You are here:
A NOVEL CELL LINE, MDA-KB2, THAT STABLY EXPRESSES AN ANDROGEN AND GLUCOCORTICOID RESPONSIVE REPORTER FOR THE DETECTION OF HORMONE RECEPTOR AGONISTS AND ANTAGONISTS
Citation:
Wilson, V S., K L. Bobseine, C R. Lambright, AND L E. Gray Jr. A NOVEL CELL LINE, MDA-KB2, THAT STABLY EXPRESSES AN ANDROGEN AND GLUCOCORTICOID RESPONSIVE REPORTER FOR THE DETECTION OF HORMONE RECEPTOR AGONISTS AND ANTAGONISTS. TOXICOLOGICAL SCIENCES 66(1):69-81, (2002).
Description:
The U.S. Environmental Protection Agency has proposed that in vitro assays for estrogen receptor (ER) and androgen receptor (AR) mediated actions be included in a Tier I screening battery to detect hormonally active chemicals. Herein we describe the development of a novel stable cell line, MDA-kb2, for screening of androgen agonist and antagonists and characterize its specificity and sensitivity to endocrine disrupting chemicals. The breast cancer cell line, MDA-MB-453, was stably transformed with the MMTV.luciferase.neo reporter gene construct. Since both GR and AR are present in the MDA-MB-453 cells and both receptors can act through the MMTV promoter, compounds that act through either AR or GR activate the MMTV-luciferase reporter. As expected, both AR agonists, such as dihydrotestosterone (DHT), and GR agonists, such as dexamethasone (DEX), corticosterone, and aldosterone induce luciferase expression at appropriate concentrations. DHT produced consistent 3 to 9 fold induction at concentrations from 0.1 to 10 nM. At 1 nM to 1000 nM, DEX induced luciferase activity 1.3 to 19.5 fold. To distinguish AR from GR mediated ligands, chemicals were assayed concurrently with the antiandrogen, hydroxyflutamide, which blocks AR but not GR mediated responses. In addition, known AR antagonists including, hydroxyflutamide, vinclozolin, vinclozolin metabolites M1 and M2, p,p'-DDE, and linuron, inhibited DHT induced luciferase gene expression at appropriate concentrations in this system. We have found that these cells are relatively easy to culture and maintain.. Responsiveness has been monitored over time and was stable for more than 80 passages. Some advantages of this assay are that it is relatively rapid (2-days), eliminates the need for transfection, 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.