Nonredundant Role of Bax and Bak in Bid-Mediated Apoptosis

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.

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax

“…apoptotic) GBM Bax ( þ ) cells. 17 Western blots analysis of proteins co-immunoprecipitated with TOM22 or Bax a showed that in resting cells TOM22 was associated with TOM40 and that it became associated with Bax a under apoptotic conditions (Figure 2c). On the other hand, Bcl-2 was co-immunoprecipitated with Bax in resting cells and in apoptotic cells (Figure 2c).…”

“…Bax a, Bax a activated by a preincubation with truncated form of Bid (p13-tBid) 17 and Bax C, a variant, which corresponds to a deletion of the first 19 N-T amino acids of Bax a. 18 We also used constructs in which the RFP was fused to two putative mitochondrial targeting signals in Bax (i.e.…”

“…Next, we used human glioblastoma multiforme (GBM) cells deficient in Bax (Bax (À) GBM) or in Bak (Bak (À) GBM). 17 These cells were microinjected with either control IgG or anti-TOM22 antibodies. Microinjection of anti-TOM22 antibodies inhibited STS-induced apoptosis in Bax ( þ ) GBM cells, while no differences in the low percentage of cell death were detected between Bax (À) GBM cells injected with anti-TOM22 and control antibodies (Figure 3d).…”

“…Microinjection. Microinjection experiments were performed as described by Cartron et al 17 Briefly, cells were seeded on glass coverslips 24 h prior microinjection using microcapillaries (femtotips II, Eppendorf) mounted on an automated microinjection system (Eppendorf). Antibodies at the indicated concentrations in PBS together with dextran (70 kDa)-conjugated lysine fixable Oregon green (0.5% final concentration; Molecular Probes) as a coinjection marker were microinjected.…”

Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells

“…Most studies addressing the impact of Bax and Bak on apoptosis sensitivity of cancer cells therefore suggest that both proteins are partially redundant. Nevertheless, recent evidence delineates that Bax and Bak exert also nonredundant activities Cartron et al, 2003;Gillissen et al, 2003;von Haefen et al, 2004;Wendt et al, 2005). At the molecular level, this may be reflected by the fact that Bax and Bak are differentially controlled by additional factors such as VDAC2 (Cheng et al, 2003;Chandra et al, 2005).…”