NADPH Oxidase-mediated Generation of Reactive Oxygen Species is Critically Required for Survival of Undifferentiated Human Promyelocytic Leukemia Cell Line HL-60

Abstract

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H²O² can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.

• NADPH oxidase
• Reactive oxygen species
• HL-60 cells
• Cell survival
• ROS, reactive oxygen species
• NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase
• FBS, fetal bovine serum
• SOD, superoxide dismutase
• CAT, catalase
• DPI, diphenylene iodonium
• APO, apocynin
• ETYA, 5,8,11,14-eicosatetraynic acid
• IM, indomethacin
• l-NAME, NG-nitro-l-arginine methylester
• AP, allopurinol
• NOS, nitric oxide synthase
• OZ, opsonified zymosan
• DMSO, Dimethyl Sulfoxide
• HRP, Horseradish peroxidase
• MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
• SDS, sodium dodecyl sulfate
• AO, acridine orange
• EB, ethidium bromide