Development of an image analysis screen for estrogen receptor alpha (ERα) ligands through measurement of nuclear translocation dynamics

Abstract

We have developed a robust high-content assay to screen for novel estrogen receptor alpha (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen utilizes a green fluorescent protein tagged-glucocorticoid/estrogen receptor (GFP-GRER) chimera which consisted of the N-terminus of the glucocorticoid receptor fused to the human ER ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands, and translocated to the nucleus in response to stimulation with ERα agonists or antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. The assay was validated with known ERα agonists and antagonists, and the Library of Pharmacologically Active Compounds (LOPAC 1280). Additionally, screening of crude natural product extracts demonstrated the robustness of the assay, and the ability to quantitate the effects of toxicity on nuclear translocation dynamics. The GFP-GRER nuclear translocation assay was very robust, with z' values >0.7, CVs <5%, and has been validated with known ER ligands, and inclusion of cytotoxicity filters will facilitate screening of natural product extracts. This assay has been developed for future primary screening of synthetic, pure natural products, and natural product extracts libraries available at the National Cancer Institute at Frederick.

FIG. 1

Quantitation of nuclear translocation of GFP-GRER. (A) The 6020 cells were plated at 3,500 cells/well into seven 384-well glass-bottom plates. The plates were treated with estradiol dose responses for 30 minutes, 1, 2, 3, 4, 5, and 6 hours. Nuclear translocation was quantitated and presented graphically as indicated in the materials and methods. Results displayed as the mean ± SD, and n=32 wells for each dose of estradiol. (B) Images depict the subcellular localization of GFP-GRER upon treatment with the controls, 0.5% DMSO, and 5 μM estradiol at the 6 hour dose. (C) Images of cells treated for 6 hours with DMSO and estradiol controls with overlays of the segmentation masks for the cytoplasmic and nuclear regions of interest (ROIs).

FIG. 2

Nuclear translocation results of estradiol dose responses diluted in various media. (A) Estradiol doses was diluted in PBS, DMEM, or complete media (containing charcoal, dextran-treated FBS), and the resulting dose response curves are depicted graphically. N=32 wells per dose on each plate and 3 plates were used per media type. (B) Estradiol dose responses were diluted in complete media containing regular FBS or charcoal, dextran-treated FBS, with corresponding dose response graphs shown. Results displayed as the mean ± SD, where N=32 wells per plate and 5 plates were used for each media type. (C) Estradiol dose responses mad e in complete media (containing charcoal, dextran-treated FBS) with or without phenol red and corresponding dose response curves are depicted. Results displayed as the mean ± SD, where n=32 well per plate and 6 plates were tested per media. (D) Estradiol dose response curves using complete media containing charcoal, dextran-treated FBS in quadruplicate 384-well plates. Results displayed as the mean ± SD, where N=32 wells per plate.

FIG. 3

Dose response curves depicting nuclear translocation dynamics of known ER ligands. Dose responses were generated for estradiol, propyl pyrazole triol (PPT), 4-hydroxy-tamoxifen (4OHT), diethylstilbestrol (DES), and genistein. Diarylpropionitrile (DPN) is a selective ERβ ligand that was used to demonstrate that the assay is specific for ERα ligands. These dose responses were dosed to 6020 cells plated in to five 96-well glass-bottom plates for 6 hours. Results displayed as the mean ± SD, where N=8 wells per dose and 5 plates were tested per ligand.

FIG. 4

Hit confirmation. DMSO and estradiol controls were retested in the 6020 (assay) cell line, and in the non-transfected parental cell line in quadruplicate. Images of DMSO and estradiol-treated cells are depicted by showing GFP and hoechst fluorescence images in both cell lines. Analysis for nuclear translocation was performed as described in the materials and methods. Images depict fluorescence of the GFP-GRER, and its subcellular location.

FIG. 5

Elimination of false positive translocation results caused by compound fluorescence. Hit compounds were retested in the 6020 (assay) cell line, and in the non-transfected parental cell line in quadruplicate. Analysis for nuclear translocation was performed as described in the materials and methods. Images depict fluorescence of the GFP-GRER, and its subcellular location. Compounds that exhibit nuclear fluorescence in the parental cell line are indicative of false positives caused by fluorescent compounds that localize to the nucleus.

FIG. 6

Cytotoxic compounds elicit false positive nuclear translocation results. The 6020 cells grown in five 96-well plates were treated with staurosporine dose responses to evaluate the effect of cytotoxicity on analysis of translocation dynamics. (A) Staurosporine dose response results were plotted and curves are depicted graphically. Results displayed as the mean ± SD, where N=8 wells per dose per plate and 5 plates were tested, and graphed individually. (B) Examination of the images for GFP-GRER and Hoechst 33342 clearly show cytotoxicity.

FIG. 7

Nuclear and cytoplasmic area measurements as a predictor of cytotoxicity. The 6020 cells grown in 96-well glass-bottom plates were treated with staurosporine dose responses for 6 hours. (A) Staurosporine dose response curves generated by quantitation of nuclear area. Results are plotted and dose response curves are shown graphically, and Hoechst 33343 fluorescent images depict the nuclear area. Results displayed as the mean ± SD, where N=8 wells per dose per plate and 5 plates were tested, and plotted individually. (B) Quantitative cytoplasmic area measurements in response to treatment with increasing doses of staurosporine are shown. Results are plotted and dose response curves are shown graphically and fluorescent images of GFP-GRER shown the morphology of the cytoplasmic area. Results displayed as the mean ± SD, where N=8 wells per dose per plate and 5 plates were tested, and graphed individually.

FIG. 8

Quantitative measurements of nuclear translocation, nuclear area, and cytoplasmic area in response to estradiol dose response treatment. 6020 cells grown in five 384-well glass-bottom plates were treated with estradiol dose responses. Results displayed as the mean ± SD, where N=32 wells per dose per plate and 5 plates were tested. (A) Nuclear translocation was quantitated in response to estradiol treatment and dose response curves were plotted. (B) Nuclear area measurements were plotted after treatment with estradiol dose responses. (C) Quantitative measurement of the cytoplasmic area after estradiol dose response treatment. (D) Fluorescent images of well F23, which has been treated with 5 μM estradiol are shown. Micrographs of the GFP-GRER and hoechst 33342 fluorescent intensities are shown with the segmentation ROIs and segmentation mask overlays for the nuclear and cytoplasmic area measurements are shown.

FIG. 9

Identification of false positives by using additional measurement parameters for cytotoxicity. Fluorescent images of GFP and hoechst staining depict the subcellular localization of the GFP-GRER. Quantitative parameters measured by the image analysis software are displayed next to the fluorescent images.