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

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins 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 to play a role in cellular responses to genotoxic stress. It is known that treatment of cells with high doses of DNA-damaging agents can cause potentially lethal consumption of NAD(+) that can be prevented by chemical inhibitors of PARP. In order to manipulate PARP enzyme activity, we had established a cell culture system with hormone-inducible overexpression of the mere DNA-binding domain of PARP acting as a dominant negative mutant of this enzyme. By using this system we and others had shown that inhibition of PARP leads to increased genetic instability and apoptosis following DNA damage. Here we show the unexpected result that despite efficient trans-dominant inhibition of poly(ADP-ribosyl)ation, there is substantial NAD(+) consumption detectable in cells exposed to high doses of gamma-irradiation, or the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).