Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.
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, non-estradiol-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 microM. 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.
D001681:Biological Assay / Q000379:methods; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured