Differential Expression in Estrogen Receptor alpha (ESRα) and beta (ESRβ) in breast (MCF-7) and prostate (PC3) cell lines

Paul Bechtel


The importance of estrogen in both men and women for cell growth and development is widely documented. Estrogen initiates alteration of cell behavior within target tissues by binding to Estrogen Receptor (ESR). The two documented estrogen receptor isoforms, alpha (ESRα) and beta (ESRβ) subsequently trigger gene activation. Because ESRís mediate all estrogen effects, they are important in oncology and specifically prostate and breast cancer. This study aimed to identify expression level differences between ESRα and ESRβ in breast (MCF-7) and prostate (PC-3) cell lines. RT-PCR was used to amplify the ESRís from purified mRNA. The PCR product was fractionated on a 1% agarose gel, purified, sequenced and aligned with the ESR mRNA sequences from the GenBank database. ESRα was expressed in the breast (MCF-7) but not in prostate (PC-3) cell lines, whereas, ESRβ was expressed in both cell lines. Amplification of ESRβ yielded more PCR product in PC-3 compared to MCF-7 in 4/5 replicates (80%). The growing literature pertaining to ESR expression in specific tissues can aid in the development of new selective estrogen receptor modulators (SERMs) for disease treatment. This experiment adds to the documented literature and provides additional replication of results found in previous studies.


Figure 1-Primer sequences used, location and expected size of amplicons

Figure 2

Figure 3-(A) Formatted alignments of purified PCR product sequence (ESR1{alpha}PCR) versus Homo sapien ESR1, mRNA (Accession # NM_000125). (B) Formatted alignments of sequenced purified PCR (ESR2{beta}PCR) versus Homo sapien ESR2, mRNA (Accession # NM_001437.1). PCR product was purified via the Rapid PCR Purification System (Marligen BioSciences Inc., Ljamsville MD), sequenced via the Sangerís dideoxy method AmpliCycleTM Sequencing Kit (Perkin Elmer, Foster City CA), compared to the NCBI-Genbank database and aligned via ClustalW in MacVector.

Estrogen is important for cell differentiation, growth and development in both men and women. A non-exhaustive list of specific roles includes the following: differentiation and development of reproductive tissues including mammary glands in women and testis, epididymus, and prostate in men; protection against osteoporosis via maintenance of bone density; reduction of lipid and cholesterol levels in blood acting as a cardioprotective hormone; regulation of reproductive behavior, homeostasis and general mood (Couse et al. 1997, Kurebayashi et al. 2000, Osborne et al. 2000, Signoretti and Loda 2001, Matthews and Gustafsson 2003, Public communication: http://newscenter.cancer.g ov/sciencebehind/,accessed 5/3/06).

In addition to its normal physiological role, the prevalence and progression of cancerous cells within the body has been linked to prolonged stimulation by higher than normal levels of estrogen (Osborne et al. 2000). In fact, anti-estrogens are used in the treatment of certain breast cancers and some prostate cancers.

Estrogens and estrogen-like molecules act on target tissues and alter cell activity and behavior by binding to estrogen receptors (ESRís). The estrogen receptor is a ligand-activated transcription factor that belongs to the nuclear receptor superfamily and mediates all biological activity of estrogen in target tissues (Matthews and Gustafsson 2003). Currently, two ESRís, alpha (ESRα) and beta (ESRβ), are documented in the literature. A thorough description of the promotor region and exons found within ESRα is provided by Kos et al. (2001). Since the cloning of ESRα in 1986 (Green et al. 1986a, Green et al. 1986b), it was believed for over a decade that ESRα was the only ESR. Then in 1996, ESRβ was first cloned from rat prostate by Kuiper et al. (1996) and then from humans by Mosselman et al. (1996). The full-length human ESRβ was isolated by Ogawa et al. (1998). Interesting to note, a novel isoform called ESR gamma may have been discovered another decade later in 2006, although the work is not yet published (Personal Communication; Brian Fox, Fordham University).

ESRβ is smaller than ESRα but both possess distinct functional domains, termed A through F, characteristic of the nuclear receptor superfamily (Couse et al. 1997). Specifically, ESRβ is homologous to ESRα in the DNA and ligand binding domains (Mosselman et al. 1996). ESRβ also binds estradiol, a prevalent form of estrogen, with similar affinity as ESR α (Kurebayashi et al. 2000).

Despite sharing high sequence homology, ESRα and ESRβ differ in many facets including tissue location, cellular function and expression level. (Krege et al. 1998, Matthews and Gustafsson 2003). Furthermore, each has several isoform variants possibly due to exon splicing, exon duplication and/or multiple start codons. These differences will be briefly discussed below with an emphasis on breast and prostate cells.

ESRα is expressed primarily in the uterus, liver, kidney and heart, whereas, ESRβ is expressed primarily in the ovary, prostate, lung, bladder and central nervous system. Both are co-expressed in a number of tissues including but not limited to mammary glands, thyroid and parts of the brain (Matthews and Gustaffson 2003).

Overall, ESR expression usually varies among tissues. Adding to the complexity, even if both ESRís are expressed in the same tissues, they may not be expressed in the same cells (Matthews and Gustafsson 2003). For example, expression of ESRβ in epithelial normal prostate cells is reproducible; however, ESRβ can exhibit both high expression (Lau et al. 2000, Horvath et al. 2001) and decreased expression (Latil et al. 2001) in prostate cancer epithelial cells. Further, Saji et al. (2000) determined that although both ESRís are expressed in rat mammary glands, their presence and cellular distribution is unique.

Adding to the complexity, both ESRís can posses multiple variant forms as well. For example, there is a 46 kDa isoform of ESRα that lacks the N-terminal 173 amino acids found in the normal 66 kDa isoform (Flouriot et al. 2000). This isoform variant is documented in MCF-7 (Flouriot et al.2000). Several ESRβ isoform variants have also been identified. Fuqua et al. (1999) was the first to characterize ESRβ expression and variant ESRβ isoforms at the protein level in breast cancer cells. Even splice variants within the ESRβ family like ERβ1 and ERβ2 are known to vary in expression and functional roles (Saunders et al. 2002). Additionally, ESRα and ESRβ can form heterodimers when both are co-expressed, however, the biological roles of these heterodimers remains unknown.

With such variation, it is not surprising that the definitive roles of estrogen receptor in the initiation, suppression or progression of prostate and breast cancer have not yet been fully defined. This gap in understanding has direct implications for effectiveness of treatments. Selective estrogen receptor modulators, (SERMs), selectively stimulate or inhibit the estrogen receptors of different target tissues. Unfortunately, even the most common SERM, Tamoxifen, is not fully effective. In fact, Tamoxifen has many of the same antagonistic effects documented for estrogen. Information on development and future goals of SERMís is found in Osborne et al. (2000).

Obviously more research is needed on the role and expression of ESR to improve upon or to develop new drugs. There is a need to document expression level differences among tissues and within variant tissue cell lines from normal to cancerous. This study was designed to provide new information on ESR expression. Specifically, the aim of this study is to characterize expression level differences between ESRα and ESRβ in two cancer cell lines, MCF-7 (breast) and PC-3 (prostate).

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Special thanks to Dr. Berish Rubin and the Fordham Biology Department for offering such a worthwhile and applied learning experience. I greatly appreciate the tireless support and guidance provided by the Teaching Assistants, Jinsong Qiu and Lisa Sarran. Thanks to Dr. Raj Kanpal for providing the MCF-7 and PC-3 cell lines and to Brian Fox for helping with RNA purification and for providing continued guidance and problem solving during this endeavor. I thank my friend Susan Hudachek, Colorado State University for long-distance support. Finally, I thank my parents for always encouraging me to take on new challenges and to learn in the process.

This document was last modified 05/16/2006.
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