Development of the superoxide-generating system during differentiation of the HL-60 human promyelocytic leukemia cell line.
Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.
D002454:Cell Differentiation; D002460:Cell Line; D004227:Dithionite / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000503:physiopathology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000502:physiology; D010587:Phagocytosis; D013481:Superoxides / Q000378:metabolism*
D013481:Superoxides; D004227:Dithionite; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases