Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells

Autor: Isuke Imada, Shikibu Muranaka, Takuzo Fujiwara, Tetsuya Ogino, Masayasu Inoue, Hirofumi Fujita, Eisuke F. Sato, Jitsuo Akiyama, Kozo Utsumi
Rok vydání: 2005
Předmět:
Time Factors
Physiology
Neutrophils
Cellular differentiation
Clinical Biochemistry
Biochemistry
Myristic Acid
Antioxidants
chemistry.chemical_compound
Onium Compounds
Staurosporine
Amines
Calcimycin
General Environmental Science
chemistry.chemical_classification
Oxidase test
NADPH oxidase
Membrane Glycoproteins
Superoxide
Cell Differentiation
Catalase
Genistein
rac GTP-Binding Proteins
N-Formylmethionine Leucyl-Phenylalanine
NADPH Oxidase 2
Tetradecanoylphorbol Acetate
Luminol
medicine.drug
Blotting
Western
Immunoblotting
HL-60 Cells
Biology
Cell Line
medicine
Humans
Molecular Biology
Reactive oxygen species
Ionophores
Acetophenones
Membrane Transport Proteins
NADPH Oxidases
Cell Biology
Phosphoproteins
Molecular biology
Hydrocarbons
chemistry
Cell culture
Apocynin
biology.protein
General Earth and Planetary Sciences
Reactive Oxygen Species
Zdroj: Antioxidantsredox signaling. 7(9-10)
ISSN: 1523-0864
Popis: 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.
Databáze: OpenAIRE
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