Cytochrome-P-450 genes in pathobiology of endometrial cancer

细胞色素-P-450 基因在子宫内膜癌病理学中的作用

• 批准号: 7409599
• 负责人: RAJVIR DAHIYA
• 金额: $ 28.37万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-10 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7409599
• 关键词: Antibodies; Benign; Biochemical; Blood; CYP1B1 gene; Cancer Patient; Catechol Estrogens; Cells; Chromatography; Cytochrome P450; Cytochromes; DNA; Data; Endometrial; Endometrial Carcinoma; Endometrium; Environmental Carcinogens; Enzymes; Estrogen Metabolism; Estrogens; Exons; Gas Chromatography; Gene Proteins; Genes; Genetic Polymorphism; Genomics; Goals; His-His-His-His-His-His; Histidine; Human; Human Cell Line; Hydroxylation; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Literature; Localized; Malignant - descriptor; Malignant Neoplasms; Mass Fragmentography; Messenger RNA; Metabolic Activation; Methods; Mixed Function Oxygenases; N-terminal; Numbers; Oligonucleotides; Pathogenesis; Patients; Pike fish; Plasmids; Polymerase Chain Reaction; Population; Principal Investigator; Production; Proteins; Publications; Publishing; Recombinant Proteins; Recurrence; Restriction fragment length polymorphism; Risk; Risk Factors; Role; Single Nucleotide Polymorphism; Site-Directed Mutagenesis; Source; Specimen; Staging; Survival Rate; System; Techniques; Testing; Time; Tissues; Variant; Western Blotting; Yin; base; cancer recurrence; enzyme activity; expression vector; in vitro Assay; mRNA Expression; novel; programs; protein expression; research study

DESCRIPTION (provided by applicant): The main goal of this project is to investigate whether the cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) genes are risk factors for pathobiology of endometrial cancer. The rationale is that CYP1A1 and CYP1B1 catalyze the hydroxylation of estrogen to highly carcinogenic 2-hydroxy catechol estrogen (2-OH-E2) and 4-hydroxy catechol estrogen (4-OH-E2) metabolites, respectively. However, such studies are lacking in endometrial cancer. Based on our preliminary data and prior publications, we hypothesize that CYP1A1 and CYP1B1 genes are risk factor for pathobiology of endometrial cancer. Specific Aim # 1. To investigate whether the CYP1A1 and CYP1B1 genes are hyper-activated in endometrial cancer. We hypothesize that the CYP1A1 and CYP1B1 genes are hyper-activated during malignant transformation of the endometrium. To test this hypothesis, we will: (a) Analyze the level of CYP1A1 and CYP1B1 mRNA expression by real-time PCR (for quantification) in benign endometrium and in different stages and grades of endometrial cancer. In addition, in situ hybridization will be used to localize CYP1A1 and CYP1B1 mRNA expression in endometrial tissues, (b) Analysis of protein expression of CYP1A1 and CYP1B1 in benign endometrium and in different stages and grades of endometrial cancer using immunohistochemistry (for localization) and western blotting for quantification. (c) Analysis of the level of CYP1A1 and CYP1B1 enzymatic activity in endometrial tissues by biochemical techniques as well as determine the level of estrogen metabolites (2-OH-E2 and 4-OH-E2) in endometrial tissues by gas chromatography methods, (d) To analyze whether CYP1A1 and CYP1B1 genes, protein, enzymatic activity and/or the levels of 2-OH-E2 and 4-OH-E2 metabolites in endometrial cancer are predictors of recurrence and patient survival. Specific Aim # 2: To investigate whether single nucleotide polymorphisms of the CYP1 Al and CYP1B1 genes are risk factors for endometrial cancer. We hypothesize that the analysis of SNPs in the CYP1A1 and CYP1B1 genes can identify populations with a higher risk of endometrial cancer. To test this hypothesis, we will: (a) Analyze SNPs of the CYP1A1 and CYP1B1 genes in blood and tissue DNA from benign and endometrial cancer patients using PCR-RFLP (restriction fragment length polymorphism) and direct genomic sequencing, (b) Analyze whether SNPs of CYP1A1 and CYP1B1 genes are associated with altered enzymatic activity and the altered production of estrogen metabolites in endometrial cancer, (c) Analyze whether SNPs of the CYP1A1 and CYP1B1 genes are associated with endometrial cancer recurrence and patient survival. Specific Aim # 3: To investigate whether polymorphic variants of the CYP1A1 and CYP1B1 genes can modulate estrogen hydroxylase activity. We hypothesize that SNPs in exon regions of CYP1A1 and CYP1B1 genes can modulate their enzymatic activity in endometrial cancer. To test this hypothesis we will express wild-type and polymorphic variant forms of CYP1A1 and CYP1B1 (which contain a NH2-terminal hexahistidine tag) using a ThioFusion expression vector (pThioHis). CYP1A1 and CYP1B1 mRNA isolated from normal endometrium will serve as the source of wild-type cDNAs, which will be inserted into the pThioHis expression vector. We will then use the wild-type pThioHis CYP1A1 and CYP1B1 expression plasmids as templates to generate polymorphic variants of CYP1A1 and CYP1B1 using site-directed mutagenesis and determine their estrogen hydroxylase activity. This project will provide us with novel mechanisms involving the pathobiology of endometrial cancer.

描述（由申请人提供）：该项目的主要目标是研究细胞色素P4501A1（CYP1A1）和细胞色素P4501B1（CYP1B1）基因是否是子宫内膜癌病理生物学的危险因素。基本原理是CYP1A1和CYP1B1分别催化雌激素的高度致癌2-羟基儿茶醇雌激素（2-OH-E2）和4-羟基雌激素（4-OH-E2）代谢物。但是，这种研究缺乏子宫内膜癌。根据我们的初步数据和先前的出版物，我们假设CYP1A1和CYP1B1基因是子宫内膜癌病理生物学的危险因素。具体目的1。研究CYP1A1和CYP1B1基因在子宫内膜癌中是否过度激活。我们假设CYP1A1和CYP1B1基因在子宫内膜的恶性转化过程中被过度激活。为了检验这一假设，我们将：（a）通过实时PCR（用于定量​​）在良性子宫内膜以及子宫内膜癌的不同阶段和等级分析CYP1A1和CYP1B1 mRNA的水平。此外，原位杂交将用于子宫内膜组织中的CYP1A1和CYP1B1 mRNA表达，（b）分析CYP1A1和CYP1B1在良性子宫内膜中的蛋白质表达，并在不同的阶段，在不同阶段和使用免疫组织化学的子宫内膜癌（用于本地化）和蛋白质化的子宫内膜癌中进行量化。 （c）通过生化技术中的CYP1A1和CYP1B1在子宫内膜组织中酶活性的水平分析，并确定子宫内膜组织中雌激素代谢物（2-OH-E2和4-OH-E2）在气体过程中通过气体形态方法中的CYP1A和CIPT1A和CIPT 1B1 BENES ORNES stute;子宫内膜癌中的2-OH-E2和4-OH-E2代谢产物是复发和患者存活的预测指标。具体目的＃2：研究CYP1 AL和CYP1B1基因的单核苷酸多态性是否是子宫内膜癌的危险因素。我们假设对CYP1A1和CYP1B1基因中SNP的分析可以鉴定患有更高的子宫内膜癌风险的种群。为了检验该假设，我们将：（a）使用PCR-RFLP（限制性片段长度多态性）和直接基因组测序的血液和组织DNA中的CYP1A1和CYP1B1基因的SNP，（b）分析CYP1A1基因的SNPS ersem ersem ersem ersem ersem ersem erse eats erse，是CYP1B1基因的改变是否相关子宫内膜癌中的代谢产物（c）分析CYP1A1和CYP1B1基因的SNP是否与子宫内膜癌复发和患者存活有关。特定目的＃3：研究CYP1A1和CYP1B1基因的多态性变体是否可以调节雌激素羟化酶活性。我们假设CYP1A1和CYP1B1基因外显子区域中的SNP可以调节其在子宫内膜癌中的酶促活性。为了检验这一假设，我们将使用硫封发表达载体（PTHIOHIS）表达CYP1A1和CYP1B1（包含NH2末端六位酰胺标签）的CYP1A1和CYP1B1（包含NH2-末端六位烷tag）的野生型和多态变体形式。从正常子宫内膜分离的CYP1A1和CYP1B1 mRNA将作为野生型cDNA的来源，将插入Pthiohis表达载体。然后，我们将使用位置定向的诱变并确定其雌激素羟化酶活性，将野生型Pthiohis pthiohis pthiohis pthiohis cyp1a1和cyp1b1表达质粒作为模板作为模板来生成CYP1A1和CYP1B1的多态性变体。该项目将为我们提供涉及子宫内膜癌病理生物学的新型机制。