Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextrancoated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5α-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [³H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated K_d for methyltrienolone (R1881) of 5 nM at 0°C. Scatchard analysis of [³H]Dx binding by RAG cytosol showed a K_d of 6 nM for Dx and 44 nM for corticosterone at 0°C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [³H]testosterone and [³H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [³H]testosterone. There was little conversion of DHT to androstanediol.