Variability of levels of specific estrogen binding in a human endometrial adenocarcinoma cell line

Previous reports from this laboratory have described marked changes in the levels of specific estradiol (E2) binders in cultured endometrial adenocarcinoma cells (HEC-1B) that occur during the first 30 h after replating. In the present study, binding levels were measured daily during a period of 8 days. HEC-1B cells were incubated with 100 nM [3H]-estradiol at 4 and 30 degrees C, in the presence or absence of 10 microM diethylstilbestrol and concentrations of bound ligand were determined in the nuclear and cytoplasmic fractions. It was found that concentrations of specific estradiol binders increased the first day after plating and declined thereafter. Saturation analysis of estrogen binding sites in cytosol of HEC-1B cells labeled with [3H]-E2 or [3H]-estriol (e3), at concentrations ranging from 0.1-100 mM, showed two plateaus for E2 binding (at about 20 and 60 nM) but only one (at about 40 nM) for E3, when dextran-coated charcoal was used to separate free and bound ligand. The constants of dissociation of E2 and E3 for the high affinity binder were about 4 and 20 nM, respectively.