CHARACTERISTICS OF THE DEXTRAN-COATED CHARCOAL ASSAY FOR ESTRADIOL RECEPTOR IN BREAST-CANCER PREPARATIONS

  • 1 January 1979
    • journal article
    • research article
    • Vol. 94  (5) , 784-798
Abstract
The measurement of E2 [estrogen] receptor (E2R) in human breast cancer cytosol is significantly influenced by conditions usually employed in the dextran-coated charcoal assay. The incubation time and temperature influence the rate of binding and stability of the receptor. Since lower temperatures preserve the integrity of the receptor, a 2 h incubation at 4.degree. C was selected as the standard incubation procedure. These conditions allow for the detection of at least 80% of the E2R. With supernatants from high-speed centrifugation of HBT [human breast tumor] biopsies or the human breast cancer cell line MCF-7, reducing agents increased the apparent E2R binding in the order: DTT [dithiothreitol] > G-SH [glutathione] > MTG [alpha-monothioglycerol]. The maximum enhancement of specific E2R binding by a given thiol agent was dependent on its concentration in the incubation medium. The optimum DTT level (7.5 mM) for MCF-7 cell homogenization and cytosol equilibrium with tritiated E2 increased E2R to 2 times control (no DTT). For the HBT 150,000 g supernatant, 1 mM DTT was required to optimize the E2R quantitation. The duration of the dextran-coated charcoal extraction of the cytosol-[3H]E2 incubation had no effect on the level of E2R up to 21 h. Minimum levels of nonspecific binding of [3H]E2 could be obtained after 4 h extraction. Maximum depletion of specific [3H]E2 binding could be obtained by adding between 200- and 1000-fold molar excess of unlabeled E2. Greater amounts of unlabeled steroid displaced the radioactive E2 from the dextran-coated charcoal, thereby artifactually increasing the apparent nonspecific binding. This phenomenon may be overcome by utilizing more dextran-coated charcoal in the extraction. There was a 9% loss of specifically bound [3H]E2 per milligram of dextran-coated charcoal (1:10 dextran to charcoal by weight) when the cytosol protein was below 90 .mu.g per incubation. Supplementation with 200 .mu.g or more albumin per incubation prevented this loss. The dextran:charcoal ratio also prevented E2R loss in the order: 1:1 > 1:10 > 1:100. One milligram of dextran-coated charcoal (1:10) has the capacity to adsorb 0.3-0.4 .mu.g of free E2. Other unlabeled competitors are capable of displacing [3H]E2 on the receptor. Although DES [diethylstilbestrol] was as effective as E2, U11, 100A and estrone were inefficient competitors. Apparently the levels of these estrogen analogues required to maximally displace [3H]E2 on receptor also eluted labeled E2 from the dextran-coated charcoal. DES was unable to displace significant quantities of the [3H]E2 from dextran-coated charcoal even at a molar excess of 50,000:1.