8 research outputs found
Use of Xenopus Laevis as a Model for Investigating in Vitro and in Vivo Endocrine Disruption in Amphibians
The estrogenic activity of 17β-estradiol (E2), α-zearalenol (α-ZEA), genistein (GEN), and 4-t-octylphenol (4-t-OP) was investigated using Xenopus laevis-based assays. All test compounds competed with [3H]E2 for binding to a recombinant Xenopus estrogen receptor (xER) with the following relative affinities: E2 \u3e α-ZEA \u3e 4-t-OP \u3e GEN. the ability of these compounds to induce xER-mediated reporter gene expression was then assessed in MCF-7 human breast cancer cells cotransfected with a Gal4-xERdef chimeric estrogen receptor and a Gal4-regulated luciferase reporter gene. Luciferase activity was increased 30- to 50-fold by 10 nM E2 relative to that in solvent control. Maximal reporter gene activity induced by 10 nM α-ZEA was 54% of that induced by E2; however, the activity did not increase following doses of up to 10 μM. a dose of 1 μM 4-t-OP induced 23% of the maximal reporter gene activity induced by E2, whereas 10 μM GEN induced activity to the same level as E2. a dose-dependent increase in vitellogenin (VTG) mRNA expression was observed in Xenopus treated intraperitoneally with E2 at 0.05 to 5 mg/kg/d for three consecutive days, with the maximal induction observed in the group receiving 1 mg/kg/d. the α-ZEA, GEN, and 4-t-OP also significantly induced VTG mRNA expression, although at higher doses. These results demonstrate the utility of X. laevis as an amphibian model to assess the estrogenic activity of endocrine disruptor
Ability of Structurally Diverse Natural Products and Sythetic Chemicals to Induce Gene Expression Mediated by Estrogen Receptors from Various Species
The ability of 14 structurally diverse estrogenic compounds to induce reporter gene expression mediated by estrogen receptors (ERs) from different species was examined. MCF-7 cells were transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and Gal4-ER chimeric receptors containing the D, E and F domains of the human α (Gal4-hERαdef), mouse α (Gal4-mERαdef), mouse β (Gal4-mERβdef), chicken (Gal4-cERαdef), green anole (Gal4-aERαdef), Xenopus (Gal4-xERdef) or rainbow trout α ERs (Gal4-rtERαdef). The efficacy of 17β-estradiol (E2) in inducing reporter gene expression was similar among the different constructs overall, with EC50 values ranging from 0.05 to 0.7 nM. However, Gal4-rtERαdef had an EC50 value at 37 °C of 28 nM, though at 20 °C an EC50 value of 1 nM was observed. Despite a similar response to E2 treatment among the ERs, many differences were observed in the magnitude of the response to other structurally diverse chemicals. For example, coumestrol induced Gal4-mERβdef- and Gal4-aERdef-mediated reporter gene expression 164- and 8-fold greater, respectively, than mediated with the other Gal4-ERs. As well, in contrast to results with other Gal4-ERs, α-zearalenol consistently induced Gal4-rtERαdef-mediated reporter gene activity at lower concentrations than did E2. Overall, the results demonstrate that selected estrogenic compounds exhibit a differential ability to induce reporter gene activity mediated by ERs from different vertebrate species. These data also highlight the importance of incubation temperature when examining rtERα-mediated activity
Estrogen Induces c-myc Gene Expression via an Upstream Enhancer Activated by the Estrogen Receptor and the AP-1 Transcription Factor
Genome-wide analysis of estrogen receptor binding sites
The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer