Nucleic Acids Research, Vol 24, Issue 5 962-969, Copyright © 1996 by Oxford University Press
JJ Pink, SQ Wu, DM Wolf, MM Bilimoria and VC Jordan
Alterations in the amino acid sequence of the estrogen receptor (ER) have
been shown to have dramatic effects on its function. Recently, mutant ERs
have been isolated from both clinical samples and established breast cancer
cell lines, primarily through the use of the polymerase chain reaction
(PCR). All previously reported mutations have given rise to either
alterations or truncations of the ER protein. We determined the structure
of a novel 80 kDa ER which is expressed in an estrogen independent subclone
of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was
initially detected by Western blot analysis using a variety of ER specific
antibodies. PCR mapping and partial PCR mediated subcloning of the ER cDNA
were used to demonstrate that this protein was an ER containing an in-frame
duplication of exons 6 and 7. This type of duplication has not been
previously described for any members of the steroid receptor superfamily.
Karyotype analysis coupled with fluorescence in situ hybridization (FISH)
demonstrated that MCF-7:2A cells contained 4-5 copies of the ER gene in
contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH
analyses in both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is
the source of the ER in normal human cells. The relative expression level
of 2:1 is consistent with DNA gene dosage analysis. Genomic PCR was then
used to demonstrate that the 80 kDa ER mRNA was not derived from the
trans-splicing of two ER mRNAs but was the result of a genomic
rearrangement in which exons 6 and 7 were duplicated in an in-frame
fashion. This variant ER may prove to be useful in elucidating the
mechanism of estrogen action in breast cancer cells.
ARTICLES
A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
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