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

László Virág, Daniel J. Marmer, Csaba Szabó

    Research output: Contribution to journalArticle

    67 Scopus citations

    Abstract

    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 μM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 μM) 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.

    Original languageEnglish (US)
    Pages (from-to)1075-1082
    Number of pages8
    JournalFree Radical Biology and Medicine
    Volume25
    Issue number9
    DOIs
    StatePublished - Dec 1 1998

      Fingerprint

    Keywords

    • Caspase
    • Free radical
    • Inflammation Apoptosis
    • Nitric oxide
    • Peroxynitrite
    • Poly(ADP-ribose) synthetase
    • Shock

    ASJC Scopus subject areas

    • Medicine(all)
    • Toxicology
    • Clinical Biochemistry

    Cite this