Androgen receptor transactivation assay using green fluorescent protein as a reporter

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpB-UbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a frilly functional transactivation assay system with P-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a sirriplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and D-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC50 values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity. (C) 2007 Elsevier Inc. All rights reserved.