| 15906006 |
Willemsen P, Scippo ML, Maghuin-Rogister G, Martial JA, Muller M: Enhancement of steroid receptor-mediated transcription for the development of highly responsive bioassays. Anal Bioanal Chem. 2005 Jun;382(4):894-905. Epub 2005 May 20.
We have previously generated several transformed human mammary cell lines for the detection of steroid receptor-mediated activities and used these cell lines to detect and characterize steroid hormone (ant) agonistic compounds. In this report, we describe the specific optimization procedures used to enhance receptor-mediated transcription through the human glucocorticoid, progesterone and androgen receptors, respectively. Sodium arsenite-induced chemical stress leads to a substantial and specific increase in the glucocorticoid receptor-mediated transcription, resulting in maximal stimulations of more than 2000-fold by the agonist dexamethasone. Similarly, a combined treatment with forskolin (an activator of adenylate cyclase) and trichostatin A (an inhibitor of histone deacetylases) leads to a synergistic enhancement of progesterone or androgen stimulation, resulting in a maximal induction of more than 200-fold or about 100-fold, respectively. The enhanced responses to specific steroids are mediated by the corresponding nuclear receptor. We show that by using these enhanced transcriptional stimulation protocols, it is possible to detect lower amounts of steroid hormones without substantially affecting the relative biological activities of various agonists. Finally, the application of these enhanced reporter cell assays to real biological samples from meat-producing animals is evaluated, and some validation parameters are presented. |
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