Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family1,2. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli3,4,5,6. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity7,8,9. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 ‘apoptosome’, which seems to amplify rather than initiate the caspase cascade.
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We thank Y. Lazebnik and P. Lassus for sharing unpublished results. We also thank P. Gruss for Apaf-1+/- mice, Y. Lazebnik and X. Opitz-Araya for monoclonal antibodies to caspases 3, 7 and 9, P. Vandenabeele and M. Kalai for the anti-mouse caspase-1 antibody, R. Anderson for the anti-HSP70 antibody and S. Nagata for the ICAD constructs. We thank E. Loza, A. Milligan, C. Tilbrook, A. Naughton and J. Merryful for animal care, F. Battye, D. Kaminaris, V. Lapatis, J. Chan and C. Tarlinton for cell sorting, S. Mifsud, L. DiRago, L.-C. Zhang and L. Tai for expert technical help and G. Filby for editorial assistance. We are grateful to S. Cory, A. Harris, K. Newton and H. Puthalakath for discussions and critical reading of this manuscript. This work was supported by fellowships and grants from the Dr Josef Steiner Cancer Research Foundation, the NHMRC, the Leukemia and Lymphoma Society (SCOR Center), the Anti-Cancer Council of Victoria, the Sylvia and Charles Viertel Charitable Foundation, the NIH, the AIRC and the Commonwealth Department of Education, Science and Training. F.C. is an Assistant Telethon Scientist.
The authors declare that they have no competing financial interests.
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