DNA-binding of androgen receptor overexpressed in mammalian cells

In order to investigate the DNA-binding properties of the rat androgen receptor (rAR) in mammalian cells after addition of androgens and antiandrogens, we established a gel-shift assay with extract from COS-1 cells (CV-1 cells transformed with the DNA-tumour virus SV40) over-expressing the rAR. First, the rAR was overexpressed in COS-1 cells. Therefore the full-length AR cDNA was inserted immediately downstream from the SV40 early promoter of pECE to generate pECE-AR. Expression of the rAR driven by the SV40 early promoter yields constant and high levels of rAR protein. In addition, the vector contains the SV40 origin of replication for obtaining high copy vector numbers in COS-1 cells. The rAR-containing expression vector was transiently transfected into COS-1 cells using Transfectam Reagent, in order to achieve high transfection efficiency. Expression of biologically active receptor was tested by analyzing the effect of the synthetic androgen R1881 on induction of transiently transfected pMMTV-CAT. Steroid binding assays were carried out to confirm overexpression of biologically active AR and to determine the binding of different hormones and antihormones to AR in COS-1 cells transiently transfected with pECE-AR. Gel-shift experiments performed with whole cell extract of those cells, containing approximately 700 fmol AR/mg protein, and labeled AR-binding GRE (glucocorticoid responsive element) showed that R1881 induced the formation of a protein-GRE complex. Furthermore, the R1881-induced formation of the protein-GRE complex could be completed by addition of unlabeled excess of GRE but not of unspecific oligonucleotides, confirming sequence-specific binding of the R1881-induced protein-GRE complex.