The Journal of Immunology, Vol 153, Issue 2 473-481, Copyright © 1994 by American Association of Immunologists

ARTICLES

Poly ADP-ribosylation of a 90-kDa protein is involved in TNF-alpha- mediated cytotoxicity

S Agarwal and NP Piesco
Division of Oral Biology, University of Pittsburgh, PA 15261.

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.