Stable transfection of the oestrogen receptor gene into a human osteosarcoma cell line.
The oestrogen receptor (ER) gene was introduced into an ER-negative osteoblast-like osteosarcoma cell line HTB 96 by transfection. A number of clones were isolated which expressed ER at levels of up to 70 fmol/mg cytosol protein as determined by immunoassay. Scatchard analysis of the binding of [3H]17 beta-oestradiol in cytosols demonstrated the presence of high affinity binding sites, with a dissociation constant of 0.08-0.13 nM at 4 degrees C. High levels of a 3 kb ER mRNA are produced by the clones, which have gene copy numbers ranging from 2 to greater than 10. Functional receptor activity has been demonstrated by co-transfection of a plasmid containing the chloramphenicol acetyl transferase (CAT) gene linked to an oestrogen response element. Induction of CAT activity is observed in the presence of added oestradiol and is concentration-dependent. The transfected ER is also able to affect endogenous cellular function as several ER-positive clones, but not HTB 96 cells, are growth inhibited by oestradiol in the concentration range 10(-9)-10(-7) M. These effects on growth are not induced by other classes of steroids and are reversible by antioestrogens. No endogenous genes have yet been identified which are oestrogen-regulated in ER-transfected clones.
Watts
C K
CK
Hormone Biochemistry Laboratories, Imperial Cancer Research Fund, London, U.K.
Parker
M G
MG
King
R J
RJ
D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004958:Estradiol / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism; D012516:Osteosarcoma; D010957:Plasmids; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D004958:Estradiol