NITRIC-OXIDE COOPERATES WITH HYDROGEN-PEROXIDE IN INDUCING DNA FRAGMENTATION AND CELL-LYSIS IN MURINE LYMPHOMA-CELLS

We examined whether NO and H2O2 could interact in inducing DNA fragmen tation and cell death. H2O2 and the NO-releasing compounds sodium nitr oprusside (SNP) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) by the mselves elicited lysis of YAC-1 murine lymphoma cells in a concentrati on-dependent manner. Exposure of the cells to a combination of sublyti c concentrations of SNP (0.78 mM) plus H2O2 (7.8 mu M) or SNAP (0.18 m M) plus H2O2 (7.8 mu M) resulted in cell death which is mediated, in p art, through apoptosis. Evidence for this direction is provided by flu orescence microscopic evaluation of the cells, which revealed the pres ence of changes in nuclear morphology characteristic of apoptosis in 3 0-40% of lymphoma cells and by the specific pattern of internucleosoma l DNA fragmentation detected by gel electrophoresis. The cytotoxic eff ect of SNP plus H2O2 could be effectively inhibited by either oxyhaemo globin, which binds NO, or catalase, which eliminates H2O2. Partial pr otection from SNP-plus-H2O2-induced cell lysis was observed with the p oly(ADP-ribose) polymerase inhibitors, nicotinamide and 3-amino-benzam ide, parallelling their ability to reverse depletion of cellular NAD() pools. These results indicate an interaction between NO and H2O2 whi ch leads to a markedly enhanced cytotoxic activity, in part, via induc tion of apoptosis and suggest that poly(ADP-ribosylation) and subseque nt NAD(+) depletion mediate, at least in part, this cytotoxic activity .