Abstract 4882: Determining endocrine receptor activity using quantitative automated image analysis and Redistribution® technology

Endocrine hormone function has been shown to regulate numerous oncogenic processes from tumor progression, promotion, and dependence to prevention, diagnosis, and treatment. Notably, estrogen and androgen activity in breast and prostate cancers have been used to diagnose steroid hormone dependence and determine therapy options. Endocrine disruptors are compounds that mimic estrogen or androgen steroid activity and affect the endocrine system by altering hormone function, providing possible therapies for endocrine-dependent cancers. Screening complex chemical libraries for endocrine disruptors and investigating the molecular and cellular effects of existing treatment strategies is accelerated by using high throughput procedures to assess endocrine activity and potential cancer therapeutic efficacy. Redistribution® technology can be used to monitor the localization of GFP-tagged proteins in response to extra-cellular stimuli such as treatment with steroid compounds or activation of signaling cascades. Redistribution® GFP-tagged steroid receptors such as estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and androgen receptor (AR) form nuclear foci in response to hormone stimulation that can be easily monitored and quantitated by automated image analysis, thereby establishing a high-throughput screen for both agonists and antagonists of ER and AR activation. Additionally, multiplexed cell and nuclear phenotypes such as receptor kinetics, cellular health, and cell cycle effects can be monitored simultaneously following direct or indirect receptor stimulation. Here we utilize Redistribution® cell lines and known endocrine agonist and antagonists to establish a screen for ER and AR disruptors. For ER screens, cells were treated with agonists including 17β-estradiol and bisphenol A with and without antagonists such as fulvestrant (ICI 182,780) and tamoxifen. AR cells were treated with dihydrotesterone (5α-androstan-17β-ol-3-one) or progesterone agonists in the presence or absence of the AR antagonist mifepristone (RU 486). Following fixation and Hoechst staining, plates were analyzed using a quantitative automated imaging platform to evaluate features such as nuclear translocation (for AR only), nuclear GFP foci count, area, and intensity, and overall nuclear morphology on a cell-by-cell basis. EC50 values were determined from dose-dependent response curves of the treatments to establish hierarchical receptor activation comparisons for both ER and AR. Z