Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Abstract

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD⁺ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD⁺ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD⁺ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD⁺ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.

ACKNOWLEDGMENTS

We thank B. Auer for providing human PARP cDNA, W. Fiers for TNF-α, D. Nicholson for recombinant caspase-3, and A. Bürkle for 10H antibody. We also thank R. Kurzbauer and S. Aigner for technical assistance. We are grateful to G. Mollon for preparation of photographs. We are also grateful to A. E. Grigoriadis, J. Hall, C. Morrison, K. Schulze-Osthoff, K. Sabapathy, and E. F. Wagner for critical comments and discussions.

Z.H. is in receipt of an IARC Special Training Award. This project was initiated at the Research Institute of Molecular Pathology, Vienna, Austria.