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

Third Division, Department of Medicine, Kobe University School of Medicine, Kobe 650, Japan

To identify the possible role of calcium ions in cell differentiation, 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 mM Ca²⁺ media. The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1,25-dihydroxyvitamin D₃, and all-trans-ß-retinoic acid inhibited the cell growth and induced mature macrophage and granulocyte phenotypes in 1.0 mM Ca²⁺ medium. 1,25-Dihydroxyvitamin D₃ and all-trans-ß-retinoic acid induced HL-60 differentiation to the same degree in 0.1 mM Ca²⁺ and 1.0 mM Ca²⁺ media. However, TPA failed to induce HL-60 differentiation or to inhibit proliferation in a 0.1 mM Ca²⁺ medium. The decrease of extracellular Ca²⁺ from 1.0 to 0.1 mM 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-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, inhibited proliferation of HL-60 cells in a dose-dependent manner. In contrast, H-7 selectively restored the proliferation of TPA-treated HL-60 cells and inhibited TPA-induced phenotypic differentiation. However, the same concentrations of 1-(5-isoquinolinylsulfonyl)-2,3-dimethylpiperazin and N-[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 D₃- and all-trans-ß-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.

¹ This research was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan.

² To whom requests for reprints should be addressed.

Received 7/ 2/85. Revised 10/15/85. Accepted 10/24/85.

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