Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage.
In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate beta-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of beta-nicotinamide adenine dinucleotide. In the cellular context, we show that Molt 3 cells respond to DNA damage by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with a dose-dependent acidification within 30 min. Whereas acidification by 0.15 pH units induced by 10 microM MNNG is reversed within 1 h, 100 , microinduced acidification by 0.5-0.6 pH units is persistent up to 7 h. Acidification is a general DNA damage response because H(2)O(2) exposure also acidifies Molt 3 cells, and MNNG causes acidification in Jurkat, U937, or HL-60 leukemia cells and in PARP(+/+) fibroblasts. Acidification is significantly decreased in the presence of PARP inhibitors or in PARP(-/-) fibroblasts, suggesting a major role for PARP activation in acidification. Inhibition of proton export through ATP-dependent Na(+)/H(+) exchanger is another major cause of acidification. Using the pH clamp method to either suppress or introduce changes in cellular pH, we show that brief acidification by 0.5-0.6 pH units may be a negative regulator of apoptosis while permitting necrotic death of cells with extensively damaged DNA.
Affar
El Bachir
el B
Laboratory for Skin Cancer Research, Hospital Research Center of Laval University (Centre Hospitalier Universitaire de Québec), Faculty of Medicine, Laval University, 2705, Laurier Boulevard, Room S16, Sainte-Foy, Quebec, Canada.
Shah
Rashmi G
RG
Dallaire
Annie-Karine
AK
Castonguay
Vincent
V
Shah
Girish M
GM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000477:Alkylating Agents / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D019169:Jurkat Cells; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D009336:Necrosis; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D013997:Time Factors; D020298:U937 Cells
D000477:Alkylating Agents; D009243:NAD; C053851:nicotinamide arabinoside adenine dinucleotide; D008769:Methylnitronitrosoguanidine; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases