Polymorphisms of estrogen-metabolizing genes in kidney cancer

描述(由申请人提供)： 背景：肾细胞癌(RCC)的发病率一直在上升，男性多于女性。肾癌的病因尚不清楚，但根据我们的初步数据和以前的文献，很明显，雌激素代谢基因(EMG)，如细胞色素P450(CYP)1b1和儿茶酚-O-甲基转移酶(COMT)参与了肾癌的发病。已有研究表明，EMG的单核苷酸多态(SNPs)可以改变酶的水平或催化活性，因此可能在肾癌的发生中起作用。研究目的：本项目的主要目的是确定肌电基因多态性是否是肾癌的危险因素。我们推测：(1)EMG SNPs参与了肾癌的发病过程，(2)EMG SNPs导致肾癌细胞色素P1B1酶活性升高，COMT酶活性降低，(3)COMT过表达或细胞色素P1B1基因沉默通过细胞周期停滞和细胞凋亡途径抑制肾细胞癌。项目设计与方法：目的1.探讨肌电SNPs是否是肾癌的危险因素。血液和肾癌组织将从患者身上以及年龄匹配的健康男性志愿者的血液中采集。将进行以下实验：(A)利用序列特异性聚合酶链式反应和基因组直接测序技术检测不同阶段和分级的肾癌患者血液中的EMG启动子和错义SNPs；(B)为了进行比较，检测年龄匹配的健康对照组血液样本中的EMG SNPs；以及(C)确定EMG SNPs是否影响肾癌组织中EMG的表达并与肿瘤的分期和分级相关。这些实验的完成将确定EMG SNPs是否是肾癌易感性的风险因素，以及它们是否参与癌症的生长和进展。目的#2.确定肌电SNPs对基因表达水平和酶活性的影响。从目标1中筛选的多态变异中，我们将分析这些SNPs的功能意义。将进行以下实验：(A)通过克隆和定点突变产生多态的EMG野生型载体，(B)将启动子区域结构导入肾细胞系并通过荧光素酶报告实验确定其对基因表达的影响，(C)将错义EMG结构转化到大肠杆菌细胞中表达和纯化改变的蛋白，并通过生化分析和高效液相色谱检测其酶活性。这些实验的完成将确定启动子区域的EMG SNPs是否会导致更高或更低的表达水平，以及错义EMG酶变异体是否会增加或降低催化活性。目的#3.探讨肌电在人肾癌细胞中的功能意义。由于SNPs可以改变细胞中基因表达或活性的水平，我们将确定EMG在肾脏细胞中的功能重要性。将进行以下实验：(A)分析EMG在正常和肿瘤肾细胞系中的表达水平；(B)根据细胞组成水平分析EMG在肾癌细胞系中的过度表达或沉默；(C)通过流式细胞仪和生物学方法检测EMG处理的肾细胞株的细胞增殖、凋亡、细胞侵袭和细胞周期分布；(D)通过基因芯片分析EMG对细胞形态和各种基因表达的影响；(E)在动物模型中监测EMG修饰的肾癌细胞的生长特性。这些实验的完成将确定肌电在肾癌中的功能意义，并可能为肾癌的潜在治疗提供参考。临床意义：这项拟议研究的成功完成将对肌电及其多态性在肾癌病因和进展中的作用产生重要的见解。这些信息可以帮助确定退伍军人患肾癌的风险较高。拟议的研究也可能对肾癌的可能治疗方法产生影响。

DESCRIPTION (provided by applicant): Background: Renal cell cancer (RCC) rates have been increasing and affect more men than women. The etiology of RCC is not known but based on our preliminary data and prior publication it is clear that estrogen- metabolizing genes (EMG) such as cytochrome P450 (CYP) 1B1 and catechol-O-methyltransferase (COMT) are involved in the pathogenesis of RCC. Single nucleotide polymorphisms (SNPs) of EMG have been shown to alter enzyme levels or catalytic activity and thus, may play a role in renal carcinogenesis. Research Objectives: The main goal of this project is to determine if polymorphisms of EMG are risk factors for RCC. We hypothesize that (1) EMG SNPs contribute to the pathogenesis of RCC, (2) EMG SNPs lead to enzyme levels and activities that are increased for CYP1B1 and decreased for COMT in RCC, and (3) Over- expression of COMT or silencing of CYP1B1 inhibit RCC through cell cycle arrest and apoptosis pathways. Project Design and Methods: Aim #1. To investigate whether SNPs of EMG are risk factors for RCC. Blood and RCC tissue will be collected from patients as well as blood from age-matched, healthy male volunteers. The following experiments will be performed: (a) Determination of EMG promoter and missense SNPs in various stages and grades of RCC patient's blood by utilizing the techniques of sequence-specific PCR and direct genomic sequencing, (b) For comparison, determine EMG SNPs in blood samples from age-matched healthy controls, and (c) Determine whether EMG SNPs affect EMG expression in RCC tissues and correlates with stage and grade of cancer. Accomplishment of these experiments will determine if EMG SNPs are risk factors for RCC susceptibility and whether they're involved in cancer growth and progression. Aim #2. To determine the effects of EMG SNPs on gene expression levels and enzyme activity. From the polymorphic variants screened in Aim #1, we will analyze the functional significance of these SNPs. The following experiments will be performed: (a) Development of EMG wild-type plasmids by cloning and generation of polymorphic variants using site-directed mutagenesis, (b) Transfection of promoter region constructs into renal cell lines and determine their effects on gene expression by using a luciferase reporter assay, (c) Transform missense constructs of EMG into E coli cells to express and purify altered proteins and determine their enzyme activity by using biochemical assays and HPLC. Accomplishment of these experiments will determine if EMG SNPs in the promoter region will lead to higher or lower expression levels and whether missense EMG enzymatic variants have increased or decreased catalytic activity. Aim #3. To investigate the functional significance of EMG in human renal cancer cells. Since SNPs may alter levels of gene expression or activity in cells, we will determine the functional importance of EMG in renal cells. The following experiments will be performed: (a) Analyze EMG expression levels in normal and cancerous renal cell lines, (b) Either over-express or silence EMG in RCC cell lines based on constitutive levels, (c) Measure cell proliferation, apoptosis, cell invasion, and cell cycle distribution in EMG-treated renal cell lines by flow cytometry and biological assays, (d) Evaluate the effects of EMG on cell morphology and expression of various genes by cDNA microarray analyses, (e) Monitor growth characteristics of EMG-modified RCC cells in animal models. Accomplishment of these experiments will determine the functional significance of EMG in RCC and may implicate potential therapeutics for RCC. Clinical Relevance: Successful completion of the proposed research will generate important insights into the role of EMG and its polymorphisms in RCC etiology and progression. Such information can lead to the identification of Veterans with higher risk for RCC. Proposed research may also lead to implications for possible therapy for RCC.