A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules.
With the aim of quickly and easily characterizing new estrogen or anti-estrogen molecules, we developed a cellular model in which estrogenic action can be detected by bioluminescence. This model is based on MCF-7 cells stably transfected with a receptor gene which allows expression of the firefly luciferase enzyme under control of the estrogen regulatory element of the Xenopus vitellogenin A2 gene. A stably transfected cell line (cultured for more than eight months without loss of the chimeric estrogenic response) was established by cotransfection of a neomycin resistance gene and cloning under selective pressure. Subcloning luminescent clones was accomplished by using a single-photon detecting camera. This cellular model allowed the study of an estrogenic activity either in whole-cell or in cell-free experiments by detection of the induced luciferase. Estradiol induced the luciferase activity in a dose-dependent manner at subnanomolar concentrations. The induced luciferase activity reached a maximum level as early as 24 hours after the cells were incubated with estradiol. The antiestrogen 4-hydroxy-tamoxifen inhibited the luciferase activity induced by estradiol. The cross-reactivity of ligands, such as dexamethasone, progesterone, testosterone, aldosterone, calcitriol, oxysterol and retinoic acid, were also studied, showing an estradiol specificity for a 24-hour incubation time.
Pons
M
M
INSERM, Montpellier, France.
Gagne
D
D
Nicolas
J C
JC
Mehtali
M
M
D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D004958:Estradiol / Q000494:pharmacology*; D015870:Gene Expression; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D010957:Plasmids; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014981:Xenopus
D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D008156:Luciferases