title: A three-tiered approach for calibration of a biosensor to detect estrogen mimics.
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-17beta binding to rhER. Each of six different concentrations of [3H]estradiol-17beta was mixed with increasing concentrations of an unlabeled putative mimic. Each of these mixtures was incubated with a constant amount of rhERalpha and then receptor-bound [[3H]estradiol-17beta was measured. This array protocol analyzes ligand binding affinities of hERalpha with a potential inhibitor over the entire range of receptor protein saturation. When either hERalpha or hERbeta 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 estrogen-responsive genes. The second tier for ligand evaluation utilized an electrophoretic mobility shift assay (EMSA), which was performed with an ERE sequence labeled with [alpha[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 rhERalpha expression plasmid (YEpE12) and a separate reporter plasmid (YRG2) containing an ERE sequence upstream of a beta-galactosidase reporter gene. Incubation of these yeast cells with an estrogenic compound allows formation of ligand-hERalpha complexes, which recognize the ERE sequence regulating beta-galactosidase expression. Estrogenic compounds, which were evaluated as calibrators for ligand-based and ERE-based biosensors, elicit varying responses in each of the three tiers of the protocol.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Louisville, Louisville, KY 40292, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Andres
authors.forename: D Alan
authors.initials: DA
authors.lastname: Kerr
authors.forename: Stefanie B
authors.initials: SB
authors.lastname: Bumpus
authors.forename: Traci L
authors.initials: TL
authors.lastname: Kruer
authors.forename: Joshua W
authors.initials: JW
authors.lastname: Thieman
authors.forename: Irina A
authors.initials: IA
authors.lastname: Smolenkova
authors.forename: James L
authors.initials: JL
authors.lastname: Wittliff
mesh_terms: D001681:Biological Assay; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D002138:Calibration; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D004967:Estrogens / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D018716:Molecular Mimicry; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D004967:Estrogens; D008024:Ligands; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D004958:Estradiol