Comparison of histochemical and biochemical assays for estrogen receptor in human breast cancer cell lines.

Abstract

Two human breast cancer lines, MCF-7 and T47D cells, were investigated for the presence of estrogen receptor (ER) by biochemical and histochemical techniques. Using the dextran-coated charcoal technique and isoelectric focusing, MCF-7 cells were ER positive, and T47D cells were ER negative. Fluorescein conjugates to 17 beta-estradiol by the sixth carbon (17 beta-estradiol-6-carboxymethyloxime:bovine serum albumin: fluorescein isothiocyanate and 17 beta-estradiol-6-iminooxyacetylfluoresceinamine) and by the 17th carbon [N-fluoresceino-N'-[17 beta-(estradiol hemisuccinamide)ethyl]thiourea, 17-FE] were prepared for cytochemical evaluation of the ER status of the two cell lines. The binding affinity of the estradiol conjugates for ER varied, the 17-FE conjugate having the highest affinity of 0.08 relative to 17 beta-estradiol. Following incubation with 10 nM 17-FE, both MCF-7 and T47D cells displayed cytoplasmic and nuclear fluorescent staining. Isoelectric focusing of MCF-7 cytosol incubated in the presence of 10 nM 17-FE revealed binding of the fluorescein conjugate to a protein species which did not bind 17 beta-[3H]-estradiol. Isoelectric focusing of T47D cytosol revealed binding of 17-FE to two protein components, neither one of which showed specific binding of 17 beta-[3H]estradiol. The results suggest different protein binding species for fluoresceinated estradiol conjugates and [3H]estradiol and help to explain reported differences in histochemical and biochemical ER analyses.