A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7.
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.
Pink
J J
JJ
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Wu
S Q
SQ
Wolf
D M
DM
Bilimoria
M M
MM
Jordan
V C
VC
D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism*; D002871:Chromosome Banding; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011960:Receptors, Estrogen / Q000235:genetics / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D011960:Receptors, Estrogen