Expression, subcellular distribution and response to phorbol esters of protein kinase C (PKC) isozymes in drug-sensitive and multidrug-resistant KB cells - Evidence for altered regulation of PKC-alpha

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 hut 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 PKC alpha (cytosol only), PKC beta (membrane only), PKC epsilon (mainly membrane associated) and PKC zeta (both fractions). PKC delta and PKC gamma were not detected. The expression levels of PKC beta, PKC epsilon and PKC zeta were unchanged in KB-V1 cells: PKC alpha was modestly increased (approximate to 65%) in the resistant cells as further determined by enzyme assay. The cytosolic nature and increased expression of PKC alpha 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 PKC alpha. KB-V1 cells were found to differ markedly from KB-3 cells with respect to the translocation and down-regulation specifically of PKC alpha 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 PKC alpha whereas 1 mu M TPA was required to deplete KB-V1 cells of PKC alpha. Similar results were obtained when phorbol-12,13-dibutyrate was used instead of TPA. Defective TPA-mediated down-regulation of PKC alpha was also observed in another PKC alpha-overexpressing MDR cell line, KB-A1. Importantly, cellular uptake of radiolabeled phorbol ester was similar for both drag-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 PKC alpha in the MDR phenotype, and suggest that this isozyme may be expressed in a modified form or be subject to on altered regulation in MDR cells.