Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line

Br J Cancer. 1997;75(1):17-27. doi: 10.1038/bjc.1997.4.

Abstract

We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogen-response element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A18 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ER77. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites / genetics
  • Breast Neoplasms / chemistry
  • Clone Cells / metabolism*
  • DNA, Complementary / genetics
  • DNA, Neoplasm / chemistry
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • Exons
  • Female
  • Humans
  • Ligands
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Receptors, Estrogen / chemistry
  • Receptors, Estrogen / isolation & purification*
  • Repetitive Sequences, Nucleic Acid
  • Transcription, Genetic
  • Transfection
  • Tumor Cells, Cultured

Substances

  • DNA, Complementary
  • DNA, Neoplasm
  • DNA-Binding Proteins
  • Ligands
  • Receptors, Estrogen