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A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7
1Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Abstract:
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.
Insights
Researchers discovered a novel 80 kDa estrogen receptor (ER) variant in breast cancer cells. This variant, caused by duplicated exons 6 and 7, offers new insights into estrogen receptor function and breast cancer mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Estrogen receptor (ER) mutations significantly impact its function.
- Mutant ERs are often found in clinical samples and breast cancer cell lines.
- Previous mutations typically resulted in altered or truncated ER proteins.
Purpose of the Study:
- To determine the structure of a novel 80 kDa ER.
- To investigate the genetic basis of this ER variant in an estrogen-independent breast cancer cell line (MCF-7:2A).
- To explore the implications of this variant for understanding estrogen action in breast cancer.
Main Methods:
- Western blot analysis using ER-specific antibodies.
- Polymerase chain reaction (PCR) mapping and subcloning.
- Karyotype analysis and fluorescence in situ hybridization (FISH).
- Genomic PCR to analyze mRNA origins.
Main Results:
- A novel 80 kDa ER variant was identified in MCF-7:2A cells.
- This variant contains an in-frame duplication of exons 6 and 7, a previously undescribed event in steroid receptors.
- MCF-7:2A cells showed increased ER gene copy number (4-5 copies) compared to control cells (2 copies).
- The ER gene was localized to chromosome 6 in both cell lines.
- Genomic rearrangement, not trans-splicing, was identified as the cause of the exon duplication.
Conclusions:
- A novel ER variant with duplicated exons 6 and 7 was characterized.
- This variant arises from a genomic rearrangement and is associated with increased ER gene copy number.
- The 80 kDa ER variant provides a new tool for studying estrogen signaling in breast cancer.