THE THIOL CROSS-LINKING AGENT DIAMIDE OVERCOMES THE APOPTOSIS-INHIBITORY EFFECT OF BCL-2 BY ENFORCING MITOCHONDRIAL PERMEABILITY TRANSITION

In several different cell lines, Bcl-2 prevents the induction of apopt osis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) b y the pro-oxidant terbutylhydroperoxide (t-BHP) but has no cytoprotect ive effect when apoptosis is induced by the thiol crosslinking agent d iazenedicarboxylic acid bis 5,N-dimethylamide (diamide), Both t-BHP an d diamide cause a disruption of the mitochondrial transmembrane potent ial Delta Psi(m), that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragm entation and poly(ADP-ribose) polymerase cleavage in these models, Bcl -2 stabilizes the Delta Psi(m) of t-BHP-treated cells but has no inhib itory effect on the Delta Psi(m) collapse induced by diamide, As compa red to normal controls, isolated mitochondria from Bcl-2 overexpressin g cells are relatively resistant to the induction of Delta Psi(m) disr uption 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 overexpressi ng Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In con trast, Bcl-2 does not confer protection against diamide-triggered Delt a Psi(m) collapse and the release of AIF and cytochrome c, This indica tes that Bcl-2 suppresses the permeability transition (PT) and the ass ociated release of intermembrane proteins induced by t-BHP but not by diamide, To further investigate the mode of action of Bcl-2, semi-puri fied PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system, Recombinant Bcl-2 or Bcl-X, proteins augment t he resistance of reconstituted PT pore complexes to pore opening induc ed by t-BHP, In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity, Agai n, Bcl-2 fails to confer protection against diamide in this experiment al system, The reconstituted PT pore complex itself cannot release cyt ochrome c encapsulated into liposomes, Altogether these data suggest t hat pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c, Furthermore, our results suggest a strategy for inducin g apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs .