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Roles of DNA Fragmentation Factor and Poly(ADP-ribose) Polymerase in an Amplification Phase of Tumor Necrosis Factor-induced Apoptosis

2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês

10.1074/jbc.m100629200

1083-351X

A. Hamid Boulares, Anna J. Zoltoski, Alexander G. Yakovlev, Ming Xu, Mark E. Smulson,

DNA Repair Mechanisms

During apoptosis, endonucleases cleave DNA into 50–300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45 −/− and control (DFF45 +/+ ) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into ∼50-kb fragments in DFF45 +/+ fibroblasts but not in DFF45 −/− cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45 −/− cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c , induced by TNF were similarly delayed in DFF45 −/− fibroblasts. DFF45 −/− and DFF45 +/+ cells were equally sensitive to the DNA-damaging agent and PARP activator N -methyl- N ′-nitro- N -nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45 +/+ cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.