Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line

Abstract

Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, nonestradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 M. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

“…As shown in Fig. 1B, TRAP220 did not enhance ER␣-mediated transcription of the 3ERE-TATA-LUC reporter gene (40), under similar transfection conditions to those used for TR␤. In contrast, co-transfection of SRC1e strongly enhanced ER␣-mediated transcription both in the presence and absence of ligand, as reported previously (4,16,44,45).…”

“…Plasmid Constructions-The following plasmids used in transient transfection experiments have been described previously: pSG5-SRC1e (16) and p3ERE-TATA-LUC (40). The reporter plasmid pMAL-TK-LUC (41) and hTR␤-RSV (42) were gifts from K. Chatterjee.…”

Estrogens in Swiss Rivers and Effluents – Sampling Matters

“…Va rious such assay systems have been developed ( e.g . E-Screen; [10] yeast estrogen screen, YES [11] and ER-CALUX [12] ). Using combinations of chemical analysis and bioassays, many countries initiated large national surveys to address the issue of estrogens in the aquatic ecosystem.…”

Comparative study of estrogenic potencies of estradiol, tamoxifen, bisphenol-A and resveratrol with two in vitro bioassays

“…These assays include competitive ligand binding assays (Kuiper et al, 1997), cell proliferation assays (Soto et al, 1992) and recombinant receptor gene assays in yeast and human cells (Routledge and Sumpter, 1996;Legler et al, 1999). Each assay measures different aspects of the initial events that may ultimately lead to estrogenic effects, but these assays also suffer from a number of drawbacks (Zacharewski, 1997).…”