| Abstract: | A method for the determination of estrogen and progesterone receptor levels in human mammary cell lines (MCF-7, Cama-1, ZR-75-1, Evsa-T and HBL-100) is described. Cells cultured as monolayers were incubated with the tritiated steroids, 3H]-17 beta-Estradiol or 3H] ORG-2058. Binding of steroids to receptors was a function of cellular uptake. Incubation periods of 50 min were sufficient to attain maximum intracellular incorporation. The binding of 17 beta-E2 and ORG-2058 to MCF-7 cells, a phenomenon which is saturable at low concentrations for the radioactive ligand, is a linear function of the number of cells assayed (Interval: 2.5 X 10(4) to 1.5 X 10(6) cells per well). Binding data and their Scatchard plot allowed for the calculation of affinity and capacity values. Thus, for ER, Kd = 2.0 +/- 0.5 X 10(-10) M and n = 3.76 +/- 0.91 Fmol/microgram DNA, and for PgR Kd = 2.0 +/- 0.2 X 10(-10) M and n = 14.02 +/- 2.30 Fmol/microgram DNA (Mean +/- SD). Binding specificity of 17 beta-Estradiol and ORG-2058 to MCF-7 cells was analysed by means of study on the inhibitory effect of increasing concentrations of unlabelled competitors: 17 beta-Estradiol, ORG-2058, Estrone, DES, R-5020, Cortisol, Androsterone and Testosterone. Only pharmacological doses of some of the mentioned molecules produce displacement of the hormonereceptor binding. This phenomenon appears to be related to the affinity of these chemical compounds for the receptor macromolecules to which estrogens and progesterone bind. |
