Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells

Breast Cancer Res Treat. 1999 Feb;53(3):229-40. doi: 10.1023/a:1006138803392.

Abstract

The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (-123 to -108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Blotting, Western
  • Breast Neoplasms / metabolism*
  • Cell Nucleus / metabolism
  • DNA / metabolism
  • Doxorubicin / pharmacology
  • Drug Resistance, Neoplasm
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • Gene Expression / drug effects
  • Humans
  • Paclitaxel / pharmacology
  • Promoter Regions, Genetic / drug effects
  • Proto-Oncogene Proteins c-fos / biosynthesis
  • Proto-Oncogene Proteins c-jun / biosynthesis
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription Factor AP-1 / metabolism*
  • Tumor Cells, Cultured
  • Up-Regulation / drug effects
  • Vinblastine / pharmacology

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • RNA, Messenger
  • Transcription Factor AP-1
  • Vinblastine
  • Doxorubicin
  • DNA
  • Paclitaxel