This component of the ARCH grant proposal will provide an accelerated mechanism for the advanced training of Dr. Ronald Thomas (FAMU) in the application of contemporary research techniques to current problems in environmental health. The impact of in situ hormone metabolism on breast cancer development and progression is an emerging concern. In the breast, free estrogens produce powerful mitogenic effects and are key factors in the growth and progression of breast cancer. To control unbridled estrogenic stimulus, estrogen receptor levels are kept to a minimum in normal breast tissue, and the enzymes that are involved in the in situ biosynthesis, metabolism, and inactivation of free estrogens are tightly regulated. The titration of intra-tissue active hormone levels has been described as """"""""intracrinology."""""""" In estrogen target tissues, estrogen sulfotransferase (SULT1E) and various cytochromes P450 metabolize estrogens and disable the mitogenic effects of free estrogens. Preliminary data suggest that SULT1E, CYP1A2, CYP2B6, and CYP3A4 are expressed in the MCF10A cell line, a model of normal breast epithelium, suggesting that these enzymes may contribute to estrogen intracrinology. The hypothesis of this proposal is that SULT1E, CYP1A2, CYP2B6, and CYP3A4 are expressed and tightly regulated in MCF10A breast epithelial cells, with each gene under the transcriptional control of a distinct set of trans-acting factors, and that these enzymes represent integral components of the breast epithelial estrogen intracrinology machinery.
The specific aims of this proposal are (1) to determine the molecular basis for differential expression of SULT1E in MCF10A (breast epithelial) and MCF7 (breast cancer) cells, (2) to define the expression of CYP1A2, CYP2B6, and CYP3A4 in MCF10A breast epithelial cells, (3) to determine the molecular basis for the breast epithelial cell-specific expression of CYP1A2, CYP2B6, and CYP3A4, and (4) to determine, through the use of microarray analysis, whether changes in the molecular phenotype of MCF10A or MCF7 cells that are produced following genetically induced alterations in CYP1A2, CYP2B6, CYP3A4, or SULT1E expression are solely attributable to effects on cellular levels of bioactive estrogen. These studies will provide essential information on both the molecular mechanisms that determine the expression of SULT1E, CYP1A2, CYP2B6, and CYP3A4 in breast epithelial cells, and the role that these enzymes play in estrogen intracrinology.
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