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

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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‐tetradecanoyl‐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.