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

Eur J Biochem. 2001 Aug;268(15):4151-7. doi: 10.1046/j.1432-1327.2001.02326.x.

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.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Blotting, Western
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / metabolism*
  • Catalysis
  • Chloramphenicol O-Acetyltransferase / metabolism
  • Drug Resistance, Neoplasm / genetics
  • Enzyme Inhibitors / pharmacology
  • Gene Deletion
  • Gene Expression Regulation, Enzymologic*
  • Genes, Reporter
  • Humans
  • Indoles / pharmacology
  • Isoenzymes / metabolism*
  • Maleimides / pharmacology
  • Phenotype
  • Promoter Regions, Genetic*
  • Protein Isoforms
  • Protein Kinase C / chemistry*
  • Protein Kinase C / metabolism*
  • Protein Kinase C-alpha
  • Protein Kinase C-theta
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription, Genetic
  • Transfection
  • Tumor Cells, Cultured

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Enzyme Inhibitors
  • Indoles
  • Isoenzymes
  • Maleimides
  • Protein Isoforms
  • Chloramphenicol O-Acetyltransferase
  • PRKCA protein, human
  • PRKCQ protein, human
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Protein Kinase C-theta
  • bisindolylmaleimide I
  • Tetradecanoylphorbol Acetate