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Inhibition of Cell Proliferation, Protein Kinase C, and Phorbol Ester-induced fos Expression by the Dihydropyridine Derivative B859-35

Institute of Medical Chemistry and Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria

The dihydropyridine derivative B859-35 inhibits phospholipid- and calcium-dependent protein kinase C (PKC) in cell-free extracts from NIH3T3 cells. Inhibition is competitive with regard to phosphatidylserine. At 1 µM phosphatidylserine, half-maximal inhibition (IC₅₀) is obtained at approximately 2.5 µM B859-35. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-dependent activation of the Na⁺/H⁺ antiporter was used to determine whether the enzyme is also affected in intact cells. The activity of the antiporter was monitored by following the dimethylamiloride-sensitive cytosolic alkalinization. It is demonstrated that B859-35 depresses the TPA-induced alkalinization with an IC₅₀ of 5 µM, indicating that PKC in intact cells and the enzyme in cell-free extracts are equally sensitive to the drug. TPA-induced expression of the c-fos gene was used as an additional marker for intracellular PKC activity. Activation of c-fos expression was determined by measuring chloramphenicol acetyltransferase (CAT) activity in cells transfected with a c-fosCAT construct in which the CAT gene is expressed under the control of the endogenous human c-fos promoter. The studies revealed that 2.5 µM B859-35, a concentration equivalent to the IC₅₀ in cell-free extracts, significantly depresses TPA-induced c-fosCAT expression. B859-35 inhibited cellular proliferation of NIH3T3 cells with an IC₅₀ of approximately 5 µM. This is close to the IC₅₀ for the anti-PKC activity of B859-35. It is suggested that the inhibition of PKC contributes to the growth inhibition following exposure to B859-35.

¹ To whom requests for reprints should be addressed, at Institut für Medizinische Chemie und Biochemie, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria.

Received 4/29/91. Accepted 8/27/91.