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Effect of the activated Raf protein kinase on the human multidrug resistance 1 (MDR1) gene promoter
1Department of Biochemistry, College of Medicine, Pusan National University, South Korea.
Abstract:
Revealing the regulatory mechanism of the multidrug resistance 1 (MDR1) gene is important to gain understanding of MDR in tumor cells. Using MDR1 deletion constructs and the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we examined the effect of the activated Raf on human MDR1 promoter activity in transient expression assay and stable transfectants of GHE-L cells. A DNA sequence exhibiting strong activation of MDR1 promoter by 22W was located between -197 and -136 containing the upstream heat shock element (HSE) motifs without other regulatory elements, whereas the MDR1 deletion construct containing downstream HSE motif showed a relatively weaker activation by 22W. We observed that the activated Raf significantly potentiated the induction of MDRCAT activity in GHE-L cells by sodium arsenite or heat shock, which stimulates heat shock factor (HSF) binding to HSE. In addition, protein kinase A inhibitor (H-87) blocked the activation of the MDR1 promoter by 22W in GHE-L cells in a dose-dependent manner. From these results, we propose the possibility that Raf- and protein kinase A-dependent pathways control the transcription of MDR1 gene via a mechanism involving the modulation of HSF activity.
Insights
Understanding multidrug resistance (MDR) in cancer cells requires knowing how the MDR1 gene is regulated. Activated Raf kinase influences MDR1 gene transcription through heat shock factor (HSF) and protein kinase A pathways.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Regulation
Background:
- Multidrug resistance (MDR) in tumor cells is a significant challenge in cancer therapy.
- The multidrug resistance 1 (MDR1) gene plays a crucial role in MDR.
- Understanding the regulatory mechanisms of MDR1 is essential for developing effective cancer treatments.
Purpose of the Study:
- To investigate the regulatory mechanism of the human MDR1 gene.
- To examine the effect of activated Raf on MDR1 promoter activity.
- To elucidate the role of heat shock factor (HSF) and protein kinase A in MDR1 gene transcription.
Main Methods:
- Utilized MDR1 deletion constructs and a constitutively active c-Raf mutant (22W).
- Performed transient expression assays and established stable transfectants (GHE-L cells).
- Analyzed DNA sequences responsible for MDR1 promoter activation and assessed the impact of inhibitors.
Main Results:
- Identified a DNA sequence between -197 and -136, containing upstream heat shock element (HSE) motifs, as critical for 22W-mediated activation.
- Demonstrated that activated Raf potentiates MDR1 promoter activity induction by sodium arsenite or heat shock.
- Showed that protein kinase A inhibitor (H-87) dose-dependently blocked 22W-induced MDR1 promoter activation.
Conclusions:
- Propose that Raf and protein kinase A-dependent pathways regulate MDR1 gene transcription.
- Suggest a mechanism involving the modulation of heat shock factor (HSF) activity.
- Highlight the potential of targeting these pathways to overcome MDR in cancer.