Sequential Activation of Poly(ADP-Ribose) Polymerase 1, Calpains, and Bax Is Essential in Apoptosis-Inducing Factor-Mediated Programmed Necrosis

Abstract

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N′-nitro-N′-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.

We thank V. L. Dawson, S. J. Korsmeyer, A. Rudensky, and Y. Tan for cells, S. Matsuyama for the pcDNA3-Bax plasmid, J. L. Fernandez-Luna for the Bcl-2 insert, S. Barbier, S. Beaucourt, M. Bras, F. J. del Castillo, N. Robert, P. Sancho, and C. Solé for helpful assistance, and M. Cohen-Salmon, G. de Murcia, G. Eberl, and M. Segade for critical reading of the manuscript.

This work was supported by institutional grants from Institut Pasteur and CNRS and by specific grants from Fondation de France Ligue Contre le Cancer, and Association pour la Recherche sur le Cancer (ARC: contract 4043) (to S.A.S.). R.S.M. received Ph.D. fellowships from Fondation Hariri and ARC. V.J.Y. was supported by a Marie Curie fellowship (contract MEIF-2003-501887).

We declare that we have no competing financial interests.