REGULATING HUMAN CYP1A1/CYPLAL/CYPLBL IN BREAST CANCER

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DESCRIPTION (Applicant's Description-edited) The importance of estrogens and estrogen metabolism in the etiology of breast cancer has long been recognized. Estrogens cause breast epithelial cells, including those of malignant phenotype, to proliferate, resulting in clonal expansion and tumorigenesis. While estrogen metabolism within mammary tissue may reduce the estrogenic effect, attenuation is highly dependent on the specific pathways of metabolism involved, as estrogenic, nonestrogenic and carcinogenic metabolites can be produced from 17-beta- estradiol (E2). Hydroxylation of E2 at C-2 terminates the estrogenic signal, whereas hydroxylation of E2 at C-4 produces a metabolite associated with carcinogenesis. Thus, a low C-2/C-4 hydroxylation ratio is prognostic for breast cancer. Recent studies in this laboratory using MCF-7 breast cancer cells, which express the estrogen receptor (ER), resulted in the identification of two cytochromes P450 (CYP) under the regulatory control of the aryl hydrocarbon receptor (AhR) that catalyze E2 hydroxylation; CYP1A1, an E2 2-, 6-alpha and 15-alpha- hydroxylase, and an E2 4-hydroxylase which appears to be CYP1B1. In studies with the MDA-MB-231 breast tumor cells, which do not express ER, AhR activation induces predominately C-4 hydroxylation of E2. The investigators hypothesize that 1) the rates of C-2 and C-4 hydroxylation of E2 in human breast epithelial cells and breast carcinomas are controlled by AhR activity and reflect the relative expression of the CYP1A1 and CYP1B1 genes, and 2) the ratio of C-2/C4 hydroxylation of E2 will correlate with expression of the ER. Our broad, long-term objectives are to characterize CYP1A1 and CYP1B1 gene expression in breast cancer and to evaluate the CYP1A1/CYP1B1 expression ratio as a prognostic biomarker of breast cancer. The specific aims of this proposal are to: 1) Determine the relationship between the expression of the CYP1A1 and CYP1B1 genes and the activity of the C-2 and C-4 hydroxylation pathways in human breast cells. The E2 metabolism catalyzed by cDNA-expressed human CYP1B1 will be characterized. The effects of AhR activation on E2 metabolism will be determined in several lines of ER-positive breast tumor, ER-negative breast tumor, immortalized nontumorigenic breast epithelial cells and in primary cultures of mammary epithelial cells. The effects of AhR activation on CYP1A1 and CYP1B1 gene expression in cell cultures will be determined by Northern blot analysis of CYP1A1 and CYP1B1 mRNA levels and by a quantitative RNA polymerase chain reaction (RNA-PCR) technique employing competitive PCR, separation of the products by capillary electrophoresis and detection by laser-induced fluorescence. 2) Determine the relationship between ER expression and the C-2/C-4 hydroxylation ratio of AhR- regulated E2 metabolism in human breast cells. Levels of ER expression in breast cell cultures used in the studies of aim 1 will be evaluated by [3H]E2 binding, RNase protection assay for ER mRNA levels and by using immunocytochemical techniques. These data will then be correlated with the E2 C-2/C4 hydroxylation ratios and CYP1A1/CYP1B1 mRNA ratios to determine the relationship between ER expression and AhR-regulated E2 metabolism in breast human cancer cells.