BID-D59A Is a Potent Inducer of Apoptosis in Primary Embryonic Fibroblasts*

Rachel SarigDagger , Yehudit ZaltsmanDagger , Richard C. Marcellus§, Richard Flavell, Tak W. Mak||, and Atan GrossDagger **

From the Dagger  Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel, § Geminx Biotechnologies Inc., Place du Parc, Montreal, Quebec H2W 2M9, Canada, the  Howard Hughes Medical Institute, Section of Immunology, Yale University School of Medicine, New Haven, Connecticut 06520, and the || Amgen Institute, Division of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada

Received for publication, October 8, 2002, and in revised form, December 20, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, 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, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 pDelta 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-- TNFalpha , 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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-beta -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).


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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.


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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.


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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).


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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-beta -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.


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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 alpha  (TNFalpha ) 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).


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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 TNFalpha (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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

We thank Stanley J. Korsmeyer for the Bax,Bak double-deficient MEFs and for many other reagents. We also thank David Wallach for the caspase-8 -/- MEFs.

    FOOTNOTES

* This work was supported in part by the Israel Science Foundation, Israel Cancer Research Fund, the Leukemia and Lymphoma Society, the Y. Leon Benoziyo Institute for Molecular Medicine, and the Willner Family Center for Vascular Biology.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.

** Incumbent of the Robert Armour Family Career Development Chair. To whom correspondence should be addressed: Dept. of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9343656; Fax: 972-8-9344116; E-mail: atan.gross@weizmann.ac.il.

Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M210296200

    ABBREVIATIONS

The abbreviations used are: Cyt c, cytochrome c; ES, embryonic stem cells; MEFs, mouse embryonic fibroblasts; GFP, green fluorescent protein; MOI, multiplicity of infection; TNF, tumor necrosis factor; nc, non-cleavable; wt, wild type; CMV, cytomegalovirus; Ab, antibody.

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