NAD(+) consumption in carcinogen-treated hamster cells overexpressing a dominant negative mutant of poly(ADP-ribose) polymerase

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear prote ins catalyzed by poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) with NAD() serving as substrate. PARP is activated upon binding of its aminoterminal DNA-binding domain to DNA double or single strand breaks, and is thought t o play a role in cellular responses to genotoxic stress. It is known that t reatment of cells with high doses of DNA-damaging agents can cause potentia lly lethal consumption of NAD(+) that can be prevented by chemical inhibito rs of PARP, In order to manipulate PARP enzyme activity, me had established a cell culture system with hormone-inducible overexpression of the mere DN A-binding domain of PARP acting as a dominant negative mutant of this enzym e. By using this system we and others had shown that inhibition of PARP lea ds to increased genetic instability and apoptosis following DNA damage. Her e we show the unexpected result that despite efficient transdominant inhibi tion of poly(ADP-ribosyl)ation, there is substantial NAD+ consumption detec table in cells exposed to high doses of gamma-irradiation, or the alkylatin g agent N-methyl-N' (C) 1999 Academic Press.