title: Isolation and characterization of a variant HL60 cell line defective in the activation of the NADPH oxidase by phorbol myristate acetate.
abstract: Promyelocytic human leukemia HL60 cells can be differentiated into neutrophil-like cells that exhibit an NADPH oxidase activity through direct stimulation of protein kinase C (PKC) with PMA or through formyl peptide receptor activation. We have isolated a variant HL60 clone that exhibited a conditional PMA-induced oxidative response depending on the agent used for the differentiation. While cells differentiated with DMSO responded to either PMA or N-formyl peptide (N-formyl-Met-Leu-Phe-Lys or fMLFK), cells differentiated with dibutyryl-cAMP (Bt2cAMP) responded to fMLFK but very poorly to PMA. However, in Bt2cAMP-differentiated cells, the expression of the different PKC isoforms was similar to that observed in DMSO-differentiated cells. Moreover, PMA was able to induce a normal phosphorylation of the cytosolic factor p47phox and to fully activate extracellular signal-regulated kinases (Erk1/2). Interestingly, Bt2cAMP-differentiated cells exhibited a strong and sustained O2- production when costimulated with PMA and suboptimal concentrations of fMLFK which were, per se, ineffective. This sustained response was only slightly reduced by the conjunction of the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor. Variant HL60 cells that were stably transfected with a constitutively active form of Rac1 were able, when differentiated with Bt2cAMP, to secrete oxidant following PMA stimulation. Altogether, the results suggest that, in addition to the phosphorylation of p47phox, the activation of NADPH oxidase requires the activation of a Rac protein through a pathway that diverges at a point upstream of MEK and that is independent of the activation of wortmannin sensitive PI3K.
authors.affiliation: Commissariat à l'Energie Atomique (CEA)/Grenoble, Laboratoire de Biochimie et de Biophysique des Systèmes Intégrés (UMR 314 CEA/Centre National de la Recherche Scientifique), Grenoble, France.
authors.forename: M
authors.initials: M
authors.lastname: Tardif
authors.forename: M J
authors.initials: MJ
authors.lastname: Rabiet
authors.forename: T
authors.initials: T
authors.lastname: Christophe
authors.forename: M D
authors.initials: MD
authors.lastname: Milcent
authors.forename: F
authors.initials: F
authors.lastname: Boulay
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008954:Models, Biological; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins
chemical_list: D000730:Androstadienes; D003994:Bucladesine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004121:Dimethyl Sulfoxide; D004791:Enzyme Inhibitors; D005419:Flavonoids; D019204:GTP-Binding Proteins; D007527:Isoenzymes; D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D009363:Neoplasm Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D013755:Tetradecanoylphorbol Acetate; D010566:Virulence Factors, Bordetella; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins; C049813:N-formylmethionyl-leucyl-phenylalanyl-lysine; C060505:neutrophil cytosolic factor 1; C482576:MAP3K1 protein, human; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one