Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death

Mitochondria have recently been shown to serve a central role in programmed cell death. In addition, reactive oxygen species (ROS) have been implicate d in cell death pathways upon treatment with a variety of agents; however, the specific cellular source of the ROS generation is unknown. We hypothesi ze that mitochondria-derived free radicals play a critical role iu apoptoti c cell death. To directly test this hypothesis, we treated murine fibrosarc oma cell lines, which expressed a range of mitochondrial manganese superoxi de dismutase (MnSOD) activities, with respiratory chain inhibitors, Apoptos is was confirmed by DNA fragmentation analysis and electron microscopy, MnS OD overexpression specifically protected against cell death upon treatment with rotenone or antimycin. We examined bcl-x(L), p53 and poly(ADP-ribose) polymerase (PARP) to identify specific cellular pathways that might contrib ute to the mitochondrial-initiated ROS-mediated tell death. Cells overexpre ssing MnSOD contained less bcl-x(L) within the mitochondria compared to con trol (NEO) cells, therefore excluding the role of bcl-x(L), p53 was undetec table by Western analysis and examination of the proapoptotic protein bax, a p53 target gene, did not increase with treatment, Activation of caspase-3 (CPP-32) occurred in the NEO cells independent of cytochrome c release fro m the mitochondria, PARP, a target protein of CPP-32 activity, was cleaved to a 64 kDa fragment in the NEO cells prior to generation of nucleosomal fr agments. Taken together, these findings suggest that mitochondrial-mediated ROS generation is a key event by which inhibition of respiration causes ce ll death, and identifies CPP-32 and the PARP-linked pathway as targets of m itochondrial-derived ROS-induced cell death.