nc/wtBID
expression using the inducible system. Of note, the levels of tBID
generated in cells infected with the constitutive wtBID virus were
similar to the levels of tBID expressed in cells infected with the tBID
virus (Fig. 3A). In this set
of experiments, the cellular location of BID and the release of Cyt
c were analyzed by immunofluorescence. Bid
/
primary
MEFs were infected with either the inducible or the constitutive
adenoviruses, fixed and stained with both anti-BID and anti-Cyt
c antibodies. The nuclei were stained by DAPI. Both ncBID
and wtBID (inducible or constitutive expression) were mainly localized
to the cytosol, and in a low percent of the cells Cyt c was
substantially released (Fig. 3B and see below). A closer examination of both ncBID and wtBID-expressing cells indicated that Cyt
c was substantially released only in cells in which BID showed a punctate staining (Fig. 3B). Prestaining of the
cells with Mitotracker indicated that the BID punctate staining
represented mitochondrial localization (Fig. 3C). 5- and 8-h
postinduction, BID was localized to mitochondria in only ~20% of the
cells expressing nc/wtBID, and only in these cells Cyt c was
substantially released (Fig. 3D, left panel).
Using the constitutive system, tBID was localized to mitochondria in
100% of the cells infected with the tBID virus, and in 73 ± 6.6% of these cells Cyt c was substantially released (Fig.
3, B and D, right panel). In contrast,
in cells infected with the wtBID virus, BID was localized to
mitochondria in only 14 ± 1.6% of the cells, and only in these
cells Cyt c was substantially released (Fig. 3D,
right panel). The fact that only 14% of the cells infected
with the wtBID virus substantially released Cyt c compared
with 73% of the cells infected with the tBID virus is surprising in
light of the fact that the levels of tBID detected in both infected
cells were similar (Fig. 3A). To compare the level of
apoptosis in cells infected with either the wtBID or tBID viruses,
apoptotic nuclei were counted in the cells from the same experiment
that was performed to monitor Cyt c release. As shown in
Fig. 4E, wtBID induced less
apoptosis than tBID at 5-h postinfection, but similar levels at 8-h
postinfection. Thus, wtBID is capable of inducing comparable levels of
apoptosis compared with tBID but with less release of Cyt
c.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
nc/wtBID are much less effective than tBID in
inducing Cyt c release but only slightly less
effective in inducing apoptosis. A, time course of
wt/tBID expression in Bid / infected MEFs. Bid / primary MEFs
were infected with recombinant adenoviruses carrying either wtBID or
tBID (constitutive system) tagged with a C-terminal HA epitope. Cell
lysates were prepared at the indicated time points postinfection, and
equal amounts of protein (20 µg per lane) were subjected to SDS-PAGE
followed by Western blot analysis using anti-BID Abs.
Asterisk denotes unidentified bands. B, ncBID is
mostly localized to the cytosol. Bid / primary MEFs were infected
with either ncBID (top three panels) or tBID (lower three
panels). Five hours postinduction/infection, the cells were fixed
and double immunostained with antibodies for both BID
(green) and Cyt c (red). The nuclei
were visualized by DAPI staining (blue). Arrows
mark cells in which BID appears in a punctate pattern, and Cyt
c was released. C, the BID punctate pattern
represents mitochondrial localization. Bid / primary MEFs were
infected with ncBID. Five hours post-BID induction, the cells were
prestained with Mitotracker red (MTR; middle),
fixed, and then immunostained with anti-BID Abs (green;
left). The right picture is a merge between the
two pictures. D, nc/wtBID are less effective than tBID in
localizing to mitochondria and in inducing Cyt c release.
Immunofluorescence studies using Bid / primary MEFs infected with
either the nc/wtBID (inducible system) or the wt/tBID (constitutive
system) were used to quantitate the percent of cells with BID localized
to mitochondria and percent of cells that substantially released Cyt
c. Only cells that were positive for BID and showed intact
nuclei were counted. At least 300 cells were counted from three time
points (3, 5, and 8 h postinduction/infection) and only the
results from the 5-h time point are shown. Mitochondrial localization
of ncBID and wtBID (inducible or constitutive) exactly correlated with
the release of Cyt c and reached only 14 ± 1.6% (for
ncBID and constitutive wtBID) and 22 ± 1.2% (for inducible
wtBID). A similar percentage/correlation was detected at 3 and 8 h
postinduction/infection (not shown). tBID was detected only in the
mitochondria, and 73 ± 6.6% of the cells that expressed tBID
released Cyt c. Similar results were obtained when wild type
primary MEFs were used for these studies. E, wtBID is only
slightly less effective than tBID in inducing apoptosis.
Condensed/fragmented (Apoptotic) nuclei were counted at the
indicated time points postinfection of Bid / primary MEFs with
wt/tBID (constitutive system). At least 300 nuclei were counted from
each time point.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 4.
nc/wtBID induce the cleavage of effector
caspases also in cells, which show a punctate/mitochondrial staining of
Cyt c. Bid / primary MEFs were infected with
the ncBID virus, treated for 6 h with doxycycline, fixed and
double immunostained with the CM-1 Ab (green) and the Cyt
c Ab (red). The nuclei were visualized by DAPI
staining (blue). Top, A, most of the
CM-1-positive cells (71 ± 4.2%) showed nuclear condensation
resulting in artificial fluorescence staining. B, 22 ± 6.5% of the CM-1-positive cells with intact nuclei released Cyt
c. C, 7 ± 2% of the CM-1-positive cells
with intact nuclei did not release Cyt c. Bottom,
a graph summarizing the percentages indicated above.
|
|
nc/wtBID Induce the Cleavage of Effector Caspases in
Cells That Seem to Retain Cyt c in Mitochondria--
A possible
explanation for the results presented in the previous section is that
nc/wtBID are actually inducing the release of Cyt c in a
larger percent of the infected cell population, but since the release
is not robust in these cells (as in the case of tBID) it may be
difficult to detect by immunofluorescence. Previous calculations have
demonstrated that ~20% of the total mitochondrial Cyt c
is sufficient to induce caspase activation (25). To address this issue
we have preformed immunofluorescence studies using the CM-1 antibody,
and searched for CM-1-positive cells, which retained Cyt c
in the mitochondria. 71 ± 4.2% of the CM-1-positive cells showed
nuclear condensation and therefore were not suitable for our analysis
(Fig. 4A). Among the CM-1-positive cells that had an intact
nuclei, 22 ± 6.5% released Cyt c and 7 ± 2%
did not (Fig. 4, B and C, respectively). These
results suggest that nc/wtBID are probably inducing low levels of Cyt c release in more than ~20% of the infected cells, which
is sufficient to induce effector caspase cleavage/activation. On the
other hand, these results might suggest that nc/wtBID could induce
caspase activation independently of Cyt c release. However,
this is probably not the case since expression of nc/wtBID in Bax, Bak
double-deficient MEFs (which are resistant to tBID-induced Cyt
c release, Ref. 13) did not result in the appearance of
CM-1-positive cells (not shown).
Apaf-1 and Caspase-9, but Not Caspase-3, Are Essential for
nc/wtBID-induced Apoptosis--
The results presented above
suggest that Cyt c release, apoptosome formation and
activation of caspase-9 and -3 are important for nc/wtBID-induced cell
death. Next, we determined whether Apaf-1, caspase-9 and caspase-3 were
essential for these forms of BID to induce cell death. Wild type
primary MEFs and primary MEFs deficient in Apaf-1, caspase-9, or
caspase-3 were infected with nc/wtBID viruses and cell death was
measured 24 h post-BID induction. Both Apaf-1
/
and caspase-9
/
cells were completely resistant to the expression of both forms
of BID (Fig. 5A). These cells remained viable even 96 h post-BID expression. In contrast,
caspase-3
/
cells were susceptible to both forms of BID but died
with delayed kinetics compared with wild-type cells (Fig.
5A). These results suggest that either caspase-6 or
caspase-7 can partially substitute for caspase-3, as previously
reported (26). To determine whether the resistance of Apaf-1
/
and
caspase-9
/
primary MEFs was due to lack of BID expression,
exogenous BID levels were analyzed by Western blot using anti-FLAG
antibodies. This analysis indicated that both wtBID and ncBID were
expressed at relatively high levels in each of these knockout primary
MEFs (Fig. 5B). Thus, both Apaf-1 and caspase-9 are
essential for nc/wtBID to induce apoptosis in primary
MEFs. We have also analyzed the Bax,Bak double-deficient primary
MEFs, which were previously reported to be resistant to tBID (13), and
found that they were also resistant to nc/wtBID (not shown).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Apaf-1 and caspase-9, but not caspase-3, are
essential for wtBID and ncBID-induced cell death. A, Apaf-1
and caspase-9-deficient primary MEFs are resistant to BID-induced cell
death. Wild type primary MEFs or primary MEFs deficient in Apaf-1,
caspase-9, or caspase-3 were infected with nc/wtBID (inducible system),
and cell death was assessed by PI dye exclusion 24 h (left
panel) or 96 h (right panel) post-BID induction.
At 24 h postinduction, the level of cell death in caspase-3 /
primary MEFs was lower than the level in wild type MEFs but eventually
reached similar levels 48 h postinduction (not shown).
B, resistance of Apaf-1 and caspase-9-deficient primary MEFs
to BID is not due to lack of BID expression. Twenty-four hours post-BID
induction cells were lysed, and an equal amount of protein (20 µg per
lane) was subjected to SDS-PAGE followed by Western blot analysis using
anti-FLAG Abs. The blot was stripped and reprobed with anti- -actin
Abs to confirm equal loading of protein (lower panel).
|
|
Apaf-1 and Caspase-9-deficient Primary MEFs Are Resistant to tBID,
while SV-40 Transformation Renders Them Susceptible to tBID but Not to
nc/wtBID--
It was recently demonstrated that tBID
induces mitochondrial dysfunction and cell death independently of
caspases in both Apaf-1 and caspase-9-deficient MEFs (27). It should be
noted that this study was performed with SV-40-transformed Apaf-1 and caspase-9-deficient MEFs. Thus, we first checked whether Apaf-1
/
and caspase-9
/
primary MEFs were susceptible to tBID. We found
that these deficient primary MEFs were not susceptible to expression of
tBID (Fig. 6A) and remained
viable even 96-h postinfection (Fig. 6B). This resistance
was not caused by lack of tBID expression, because relatively high
levels of tBID were detected 96-h postinfection (not shown). Thus,
Apaf-1 and caspase-9 are essential for tBID-induced cell death in
primary MEFs.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
Apaf-1 and caspase-9 are essential for
tBID-induced cell death. A, Apaf-1 and caspase-9-deficient
primary MEFs are resistant to tBID-induced cell death. Primary MEFs
deficient in either BID, caspase-8, caspase-3, caspase-9, or Apaf-1
were infected with wt/tBID (constitutive system). Twenty-four hours
postinfection the cells were collected and the level of cell death was
assessed by PI dye exclusion. B, tBID induces cell death in
SV-40-transformed Apaf-1 and caspase-9-deficient MEFs but wtBID does
not. Primary or SV-40 transformed Apaf-1 / and caspase-9 / MEFs
were infected with wt/tBID viruses (constitutive system). Ninety-six
hours postinfection the cells were collected, and the level of cell
death was assessed by PI dye exclusion.
|
|
Next, we determined whether SV-40 cell transformation renders either
Apaf-1 or caspase-9-deficient MEFs susceptible to wt/tBID. As
previously demonstrated, both deficient MEFs were susceptible to tBID
(Fig. 6B). Surprisingly, however, these deficient MEFs were
resistant to wtBID. Western blot analysis indicated that this
resistance to wtBID was not caused by low levels of BID expression (not
shown). Thus, SV-40 cell transformation enables tBID but not wtBID to
induce cell death independently of Apaf-1 or caspase-9.
Expression of Non-apoptotic Levels of Either ncBID or wtBID in Bid
/
MEFs Induces a Similar Enhancement in Apoptosis in
Response to a Variety of Death Signals--
The results presented
above indicate that ncBID is a potent inducer of the mitochondrial
apoptotic pathway when overexpressed in primary MEFs. Next, we wanted
to determine the cellular death pathways that involve full-length BID.
For this purpose we used the inducible expression system to express
low, non-apoptotic levels of ncBID in Bid
/
MEFs, and analyzed
the response to several death stimuli. As reference cells, we have used
Bid
/
MEFs expressing low, non-apoptotic levels of wtBID and Bid
/
MEFs that were not infected but treated with each of the stimuli. We examined several signals proposed to cause Cyt c release
from mitochondria: staurosporine (STS; kinase inhibitor), etoposide (Etop; topoisomerase II inhibitor), ultraviolet radiation (UVB), and
cisplatin (Cis; forms covalent adducts with the DNA). We also examined
the effect of tumor necrosis factor
(TNF
) plus actinomycin D
(ActD) and the effect of stress signaling from the endoplasmic reticulum (ER) induced by thapsigargin (which inhibits the
Ca2+ adenosine triphosphate pump). Following doxycycline
treatment (only 2 h) and its removal, cells were treated with each
of the stimuli for 22 h, and cell death levels were assessed by PI
dye exclusion. With all six stimuli, expression of nc/wtBID induced an
enhancement in cell death, which was most prominent with DNA-damaging agents (Fig. 7A,
top). In correlation with the overexpression experiments,
expression of ncBID was as potent as the expression of wtBID. To
determine whether treatment with either of these death stimuli resulted
in the cleavage of ncBID, we performed Western blot analysis of whole
cell lysates using anti-BID antibodies. This analysis indicated that
ncBID was not cleaved in cells treated with the death signals examined
(Fig. 7A, bottom).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7.
Expression of non-apoptotic levels of ncBID
induces an enhancement in apoptosis in response to a variety of death
signals. A, expression of non-apoptotic levels of either
ncBID or wtBID results in a similar enhancement in cell death in
response to divergent death stimuli. Top, Bid / primary
MEFs were infected with adenoviruses carrying either ncBID or wtBID or
left untreated (N/T). Two hours after the addition of doxycycline, the
cultures were washed three times and either left untreated ( ) or
treated with the indicated death stimuli: cisplatin (Cis; 50 µM), etoposide (Etop; 100 µM),
UVB (200 J/m2), thapsigargin (Thap; 2 mM), staurosporine (STS; 4 µM),
and TNF (40 ng/ml) together with actinomycin D (ActD; 2 µg/ml). Cells were collected 22 h later, and the level of cell death was
assessed by PI dye exclusion. Bottom, ncBID is not cleaved
in response to a variety of death signals. The cells were treated as
described above and collected 10 h after treatment with the death
signals. Whole cell lysates were prepared, and equal amounts of protein
(20 µg per lane) were subjected to SDS-PAGE followed by Western blot
analysis using anti-BID Abs. B, etoposide, cisplatin, and
thapsigargin induce the translocation of full-length BID to
mitochondria, leading to a substantial increase in Cyt c
release. Apaf-1 / primary MEFs were infected with the ncBID
adenovirus. Three hours after the addition of doxycycline, the cells
were washed three times and either treated with each of the death
signals indicated (Etop/Cis/Thap + ncBID) or left untreated (ncBID) for
22 h. As reference cells, we used non-infected cells that were
treated with each of the death signals (Etop/Cis/Thap). The cells were
immunostained as described in the legend to Fig. 3, and then used to
quantitate the percent of cells with ncBID localized to mitochondria
and percent of cells that substantially released Cyt c.
Bottom right, treatment with each of the death signals does
not result in the cleavage of either ncBID or endogenous BID. Cells
were treated and analyzed as described in A.
|
|
Next we assessed whether part of the death signals used above induced
the translocation of BID to mitochondria and the release of Cyt
c. In order to increase the number of cells that have
released Cyt c and retained an intact nuclei, we have used
the Apaf-1-deficient MEFs. Following infection with the
adenovirus-carrying ncBID, treatment with doxycycline and its removal,
cells were treated with cisplatin, etoposide, or thapsigargin, and the
cellular locations of BID and Cyt c were analyzed by
immunofluorescence. In all three cases, the addition of the death
signal significantly enhanced BID localization to the mitochondria and
the release of Cyt c (Fig. 7B). To ensure that
neither of these death stimuli resulted in the cleavage of either ncBID
or endogenous BID in the Apaf-1-deficient MEFs, we performed Western
blot analysis of whole cell lysates using anti-BID antibodies. This
analysis indicated that neither ncBID nor endogenous BID were cleaved
in cells treated with each of the death signals (Fig. 7B,
bottom).
 |
DISCUSSION |
In this study we demonstrate that expression of a caspase-8
non-cleavable (nc) BID mutant in primary MEFs induces
apoptosis. Moreover, ncBID and wtBID showed a similar and
significant ability to enhance apoptosis in response to a variety of
death signals. Both forms of BID induced the release of Cyt
c, which led to caspase activation. nc/wtBID mainly
localized to the cytosol and were less effective than tBID in inducing
Cyt c release, but were similarly effective in inducing
apoptosis. Finally, Apaf-1, caspase-9 and both BAX and BAK, but not
caspase-3 or caspase-8 were essential for both nc/wtBID and
tBID-induced apoptosis in primary MEFs.
Several experiments in this study indicate that cleavage of BID by
caspase-8 is not essential for triggering its apoptotic activity.
First, the ncBID mutant, which is resistant to caspase-8 cleavage,
induced apoptosis in Bid-deficient and wild type primary MEFs (Figs. 1
and 5). Second, wtBID induced apoptosis in caspase-8
/
primary MEFs
(Fig. 1). Third, the ncBID mutant induced Cyt c release,
caspase activation and apoptosis in a similar time course and was
similarly effective as the cleavable wtBID (Fig. 2). Fourth, expression
of non-apoptotic levels of either ncBID or wtBID in Bid
/
MEFs
induced a similar enhancement in apoptosis in response to divergent
death stimuli (Fig. 7).
Caspase-3 was also demonstrated to cleave BID at Asp-59 (7, 9, 23).
However, caspases are not the only proteases that have been
demonstrated to cleave BID. Several studies have reported that granzyme
B, lysosomal proteases and calpain cleave BID at sites close but
distinct of Asp-59, leading to its activation (28-30). Thus, each of
these enzymes is potentially capable of cleaving the ncBID mutant.
However, using Western blot analysis we could not detect
any BID cleavage products in Bid
/
MEFs infected with the ncBID
adenovirus (Fig. 1). Moreover, treatment with several death signals did
not result in a detectable cleavage of ncBID (Fig. 7). Finally, the
fact that BID is cleaved only at Asp-59 (Fig. 1) seems to exclude these
proteases as cleavers in our biological setting.
The use of a variety of primary deficient MEFs revealed that the core
components of the apoptosome (Apaf-1 and caspase-9) were absolutely
essential for nc/wtBID-induced cell death (Fig. 5). It was recently
demonstrated that tBID induces caspase-independent cell death in
SV-40-transformed Apaf-1
/
and caspase-9
/
MEFs (27).
Surprisingly, in primary MEFs, Apaf-1, and caspase-9 were essential for
tBID-induced cell death (Fig. 6). Thus, SV-40 transformation enables
tBID but not nc/wtBID to bypass the requirement for Apaf-1 and
caspase-9. Our preliminary studies with the SV-40 transformed Apaf-1-deficient MEFs showed that tBID was much more potent than wtBID
in depolarizing mitochondria (data not shown). Thus, substantial mitochondrial dysfunction may be sufficient to induce
apoptosome/caspase-independent cell death.
Why does wtBID, which is cleaved to generate tBID, act like ncBID and
not like tBID? This phenomenon is particularly intriguing in light of
the fact that the levels of tBID generated in cells infected with the
wtBID virus were similar to the levels of tBID expressed in cells
infected with the tBID virus (Fig. 3). It was previously shown that
following the cleavage of BID and its translocation to mitochondria,
the p7 N-terminal part remains tightly associated to the p15 tBID
C-terminal part (31). Thus, in the case of the cleaved wtBID, the p7
fragment may prevent from tBID to efficiently insert into the membrane
and induce large-scale mitochondrial changes, which are induced by the
"free" tBID. One may suspect that cleaved wtBID may act like ncBID
in vivo since release of a small amount of Cyt c
is sufficient to induce caspase activation but not sufficient to induce
large-scale mitochondrial dysfunction. Maintaining normal mitochondrial
function to maintain the intracellular levels of ATP seems to be
essential for ensuring the manifestation of apoptotic and not necrotic
cell death (32). In this respect it was previously proposed that the
release of Cyt c following intrinsic damage signals occurs
in two distinct stages: an initial stage involving the release of small
amounts of Cyt c, which precedes the activation of caspases,
and a 2nd stage that involves caspase activation, the release of large
amounts of Cyt c, and large scale mitochondrial dysfunction
(33, 34). Interestingly, BID was proposed to be involved in the 2nd
stage since its cleavage during cytotoxic drug and UV radiation-induced
apoptosis occurs downstream of mitochondria and is catalyzed by
caspase-3 (23).
Our results clearly demonstrate that ncBID can significantly enhance
apoptosis in response to a variety of death signals (Fig. 7). Treatment
with these death signals did not result in detectable cleavage of
ncBID, indicating that cell death enhancement was due to full-length
BID. Importantly, these death signals induced enhanced localization of
BID to mitochondria, which was accompanied by a substantial increase in
Cyt c release. Thus, full-length BID can act as an initiator
of the mitochondrial apoptotic pathway in response to divergent
apoptotic death signals. Moreover, apoptotic death signals convert
full-length BID into a "tBID-like" molecule, to become a much
better inducer of the mitochondrial apoptotic program. Future studies
will determine the region in BID that "receives" the death signal
and define how this signal activates BID. It will also be a challenge
to identify the cellular pathways that involve endogenous full-length
BID and to clearly define whether the non-cleaved form of BID plays a
role during apoptosis in vivo.