Dietary phytoestrogens (PEs) have been given considerable attention due to their protective role in certain hormone-dependent cancers. However, the current scientific consensus is that the outcome from increased phytoestrogen intake is unpredictable partly due to a poor understanding of their mechanisms of action in breast cells. Thus, developing a model system for phytoestrogens in breast cancer cells is a critical step in reconciling the possibility of both adverse and beneficial effect of these compounds. The potential mechanisms by which dietary PE may exert cancer protective or anti-estrogenic effects are unknown. Thus, elucidation of these mechanisms is essential in understanding their cancer-protective effects. Currently, no methodology is available to distinguish the specific cancer-protective effects of PE from their well-known estrogenic-like effects. Therefore, to understand the molecular mechanisms of phytoestrogenic chemicals in breast cancer, we have identified novel PE-responsive genes using differential display RT-PCR, representational difference analysis of cDNA, and quantitative RT-PCR. Using these methodologies, we have characterized the expression of one of these genes, PE13.1, that has not previously known to be regulated by PE. This gene is selectively activated by genistein and coumestrol but not by estradiol. Furthermore, its expression is PE specific and estrogen receptor (ER) dependent. Thus, PE13.1 is an ideal candidate to use as a model in understanding the role dietary estrogen plays in cancer prevention at the molecular level. In this proposal, we will test the hypothesis that differences in PE13.1 expression result from activity on the PE-driven upstream enhancer present in the gene and from interaction with a PE/ER complex. Therefore, we will begin to elucidate the mechanisms of regulation of the PE13.1 gene at the molecular level by characterizing PE13.1 regulatory elements (aim 1) and, in particular, how PEs in conjunction with the ER lead to PE13.1 transcriptional activation in a cell (aim 2). The key cis-regulatory elements and trans-acting factors will be identified through sequence analysis of the PE13.1 gene, particularly the 5'-flanking region, and through transfection of luciferase reporter constructs into ER-positive (MCF-7 ER+) and ER-negative (MDN-ER-) cells. We will determine which ER subtype is responsible for PE13.1 transcriptional activity and establish if differences in PE13.1 expression parallel differences in ER status in breast tumor cell lines. These experiments will begin to provide a basis of a biochemical model in which to better understand the estrogenic effect of phytoestrogen in breast cancer cells.
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