A method is described for the determination of binding capacity (Ro) and dissociation constant (Kd) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [3H]dihydrotestosterone3 3 The following trivial names were used: dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), methyltrienolone (R1881, 17β-hydroxy-17-methyl-estra-4,9,11-trien-3-one), R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R2956 (17β-hydroxy-2,2,17-trimethylestra-4,9, 11-trien-3-one), androstanediol (5α-androstane-3α,17β-diol), cyproterone acetate (6-chloro-17 acetoxy-1, 2α-methylene pregna-4,6-diene-3,20-diene), and dexamethasone (9-fluoro-11β, 17,21-trihydroxy-16α-methylpregna-1,4-diene-3, 20-dione). (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (Ro = 12,105 ± 4305 (S.D.) sites/cell; Kd = 1.0 ± 0.7 (S.D.) × 10-9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.
ASJC Scopus subject areas
- Molecular Biology