Induction of cytosolic activation factor for NADPH oxidase in differentiated HL-60 leukemia cells.
NADPH oxidase was induced in HL-60 human promyelocytic leukemia cells when these cells were treated with 10(-7) M 1,25-dihydroxyvitamin D3 (VD3) for 4 days. The treated cells were disrupted by sonication, and the postnuclear fraction was separated into 48,000 X g supernatant (cytosol) and precipitate (membrane) fractions. Membrane-bound NADPH oxidase was activated in vitro with SDS and cytosol. However, the cytosol from untreated HL-60 cells could not activate NADPH oxidase. The cytosolic activity was induced 2 days after VD3 treatment and fully expressed on day 4. The activity was heat-sensitive and destroyed by trypsin. The possibility that the cytosolic activation factor is a protein kinase C (PKC) was then tested. Ca2+- and phospholipid-dependent PKC activity was low in the cytosol of untreated HL-60 cells but increased in the cytosols of VD3-treated cells 4 and 11 times, respectively, 2 days and 4 days after treatment. H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], inhibited PKC activity in a dose-dependent manner at 1-100 microM. Cytosolic activity of NADPH oxidase was not inhibited at all at those concentrations. Furthermore, PKC activity was lost when Ca2+ was omitted from the assay mixture, but NADPH oxidase was activated in the presence of EGTA. These results indicated that the cytosolic factor is not a PKC, and that NADPH oxidase in this cell-free system is activated by a mechanism that does not involve PKC.
Nozawa
R
R
Department of Bacteriology, Juntendo University School of Medicine, Tokyo.
Kato
H
H
Yokota
T
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000502:physiology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors
D007546:Isoquinolines; D009363:Neoplasm Proteins; D010879:Piperazines; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D002117:Calcitriol; D002118:Calcium