Tamoxifen inhibits thyrotropin releasing hormone stimulation of transcription of a rat prolactin promoter containing fusion gene in GH, cells

Triphenylethylene anti-oestrogens. such as tamoxifen, are thought to reverse oestrogen stimulated events by a mechanism which involves their occupying the oestrogen receptor. However. there is evidence to suggest tamoxifen may have other effects such as inhibiting calmodulin-mediated events (Lam, 1984). We have investigated the effect of tamoxifen on thyrotropin releasing hormone (TRH) induction of rat prolactin (Prl) gene transcription in GH, cells and observe that i t inhibits it: it is possible that this effect is mediated by an oestrogen-receptor-independent mechanism. Transcriptional studies were carried out using a plasmid prPrl-CAT (chloramphenicol acetyl transferase) (T. Lufkin & C. Bancroft, unpublished work) which contains 2 kb of rat prolactin gene immediate 5'-flanking sequence and the CAT construct from pRSV-CAT which can be expressed in eukaryotic cells (Gorman et a/., 1982). Transient transfection of prPrl-CAT into GH, cells (2 x 10' cells per 60mm dish) was carried out by placing the plasmid (10 pgt-DEAE dextran mixture on the cells for 1 h at 37°C. This solution was then aspirated, the cells exposed to 10% ( v i v ) glycerol for 1 min. washed with Dulbecco's modified Eagle's medium (DMEM) three times and then incubated with DMEM containing 12.5% horse serum and 2.5% (v/v) fetal calf serum at 37°C in a humidified atmosphere containing 9% CO?, for 48 h. In some experiments the same medium/serum preparation which had been 'hormonedepleted' by ion-exchange resin and charcoal treatment was employed. TRH (27 n ~ ) , oestradiol (10 '-10 "'M) and/or tamoxifen (10 or 10 ' M ) were incorporated into the culture medium. To assess chloramphenicol acetyl transferase activity the cells were harvested after 48 h and sonicated, and a cytosolic extract isolated by centrifugation. This was