A Double-Labeling Assay for Simultaneous Estimation and Characterization of Estrogen and Progesterone Receptors using Radioiodinated Estradiol and Tritiated Org 2058

Abstract

Estrogen (ER) and progesterone receptors (PgR) appear to be a prerequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g., dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, we have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using ¹²⁵I-E₂ and ³H-Org 2058 as tracers. The interaction of ¹²⁵I-E₂ with ER and plasma proteins in comparison to ³H-E₂ was studied in terms of specificity, time course, affinity binding and sedimentation pattern. ¹²⁵I-E₂ bound the same molecular forms displayed by ³H-E₂ (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of ¹²⁵I and ³H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous result was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV < 8.0 %). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less purturbation of sedimenting receptor molecules. Furthermore, a biochemical application of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease.