Poly ADP-ribosylation of a 90-kDa protein is involved in TNF-alpha-mediated cytotoxicity.
Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ribosylation plays a crucial role in this reaction. We have studied the role of ADP-ribosylation during TNF-alpha action in TNF-alpha-sensitive and TNF-alpha-resistant cells. Treatment of 3T3 cells with TNF-alpha, in the presence of [adenylate-32P]NAD followed by SDS-PAGE, revealed the involvement of specific ADP-ribosylation of a 90-kDa protein in TNF-alpha-mediated cytotoxicity. The stability of the ADP-ribosyl linkage on the 90 kDa protein in 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid at pH 9.0 confirmed that ADP-ribosylation of the 90 kDa protein was mediated by an enzymatic reaction. Analysis of ADP-ribose residues by phosphodiesterase hydrolysis showed that the 90-kDa protein was modified by poly ADP-ribosylation. Poly ADP-ribosylation of the 90-kDa protein concomitant with cytotoxicity was observed in all TNF-alpha-sensitive but not TNF-alpha-resistant cells. Inhibition of ADP-ribosylation of the 90-kDa protein by benzamide but not by benzoic acid abrogated cytotoxicity, which further suggested that the poly-ADP-ribosylation of the 90-kDa protein is causally related to TNF-alpha-induced cell death. Our results demonstrate that TNF-alpha modifies a specific protein by poly-ADP-ribosylation during its action. Furthermore, ADP-ribosylation of specific proteins may be yet another mechanism regulating protein function during cellular metabolism.
Division of Oral Biology, University of Pittsburgh, PA 15261.
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Agarwal
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Piesco
D016475:3T3 Cells; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D001549:Benzamides; D011506:Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000246:Adenosine Diphosphate Ribose; C037689:benzamide