ECC-1, an established epithelial cell line derived from an adenocarcinoma of human endometrial lining, was examined for growth optimization, steroid hormone receptor- and Ah receptor content, and dioxin modulation of estrogen receptor function. Proliferation of ECC-1 cells was accelerated by growth on a lethally irradiated feeder layer of murine 3T3 fibroblasts. Immunoblot analysis demonstrated the presence of Ah receptor, an intracellular protein that binds and regulates the toxic action of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). The Ah receptor was functional in these cells as assessed by concentration and kinetic patterns of CYP1A1-mediated 7- ethoxycoumarin O-deethylase (ECOD) induction. The half-maximal effective concentration (EC50) for TCDD was 0.2 nM, and maximal activity appeared after 24-h exposure. A limited structure-activity examination of ECOD activity provided additional evidence for Ah receptor involvement. Competitive binding assays were performed to examine kinetic parameters for estrogen, progesterone, and glucocorticoid receptors. Binding parameters of dissociation constant (K(d)) and number of binding sites (B(max)) derived from Scatchard analysis were: estrogen, K(d) = 0.67 nM; B(max) = 321 fmol/mg cytosolic protein; progesterone, K(d) = 1.31 nM; B(max) = 258 fmol/mg cytosolic protein; dexamethasone, K(d) = 1.75 nM, B(max) = 128 fmol/mg cytosolic protein. Exposure of ECC-1 cells to TCDD reduced the estrogen receptor level by 40% without affecting the K(d) value, and reduced estrogen receptor-mediated transcription by 50% assessed by transient transfection of an estrogen-responsive reporter plasmid. These data suggest that the ECC-1 cell line is a useful model system for examining the action of dioxin in human endometrial tissue. Both the estrogen receptor and Ah receptor have been implicated in diseases of the endometrium, and examining their interactions may elucidate mechanisms of uterine disease etiology, as well as potential targets for disease prevention.
|Original language||English (US)|
|Number of pages||7|
|Journal||In Vitro Cellular and Developmental Biology - Animal|
|State||Published - Apr 1999|
- Cytochrome P450