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. 2003 Jul;23(13):4701-12.
doi: 10.1128/MCB.23.13.4701-4712.2003.

Nonredundant role of Bax and Bak in Bid-mediated apoptosis

Pierre-François Cartron  1 , Philippe Juin, Lisa Oliver, Stéphane Martin, Khaled Meflah, François M Vallette
Affiliations

Nonredundant role of Bax and Bak in Bid-mediated apoptosis

Pierre-François Cartron et al. Mol Cell Biol. 2003 Jul.

Abstract

Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM). Using an antisense strategy, we generated Bak(-) BeGBM and Bak(-) BdGBM, which enabled us to show that the remaining sensitivity of the BdGBM to apoptosis was due to the overexpression of Bak. Bax/Bak single or double deficiency had no influence on either the clonogenicity or the growth of tumors in Swiss nude mice. Of note, Bak(-) BeGBM cells were resistant to apoptosis induced by caspase 8 (C8) but not to that induced by granzyme B (GrB). Cells lacking both Bax and Bak (i.e., Bak(-) BdGBM) were completely resistant to all stimuli including the microinjection of C8 and GrB. We show that GrB-cleaved Bid and C8-cleaved Bid differ in size and utilize preferentially Bax and Bak, respectively, to promote cytochrome c release from mitochondria. Our results suggest that Bax deficiency is compensated by an increase of the expression of Bak in GBM and show, for the first time in human cancer, that the double Bax and Bak deficiency severely impairs the apoptotic program.

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Figures

FIG. 1.
FIG. 1.
Absence of expression of Bax in a subset of GBM. (a) Confocal microscopy analysis of in vitro Bax expression in BdGBM and in BeGBM cells using the GFAP as glial marker and as a control of the level of protein expression. (b) Immunoblot analysis of Bax confirming the absence of the protein in BdGBM cells in vitro. Representative immunoblots were done with 50 μg of BeGBM and 150 μg of BdGBM loaded on SDS-PAGE. (c) mRNA encoding Bax was examined by RT-PCR using the SMART system as described in Materials and Methods using β2-macroglobulin as a control as described in reference . (d) Methylation specific PCR analysis of the Bax promoter region in BeGBM and in BdGBM cells. PCR was performed on genomic DNA purified from BdGBM or BeGBM cells. The DNA was untreated (U) or modified (M) by bisulfite sodium as originally described by Herman et al. (16). Pos control, ummethylated DNA; Normal control, methylated DNA. A gel representative of results from three independent experiments performed on each cell line is illustrated. (e) 5aza2dc treatment of GBM cells restored the expression of Bax in BdGBM cells as shown by immunoblotting with anti-Bax antibodies using actin as an internal control (50 μg of protein was loaded in each lane).
FIG. 1.
FIG. 1.
Absence of expression of Bax in a subset of GBM. (a) Confocal microscopy analysis of in vitro Bax expression in BdGBM and in BeGBM cells using the GFAP as glial marker and as a control of the level of protein expression. (b) Immunoblot analysis of Bax confirming the absence of the protein in BdGBM cells in vitro. Representative immunoblots were done with 50 μg of BeGBM and 150 μg of BdGBM loaded on SDS-PAGE. (c) mRNA encoding Bax was examined by RT-PCR using the SMART system as described in Materials and Methods using β2-macroglobulin as a control as described in reference . (d) Methylation specific PCR analysis of the Bax promoter region in BeGBM and in BdGBM cells. PCR was performed on genomic DNA purified from BdGBM or BeGBM cells. The DNA was untreated (U) or modified (M) by bisulfite sodium as originally described by Herman et al. (16). Pos control, ummethylated DNA; Normal control, methylated DNA. A gel representative of results from three independent experiments performed on each cell line is illustrated. (e) 5aza2dc treatment of GBM cells restored the expression of Bax in BdGBM cells as shown by immunoblotting with anti-Bax antibodies using actin as an internal control (50 μg of protein was loaded in each lane).
FIG. 2.
FIG. 2.
Bak is overexpressed in BdGBM. (a) Immunoblot analyses of Bcl-2, Bcl-XL and Bid expression in BeGBM and in BdGBM cells. Immunoblotting with a Bak antibody revealed that this protein was overexpressed in BdGBM cells; 50 μg of protein was loaded in each lane and actin was used as a control. (b) The ratio between Bak and actin in the resected tumors was calculated from immunoblots and represented as a histogram.
FIG. 3.
FIG. 3.
Bak overexpression in BdGBM cells can be inhibited by full-length Bak antisense transfection. The inhibition of Bak expression by the full-length Bak antisense in the cell lines was analyzed by immunoblotting using actin and Bax as controls (a) and by confocal microscopy with mitochondria labeled with Mitotrackter green (mito) as a control (b).
FIG. 4.
FIG. 4.
Single or double Bax and/or Bak deficiency influenced neither GBM cell clonogenicity nor their growth in the nude mice. (a) The clonogenicity assays were performed as described in Materials and Methods; histograms were drawn from the results obtained from six independent experiments. Data are given as numbers of UFC (colony-forming units) ± standard deviations. (b) Tumoral growth in Swiss nude mice (data shown were obtained from six different mice for each tumor). Standard deviations are shown. Bax+/Bak+ = BeGBM, Bax−/Bak+ = BdGBM, Bax+/Bak− = antisense Bak-transfected BeGBM cells, and Bax−/Bak− = antisense Bak-transfected BdGBM cells.
FIG. 5.
FIG. 5.
Double Bax/Bak deficiency abolished sensitivities toward apoptosis in GBM. Bax+/Bak+ (BeGBM), Bax−/Bak+ (BdGBM), Bax+/Bak− (antisense Bak-transfected BeGBM cells) and Bax−/Bak− (antisense Bak-transfected BdGBM) cells were treated with UV irradiation (10 s to 120 s) and increasing concentrations of DOXO (up to 20 ng/ml) or STS (up to 20 μM). Cells were transfected with increasing concentrations of plasmid encoding human C3 (up to 20 μg of plasmid) or an empty plasmid (20 μg) used as a control. It should be noted that no significant cell death was recorded in mock-transfected cells after selection. Cell death was quantified after 24 h using the release of LDH (measured as described in Materials and Methods) in the cell culture medium. Representative results from one of 3 independent experiments are illustrated for each treatment.
FIG. 6.
FIG. 6.
Sensitivity of single or double Bax/Bak-deficient GBM cells toward immune-induced apoptosis. (a) Bax+/Bak+ (BeGBM), Bax−/Bak+ (BdGBM), Bax+/Bak− (antisense Bak-treated BeGBM cells) and Bax−/Bak− (antisense Bak-treated BdGBM) cells were treated with increasing concentrations of FasL (from 0 to 20 μg/ml) or were transfected with increasing concentrations of plasmid (up to 200 μg of plasmid) encoding GrB. Cell death was quantified after 24 h of treatment using the release of LDH as an assay. Representative results from one of three independent experiments are illustrated for each treatment. (b) BeGBM or BeGBM cells treated with UVB irradiation (60 s) or FasL (10 μg/ml) or transfected with a plasmid encoding GrB (10 μg) were harvested 24 h after the start of the treatment. Cell extracts (50 μg) were analyzed for pro-caspase 8 (proC8) cleavage into the large subunit of caspase 8 (C8 LS) as a marker. The processing of fl-Bid (p22Bid) into p15-tBid or p13-tBid was analyzed in the same extracts using a 12 to 20% gradient SDS-PAGE.
FIG. 7.
FIG. 7.
Different forms of truncated Bid generated by C8 and GrB affect differently the single or double deficient GBM cells. (a) GBM cells with distinct Bax and Bak phenotypes were microinjected with GrB and C8 concentrations exhibiting similar IETDase activities (5 U) and mixed with Oregon green dextran as a microinjection marker. Cell death was assessed morphologically by fluorescent microscopy as described by Juin et al. (22). (b) Specific cleavage of fl-Bid (4 fmol) into p15-tBid by C8 (300 U of IETDase) and into p13-tBid by GrB (300 U of IETDase) in a cell-free assay. (c) Similar amounts of p13-tBid or p15-tBid (0.1 ng/μl) were microinjected, and cell death was quantified as in panel a.
FIG. 8.
FIG. 8.
Preferential cooperation of p15-tBid with Bak and p13-tBid with Bax in a cell-free assay. (a) 35S-fl-Bid was digested by C8 or GrB as described in Fig. 7, and 4 fmol of 35S-C8-cleaved fl-Bid (p15-tBid) or 35S-GrB-cleaved Bid (p13-tBid) was incubated for 30 min at 37°C with 50 μg of mitochondria before centrifugation for 10 min at 10,000 × g at 4°C. The binding of 35S-Bid to mitochondria was analyzed by SDS-PAGE and autoradiography of the mitochondrial pellet. One femtomole (25% of the initial input of 35S-labeled protein [i.e., 25%]), was added to the mitochondrial binding assay mix as a loading control. The influence of the different forms of Bid on Bax (b) and Bak (c) association in the cell-free system was assessed by the association of 35S-Bax and 35S-Bak with mitochondria after incubation with equimolar concentrations of cold Bid (4 fmol). Mitochondrial pellets were analyzed after SDS-PAGE and autoradiography as described for panel a. p13-tBid and p15-tBid involvement in Bax- (d) or Bak- (e) induced CYT-C release from mitochondria. After binding of Bax and Bak to mitochondria in the absence or the presence of different forms of Bid, mitochondria were pelleted for 10 min at 10,000 × g at 4°C and the presence of CYT-C in the supernatant was analyzed by immunoblotting. Data (± standard deviations) were obtained from at least 3 independent experiments.
FIG. 9.
FIG. 9.
Schematic representation of the cross talks between Bak and the death receptor pathway and Bax and the cytotoxic pathway. The immune system can trigger cell death in target GBM cells by the death receptor pathway (i.e., interaction of FasL with its plasma membrane receptor Fas) or by the cytotoxic pathway (i.e., the translocation of GrB throughout the target plasma membrane mediated by the perforin proteins). The activation of C8 is achieved by activation of the death receptor pathway or by GrB (cf. Fig. 6b). In the latter case, fl-Bid is cleaved into two fragments: p13-tBid directly by GrB and p15-tBid by the GrB-activated C8 (Fig. 6b). Hence, GrB generates both products of cleavage of fl-Bid and C8 generates only p15-tBid (Fig. 7a). p13-tBid has a preferential interaction with Bax and triggers its association with mitochondria while p15 appears to preferentially interact with Bak to induce the release of CYT-C from mitochondria and the subsequent activation of the execution phase of apoptosis (Fig. 7c and 8).
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References

    1. Adams, J. M., and S. Cory. 2002. The Bcl-2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer 2:647-656. - PubMed
    1. Barry, M., J. A. Heibein, M. J. Pinkoski, S. F. Lee, R. W. Moyer, D. R. Green, and R. C. Bleackley. 2000. Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol. Cell. Biol. 20:3781-3794. - PMC - PubMed
    1. Bayling, S. B., M. Esteller, M. R. Rountree, K. E. Bachman, and J. G. Herman. 2001. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum. Mol. Genet. 10:687-692. - PubMed
    1. Bouillet, P., and A. Strasser. 2002. BH3-only proteins—evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death. J. Cell Sci. 115:1567-1574. - PubMed
    1. Cartron, P. F., C. Moreau, L. Oliver, E. Mayat, K. Meflah, and F. M. Vallette. 2002. Involvement of the N-terminus of Bax in its intracellular localization and function. FEBS Lett. 512:95-100. - PubMed

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