Characterization of stably transfected fusion protein GFP-estrogen receptor-alpha in MCF-7 human breast cancer cells.
Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.
Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Winnipeg, Canada R3E 0V9.
Helen
H
Zhao
Laura L
LL
Hart
Ulrike
U
Keller
Laurel T
LT
Holth
James R
JR
Davie
D001943:Breast Neoplasms / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002945:Cisplatin / Q000378:metabolism; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006207:Half-Life; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D047628:Estrogen Receptor alpha; D004967:Estrogens; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline; D002945:Cisplatin