The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

Abstract

In several di erent cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant terbutylhydroperoxide (t-BHP) but has no cytoprotective e ect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid bis 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential DC m that is not inhibited by the broad spectrum caspase inhibitor Z-VAD.fmk, although Z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the DC m of t-BHP-treated cells but has no inhibitory e ect on the DC m collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of DC m disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamidetriggered DC m collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-puri®ed PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X L proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PTmodulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their e ects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.

α-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate

“…This reinforces the concept that mitochondrial destabilisation constitutes a central event in the programmed cell death such as apoptosis and autoschizis (Verrax et al, 2005). Again, this evokes the possibility that primary thiol oxidation of mitochondrial proteins, derived from an oxidative shift in the cellular redox potential , can induce mitochondrial membrane permeabilisation (Zamzami et al, 1998). Moreover, oxidation of critical thiols within the catalytic centre of caspases annihilates their latent proteolytic potential and, thus, preclude their auto-activation (Mannick et al, 2001).…”

Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins

“…Correlating with its cytoprotective e ect, Bcl-2 has no e ect on diamide induced PT, both in mitochondria and in PT pore complexes reconstituted into liposomes Zamzami et al, 1998). Similarly, Bcl-2 and Bcl-X L have a limited inhibitory e ect on PT pore complexes treated with recombinant caspases.…”

“…We have recently puri®ed complexes of hexokinase-associated proteins which we have incorporated into liposomes to reestablish the function of the PT pore in vitro Zamzami et al, 1998). Biochemical and functional data indicate that the hexokinase-associated polyprotein complex enriched from brain homogenates contains the proapoptotic Bcl-2 homolog Bax (but not Bcl-2 and Bcl-X L ), in addition to proteins previously suggested to participate in the regulation of PT (ANT, VDAC, cyclophilin D, and hexokinase).…”

“…Membrane hexokinase-associated protein complexes from the rat brain are incorporated into phosphatidylcholine/cholesterol liposomes. Thereafter the capacity of retaining the¯uorochrome DiOC 6 (3) after stimulation with di erent inducers of PT pore opening is assessed using a¯ow cytometric approach Zamzami et al, 1998). Incorporation of recombinant Bcl-2 or Bcl-X L prevents DiOC 6 (3) release induced by a number of di erent agents that act to induce PT in control liposomes lacking Bcl-2 or Bcl-X L .…”

“…Based on three di erent ®ndings, we thus tend to believe that Bcl-2 regulates cytochrome c release indirectly, via an e ect on the PT pore ( Figure 5). First, Bcl-2 directly acts on the reconstituted PT pore complex Zamzami et al, 1998). Second, recombinant Bax added to isolated mitochondria induces both PT and cytochrome c release, and this e ect is prevented by the PT inhibitors cyclosporin A and bongkrekic acid (manuscript in preparation).…”