Progesterone receptor quantification with radiolabeled promegestone (R 5020) in frozen sections of endometrium and breast cancer tissue

• Published in: Journal of steroid biochemistry Vol. 29; no. 5; pp. 465 - 474
• Main Authors: De Goeij, Antony F.P.M., Scheres, Hubert M.E., Rousch, Mat J.M., Hondius, Gerry G., Bosman, Fred T.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier B.V 01.05.1988; New York, NY Pergamon
• Subjects: Animal tumors. Experimental tumors; Animals; Biological and medical sciences; Breast Neoplasms - analysis; Carcinoma - analysis; Cattle; Endometrium - analysis; Experimental genital and mammary tumors; Female; Freezing; Humans; Kinetics; Lipids; Medical sciences; Promegestone - metabolism; Receptors, Progesterone - analysis; Receptors, Progesterone - metabolism; Solubility; Tumors; Radiolabelling; Cryopreservation; Female genital system; Quantization; Tumor; Mammary gland; Progesterone; Technique; Sex steroid hormone; Histological section; Endometrium; Hormonal receptor
• ISSN: 0022-4731
• DOI: 10.1016/0022-4731(88)90180-X

A technique for the determination of the progesterone receptor content in cryostat sections was developed. Series of coverglass-mounted unfixed frozen sections were incubated with [ 3H]R5020 only, to determine total binding, or with excess unlabeled R5020, to determine non-specific binding. Ligand binding in the tissue sections was measured by liquid scintillation counting after repeated washing of the coverslips. Elution of ligand binding proteins into the incubation buffer was quantitated with the dextran-coated charcoal method. Specific ligand binding was related to the total tissue protein content which was determined on parallel, unmounted sections. Scatchard analysis showed specific saturable and high affinity ( K d = 0.01−2 nM) section-bound and soluble binding sites in cryostat sections of calf uterus, human endometrium and breast cancer samples. Ligand specificity was studied by competition of [ 3H]R5020 with a 100-fold excess of various steroid receptor ligands. The competition was excellent for R5020 and progesterone, negligible for estrogens and slight for androgens and corticosteroids. These binding characteristics provide evidence that with this assay progesterone receptors are determined. Exchange experiments showed that with this method total, free as well as occupied, progesterone receptors can be measured. A highly significant linear correlation, and agreement in PR status classification between assay on cytosol and sections was obtained for a series of 21 breast cancer samples. Finally, progesterone receptor analysis using cryostat sections results in the recovery of 2–3 times more PR from the same amount of tissue as compared to the use of cytosol. These results indicate that progesterone receptors can be reliably assayed with Scatchard analysis using cryostat sections, which requires less tissue than the cytosol assay. This method, which is simple and easy to perform could be of practical importance, particularly when only small tissue samples (which also have to be analyzed morphologically or histochemically) are available and when quantitative radiochemical progesterone receptor data are required for direct comparison with (immuno-) histochemical information.