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A Three-tiered Approach for Calibration of a Biosensor to Detect Estrogen Mimics

  • Sarah A. Andres
  • D. Alan Kerr
  • Stefanie B. Bumpus
  • Traci L. Kruer
  • Joshua W. Thieman
  • Irina A. Smolenkova
  • James L. Wittliff
Part of the Advances In Experimental Medicine And Biology book series (AEMB, volume 614)

Abstract

A three-tiered approach was developed to determine the influence of a chemically-diverse group of compounds exhibiting estrogen mimicry using recombinant human estrogen receptor (rhER) activity to calibrate a receptor protein-based biosensor. In the initial tier, a ligand competition array was developed to evaluate compounds inhibiting [3H]estradiol-17β binding to rhER. Each of six different concentrations of [3H]estradiol-17β was mixed with increasing concentrations of an unlabeled putative mimic. Each of these mixtures was incubated with a constant amount of rhERα and then receptorbound [[3H]estradiol-17β was measured. This array protocol analyzes ligand binding affinities of hERα with a potential inhibitor over the entire range of receptor protein saturation.

When either hERα or hERβ binds to an estrogenic ligand, the receptor monomer forms both homo- and hetero-dimers. Then the ligand-receptor dimer complex activates transcription by associating with an estrogen response element (ERE), which is a specific DNA sequence located upstream of estrogenresponsive genes. The second tier for ligand evaluation utilized an electrophoretic mobility shift assay (EMSA), which was performed with an ERE sequence labeled with [α[32]P]dATP and incubated with rhER in the presence or absence of unlabeled ligand. ERE-hER complexes were separated by electrophoresis and analyzed using phosphor imaging technology.

To assess biological effects of an estrogen mimic on expression of an ER-target gene, a yeast cell-based bioassay was constructed with recombinant DNA technology using Saccharomyces cerevisiae. Each of these engineered yeast cells contained a rhERα expression plasmid (YEpE12) and a separate reporter plasmid (YRG2) containing an ERE sequence upstream of a β-galactosidase reporter gene. Incubation of these yeast cells with an estrogenic compound allows formation of ligand-hERα complexes, which recognize the ERE sequence regulating β-galactosidase expression. Estrogenic compounds, which were evaluated as calibrators for ligand-based and EREbased biosensors, elicit varying responses in each of the three tiers of the protocol.

Keywords

Estrogenic Compound Estrogen Response Element Human Estrogen Receptor Competition Curve Estrogen Mimic 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Sarah A. Andres
    • 1
  • D. Alan Kerr
    • 1
  • Stefanie B. Bumpus
    • 1
  • Traci L. Kruer
    • 1
  • Joshua W. Thieman
    • 1
  • Irina A. Smolenkova
    • 1
  • James L. Wittliff
    • 1
  1. 1.Department of Biochemistry & Molecular BiologyUniversity of LouisvilleLouisvilleUSA

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