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STANDARDIZATION AND VALIDATION OF ADENOVIRAL TRANSDUCTION OF AN ANDROGEN RECEPTOR POSITIVE CELL LINE WITH AN MMTV-LUC REPORTER FOR ENDOCRINE SCREENING
Citation:
Hartig, P C., K L. Bobseine, M C. Cardon, C R. Lambright, AND L E. Gray. STANDARDIZATION AND VALIDATION OF ADENOVIRAL TRANSDUCTION OF AN ANDROGEN RECEPTOR POSITIVE CELL LINE WITH AN MMTV-LUC REPORTER FOR ENDOCRINE SCREENING. Presented at Society for the Study of Reproduction, Madison, WI, July 15-18, 2000.
Description:
Standardization and Validation of Adenoviral Transduction of an Androgen Receptor Positive Cell Line with an MMTV-Luc Reporter for Endocrine Screening P. Hartig, K . Bobseine,
M. Cardon, C. Lambright and L. E. Gray, Jr. USEPA, Reproductive Toxicology Division, NHEERL, RTP, NC
The discovery that xenobiotics interfere with androgen activity has stimulated the need to assess chemicals for their ability to modulate androgen receptor binding. Transduction, the ability of replication defective viruses to deliver biologically competent genes, is a well studied process. Here we report the novel use of viral transduction followed by an assessment of chemical modulation of DHT-induced gene activation.
Human breast carcinoma cells ((MDA-MB-453) AR+,GR+, ER a-,PR-) were subcultured in 96 well plates, transduced with replication defective human adenovirus type 5 (containing the luciferase reporter gene driven by the AR and GR responsive glucocorticoid-inducible response element found with the MMTV LTR (Ad/MLUC7)), exposed to putative AR and GR ligands, incubated 48 hrs, lysed and assayed for luciferase. Luc gene expression was induced in a concentration dependent manner by DHT, E2, dexamethasone and high concentrations of hydroxyflutamide (OHF) and M2, but inhibited by AR antagonists, Bis-hydroxy-DDE (OH-DDE), 2,2-bis (p-hydroxyophenyl)-1,1,1-trichloroethane (HPTE), M1 and low concentrations of M2 and E2. E2 induced Luc expression was blocked with OHF, but not with the anti-estrogen, ICI 182.780, confirming E2 is an AR ligand. Results following transduction of MDA-MB-453 cells with MMTV Luc are nearly identical to those obtained with cell lines stably expressing MMTL-Luc. In summary, this assay correctly identified all chemicals examined and induction was found to be sensitive and significant (approx 100 fold induction). Transduction can be rapidly applied to other cell lines and utilized to deliver receptors and reporter genes (This does not reflect EPA policy).