title: Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.
abstract: 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 approximately 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
authors.forename: A H
authors.initials: AH
authors.lastname: Boulares
authors.forename: A J
authors.initials: AJ
authors.lastname: Zoltoski
authors.forename: A
authors.initials: A
authors.lastname: Yakovlev
authors.forename: M
authors.initials: M
authors.lastname: Xu
authors.forename: M E
authors.initials: ME
authors.lastname: Smulson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D006720:Homozygote; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D003574:Cytochrome c Group; D011506:Proteins; D014409:Tumor Necrosis Factor-alpha; C109664:caspase-activated DNase inhibitor; D008769:Methylnitronitrosoguanidine; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases