The activation of PI 3-K and PKC ζ in PMA-induced differentiation of HL-60 cells

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol(3)P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 μM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC ζ is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC ζ during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC ζ activation. The activity of PI 3-K is essential for PKC ζ activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC ζ during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC ζ in the process of PMA-induced monocytic differentiation.