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

Increased levels of the protein kinase C (PKC) isoenzymes alpha and theta o ccur in conjunction with MDR1 gene expression in cells and tissues that hav e acquired a multidrug resistance (MDR) phenotype. Studies using PKC activa tors or antisense strategies against PKC suggest that activation of PKC eng enders MDR1 gene transcription. In this study the potential roles of PKC-al pha and PKC-theta in MDR1 gene transcriptional regulation were explored. Hu man-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-alpha or PKC-theta at detectable levels were transfected with full-leng th PKC-alpha or PKC-theta genes driven by the ecdysone promoter. Stable tra nsfectants were selected by use of the appropriate antibiotics. Treatment o f these cells with ponasterone A induced expression of PKC that was catalyt ically active and underwent translocation and downregulation on exposure to 12-O-tetradecanoyl-13-phorbol acetate (TPA), These cells were used to anal yse PKC-mediated regulation of the MDR1 promoter by further transient trans fection 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-alpha expressing cells TPA caused activation of all promoter fragments to -29 bp. This finding suggests that TPA-inducible MDR 1 transcription mediated through the TPA responsive factor early growth res ponse 1 (EGR-1) in this region of the promoter may be due to activation of PKC-alpha. In contrast, PKC-theta activated only two MDR1 fragments, -982 a nd -612 bp. The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X. These data suggest that MDR1 promoter transc ription can be regulated by PKC-alpha and PKC-theta. The results support th e search for therapeutic strategies directed specifically against PKC-theta to ameliorate resistance of tumours against cytotoxic agents.