Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen recep tor (AR) expressing human breast cancer cell line T47D was stably transfect ed with a luciferase gene under transcriptional control of the PB-ARE-2 and rogen response element. The application of this cell line in an endogenous Androgen Receptor-mediated LUciferase eXpression assay (ARLUX) was validate d. Ain EC50 value of 86 pM was determined for the standard androgen R1881 w ith a detection limit of 46 pM. Other androgens like dihydrotestosterone, 1 7 beta -trenbolone, and bolasterone also induced luciferase expression, whi le anti-androgens suppressed these responses. As expected, AP-mediated resp onses were also elicited by high concentrations of the steroids progesteron e, 17 beta -estradiol, d-aldosterone, and dexamethasone, with observed EC50 values 10 to 350,000 times higher than that for R1881. A unique feature of the AR-LUX assay is that effects on modulation of active endogenous AR-lev els are reliably reflected in the luciferase induction response, as exempli fied by vitamin D, all-trans-retinoic acid, epigallocatechin gallate, and f orskolin. This feature is especially useful when assessing complex mixtures , e.g., environmental samples or natural compound libraries. From these dat a it is concluded that the AR-LUX assay is a reliable in vitro test system for the detection and quantification of AR-mediated biological effects. The 96-well plate format makes the assay particularly suitable for high-throug hput screening. (C) 2001 Academic Press.