Rare Variants in theABCG2Promoter Modulate In Vivo Activity

Abstract

Downloaded fromDMD# 79541 Page 3 forkhead homologue; HNF, hepatic nuclear factor; NRE, nuclear response element; RXR, retinoid X receptor; SNP, single nucleotide polymorphism.This article has not been copyedited and formatted. The final version may differ from this version. Abstract ABCG2 encodes the breast cancer resistance protein (BCRP), an efflux membrane transporter important in detoxification of xenobiotics. In the present study, the basal activity of the ABCG2 promoter in liver, kidney, intestine and breast cell lines was examined using luciferase reporter assays. The promoter activity of reference and variant ABCG2 sequences were compared in HepG2, HEK293T, HCT116 and MCF-7 cell lines. The ABCG2 promoter activity was strongest in the kidney and intestine cell lines. Four variants in the basal ABCG2 promoter (rs76656413, rs66664036, rs139256004 and rs59370292) decreased the promoter activity by 25-50% in at least three of the four cell lines. The activity of these four variants were also examined in vivo using the hydrodynamic tail vein assay, and two SNPs (rs76656413 and rs59370292) significantly decreased in vivo liver promoter activity by 50-80%. Electrophoretic mobility shift assays confirmed a reduction in nuclear protein binding to the rs59370292 variant probe, while the rs76656413 probe had a shift in transcription factor binding specificity. While both rs59370292 and rs76656413 are rare variants in all populations, they could contribute to patientlevel variation in ABCG2 expression in the kidney, liver and intestine.

Reporters of Cancer Stem Cells as a Tool for Drug Discovery

“…One of the widely used reporter construct for ABCG2 is ABCG2-Luc, which is used to mark ABCG2 expression and CSCs in different cancers including solid tumors and large Bcell lymphoma (143)(144)(145). To study the effect of methylation in the transcriptional regulation of ABCG2, luciferase constructs driven by ABCG2 promoter (pGL3-abcg2-Luc) and in vitro methylated ABCG2 promoter was used.…”