DNA fragmentation and NAD depletion. Their relation to the turnover of endogenous mono(ADP-ribosyl) and poly(ADP-ribosyl) proteins.

Abstract

Treatment of Ehrlich ascites tumor cells with the trifunctional alkylating agent 2,3-5-tris(ethyleneimino)benzoquinone-1,4 (triaziquonum) led to rapid fragmentation of DNA and depletion of NAD while poly(ADP-ribose) synthetase activity showed a retarded increase. Poly(ADP-ribosyl) residues in treated cells increased 4- to 30-fold, but transiently, and in a dose-dependent manner, exhibiting the same initial kinetics as the loss of NAD and the appearance of DNA strand breaks when determined by the nucleoid method. Although the amounts of "activated ADP-ribosyl" groups present in the substrate NAD (80 nmol/10(8) cells) exceeded by far basal and triaziquonum-induced poly(ADP-ribosyl) groups (up to 250 pmol/10(8) cells), accelerated formation of the polymer, nevertheless, may explain at least partially the loss of NAD seen under these conditions. Addition of benzamide, a potent inhibitor of poly(ADP-ribose) synthetase, to triaziquonum-treated cells effected an immediate drop of poly(ADP-ribose) to basal values. The data indicate a biphasic decay, the half-life of greater than 85% of the polymeric ADP-ribosyl groups exhibiting a t1/2 less than 1 min under these conditions, while the residual fraction died away with t1/2 approximately 6 min. Treatment with the DNA fragmenting agent also led to a 9-fold increase of nuclear mono(ADP-ribosyl) groups, while cytoplasmic mono(ADP-ribosyl) protein conjugates were not significantly affected. The apparent half-life of nuclear mono (ADP-ribosyl) protein conjugates (8-10 min) at peak elevation was definitely longer than that of poly(ADP-ribosyl) residues. This result is consistent with the interpretation that accumulation of mono(ADP-ribosyl) groups is due to a retarded removal of the primary ADP-ribosyl group from the acceptor protein by a separate mono(ADP-ribosyl) protein glycohydrolase, being the rate-limiting step in the overall turnover of poly(ADP-ribosyl) residues.