A simple and sensitive high-throughput assay for steroid agonists and antagonists.
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.
White
J H
JH
Department of Physiology, McGill University, Montreal, Que., Canada.
McCuaig
K A
KA
Mader
S
S
D001943:Breast Neoplasms / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D010957:Plasmids; D011374:Progesterone / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D013256:Steroids / Q000819:agonists* / Q000037:antagonists & inhibitors*; D014162:Transfection; D014407:Tumor Cells, Cultured
D013256:Steroids; D013196:Dihydrotestosterone; D011374:Progesterone; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase