Inhibition of cell proliferation, protein kinase C, and phorbol ester-induced fos expression by the dihydropyridine derivative B859-35.
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 microM phosphatidylserine, half-maximal inhibition (IC50) is obtained at approximately 2.5 microM 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 microM, 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 microM 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 microM. 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.
Uberall
F
F
Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Austria.
Maly
K
K
Egle
A
A
Doppler
W
W
Hofmann
J
J
Grunicke
H H
HH
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004095:Dihydropyridines / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D004095:Dihydropyridines; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D015500:Chloramphenicol O-Acetyltransferase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine; C054074:niguldipine