Altered estrogen receptor system in estrogen-unresponsive human endometrial adenocarcinoma cells.
Previous studies from our laboratories demonstrated that cells from a human endometrial adenocarcinoma cell line (Ishikawa) responded to estradiol whereas cells from another endometrial cancer line (HEC-50) did not. In an attempt to identify factors responsible for the observed estrogen insensitivity we compared the characteristics of the estradiol receptor (ER) systems in Ishikawa and HEC-50 cells. Saturation analyses of cytosolic estrogen binders were performed over a 0.1-70 nM range of [3H]estradiol concentrations. Equilibrium dissociation constants and number of binding sites were determined by graphic analysis of Scatchard plots or computed by applying Fourier-derived affinity spectrum analysis (FASA) of the binding data. No significant differences were noted in the dissociation constants (Kd approx. 0.6 nM) or number of binding sites (approx. 6-10 fmol/mg protein) for the single binder that could be evaluated by the graphic method in cytosol from the two cell lines. However, 2 binders in Ishikawa cells (Kd approx. 0.2 and 6 nM) could be detected by the FASA method; the higher affinity binder in HEC-50 cells could not be clearly demonstrated. Structural differences in the specific estrogen binders which might distinguish HEC-50 from Ishikawa cells or normal endometrial tissue were investigated by using the anti-ER monoclonal antibody JS 34/32. Interaction of the antibody with [3H]estradiol binders of estrogen-responsive cells and tissue was evident from the formation of labeled complexes that were shown to sediment faster in glycerol density gradients and could be immunoprecipitated with Protein A attached to Sepharose beads. In contrast, the antibody did not recognize labeled specific binders in the HEC-50 cells. Furthermore, [3H]estradiol receptors in Ishikawa cells could be transformed into a species that exhibited increased hydrophilicity, evident from its binding to DNA-cellulose, whereas binders from HEC-50 could not. These results indicate that the lack of responsiveness of HEC-50 cells to estrogens might be due to structural or functional alterations in the ER protein resulting in a loss of its capability to undergo estrogen-directed conformational changes required for biological activity.
Department of Obstetrics, Gynecology and Reproductive Science, Mount Sinai School of Medicine, City University of New York, New York 10029.
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Kassan
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Mechanick
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Gurpide
D000230:Adenocarcinoma / Q000378:metabolism*; D000328:Adult; D000911:Antibodies, Monoclonal; D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000031:analogs & derivatives; D004958:Estradiol / Q000378:metabolism; D004967:Estrogens / Q000502:physiology; D005260:Female; D006801:Humans; D011960:Receptors, Estrogen / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C033190:DNA-cellulose; D004967:Estrogens; D011960:Receptors, Estrogen; D004958:Estradiol; D002482:Cellulose; D004247:DNA