REGULATING HUMAN CYP1A1/CYPLAL/CYPLBL IN BREAST CANCER

描述(申请人的描述-编辑)重要性 雌激素和雌激素代谢在乳腺癌病因学中的作用 很久以前就被认可了。雌激素导致乳腺上皮细胞， 包括恶性表型的，以增殖，导致 克隆性扩张与肿瘤发生。而体内的雌激素代谢 乳腺组织可能会降低雌激素的作用，衰减率很高。 取决于所涉及的特定代谢途径，如雌激素， 非雌激素和致癌代谢物可以从17-β- 雌二醇(E_2)。雌二醇在C-2位的羟基化终止雌激素 信号，而E2在C-4上的羟基化产生代谢物 与致癌有关。因此，低的C-2/C-4羟化比 是乳腺癌的预后指标。这个实验室的最新研究 使用表达雌激素受体的MCF-7乳腺癌细胞 (Er)，结果鉴定出两种细胞色素P450(CYP) 在芳烃受体(AhR)的调控下， 催化雌二醇羟化；细胞色素P1A1，雌二醇2-，6-α和15-α- 羟基酶，以及一种似乎是CYP1B1的E2 4-羟基酶。在……里面 以不表达ER的乳腺肿瘤细胞MDA-MB-231为研究对象， AHR的激活主要诱导E2的C-4羟化。这个 研究人员假设1)C-2和C-4羟化的速率 人类乳腺上皮细胞和乳腺癌中的雌二醇组 受AhR活性控制，并反映了 2)C-2/C4羟化比率； 雌二醇与雌激素受体的表达相关。我们广泛的、长期的 目的是研究细胞色素P1A1和细胞色素P1B1基因的表达。 并评估细胞色素P1A1/细胞色素P1B1的表达比率为 乳腺癌的预后生物标志物。这样做的具体目的是 建议是：1)确定表达式之间的关系 细胞色素P1A1和细胞色素P1B1基因与C-2和C-4活性的关系 人乳腺细胞中的羟化途径。雌二醇代谢 催化表达的人细胞色素P1B1将被鉴定。这个 AhR激活对E2代谢的影响将在几个方面确定 ER阳性乳腺肿瘤，ER阴性乳腺肿瘤， 永生化非致瘤乳腺上皮细胞及原代培养 乳腺上皮细胞培养。血管紧张素转换酶激活对血管紧张素转换酶活性的影响 将测定细胞培养物中细胞色素P1A1和细胞色素P1B1基因的表达 通过Northern印迹分析细胞色素P1A1和细胞色素P1B1的mRNA水平，并通过 定量RNA聚合酶链式反应(RNA-PCR)技术 竞争性聚合酶链式反应，毛细管电泳法分离产物 激光诱导荧光检测。2)确定 雌激素受体表达与C-2/C-4羟化比率的关系 人乳腺细胞中AhR调节的E2代谢的研究。ER水平 在AIM 1研究中使用的乳腺细胞培养中的表达将是 用[~3H]E_2结合核糖核酸酶保护法检测ER基因表达水平 并使用免疫细胞化学技术。然后这些数据将被 与E_2、C_2/C_4羟化比率和细胞色素P1A1/细胞色素P1B1的相关性 用信使核糖核酸比率确定雌激素受体表达与细胞周期的关系 AHR对人乳腺癌细胞中E2代谢的调节。

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.