Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: inhibition of cat expression by anti-androgens.
A hormone-inducible transcriptional 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.
Fuhrmann
U
U
Research Laboratories of Schering AG, Berlin, Germany.
Bengtson
C
C
Repenthin
G
G
Schillinger
E
E
D000726:Androgen Antagonists / Q000494:pharmacology*; D000728:Androgens / Q000378:metabolism*; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D002999:Clone Cells; D004351:Drug Resistance / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011944:Receptors, Androgen / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D015533:Transcriptional Activation; D014162:Transfection
D000726:Androgen Antagonists; D000728:Androgens; D011944:Receptors, Androgen; D015500:Chloramphenicol O-Acetyltransferase; D009355:Neomycin