Skip Navigation


ToxSci Advance Access originally published online on May 27, 2004
Toxicological Sciences 2004 81(1):69-77; doi:10.1093/toxsci/kfh180
This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
81/1/69    most recent
kfh180v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (15)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wilson, V. S.
Right arrow Articles by Gray, L. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wilson, V. S.
Right arrow Articles by Gray, L. E., Jr.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

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

Vickie S. Wilson1, Kathy Bobseine and L. Earl Gray, Jr.

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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
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]

Home page
 J Mol EndocrinolHome page
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]

Home page
 EndocrinologyHome page
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]

Home page
 Toxicol SciHome page
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]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.