Virág L, Marmer D J, Szabó C
Division of Critical Care, Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Free Radic Biol Med. 1998 Dec;25(9):1075-82. doi: 10.1016/s0891-5849(98)00139-7.
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.
过氧亚硝酸盐是超氧化物和一氧化氮反应生成的一种细胞毒性氧化剂，已知其可导致程序性细胞死亡。然而，过氧亚硝酸盐诱导细胞凋亡的机制尚不清楚。本研究旨在阐明过氧亚硝酸盐诱导HL-60细胞凋亡的分子机制，特别关注半胱天冬酶的作用。通过荧光肽切割测定，过氧亚硝酸盐可诱导凋亡蛋白酶/半胱天冬酶-3的激活，但不能诱导ICE/半胱天冬酶-1的激活。考虑到过氧亚硝酸盐的半衰期较短以及半胱天冬酶-3激活的动力学（过氧亚硝酸盐处理后3-4小时开始），该酶不太可能直接被氧化剂激活。半胱天冬酶-3的激活被证明对DNA片段化至关重要，因为用特异性四肽抑制剂DEVD-fmk预处理细胞可完全阻断过氧亚硝酸盐诱导的DNA片段化。半胱天冬酶-3的抑制也显著改变了过氧亚硝酸盐诱导的细胞毒性，而磷脂酰丝氨酸暴露不受DEVD-fmk处理的影响。由于过氧亚硝酸盐的许多作用是由聚（ADP-核糖）合成酶（PARS）激活介导的，我们还研究了PARS抑制对过氧亚硝酸盐诱导的细胞凋亡的影响。我们发现PARS抑制可调节HL-60细胞中过氧亚硝酸盐诱导的凋亡DNA片段化。PARS抑制剂3-氨基苯甲酰胺和5-碘-6-氨基-1,2-苯并吡喃的作用取决于所用过氧亚硝酸盐的浓度。虽然PARS抑制在低剂量（15 microM）过氧亚硝酸盐时导致DNA片段化增加，但在高剂量（60 microM）过氧亚硝酸盐时发现DNA片段化减少。通过流式细胞术测定，PARS抑制对细胞活力有负面影响。这些数据表明半胱天冬酶-3在介导暴露于过氧亚硝酸盐的HL-60细胞凋亡DNA片段化中起关键作用。
