Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes α and Θ

Abstract

Increased levels of the protein kinase C (PKC) isoenzymes α and Θ occur in conjunction with MDR1 gene expression in cells and tissues that have acquired a multidrug resistance (MDR) phenotype. Studies using PKC activators or antisense strategies against PKC suggest that activation of PKC engenders MDR1 gene transcription. In this study the potential roles of PKC-α and PKC-Θ in MDR1 gene transcriptional regulation were explored. Human-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-α or PKC-Θ at detectable levels were transfected with full-length PKC-α or PKC-Θ genes driven by the ecdysone promoter. Stable transfectants were selected by use of the appropriate antibiotics. Treatment of these cells with ponasterone A induced expression of PKC that was catalytically active and underwent translocation and down-regulation on exposure to 12-O-etradecanoyl-13-phorbol acetate (TPA). These cells were used to analyse PKC-mediated regulation of the MDR1 promoter by further transient transfection with either 1073 bp of the MDR1 gene promoter or deletion fragments thereof to -8 bp, each linked to a chloramphenicol acetyl transferase (CAT) reporter gene. In PKC-α expressing cells TPA caused activation of all promoter fragments to -29 bp. This finding suggests that TPA-inducible MDR1 transcription mediated through the TPA responsive factor early growth response 1 (EGR-1) in this region of the promoter may be due to activation of PKC-α. In contrast, PKC-Θ activated only two MDR1 fragments, -982 and -612 bp. The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X. These data suggest that MDR1 promoter transcription can be regulated by PKC-α and PKC-Θ. The results support the search for therapeutic strategies directed specifically against PKC-α to ameliorate resistance of tumours against cytotoxic agents.