Activation of c-Jun NH2-terminal kinase and subsequent CPP32/Yama during topoisomerase inhibitor beta-Lapachone-induced apoptosis through an oxidation-dependent pathway

beta-Lapachone (beta-Lap) has been found to inhibit DNA topoisomerases (Top os) by a mechanism distinct from that of other commonly known Topo inhibito rs. Here, we demonstrated a pronounced elevation of H2O2 and O-2(-) in huma n leukemia HL-60 cells treated with beta-Lap. Treatment with other Topo poi sons, such as camptothecin (CPT), VP-16, and GL331, did not have the same e ffect. On the other hand, antioxidant vitamin C (Vit C) treatment effective ly antagonized beta-Lap-induced apoptosis. This suggested that a reactive o xygen species (ROS)-related pathway was involved in beta-Lap-induced apopto sis program. We also found that c-Jun NH2-terminal kinase (JNK) but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 was persistently activated in apoptosis induced by beta-Lap. Overexpres sion of a dominant-negative mutant mitogen-activated protein kinase kinase kinase I (MEKK1-DN) or treatment with JNK-specific antisense oligonucleotid e or Vit C all prevented beta-Lap-induced JNK activation and the subsequent apoptosis. Only the expression of MEKK1-DN, not Vit C treatment, blocked t he JNK activity induced by CPT, VP-16, or GL331. These results confirm agai n that ROS acts as a mediator for JNK activation during beta-Lap-induced ap optosis. Furthermore, we found that beta-Lap can stimulate CPP32/Yama activ ity, which was, however, markedly inhibited by the MEKK1-DN expression or V it C treatment. Again, CPT-induced CPP32/Yama activation can be abolished b y MEKK1-DN but not by Vit C treatment. Taken together, these results indica te that beta-Lap but not other Topo inhibitors triggers apoptosis signaling , i.e., JNK and subsequent CPP32/Yama activation are mediated by the genera tion of ROS.