ToxSci Advance Access originally published online on May 27, 2004
Toxicological Sciences 2004 81(1):69-77; doi:10.1093/toxsci/kfh180
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Toxicological Sciences vol. 80 no. 1 © Society of Toxicology 2004; all rights reserved.
Development and Characterization of a Cell Line That Stably Expresses an Estrogen-Responsive Luciferase Reporter for the Detection of Estrogen Receptor Agonist and Antagonists
U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, North Carolina 27711
Received March 17, 2004; accepted May 25, 2004
Recently several advisory committees (EDSTAC, ICCVAM) have recommended that stable estrogen-dependent gene expression assays be developed for screening chemicals for estrogenic activity because of the high degree of specificity of the response and potential for use in a high-throughput mode. In this paper we describe a specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities. T47D human breast cancer cells, which naturally express estrogen receptor (ER) alpha and beta, were stably transfected with a triplet ERE (estrogen-responsive elements)–promoter–luciferase reporter gene construct. The transformed cells were named T47D-KBluc. These cells are sensitive to the potent estrogens, 17ß-estradiol, ethynyl estradiol, and diethylstibesterol, and well-characterized weaker environmental estrogens like genistein, HPTE (an estrogenic pesticide metabolite), and 4-nonylphenol. The EC50 for estradiol was about 0.01 nM, reaching maximal induction at 0.1 nM. The antiestrogen, ICI 182,780, was able to completely inhibit the induction of luciferase expression by 0.1 nM estradiol at 10 nM, with an IC50 of 1 nM. In addition, we were able to replicate, in this in vitro assay, the observation that low concentrations of cadmium were able to induce estrogen-dependent gene expression, an effect that was completely inhibited by the potent antiestrogen ICI 182,780. The potent glucocorticoid receptor agonist, dexamethasone, was without effect as an ER agonist at concentrations up to 10 nM, whereas the potent androgen, dihydrotestosterone (DHT), showed no induction at concentration of 50 µM, but was a partial agonist at high concentrations of 0.2 mM and above. In summary, we have developed a specific, sensitive estrogen-responsive gene expression assay in a stable cell line that could possibly be adapted for high throughput screening of large numbers of chemicals for estrogenic and antiestrogenic activity. In addition, herein we also provide key protocol recommendations necessary to identify and eliminate common problems encountered in in vitro screening for estrogenicity.
Key Words: estrogenicity; in vitro screening assay; estrogen-responsive luciferase reporter.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Bitto, F. Polito, B. Burnett, R. Levy, V. Di Stefano, M. A. Armbruster, H. Marini, L. Minutoli, D. Altavilla, and F. Squadrito Protective effect of genistein aglycone on the development of osteonecrosis of the femoral head and secondary osteoporosis induced by methylprednisolone in rats J. Endocrinol., June 1, 2009; 201(3): 321 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Hotchkiss, C. V. Rider, C. R. Blystone, V. S. Wilson, P. C. Hartig, G. T. Ankley, P. M. Foster, C. L. Gray, and L. E. Gray Fifteen Years after "Wingspread"--Environmental Endocrine Disrupters and Human and Wildlife Health: Where We are Today and Where We Need to Go Toxicol. Sci., October 1, 2008; 105(2): 235 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Wilkinson, S. Hayes, D. Thompson, P. Whitney, and K. Bi Compound Profiling Using a Panel of Steroid Hormone Receptor Cell-Based Assays J Biomol Screen, September 1, 2008; 13(8): 755 - 765. [Abstract] [PDF]
|
|
|
|
 |
|
 C. Mao, N. M. Patterson, M. T. Cherian, I. O. Aninye, C. Zhang, J. B. Montoya, J. Cheng, K. S. Putt, P. J. Hergenrother, E. M. Wilson, et al. A New Small Molecule Inhibitor of Estrogen Receptor {alpha} Binding to Estrogen Response Elements Blocks Estrogen-dependent Growth of Cancer Cells J. Biol. Chem., May 9, 2008; 283(19): 12819 - 12830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Sabo-Attwood, J. L Blum, K. J Kroll, V. Patel, D. Birkholz, N. J Szabo, S. Z Fisher, R. McKenna, M. Campbell-Thompson, and N. D Denslow Distinct expression and activity profiles of largemouth bass (Micropterus salmoides) estrogen receptors in response to estradiol and nonylphenol J. Mol. Endocrinol., October 1, 2007; 39(4): 223 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Penza, C. Montani, A. Romani, P. Vignolini, B. Pampaloni, A. Tanini, M. L. Brandi, P. Alonso-Magdalena, A. Nadal, L. Ottobrini, et al. Genistein Affects Adipose Tissue Deposition in a Dose-Dependent and Gender-Specific Manner Endocrinology, December 1, 2006; 147(12): 5740 - 5751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pillet, M. D'Elia, J. Bernier, J.-M. Bouquegneau, M. Fournier, and D. G. Cyr Immunomodulatory Effects of Estradiol and Cadmium in Adult Female Rats Toxicol. Sci., August 1, 2006; 92(2): 423 - 432. [Abstract] [Full Text] [PDF]
|
|