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
Ba. Cloudheflin et al., 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, European journal of biochemistry, 239(3), 1996, pp. 796-804
Protein kinase C (PKC) comprises a family of related phospholipid-depe
ndent 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 compare
d tile expression and subcellular distribution, and membrane associati
on and down-regulation induced by phorbol esters, of individual PKC is
ozymes in drug-sensitive KB-3 and multidrug-resistant KB-V1 human carc
inoma cell lines. Immunoblotting with isozyme-specific antibodies indi
cated the presence of PKC alpha (cytosol only), PKC beta (membrane onl
y), PKC epsilon (mainly membrane associated) and PKC zeta (both fracti
ons). 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 ce
lls 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 t
o 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 wh
ereas 1 mu M TPA was required to deplete KB-V1 cells of PKC alpha. Sim
ilar results were obtained when phorbol-12,13-dibutyrate was used inst
ead of TPA. Defective TPA-mediated down-regulation of PKC alpha was al
so 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 Im
portant in activation-induced translocation. These findings further hi
ghlight 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.