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A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS
Citation:
Wilson, V S., K L. Bobseine, C R. Lambright, AND L. E. Gray Jr. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS. Presented at Society of Toxicology, San Francisco, CA, March 25 - 29, 2001.
Description:
The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of androgen agonist and antagonists. The parent breast cancer cell line, MDA-MB-453, which contains both endogenous AR and glucocorticoid receptor (GR), was stably transfected with the MMTV.neo.luciferase reporter gene construct. The purpose of this study was to characterize the specificity and sensitivity of this cell line. Since both GR and AR can act through this response element, compounds that act through either receptor should activate the luciferase reporter. We have tested the AR agonists, dihydrotestosterone (DHT) and medroxyprogesterone acetate (MPA), and GR agonists, dexamethasone (DEX), corticosterone, and aldosterone. Cells display appropriate sensitivity to agonists. DHT produced consistent 3 to 9 fold induction over background at concentrations from 0.1 to 10 nM. DEX induced luciferase activity from 1.3 to 19.5 fold at concentrations from 1 nM to 1000 nM. To distinguish between AR and GR mediated ligands, chemicals were assayed concurrently with hydroxyflutamide, which blocked AR, but not GR mediated responses. Furthermore, several known AR antagonists including, hydroxyflutamide, vinclozolin, vinclozolin metabolites M1 and M2, p,p=-DDE, and linuron, all inhibited DHT induced luciferase gene expression in this system. These cells are genetically identical and easy to culture, maintain, and expand. Some advantages of this assay are that it is relatively rapid (2-days), can be conducted in 96-well plate format, and produces consistent reproducible results. In summary, we have developed a cell line that can be used to screen chemicals not just for AR mediated activity but GR activity as well.
NCSU/EPA Cooperative Training Agreement CT826512010. DISCLAIMER: This is an abstract of a proposed presentation and does not necessarily reflect USEPA policy.