CUPRIC NITRILOTRIACETATE INDUCES OXIDATIVE DNA-DAMAGE AND APOPTOSIS IN HUMAN LEUKEMIA HL-60 CELLS

Recent reports have implicated a possible role of reactive oxygen spec ies (ROS) in the induction and mediation of apoptosis and DNA damage. Oxidative DNA base modification induced by cupric nitrilotriacetate (C u-NTA) and the following apoptosis were observed in human promyelocyti c leukemia HL-60 cells. We measured the level of ROS in the cells by u sing a fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-D A), and the amount of a modified DNA base, 8-hydroxydeoxyguanosine (8- OHdG) by HPLC-ECD. It was found that Cu-NTA exposure significantly enh anced ROS and 8-OHdG formation in the cells. Meanwhile, we observed bo th DNA fragmentation and morphological changes characteristic of apopt osis, which was also determined quantitatively by flow cytometry and s howed dose- and time-dependent manners. Furthermore, several antioxida nts such as dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), and catalase were used to detect whether the apoptosis could be blocked. Only DMSO protected against this form of cell death. To elucidate mole cular events in the apoptosis, expressions of Bcl-2 protein family mem bers, such as Bcl-2, Bcl-X and Bar, and heat shock protein 70 (HSP-70) were measured by western blotting using specific antibodies. The leve ls of Bar and Bcl-X, remained largely unchanged, but the Bcl-2 and Bcl -X, expression showed down-regulation. After 24 h incubation in the pr esence of copper, the levels of Bcl-2 and Bcl-X, reduced about 33.8% a nd 51.1% compared with untreated cells, respectively. Furthermore, aft er 16 h incubation, the level of HSP-70 expression was about 3.4-fold greater than that in untreated cells, suggesting that HSP-70 is import ant in increasing resistance to oxidative stress induced by Cu-NTA. Bu t overexpression of HSP-70 failed to protect HL-60 cells from apoptosi s induced by Cu-NTA. We inferred that Cu-NTA may induce oxidative DNA damage through free radical injuries, which may turn on the apoptosis in HL-60 cells. (C) 1998 Elsevier Science Inc.