THE ESTABLISHMENT OF THE LONG TERMINAL REPEAT OF THE MOUSE MAMMARY-TUMOR VIRUS INTO CV-1 CELLS ALLOWS A FUNCTIONAL-ANALYSIS OF STEROID-RECEPTORS

To analyze in situ the effects of mineralocorticoid receptor (MR) on t he nucleo-protein organization of the target MMTV promoter, we have es tablished a new cell line by integrating in CV-1 cells a construct con taining the long terminal repeat of the mouse mammary tumor virus (MMT V-LTR). The MMTV-LTR contains glucocorticoid response elements (GREs), known to interact with MR. CV-1 cells were selected because they lack glucocorticoid receptor (GR). The absence of GR in the host cell line allows the selective analysis of transcription activation by aldoster one in cells expressing MR transiently. The CV-1 cells were transfecte d with the construct pMAMneoCAT, a plasmid containing the MMTV promote r driving the chloramphenicol acetyl transferase (CAT) gene and a gene for neomycin selection. A neomycin-resistent clone (M8), which contai ns two copies of the unrearranged construct was characterized. The int egrated MMTV promoter is functional, as demonstrated by the induction of the CAT activity upon addition of aldosterone, dexamethasone, and R 5020 to M8 cells transiently transfected with MR, GR, and progesterone receptor (PR) expression vectors, respectively. Induction of the CAT activity by dexamethasone or progesterone was 2 to 3-fold higher than by aldosterone. These differences in CAT activities were not related t o differences in the levels of receptor expression. In the transiently transfected M8 cells, MR and PR contents were similar (50-70 fmol/mg protein) while GR content was higher (250 fmol/mg protein). Thus, this new cell line Ms, provides a useful tool for selectively studying the effect of MR on a target promoter organized into chromatin.