Analysis of the estrogen receptor (ER) gene, transcript and protein in ER-positive and -negative breast cancer cell lines

L. E. Yaich, N. Roodi, L. R. Bailey, C. S. Verrier, C. J. Yee, D. R. Cavener, F. F. Parl

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

The estrogen receptor (ER) content of breast cancers predicts the likelihood of benefit from antiestrogen therapy. Tumors with an ER content of less than 10 fmol/mg are considered to be ER-negative and have a poor response rate to tamoxifen. The molecular basis of the ER-negative phenotype is not well understood. This study was conducted in five ER-positive (MCF-7, T47D, ZR-75-1, MDA-MB-134, MDA-MB-361) and five ER-negative (BT-20, HBL-100, MDA-MB-157, MDA-MB-231, MDA-MB-468) human breast cancer cell lines to determine whether the ER-negative phenotype results from mutations in the coding region of the ER gene or is due to a deficiency of the transcriptional or post-transcriptional regulation of ER expression. The coding region was examined for mutations by denaturing gradient gel electrophoresis, single-strand conformation polymorphism and DNA sequence analysis. The presence and integrity of ER mRNA were investigated by Northern blotting and reverse transcription (RT)-PCR amplification. The presence and concentration of ER protein were assessed by Western blotting and hormone-binding assay. Both ER-negative and -positive cell lines contained silent mutations in codons 10, 325 and 594 of the ER gene, all of which probably represent neutral polymorphic sites. No missense or nonsense mutations were identified in any of the cell lines. About 2 kb of the 5' upstream region and 4.3 kb of the 3' untranslated region of the ER gene were present and grossly intact in all cell lines. By Northern blotting, ER mRNA was shown to be present in all ER-positive cell lines and, with the exception of BT-20 cells, absent in all ER-negative cell lines. However, ER mRNA was found to be detectable in all ER-negative cell lines when using the more sensitive RT-PCR. ER protein was undetectable by Western blotting in all cell lines that were ER-negative by ligand-binding assay. This study indicates that the ER-negative phenotype of breast cancer cell lines is not the result of ER gene mutations. It is due to the actual absence of ER protein which results from deficient ER expression at the transcriptional or post-transcriptional level.

Original languageEnglish (US)
Pages (from-to)293-309
Number of pages17
JournalEndocrine-Related Cancer
Volume2
Issue number4
DOIs
StatePublished - Jan 1 1995

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Oncology
  • Endocrinology
  • Cancer Research

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