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.

miR-342 Regulates BRCA1 Expression through Modulation of ID4 in Breast Cancer

“…ER and PR status was routinely evaluated at time of diagnostic procedure according to the EORTC recommendations and within national [18] and international quality control programs by a ligand binding assay [19] and expressed as fmol mg −1 of protein. Tumors with an ER concentration higher than 10 fmol mg −1 of protein or with a PR concentration higher than 25 fmol mg −1 of protein were defined as ER-positive or PR-positive, respectively.…”

Axillary Dissection Versus No Axillary Dissection in Elderly Patients with Breast Cancer and No Palpable Axillary Nodes: Results After 15 Years of Follow-Up

“…Tumor specimens were assayed for estrogen receptor (ER) and progesterone receptor (PgR) levels using the dextran-coated charcoal technique according to Ronchi et al16 Receptors levels were expressed in fmol mg −1 cytosol protein. Tumors with ER concentration ≤10 fmol cytosol protein were considered ER negative (ER−); those with PgR concentration ≤25 fmol cytosol protein were considered PgR negative (PgR−).…”

Cell proliferation in 3,800 node-negative breast cancers: Concistency over time of biological and clinical information provided by3H-Thymidine labelling index

“…Immediately after surgery, part of the tumour material was incubated at 37°C with 3 H-thymidine ( 3 H-dT) for determination of cell proliferation (Silvestrini, 1991), and part was frozen in liquid nitrogen and stored at 280°C for the determination of hormone receptor status (Ronchi et al, 1986).…”

“…Hormone receptor determination. Estrogen (ER) and progesterone (PgR) receptor concentrations were measured by the dextrancoated charcoal technique (Ronchi et al, 1986). Ten and 25 fmol/mg of cytosol proteins were used as cutoff values for ER and PgR, respectively (Table I).…”