Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells.
It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.
Muranaka
Shikibu
S
Institute of Medical Science, Kurashiki Medical Center, Kurashiki, Japan.
Fujita
Hirofumi
H
Fujiwara
Takuzo
T
Ogino
Tetsuya
T
Sato
Eisuke F
EF
Akiyama
Jitsuo
J
Imada
Isuke
I
Inoue
Masayasu
M
Utsumi
Kozo
K
D000098:Acetophenones / Q000494:pharmacology; D000588:Amines / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019833:Genistein / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D006838:Hydrocarbons; D015151:Immunoblotting; D007476:Ionophores / Q000494:pharmacology; D008165:Luminol / Q000031:analogs & derivatives / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D019814:Myristic Acid / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D017382:Reactive Oxygen Species; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
D000098:Acetophenones; D000588:Amines; D000975:Antioxidants; D000001:Calcimycin; D002374:Catalase; D019833:Genistein; D006838:Hydrocarbons; D007476:Ionophores; D008165:Luminol; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D019814:Myristic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009861:Onium Compounds; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D013755:Tetradecanoylphorbol Acetate; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C079789:neutrophil cytosol factor 67K; C081614:L 012; C007517:diphenyleneiodonium; C056165:acetovanillone; C056558:CYBB protein, human; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; C013553:hexadecylamine