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

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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.

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Authors and Affiliations

  1. Cellular Defense and Carcinogenesis Section, Basic Research Laboratory, National Cancer Institute, NIH, [H.P.C., C.A.P., G.C.Y, Frederick, MD, USA

    Phillip J. Daschner, Henry P. Ciolino, Cathie A. Plouzek & Grace Chao Yeh

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  1. Phillip J. Daschner
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  4. Grace Chao Yeh
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Daschner, P.J., Ciolino, H.P., Plouzek, C.A. et al. Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells. Breast Cancer Res Treat 53, 229–240 (1999). https://doi.org/10.1023/A:1006138803392

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