Protein kinase C (PKC) comprises a family of related phospholipid‐dependent serine/threonine protein kinases. PKC has been implicated in the induction and maintenance of the multidrug‐resistance ...(MDR) phenotype but the role of different isozymes is not well understood. We compared the expression and subcellular distribution, and membrane association and down‐regulation induced by phorbol esters, of individual PKC isozymes in drug‐sensitive KB‐3 and multidrug‐resistant KB‐V1 human carcinoma cell lines. Immunoblotting with isozyme‐specific antibodies indicated the presence of PKCα (cytosol only), PKCβ (membrane only), PKCɛ (mainly membrane associated) and PKCζ (both fractions). PKCδ and PKCγ were not detected. The expression levels of PKCβ, PKCɛ and PKCζ were unchanged in KB‐V1 cells; PKCα was modestly increased (≈ 65%) in the resistant cells as further determined by enzyme assay. The cytosolic nature and increased expression of PKCα were confirmed by immunofluorescent localization studies. Revertant cells, obtained by culturing KB‐V1 cells in a drug‐free medium, regained drug sensitivity with a loss of P‐glycoprotein and a concomitant decrease in expression of PKCα. KB‐V1 cells were found to differ markedly from KB‐3 cells with respect to the translocation and down‐regulation specifically of PKCα upon exposure to 12‐O ‐tetradecanoyl‐1‐phorbol‐13‐acetate (TPA). Treatment with 30 nM TPA for 24 h completely depleted KB‐3 cells of PKCα whereas 1 μM TPA was required to deplete KB‐V1 cells of PKCα. Similar results were obtained when phorbol‐12,13‐dibutyrate was used instead of TPA. Defective TPA‐mediated down‐regulation of PKCα was also observed in another PKCα‐overexpressing MDR cell line, KB‐A1. Importantly, cellular uptake of radiolabeled phorbol ester was similar for both drug‐sensitive and MDR cells. Sensitive and resistant cells exhibited similar expression levels of RACK1, a PKC‐binding protein important in activation‐induced translocation. These findings further highlight the importance of PKCα in the MDR phenotype, and suggest that this isozyme may be expressed in a modified form or be subject to an altered regulation in MDR cells.
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