Abstract #912: Regulatory role of Notch1 in the expression of the multidrug resistance protein, ABCC1/MRP1

Multidrug resistance (MDR) is a major impediment to a successful chemotherapeutic outcome. Although the overexpression of ATP binding cassette (ABC) drug transporters is an evident phenotypic property in MDR, mechanisms behind the upregulation of these genes and proteins are not entirely understood. In our previous report (# 2300, Proc AACR, 2007), we presented data suggesting the potential involvement of intracellular Notch1 (Notch1^IC) in the overexpression of ABCC1/MRP1. We have now studied the regulatory role of Notch1^IC in the overexpression of ABCC1/MRP1 in MCF7/VP cells that overexpress this gene. We treated these cells with a \#947;-secretase inhibitor (GSI), DAPT, to prevent generation of Notch1^IC from Notch1^TM. Under these conditions, the expression of ABCC1/MRP1 was reduced ~28% at 50\#956;M DAPT. In complementary experiments, we used the parental cells of MCF7/VP and established MCF7/WT sublines stably overexpressing Notch1^IC. By immunoblot, these cells expressed increased levels of ABCC1/MRP1. Of considerable interest, these cells were as resistant to etoposide as the MCF7/VP cells, with an IC₅₀-value of 15.8±1.3 \#956;M, compared to 17.3±1.2 \#956;M for MCF7/VP, as determined by MTT assay. The control MCF7/WT-pcDNA3 cells had an IC₅₀ -value of 3.1±1.6 \#956;M. Based on these observations, we hypothesized that Notch1^IC is a regulator of the expression of ABCC1/MRP1 in MDR cells. To test this, we transfected MCF7/VP cells with a full-length ABCC1/MRP1 promoter-luciferase construct that covers the promoter region of ABCC1/MRP1 from -2008 to +103 bp (relative to the transcriptional start site), and treated these cells with 1, 10, and 50 \#956;M DAPT. We found that DAPT treatment decreased transcriptional activity of the ABCC1/MRP1 promoter to 87.8 ± 3.7, 82.8 ± 7.0, and 66.8 ± 7.8 percent, respectively, compared to transfection control cells. Of interest, ABCC1/MRP1 promoter activity in MCF7/WT cells stably expressing Notch1^IC (ICN1-c4) revealed increased transcriptional activity of 21.3 ± 3.1 relative luciferase unit (RLU) compared to 5.8 ± 1.1 RLU in the controls. By measuring luciferase activities in a series of MCF7/VP cells expressing ABCC1/MRP1 promoter-deletion constructs, we identified a promoter region between -91 and -411 bp that contains putative CBF1 binding sequences (GTGGAGA) that compare favorably to the consensus sequence (GTGGGAA) for this, a transcriptional coactivator that combines with Notch1^IC. In conclusion, our results suggest that Notch1^IC may play a role in regulating the overexpression of ABCC1/MRP1 in MDR cells. (Supported in part by CA-40570 from NCI [to WTB] and in part by UIC)

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 912.