title: Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes alpha and theta.
abstract: Increased levels of the protein kinase C (PKC) isoenzymes alpha and theta 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-alpha and PKC-theta in MDR1 gene transcriptional regulation were explored. Human-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-alpha or PKC-theta at detectable levels were transfected with full-length PKC-alpha or PKC-theta 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-alpha 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-alpha. In contrast, PKC-theta 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-alpha and PKC-theta. The results support the search for therapeutic strategies directed specifically against PKC-alpha to ameliorate resistance of tumours against cytotoxic agents.
authors.affiliation: MRC Toxicology Unit, Leicester, UK.
authors.forename: P K
authors.initials: PK
authors.lastname: Gill
authors.forename: A
authors.initials: A
authors.lastname: Gescher
authors.forename: T W
authors.initials: TW
authors.lastname: Gant
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D020033:Protein Isoforms; D015500:Chloramphenicol O-Acetyltransferase; C496628:PRKCA protein, human; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate