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（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学的危险因素。其基本原理是CYP1A1和CYP1B1分别催化雌激素羟基化为高度致癌的2-羟基儿茶酚雌激素(2-OH-E2)和4-羟基儿茶酚雌激素(4-OH-E2)代谢物。然而，子宫内膜癌缺乏此类研究。根据我们的初步数据和之前的出版物，我们假设 CYP1A1 和 CYP1B1 基因是子宫内膜癌病理学的危险因素。具体目标#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 基因在子宫内膜恶性转化过程中过度激活。为了检验这一假设，我们将： (a) 通过实时 PCR（定量）分析良性子宫内膜以及不同阶段和级别的子宫内膜癌中 CYP1A1 和 CYP1B1 mRNA 的表达水平。此外，将利用原位杂交定位子宫内膜组织中CYP1A1和CYP1B1 mRNA的表达，(b)使用免疫组织化学分析良性子宫内膜和不同阶段和级别的子宫内膜癌中CYP1A1和CYP1B1的蛋白表达（用于定位）和蛋白质印迹法进行定量。 (c)采用生化技术分析子宫内膜组织中CYP1A1和CYP1B1酶活性水平，并采用气相色谱法测定子宫内膜组织中雌激素代谢物(2-OH-E2和4-OH-E2)水平，( d) 分析CYP1A1和CYP1B1基因、蛋白质、酶活性和/或2-OH-E2和子宫内膜癌中的 4-OH-E2 代谢物是复发和患者生存的预测因子。具体目标#2：研究 CYP1A1 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 基因中的 S​​NP 进行分析可以识别子宫内膜癌风险较高的人群。为了检验这一假设，我们将：(a) 使用 PCR-RFLP（限制性片段长度多态性）和直接基因组测序分析良性和子宫内膜癌患者血液和组织 DNA 中 CYP1A1 和 CYP1B1 基因的 SNP，(b) 分析是否CYP1A1 和 CYP1B1 基因的 SNP 与子宫内膜癌中酶活性的改变和雌激素代谢物的产生改变相关，(c) 分析CYP1A1 和 CYP1B1 基因的 SNP 是否与子宫内膜癌复发和患者生存相关。具体目标#3：研究 CYP1A1 和 CYP1B1 基因的多态性变异是否可以调节雌激素羟化酶活性。我们假设 CYP1A1 和 CYP1B1 基因外显子区域的 SNP 可以调节它们在子宫内膜癌中的酶活性。为了测试这一假设，我们将使用 ThioFusion 表达载体 (pThioHis) 表达 CYP1A1 和 CYP1B1 的野生型和多态性变体形式（包含 NH2 末端六组氨酸标签）。从正常子宫内膜分离的 CYP1A1 和 CYP1B1 mRNA 将作为野生型 cDNA 的来源，将其插入 pThioHis 表达载体中。然后，我们将使用野生型 pThioHis CYP1A1 和 CYP1B1 表达质粒作为模板，通过定点诱变生成 CYP1A1 和 CYP1B1 的多态性变体，并测定其雌激素羟化酶活性。该项目将为我们提供涉及子宫内膜癌病理学的新机制。