A Simple and Sensitive High–Throughput Assay for Steroid Agonists and Antagonists

• Published in: Bio/Technology 1994-10, Vol.12 (10), p.1003-1007
• Main Authors: White, John H., McCuaig, Kimberly A., Mader, Sylvie
• Format: Article
• Language: English
• Subjects: Agriculture; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Breast Neoplasms - metabolism; Chloramphenicol O-Acetyltransferase - genetics; Dexamethasone - pharmacology; Dihydrotestosterone - pharmacology; Epstein-Barr virus; Fundamental and applied biological sciences. Psychology; Gene Expression - drug effects; Gene Transfer Techniques; Genes, Reporter; Genetic engineering; Genetic technics; HeLa Cells; Herpesvirus 4, Human - genetics; Humans; Life Sciences; Methods. Procedures. Technologies; Plasmids; Progesterone - pharmacology; Promoter Regions, Genetic; Steroids - agonists; Steroids - antagonists & inhibitors; Transfection; Tumor Cells, Cultured; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0733-222X; 1546-1696; 2331-3684
• DOI: 10.1038/nbt1094-1003

We have developed a simple and highly sensitive tissue culture–based assay for the biological activity of steroids and synthetic steroidal compounds. A DNA cassette, containing a synthetic steroid–inducible promoter controlling the expression of a bacterial chloramphenicol acetyltransferase gene (GRE5–CAT), was inserted into an Epstein–Barr virus (EBV) episomal vector which replicates autonomously in primate and human cells. We then used this promoter/reporter system to generate two stably transfected human cell lines. In the cervical carcinoma cell line HeLa, which expresses high levels of glucocorticoid receptor, the GRE5 promoter is inducible over 100–fold by the synthetic glucocorticoid dexamethasone. In the breast carcinoma cell line T47D, which expresses progesterone and androgen receptors, the GRE5 promoter is inducible over 100–fold by either progesterone or dihydrotestosterone. In both cell lines basal expression of CAT activity is strictly dependent on the presence of steroid, so that very low levels of induction can be detected. Thus, the cell lines can be used to test for low levels of agonist activity in steroid antagonists. These cell lines can be used to screen compounds for steroid agonist or antagonist activity by testing extracts of cells grown in microtiter wells directly using a colorimetric CAT assay. This system should provide a sensitive and efficient method for screening and analysis of the activity of large numbers of natural or synthetic steroid agonists or antagonists.