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 the nucleo-protein organization of the target MMTV promoter, we have established a new cell line by integrating in CV-1 cells a construct containing the long terminal repeat of the mouse mammary tumor virus (MMTV-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 aldosterone in cells expressing MR transiently. The CV-1 cells were transfected with the construct pMAMneoCAT, a plasmid containing the MMTV promoter driving the chloramphenicol acetyl transferase (CAT) gene and a gene for neomycin selection. A neomycin-resistant clone (M8), which contains two copies of the unrearranged construct was characterized. The integrated MMTV promoter is functional, as demonstrated by the induction of the CAT activity upon addition of aldosterone, dexamethasone, and R5020 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 to 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 M8, provides a useful tool for selectively studying the effect of MR on a target promoter organized into chromatin.
Couette
B
B
INSERM U246, Faculté de Médecine Xavier Bichat, Paris, France.
Le Ricousse
S
S
Fortin
D
D
Rafestin-Oblin
M E
ME
Richard-Foy
H
H
D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D011397:Promegestone / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D018161:Receptors, Mineralocorticoid / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015398:Signal Transduction; D013256:Steroids / Q000494:pharmacology*; D014158:Transcription, Genetic
D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D011980:Receptors, Progesterone; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D013256:Steroids; D000450:Aldosterone; D003907:Dexamethasone; D011397:Promegestone