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Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells
P J Daschner1, H P Ciolino, C A Plouzek
1Intramural Research Support Program, SAIC, NCI-FCRDC, Frederick, MD 21702, USA.
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.
Insights
Increased activator protein 1 (AP-1) activity, including c-jun and c-fos expression and DNA binding, correlates with multidrug resistance (MDR) in breast cancer cells. This suggests AP-1 regulates MDR1 gene expression, contributing to drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Multidrug resistance (MDR) in cancer is a significant clinical challenge.
- The MDR1 gene encodes P-glycoprotein (Pgp), a key efflux pump in MDR.
- The role of transcription factors in MDR development requires further elucidation.
Purpose of the Study:
- To investigate the role of activator protein 1 (AP-1) in the development of multidrug resistance (MDR) in MCF-7 human breast cancer cells.
- To determine if AP-1 activity, DNA binding, and transactivating function are altered in MDR cells.
- To assess the impact of AP-1 on the expression of the MDR1 gene.
Main Methods:
- Analysis of c-jun and c-fos mRNA levels using RT-PCR.
- Electrophoretic mobility shift assays (EMSA) to assess AP-1 DNA binding activity.
- Luciferase reporter assays to measure AP-1 transactivating function and MDR1 promoter activity.
- Western blot analysis to determine Pgp levels.
Main Results:
- MDR MCF-7 cells exhibited increased c-jun and c-fos mRNA expression compared to drug-sensitive cells.
- AP-1 DNA binding activity was significantly elevated in nuclear extracts from MDR cells.
- AP-1 mediated gene expression and binding to the MDR1 promoter were enhanced in resistant cells.
- Overexpression of c-jun in sensitive cells led to increased MDR1 mRNA and Pgp levels.
Conclusions:
- Increased AP-1 activity, including enhanced DNA binding and transactivation, is associated with multidrug resistance in MCF-7 breast cancer cells.
- AP-1 directly binds to the MDR1 promoter and activates its transcription.
- Elevated AP-1 activity is a potential key mediator of MDR by regulating MDR1 gene expression.