Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: Inhibition of cat expression by anti-androgens

Abstract

A hormone-inducible transriptional system has been established, based on the stable transfection of the rat androgen receptor (rAR) and a reporter plasmid containing the mouse mammary tumour virus promoter linked to the chloramphenicol acetyltransferase gene (pMMTV-CAT) into steroid receptor-negative CV-1 cells. First, the rAR was stably introduced into CV-1 cells. Single clones were tested for stable expression of functionally active AR by analysing the effect of dihydrotestosterone on induction of transiently transfected pMMTV-CAT. Stable transfection and the expression of AR was confirmed by steroid-binding assays. In a second step, a clone expressing physiological amounts of AR protein (30 fmol/mg protein) was stably transfected with pMMTV-CAT to yield a permanent cell line that stably expresses functional AR and MMTV-CAT sequences. This cell line provides a powerful tool for the efficient and accurate determination and quantification of the effects of androgens and anti-androgens on reporter gene transcription. This was demonstrated by investigating the action of the three anti-androgens hydroxyflutamide, casodex and cyproterone acetate. The three compounds were shown to reverse the effects of the androgen R1881 on gene expression but were themselves devoid of agonistic activity.