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 microM hydrogen peroxide (H2O2) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H2O2-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-Ile-Asp-aldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H2O2-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered apoptosis.
Matsura
T
T
Department of Biochemistry, Faculty of Medicine, Tottori University, Yonago, Japan. tmatsura@grape.med.tottori-u.ac.jp
Kai
M
M
Fujii
Y
Y
Ito
H
H
Yamada
K
K
D000590:Amino Acid Chloromethyl Ketones / Q000008:administration & dosage / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009842:Oligopeptides / Q000008:administration & dosage / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; D017382:Reactive Oxygen Species; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases