NIH Chemical Genomics Center [NCGC]
U.S. Environmental Protection Agency [EPA]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

NCGC Assay Overview

Estrogen receptor alpha (ER-alpha) is an important member of nuclear hormone receptors. ER-alpha plays important role in breast cancer as well as in other women's health issues. The majority of breast cancers is estrogen receptor (ER) positive and depends on estrogen for growth. There is also growing concern for some of environmental chemicals which may contribute the increasing the incidence of breast cancer. Therefore, it is important to understand the effect of environmental chemicals in ER-alpha signaling pathway.

NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of the ER response element, a cell-based assay [ER-alpha-UAS-bla GripTiteTM cell-Based Assay from Invitrogen] was used to measure ER-alpha signaling pathway in agonist mode. ER-alpha-UAS-bla-GripTite HEK 293 cells were used with assay medium containing 2% charcoal/dextran treated FBS, 0.1 mM NEAA and 1 mM sodium pyruvate. Cells were cultured in this assay medium overnight in the flasks before the assay. The assay was performed in clear bottom black Greiner 1536-well plates. 17-beta-estradiol was used as a positive control in the screen. Library compounds were measured for their ability to either stimulate the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to 17-beta-estradiol control (20 nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal stimulation (EC50) and maximal response (efficacy) values.

qHTS protocol for ER-alpha beta-lactamase assay in agonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 6 uL; 5000 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (17-beta-estradiol); 23 nL
- Dose response, start 1uM, 1:3 dilutions with duplicates
- Two columns with 20 and 0.5 nM (final) concentration.
5. Incubation; 37 C for 18 hrs
6. Detection reagent; 1 uL of CCF4-dye
7. Incubation; RT for 2 hrs
8. Detection; Fluorescence; Envision plate reader or Acumen plate reader

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.