Inhibition of Phorbol Ester-induced Phenotypic Differentiation of HL-60 Cells by 1-(5-lsoquinolinylsulfonyl)-2-methylpiperazine, a Protein Kinase Inhibitor1

To identify the possible role of calcium ions in cell differentia tion, we studied the extracellular Ca2+requirement and the effect of Ca2+/phospholipid-dependent protein kinase (protein kinase C) inhibitor on proliferation and differentiation of human promyelocytic leukemic HL-60 cells. HL-60 cells grew equally well in 0.1 and 1.0 mw Ca2+ media. The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1,25-dihydroxyvitamin D3, and allfrans-0-retinoic acid inhibited the cell growth and induced mature macrophage and granulocyte phenotypes in 1.0 mw Ca2+ me dium. 1,25-Dihydroxyvitamin D3 and all-fra/7S-/3-retinoic acid in duced HL-60 differentiation to the same degree in 0.1 ITÃŒM Ca2+ and 1.0 rriM Ca2+ media. However, TPA failed to induce HL-60 differentiation or to inhibit proliferation in a 0.1 mw Ca2+medium. The decrease of extracellular Ca2+from 1.0 to 0.1 mw caused a significant drop in the intracellular Ca2+ level in undifferentiated and TPA-treated HL-60 cells, although no rapid change in cytosolic Ca2+ was detected in response to TPA addition. 1-(5lsoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, inhibited proliferation of HL-60 cells in a dose-de pendent manner. In contrast, H-7 selectively restored the prolif eration of TPA-treated HL-60 cells and inhibited TPA-induced phenotypic differentiation. However, the same concentrations of 1-(5-isoquinolinylsulfonyl)-2,3-dimethylpiperazin and A/-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, analogues of H-7 that inhibit protein kinase C more weakly, had no effect on the proliferation or differentiation induction. H-7 also suppressed 1,25-dihydroxyvitamin D3and all-frans-0-retinoic acid-induced phenotypic changes of HL-60 cells but did not eliminate the growth inhibition by these inducers. These results demonstrate the Ca2+ requirement and the protein kinase C involvement in phorbol ester-induced phenotypic differentiation of HL-60 cells.