Estrogen receptor alpha and beta (ERα and -β) gene and protein expression in breast cancer cell lines: verification by RT-PCR and Western blotting
MD Al-Bader, CHJ Ford, J Jacob, LJ Jacob & SS Mohan
Two estrogen receptor isoforms are known to exist, ERα and ERβ. The expression of ER isoforms in breast cancer cell lines was studied to see whether both ERs, as well as any other variants, are expressed at the mRNA and protein level. Three breast cell lines: two which are known to be ER+ve (MCF7 and T47D) and one reported as ER-ve (MDA-MB231) were used in this experiment. For gene expression studies RT-PCR methodology was applied; primers were used that detect ERα and ERβ isoforms and their variants. For protein measurements Western Blotting was the method of choice and the antibodies used were: two monoclonal antibodies raised against the steroid binding domain or the hinge region of the ERα, named ERα -S and ERα -H, respectively, and a polyclonal antibody against the ERβ. All cell lines expressed the ERα and ERβ gene and protein isoforms, however, the MDA-MB231 cell line showed a different pattern of expression of the variants whereby some variants were expressed only in this cell line and not in the ER+ve cell lines. Cell lines that have been reported to be positive for ERα (MCF7 and T47D) are also positive for ERβ. Interestingly the cell line which has been reported to be ER-ve showed positivity for both ER isoforms. The presence of ER variants in breast cancer cell lines, created by alternative splicing, where entire exons are skipped, has promoted the hypothesis that tamoxifen resistance and estrogen independent tumor growth could be caused by these variants which may explain why many estrogen receptor positive tumors develop resistance to anti-estrogens such as tamoxifen. We are currently working on the expression of these isoforms and their variants in breast cancer tissue to try and highlight what this means in development, progression and treatment of breast cancer.