Mechanisms of hormonal regulation of CAD gene expression and inhibition by Aryl hydrocarbon receptor agonist in human breast cancer cells
Year: 2007
Type: dissertation
Abstract: The CAD gene is trifunctional and expresses carbamoylphosphate\nsynthetase/aspartate carbamyltransferase/dihydroorotase, which are required for\npyrimidine biosynthesis. CAD gene activities are induced in MCF-7 human breast cancer\ncells, and treatment of MCF-7 or ZR-75 cells with 17b-estradiol (E2) resulted in a 3-5\nfold increase in CAD mRNA levels in both cell lines. E2 induced reporter gene activity\nin MCF-7 and ZR-75 cells transfected with a construct containing the growth-responsive\n-90/+115 (pCAD1) region of the CAD gene promoter, which contains three upstream\nGC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD\ngene promoter demonstrated that only the GC boxes that bind Sp1 protein were required\nfor E2-responsiveness. Results of gel shift and chromatin immunoprecipitation (CHIP)\nassays show that both Sp1 and estrogen receptor a (ERa) interact with the GC-rich\nregion of the CAD gene promoter. Moreover, hormone-induced transactivation of\npCAD1 was inhibited by cotransfection with dominant-negative Sp1 expression plasmid\nand small inhibitory RNA for Sp1. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the CAD gene is also\nregulated by a nonclassical ERa/Sp1-mediated pathway.\n2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon\nreceptor (AhR) ligands suppress several E2-induced responses in the rodent uterus and\nmammary tumors and in human breast cancer cells. TCDD inhibited hormone-induced\nactivation of CAD mRNA levels and reporter gene activity in MCF-7 and ZR-75 cells\ntransfected with E2-responsive pCAD promoter constructs. E2-mediated transactivation\nof pCAD constructs with a mutant inhibitory dioxin responsive element DRE (iDRE)\nwere also inhibited by TCDD suggesting that inhibitory AhR-ERa/Sp1 crosstalk was\niDRE-independent. It was not possible to determine whether the levels of ERa in cells\ncotreated with E2 plus TCDD were limiting since the proteasome inhibitor MG132 itself\ndirectly decreased CAD mRNA levels. Using fluorescence resonance energy transfer\n(FRET), it was shown that both E2 and TCDD enhanced AhR-ERa interactions. E2 also\ninduced interactions between ERa and Sp1. However cotreatment with TCDD abrogated\nthis effect. Results of this study demonstrate a unique model of AhR-ERa crosstalk\nwhere the liganded AhR inhibits ERa-Sp1 interactions and also recruits ERa to Ahresponsive\ngene promoters such as CYP1A1.
Author: Shaheen Khan
Language: English
Subfield: Endocrinology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2738716042