Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 mu M hydrogen peroxide (H2O2) for 4 h indu ced the morphological changes such as condensed and/or fragmented nuclei, i ncrease in caspase-3 subfamily protease activities, reduction of the procas pase-3 and a DNA fragmentation. To determine the role of caspases in H2O2-i nduced apoptosis, caspase inhibitors,acetyl-Tyr-Val-Ala-Asp-chloromethyl ke tone (Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acety l-Val-Glu-Ile-Asp-aldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily , caspase-3 subfamily and caspase-6, respectively, were loaded into the cel ls using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfa mily protease activation and DNA ladder formation in H2O2-treated HL-60 cel ls. This inhibitory effect was dose-dependent. These results suggest that c aspase-3, but not caspase-1 is required for commitment to ROS-triggered apo ptosis.