Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis. |
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Authors: | A Hamid Boulares Anna J Zoltoski Zaki A Sherif Alexander Yakovlev Mark E Smulson |
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Institution: | Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA. |
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Abstract: | 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. |
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Keywords: | apoptosis PARP-1 DFF TNF fibroblasts 50-kb DNA fragments cytochrome c |
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