A SIMPLE AND SENSITIVE HIGH-THROUGHPUT ASSAY FOR STEROID AGONISTS ANDANTAGONISTS

We have developed a simple and highly sensitive tissue culture-based a ssay for the biological activity of steroids and synthetic steroidal c ompounds. A DNA cassette, containing a synthetic steroid-inducible pro moter controlling the expression of a bacterial chloramphenicol acetyl transferase gene (GRE5-CAT), was inserted into an Epstein-Barr virus ( EBV) episomal vector which replicates autonomously in primate and huma n cells. We then used this promoter/reporter system to generate two st ably transfected human cell lines. In the cervical carcinoma cell line HeLa, which expresses high levels of glucocorticoid receptor, the GRE 5 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 o ver 100-fold by either progesterone or dihydrotestosterone. In both ce ll lines basal expression of CAT activity is strictly dependent on the presence of steroid, so that very low levels of induction can be dete cted. Thus, the cell lines can be used to test for low levels of agoni st activity in steroid antagonists. These cell lines can be used to sc reen compounds for steroid agonist or antagonist activity by testing e xtracts of cells grown in microtiter wells directly using a colorimetr ic CAT assay. This system should provide a sensitive and efficient met hod for screening and analysis of the activity of large numbers of nat ural or synthetic steroid agonists or antagonists.