title: Development of a stable dual cell-line GFP expression system to study estrogenic endocrine disruptors.
abstract: Environmental estrogenic endocrine disruptors are a health concern. Here we constructed a dual cell-line green fluorescence protein (GFP) expression system to identify and study endocrine disrupting compounds with activities of estrogen receptor agonists or antagonists. Human breast cancer MCF-7 cells and endometrial carcinoma Ishikawa cells were infected with a two tandem estrogen response elements--E4 promoter-GFP reporter gene construct. The use of GFP reporter enabled direct and simple evaluations of cell responses. GFP intensity in stably transfected MCF7-GFP and Ishikawa-GFP cells was dose-responsive to 17-beta-estradiol, diethylstilbestrol, 2-hydroxyestradiol, and environmental toxins bisphenol A, genistein and o-p'-DDT. Raloxifene and tamoxifen were effective antiestrogens in MCF7-GFP cells, but acted as partial estrogen receptor agonists in Ishikawa-GFP cells at concentrations of 0.1 nM and above. No synergistic effect was observed in chemical combinations between organochlorine pesticides methoxychlor, o-p'-DDT, p-p'-DDT, nor between estradiol and estrone. In summary, for the first time the effects of estrogen receptor agonists or antagonists were compared between mammary and endometrial cancer cells both stably expressing identical plasmids with GFP reporter genes under the control of tandem estrogen response elements. This dual cell-line system provides a rapid method and sensitive assay to identify environmental estrogens, antiestrogens, selective estrogen receptor modulators and to study their tissue specific effects and chemical interactions. Such a system is especially useful for direct and parallel toxicity assessments with a microfluidic cell culture device.
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
authors.forename: W Lee
authors.initials: WL
authors.lastname: Kraus
authors.forename: Michael L
authors.initials: ML
authors.lastname: Shuler
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D052244:Endocrine Disruptors / Q000494:pharmacology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D010957:Plasmids; D011960:Receptors, Estrogen / Q000819:agonists* / Q000037:antagonists & inhibitors*; D014162:Transfection
chemical_list: D052244:Endocrine Disruptors; D011960:Receptors, Estrogen; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins