Androgen Receptor Assay with [3H]Methyltrienolone (R1881) in the Presence of Progesterone Receptors*

Abstract

The synthetic radiolabeled androgen methyltrienolone-17βhiydroxy-17α-methyl-estra-4,9,ll-trien-3-one (R1881) is superior to the native ligand 5α-dihydrotestosterone (DHT) in the measurement of androgen receptor (AR), especially in tissues where radiolabeled DHT binds with high affinity to plasma proteins in addition to receptor or is rapidly metabolized to inactive derivatives. However, R1881 also binds progesterone receptor (PgR), as shown by its ability to complete for [³]-promestone-17α,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020) binding to 8S PgR on sucrose density gradients, resulting in an overestimation of AR if PgR is present. Therefore, we have devised a method by which PgR binding of R1881 can be blocked without interfering with AR binding. The glucocorticoid triamcinolone acetonide (TA) was found to bind PgR (competition for 8S [³]R5020 binding) but not AR (no competition for 8S [³H]DHT binding). In cytosols containing both receptors, a 500-fold excess of unlabeled TA was sufficient to eliminate [³H]R1881 binding to PgR, leaving only [³H]R1881 binding to AR, which was not affected even by larger TA doses. Scatchard plots of [³H]R1881 binding in such cytosols were reduced by TA to agree with [³H]DHT binding to AR. In rat prostate, which contains little if any PgR, [³H]R1881 binding was slightly greater than that of [³H]DHT, and neither was affected by a 500-fold excess of TA. In a human prostate tumor, however, [³H]R1881 revealed a substantial 8S peak, shown to be AR by the failure of TA to compete, even though [³H]DHT was unable to show 8S binding in the same cytosol. The use of TA to selectively block [³H]R1881 binding to PgR in AR assays has been validated using a variety of methods (sucrose gradient, protamine sulfate, and dextran charcoal) and systems (MCF-7 human breast cancer cells, rat uterus, and human prostate cancer).