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.

The roles of poly(ADP-ribose)-metabolizing enzymes in alkylation-induced cell death

“…In agreement with recent reports [19,20,32], PAR induces AIF release, which then translocates from mitochondria to the nucleus, leading to nuclear condensation and programmed cell death. Moreover, whether highdose-MNNG-mediated necrotic cell death is induced [20,32] or whether apoptotic cell death by low doses of the alkylating agent was analyzed, this did not change the death link between PARP-1 and mitochondrial AIF release. Thus, the PARP-1-generated death signal operates in both death pathways, although the pathways differ in substantial intermediary steps, such as the involvement of caspases.…”

“…In contrast to recently published data showing that MNNG-mediated necrosis proceeds in a caspaseindependent manner [32], we observed that PAR polymer-induced cell death in HeLa cells after low doses of MNNG treatment could be prevented by caspase inhibition. In addition, we observed caspase cleavage of PARP-1 as well as caspases-7 and -9 activation, in combination with the results of the ADP/ATP-ratio assay, further corroborating an apoptotic form of cell death after low doses of MNNG.…”

“…In particular, PAR was found to mediate between DNA damage and the translocation of AIF from mitochondria to the nucleus. Another study focusing on MNNG-induced cell death and AIF release, suggested sequential activation of PARP-1, calpains, Bax and AIF in a single cell death pathway [32]. All of these studies were performed with high doses of MNNG (500 mM) inducing a caspase-independent necrotic form of programmed cell death.…”

A systems level strategy for analyzing the cell death network: implication in exploring the apoptosis/autophagy connection

“…Consistent with this phenotype, molecular analysis indicated that etoposide induced PAR ribosylation of proteins and the release of HMGB1 from the cells into the culture media (Figures 3b and c), two previously characterized hallmarks of programmed necrosis. 4,[18][19][20] In addition, the canonical apoptotic markers developed in response to the drug, including activation of the upstream initiator caspases2 and 9, and of the executioner caspases3 and 7 and the cleavage of their two substrates, PARP-1 and ICAD (Figure 3b and d). Also, fragmented nuclei and the release of cytochrome c from mitochondria to the cytosol were frequently detected (Supplementary Figure S5).…”

“…15 Next, the different components of this pathway (i.e., Atg5, DEDD, RHPN2) were knocked-down to assess their functional link to caspase3 activation. To this end, HeLa cells, which are rich in Cytokeratin, 18 were exposed to staurosporine, and caspase3 activation was quantified by the CaspaseGlo luminescent assay. We found that in this cellular setting the knock down of Atg5 reduced caspase3 activation, thus expanding the proapoptotic effects of Atg5 to other cell lines, and to another cell death-inducing agent (Figure 5d).…”