Crucial role of apopain in the peroxynitrite-induced apoptotic DNA fragmentation

Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechani sms of peroxynitrite-induced apoptosis are poorly defined. The present stud y was designed to characterize the molecular mechanisms by which peroxynitr ite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but no t ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Consid ering the short half-life of peroxynitrite and the kinetics of caspase-3 ac tivation (starting 3-4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation pr oved to be essential for DNA fragmentation, because pretreatment of the cel ls with the specific tetrapeptide inhibitor DEVD-fmk completely blocked per oxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity wa s also significantly altered by the inhibition of caspase-3, whereas phosph atidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of peroxynitrite are mediated by poly(ADP-ribose) synthetase ( PARS) activation, we have also investigated the effect of PARS-inhibition o n peroxynitrite-induced apoptosis. We have found that PARS-inhibition modul ates peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2- benzopyrone were dependent on the concentration of peroxynitrite used. Whil e PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 mu M) Of peroxynitrite, a decreased DNA-fragmentation was found at high dos es (60 mu M) of peroxynitrite. PARS inhibition negatively affected viabilit y as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells expose d to peroxynitrite. (C) 1998 Elsevier Science Inc.