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 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).

Küpper JH ，Müller M ，Wolf I 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates carcinogen induced gene amplification in SV40-transformed Chinese hamster cells.

Kupper JH ，Müller M ，Bürkle A 《CANCER RESEARCH》

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase.

Gao H ，Coyle DL ，Meyer-Ficca ML ，Meyer RG ，Jacobson EL ，Wang ZQ ，Jacobson MK ... -  《EXPERIMENTAL CELL RESEARCH》

Ex vivo supplementation with nicotinic acid enhances cellular poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood mononuclear cells.

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD(+) as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD(+) depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD(+) precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD(+) levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N'-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD(+) levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death.

Weidele K ，Kunzmann A ，Schmitz M ，Beneke S ，Bürkle A ... -  《-》

Functional interactions of p53 with poly(ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: covalent poly(ADP-ribosyl)ation of p53 by exogenous PARP and noncovalent binding of p53 to the M(r) 85,000 proteolytic fragment.

Kumari SR ，Mendoza-Alvarez H ，Alvarez-Gonzalez R 《CANCER RESEARCH》