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，男性比女性更受影响。RCC的病因尚不清楚，但基于我们的初步数据和先前的出版物，清楚的是雌激素代谢基因（EMG）如细胞色素P450（CYP 1B 1）和儿茶酚-O-甲基转移酶（COMT）参与RCC的发病机制。EMG的单核苷酸多态性（SNP）已被证明可以改变酶水平或催化活性，因此可能在肾癌发生中起作用。研究目的：本研究的主要目的是确定肌电图多态性是否是肾细胞癌的危险因素。我们推测：（1）EMG SNPs参与了RCC的发病机制，（2）EMG SNPs导致RCC中CYP 1B 1的酶水平和活性升高，而COMT的酶水平和活性降低，（3）COMT的过表达或CYP 1B 1的沉默通过细胞周期阻滞和凋亡途径抑制RCC。项目设计和方法：目标#1。探讨肌电图单核苷酸多态性是否是肾细胞癌的危险因素。将从患者以及年龄匹配的健康男性志愿者中采集血液和RCC组织。将进行以下实验：（a）通过利用序列特异性PCR和直接基因组测序技术，确定RCC患者血液的各个阶段和等级中的EMG启动子和错义SNP。（B）为了比较，确定来自年龄匹配的健康对照的血液样品中的EMG SNP，和（c）确定EMG SNPs是否影响RCC组织中的EMG表达并与癌症的分期和分级相关。这些实验的完成将确定EMG SNPs是否是RCC易感性的风险因素，以及它们是否参与癌症的生长和进展。目标2。确定EMG SNPs对基因表达水平和酶活性的影响。从目标#1中筛选的多态性变体，我们将分析这些SNP的功能意义。将进行以下实验：（a）通过克隆和使用定点诱变产生多态性变体来开发EMG野生型质粒，（B）将启动子区构建体转染到肾细胞系中，并通过使用荧光素酶报告基因测定来确定它们对基因表达的影响，（c）将EMG的错义构建体转化到大肠杆菌细胞中，以表达和纯化改变的蛋白质，并通过使用生物化学测定和HPLC测定其酶活性。这些实验的完成将确定启动子区域中的EMG SNP是否会导致更高或更低的表达水平，以及错义EMG酶变体是否具有增加或降低的催化活性。目标3。探讨人肾癌细胞肌电图的功能意义。由于SNPs可能改变细胞中基因表达或活性的水平，我们将确定EMG在肾细胞中的功能重要性。将进行以下实验：（a）分析正常和癌性肾细胞系中的EMG表达水平，（B）基于组成性水平在RCC细胞系中过度表达或沉默EMG，（c）通过流式细胞术和生物测定法测量EMG处理的肾细胞系中的细胞增殖、凋亡、细胞侵袭和细胞周期分布，（d）通过cDNA微阵列分析评估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RCC细胞的生长特性。这些实验的完成将确定EMG在RCC中的功能意义，并可能涉及RCC的潜在治疗方法。临床相关性：成功完成拟议的研究将产生重要的洞察力的作用，肌电图及其多态性在肾细胞癌的病因和进展。这些信息可以导致识别具有RCC较高风险的退伍军人。拟议的研究也可能导致对RCC的可能治疗的影响。

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.