Isolation and characterization of a variant HL60 cell line defective in the activation of the NADPH oxidase by phorbol myristate acetate

Promyelocytic human leukemia HL60 cells can be differentiated into neutroph il-like cells that exhibit an NADPH oxidase activity through direct stimula tion of protein kinase C (PKC) with PMA or through formyl peptide receptor activation. We have isolated a variant HL60 clone that exhibited a conditio nal PMA-induced oxidative response depending on the agent used for the diff erentiation, While cells differentiated with DMSO responded to either PMA o r N-formyl peptide (N-formyl-Met-Leu-Phe-Lys or fMLFK), cells differentiate d with dibutyryl-cAMP (Bt(2)cAMP) responded to fMLFK but very poorly to PMA , However, in Bt(2)cAMP-differentiated cells, the expression of the differe nt PKC isoforms was similar to that observed in DMSO-differentiated cells, Moreover, PMA was able to induce a normal phosphorylation of the cytosolic factor p47(phox) and to fully activate extracellular signal-regulated kinas es (Erk1/2). Interestingly, Bt(2)cAMP-differentiated cells exhibited a stro ng and sustained O-2(-) production when costimulated with PMA and suboptima l concentrations of fMLFK which were, per se, ineffective. This sustained r esponse was only slightly reduced by the conjunction of the mitogen-activat ed protein (MAP) kinase kinase (MEK) inhibitor PD98059 and wortmannin, a ph osphatidylinositol-3 kinase (PI3K) inhibitor, Variant HL60 cells that were stably transfected with a constitutively active form of Rad were able, when differentiated with Bt(2)cAMP, to secrete oxidant following PMA stimulatio n. Altogether, the results suggest that, in addition to the phosphorylation of p47(phox), th, 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.