ACTIVATION OF HUMAN LEUKEMIA PROTEIN-KINASE-C BY TUMOR PROMOTERS AND ITS INHIBITION BY N-TRIFLUOROACETYLADRIAMYCIN-14-VALERATE (AD32)

N-Trifluoroacetyladriamycin-14-valerate (AD 32), a lipophilic, DNA non-binding analog of Adriamycin(R) (ADR), was found to be a potent inhibitor of the membrane-bound enzyme, protein kinase C (PKC). PKC was isolated and purified from human leukemia ML-1 cells, and the enzyme activity was shown to be activated by the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu). AD-32, nevertheless, inhibited the activation of PKC by TPA or PDBu. The IC50 values for AD 32 inhibition of PKC activation were 0.85-mu-M for TPA and 1.25-mu-M for PDBu. Under the same assay conditions, ADR demonstrated much higher IC50 values: 550-mu-M for TPA and > 350-mu-M for PDBu. The inhibition of PKC by AD 32 was further shown to be competitive in nature; AD 32 inhibited the binding of [H-3]PDBu to PKC. Therefore, AD 32 competes with the tumor promoter for the PKC binding site and prevents the latter from both interacting with the phospholipid and binding to PKC. These effects of AD 32 were reproduced in situ; incubation of human leukemia ML-1 cells with TPA showed an increased phosphorylation of cellular proteins, and the TPA-induced protein phosphorylation was inhibited by the addition of AD 32 to the cultured cells.