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Mechanisms of hormonal regulation of CAD gene expression and inhibition by Aryl hydrocarbon receptor agonist in human breast cancer cells

The CAD gene is trifunctional and expresses carbamoylphosphate
synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for
pyrimidine biosynthesis. CAD gene activities are induced in MCF-7 human breast cancer
cells, and treatment of MCF-7 or ZR-75 cells with 17b-estradiol (E2) resulted in a 3-5
fold increase in CAD mRNA levels in both cell lines. E2 induced reporter gene activity
in MCF-7 and ZR-75 cells transfected with a construct containing the growth-responsive
-90/+115 (pCAD1) region of the CAD gene promoter, which contains three upstream
GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD
gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required
for E2-responsiveness. Results of gel shift and chromatin immunoprecipitation (CHIP)
assays show that both Sp1 and estrogen receptor a (ERa) interact with the GC-rich
region of the CAD gene promoter. Moreover, hormone-induced transactivation of
pCAD1 was inhibited by cotransfection with dominant-negative Sp1 expression plasmid
and 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
regulated by a nonclassical ERa/Sp1-mediated pathway.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon
receptor (AhR) ligands suppress several E2-induced responses in the rodent uterus and
mammary tumors and in human breast cancer cells. TCDD inhibited hormone-induced
activation of CAD mRNA levels and reporter gene activity in MCF-7 and ZR-75 cells
transfected with E2-responsive pCAD promoter constructs. E2-mediated transactivation
of pCAD constructs with a mutant inhibitory dioxin responsive element DRE (iDRE)
were also inhibited by TCDD suggesting that inhibitory AhR-ERa/Sp1 crosstalk was
iDRE-independent. It was not possible to determine whether the levels of ERa in cells
cotreated with E2 plus TCDD were limiting since the proteasome inhibitor MG132 itself
directly decreased CAD mRNA levels. Using fluorescence resonance energy transfer
(FRET), it was shown that both E2 and TCDD enhanced AhR-ERa interactions. E2 also
induced interactions between ERa and Sp1. However cotreatment with TCDD abrogated
this effect. Results of this study demonstrate a unique model of AhR-ERa crosstalk
where the liganded AhR inhibits ERa-Sp1 interactions and also recruits ERa to Ahresponsive
gene promoters such as CYP1A1.
Date25 April 2007
CreatorsKhan, Shaheen Munawar Ali
ContributorsSafe, Stephen H.
PublisherTexas A&M University
Source SetsTexas A and M University
Detected LanguageEnglish
TypeBook, Thesis, Electronic Dissertation, text
Format1704362 bytes, electronic, application/pdf, born digital

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