Abstract
The multidrug resistance (MDR) phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells. Protein kinase C (PKC) has been implicated in the regulation of the MDR phenotype. In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the expression and activity of PKC isoenzymes in the human breast cancer cell line, MCF-7-WT, and an MDR subline, MCF-7-MDR, were examined. The MDR phenotype was associated with a 10-fold increase in calcium-dependent PKC activity as well as a 10-fold decrease in calcium-independent activity was due to a selective increase in the activity was due to a selective increase in the expression of PKC alpha as determined by Western blot analysis and hydroxylapatite chromatography. This increase in expression of PKC alpha was regulated at the message level as demonstrated by Northern blot analysis. The decrease in calcium-independent activity was caused by a decrease in the expression of PCK delta and epsilon. The significance of the increase in PKC alpha expression was then demonstrated by a commensurate 11-fold increase in the basal and stimulated phosphorylation of the myristolated alanine-rich C kinase substrate. Phosphorylation of P-glycoprotein, the cellular mediator of the MDR phenotype, was increased > 20-fold in the unstimulated MCF-7-MDR cell line and its phosphorylation was further increased 2-fold in response to phorbol 12-myristate 13-acetate. These changes paralleled the increases in P-glycoprotein pump function and the MDR phenotype underscoring the role for PKC alpha in regulating P-glycoprotein phosphorylation and function.
Highlights
From theDivisions of $Hematology/Oncology and **Geriatrics,Departments of Medicine and Cell Biology, Duke Universityand Durham Veterans Administration Medical Centers, Durham, North Carolina 27710, §Research Department, Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland, YMar-Delbruck-Labor in der Max-Planck-Gesellshaft, Carl-von-LinneWeg 10, 0-5000 Kaln30, Germany, and IlLuboratory of Molecular and Integrated Neuroscience, National Instituteof Environmental Health Sciences, Research Triangle Park, Durham, NorthCarolina 27709
Multidrug resistance (MDR)' is a phenotype expressed by In order to define the role of individual Protein kinase C (PKC) isoenzymesin drug resistance, we examined the expression and activity of
PKCActiuity in MCF-7- WT and MCF-7-multidrug resistance (MDR) Cell LinesIn theMCF-7 cell line, induction of the MDR phenotype has been associated with increases in total PKC activity [5]
Summary
The cell lysate was h, and thefilter was prehybridized for 3 h at 42 "Cin 50% formamide, centrifuged at 14,000 X g for 15 min in a microcentrifuge and the 2 X SSC, 50 mM (Na)2P04,0.1% SDS, 10 X Denhardt's solution, and supernatant was collected as the cytosolic fraction This cytosolic fraction (25 pl) was used as a source of PKC andassayed using Triton. Cium-dependent activity was determined in the presence of 0.1 mM 32P-hbelingof MCF-7-WT and MCF-7-MDR Cell Lines-Nearcalcium, and calcium-independent activity was assayed in the pres- confluent cell monolayers were washed two times with DPBS and ence of 10 mM excess EGTA.
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