| Autor: |
Sarah A, Andres, D Alan, Kerr, Stefanie B, Bumpus, Traci L, Kruer, Joshua W, Thieman, Irina A, Smolenkova, James L, Wittliff |
| Rok vydání: |
2008 |
| Předmět: |
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| Zdroj: |
Advances in experimental medicine and biology. 614 |
| ISSN: |
0065-2598 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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