The thiol crosslinking 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 apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(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 delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(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 diamide-triggered delta psi(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-purified 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 PT-modulatory 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 effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.
Zamzami
N
N
Centre National de la Recherche Scientifique, Unité Propre de Recherche 420, Villejuif, France.
Marzo
I
I
Susin
S A
SA
Brenner
C
C
Larochette
N
N
Marchetti
P
P
Reed
J
J
Kofler
R
R
Kroemer
G
G
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003958:Diamide / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D010539:Permeability / Q000187:drug effects; D010545:Peroxides / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D017382:Reactive Oxygen Species; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D020122:tert-Butylhydroperoxide
D003432:Cross-Linking Reagents; D010545:Peroxides; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D013439:Sulfhydryl Reagents; D003958:Diamide; D020122:tert-Butylhydroperoxide