Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

Abstract

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 μM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The K d for the high affinity binding site was 2 × 10 −9 M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5α-androstane-3α,17β-diol, testosterone, estradiol and estrone. Progesterone, 5α-androstane-3β,17β-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 ± 7.6fmol of bound R1881 per 10 6 cells ( n = 34) or 2075 ± 1434fmol per mg of DNA; these values correspond to an average of 6624 ± 4577 sites per cell. Thus, using this whole cell assay system, specific androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.