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 mechanisms of peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of peroxynitrite and the kinetics of caspase-3 activation (starting 3-4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine 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 on peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates 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. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 microM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 microM) of peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to peroxynitrite.
Virág
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Division of Critical Care, Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Marmer
D J
DJ
Szabó
C
C
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018922:HL-60 Cells / Q000378:metabolism*; D006801:Humans; D009566:Nitrates / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors
D004791:Enzyme Inhibitors; D009566:Nitrates; D010455:Peptides; D010718:Phosphatidylserines; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C039642:peroxynitric acid; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases