Identification of estrogen receptor beta expression in Chinese hamster ovary (CHO) cells and comparison of estrogen-responsive gene transcription in cells adapted to serum-free media.
Most cultured cell lines require addition of serum to the medium to maintain their proliferative capacity. For studies examining the cellular effects of estrogens serum is charcoal-stripped to remove steroids. Nonetheless, addition of the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) inhibits the basal transcriptional activity of estrogen receptors alpha or beta (ERalpha or ERbeta) in transfected cells. We tested the hypothesis that elimination of serum from the culture medium will block 4-OHT's repression of basal activity. Chinese hamster ovary (CHO-K1) cells adapted to serum-free medium exhibited estrogen responsiveness that was identical with that of the cells grown in serum-containing media. 4-OHT-suppressed basal transcription of an estrogen response element (ERE)-reporter in ERalpha-transfected cells even in the absence of serum, indicating that the 4-OHT suppressive activity is not mediated by blocking ER interaction with serum estrogens. We speculate that 4-OHT-ER recruits co-repressors to suppress basal transcription. We discovered that CHO-K1 cells express ERalpha and ERbeta mRNA. However only ERbeta protein was expressed and use of ERbeta-selective 2,3-bis(4-hydroxy-phenyl)propionitrile (DPN) and ERalpha-selective 4-propyl-1,3,5-tris(4-hydroxy-phenyl)pyrazole) (PPT) revealed that only ERbeta was transcriptionally active. In conclusion, growing CHO-K1 in serum-free medium does not impact the estrogen responsiveness and this cell line expresses functional ERbeta.
Thomas
Padmaja B
PB
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
Risinger
Kelly E
KE
Klinge
Carolyn M
CM
D000818:Animals; D016466:CHO Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D020847:Estrogen Receptor Modulators / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D000431:Ethanol / Q000494:pharmacology; D000077267:Fulvestrant; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D009570:Nitriles / Q000737:chemistry / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011960:Receptors, Estrogen / Q000819:agonists / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects / Q000235:genetics; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D016895:Culture Media, Serum-Free; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D009570:Nitriles; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D000077267:Fulvestrant; D000431:Ethanol; D004958:Estradiol; D008156:Luciferases