Abstract
The mitochondrial (MT) death pathway that involves cytochrome c (Cytc) release under regulation by Bcl-2 family proteins plays a crucial role in p53-dependent IR apoptosis. The Cytc-conducting pore remains controversial and unidentified in cells, despite several in vitro models presented. We aimed to delineate the role for Bax (Bak)/VDAC1 hybrids in Cytc release in vivo. For that purpose, we used the p53/Bax-expressing Black93 cells and the Bcl2/Bak-expressing, p53/Bax-deficient Reh cells. The p53-Bax-dependent MT death pathway determined Cytc release and apoptosis in 5 Gy-exposed Black93 cells, but Reh cells lacking p53/Bax despite Bak expression showed neither Cytc release nor apoptosis. The protein cross-linking and 2-step IP studies for isolated MT indicated that IR induced mono- and dimeric Bax/VDAC1 hybrids in Black93 MT, but only monomeric Bak/VDAC1 hybrid in Reh MT. The Bax and Bak oligomers were invariable with time post-IR. In Black93 cells, clotorimazol or a synthetic N-terminal hexokinase peptide recruited Bax to VDAC1, enhanced IR-induced Cytc release and apoptosis, but a VDAC inhibitor DIDS suppressed all of such events. On the other hand, a chemical BH3 peptide-like apoptosis agonist HA14-1 that binds to the Bcl2 pocket induced the Bak/VDAC1 hybrid dimer in Bak/Bcl2-expressing Reh cells, which, otherwise not, rendered the cells highly susceptible to Cytc release and apoptosis. These results demonstrate a new evidence for the important in vivo role of Bax (Bak)/VDAC1 hybrid dimer in apoptosis
