Analysis of the upstream regulatory region of the estrogen receptor gene in ER-positive and ER-negative breast cancer cell lines
Description
The estrogen receptor (ER) is a ligand dependent transcription factor that regulates the expression of a number of gene products involved in the proliferation and progression of breast neoplasms. Given the critical functions of the ER in the breast was well as the prognostic relationships between ER expression and breast cancer, it is necessary to elucidate the mechanism(s) responsible for the failed expression of the ER gene in nearly 40% of all breast tumors. Based upon information from other eukaryotic genes, it is known that the maintenance of the integrity of cis-elements located in the upstream regulatory region is crucial for the proper regulation of gene expression. Thus, we have hypothesized that sequence alteration in the upstream regulatory region of the ER gene may account for the failure of the ER gene to be transcribed in a number of breast tumors Sequence analyses of the upstream regulatory region of the ER gene from both the ER-positive MCF-7 and the ER-negative MDA-MB-231 breast cancer cell lines revealed a number of alterations/variations, These sequence alterations/variations included base pair additions, deletions, and substitutions. When the functional importance of these alterations/variations was tested utilizing chloramphenicol acetyl transferase (CAT) assays, two of the alterations located in the upstream region of the ER-negative MDA-MB-231 cells, a guanine insertion at -1976 and a cytosine to thymine transition at -1752, were determined to be associated with a significant 50% decrease in transcriptional activity upon comparison to control levels. From subsequent studies, this decrease in transcriptional activity was determined to be dependent upon the concurrent presence of both alterations as well as upon the exposure to estrogen and progesterone. Gel mobility analyses of DNA segments containing either of these alterations revealed no variations in binding patterns, thereby suggesting that these alterations do not independently affect the binding of trans-acting factors In addition, a number of alterations were identified between these two breast cancer cell lines and the sequence that has been previously published for leukocytes. The most prominent of these was a cluster of 17 base pair insertions located approximately 300 base pairs upstream of the Exon 1' upstream open reading frame (ORF). The role of these base pair insertions in the regulation of the expression of Exon 1' sequences was tested utilizing Northern blot analysis, RNase protection analysis, RT-PCR, and subsequent sequencing analysis upon additional breast cancer cell lines. The results of these efforts indicate that the cluster of 17 base pair insertions is not a factor in determining the expression of the Exon 1' transcript. However, the expression of this alternative ER transcript does appear to be related to tissue culture conditions and cell passage number