A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists.
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 to 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, AR agonists such as dihydrotestosterone (DHT), and GR agonists such as dexamethasone (DEX), corticosterone, and aldosterone induce luciferase expression at appropriate concentrations. DHT consistently produced 3-9-fold induction at concentrations from 0.1 to 10 nM. At 1 to 1000 nM, DEX induced luciferase activity 1.3-19.5-fold. To distinguish AR- from GR-mediated ligands, chemicals were assayed concurrently with the antiandrogen, hydroxyflutamide (OHF), 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 was 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 a 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- but for GR-mediated activities as well.
Wilson
Vickie S
VS
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD-72, Research Triangle Park, North Carolina 27711, USA. wilson.vickie@epa.gov
Bobseine
Kathy
K
Lambright
Christy R
CR
Gray
L E
LE
D000726:Androgen Antagonists / Q000494:pharmacology*; D059002:Androgen Receptor Antagonists; D000728:Androgens; D003345:Corticosterone / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D015394:Molecular Structure; D010575:Pesticides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011374:Progesterone / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011944:Receptors, Androgen / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000726:Androgen Antagonists; D059002:Androgen Receptor Antagonists; D000728:Androgens; D004965:Estrogen Antagonists; D005938:Glucocorticoids; D010575:Pesticides; D011944:Receptors, Androgen; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D011374:Progesterone; D004958:Estradiol; D003907:Dexamethasone; D008156:Luciferases; D003345:Corticosterone