Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis.
DNA fragmentation factor (DFF) comprises DFF45 and DFF40 subunits, the former of which acts as an inhibitor of the latter (the catalytic subunit) and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks the generation of 50-kb DNA fragments and confers resistance to apoptosis. We recently suggested that the early fragmentation of DNA by DFF and the consequent activation of poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial dysfunction, and activation of caspase-3 contribute to an amplification loop in the apoptotic process. To verify the existence of such a loop, we have now examined the effects of restoring DFF expression in DFF45-deficient fibroblasts. Co-transfection of mouse DFF45(-/-) fibroblasts with plasmids encoding human DFF40 and DFF45 reversed the apoptosis resistance normally observed in these cells. The DFF45(-/-) cells regained the ability to fragment their DNA into 50-kb pieces in response to TNF, which resulted in a marked activation of PARP-1 and a concomitant depletion of intracellular NAD. DFF expression also resulted in an increase both in cytochrome c release into the cytosol and in caspase-3 activation triggered by TNF. These results support the importance of DFF, PARP-1, mitochondria, and caspase-3 in an amplification phase of TNF-induced apoptosis.
Boulares
A Hamid
AH
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Zoltoski
Anna J
AJ
Sherif
Zaki A
ZA
Yakovlev
Alexander
A
Smulson
Mark E
ME
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects / Q000235:genetics; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008817:Mice, Mutant Strains; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D021122:Protein Subunits; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D051017:Apoptosis Regulatory Proteins; D003574:Cytochrome c Group; D047908:Intracellular Signaling Peptides and Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D021122:Protein Subunits; D011506:Proteins; D014409:Tumor Necrosis Factor-alpha; C109664:caspase-activated DNase inhibitor; D009243:NAD; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; C113696:DFFB protein, human; D003851:Deoxyribonucleases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases