FUNCTIONAL CHARACTERIZATION OF AGT GENETIC POLYMORPHISM

AGT 遗传多态性的功能表征

• 批准号: 6178627
• 负责人: Jun-Yan Hong
• 金额: $ 29.05万
• 依托单位: UNIV OF MED/DENT NJ-SCH OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178627
• 关键词: DNA repair; alkylating agents; alkyltransferase; chemical carcinogen; environmental toxicology; enzyme activity; gene expression; genetic polymorphism; genetic susceptibility; genetic transcription; human subject; lymphocyte; nucleic acid sequence; oral mucosa; reporter genes; single strand conformation polymorphism; site directed mutagenesis; tissue /cell culture; transfection

A large number of environmental carcinogens and toxicants are alkylating agents. Human O6-alkylguanine-DNA alkyltransferase (AGT, EC 2.1.1.63) is an important DNA repair protein which specifically repairs O6- alkylguanine in DNA, a major premutagenic lesion produced by environmental alkylating agents. AGT plays a critical role in protecting cells against mutation and toxic response induced by alkylating agents, including alkylating chemotherapeutic drugs. Significant interindividual variations (greater than 10 fold) in human AGT activity levels have been reported. However, little is known about the molecular basis of these variations and their importance in an individual's susceptibility to environmental carcinogenesis and toxicity related to alkylating agents. Recently, we have identified two novel forms of genetic polymorphism of human AGT. One missense alteration occurs at the codon 143 (isoleucine to valine). The codon 143 (Ile143) is adjacent to the alkyl acceptor Cys145 of the AGT active site and is conserved among the mammalian AGT. The other polymorphic alteration is localized in the promoter region of the AGT gene, which creates a new BanI restriction site. Our working hypothesis is that genetic polymorphism of AGT, particularly those occurring near the enzyme active sites or within an important regulatory region, is a major reason for the reported large interindividual variations of AGT activity in humans. As a consequence, AGT genetic polymorphism may play a significant role in determining an individual's susceptibility to environmental alkylating carcinogens and response to alkylating chemotherapeutic drugs. The present proposal will test our hypothesis by focusing on the functional characterization of genetic polymorphism of human AGT (known or to be identified) with the following specific aims: 1. To elucidate the functional significance of AGT genetic polymorphism. Initial work will focus on the codon 143 and BanI polymorphisms we recently identified. This will be accomplished by: (a) biochemical studies on AGT missense polymorphic variants; (b) comparison of the toxic response to alkylating agents between human cells expressing wild-type and missense variant AGT; and (c) transcriptional analysis of the BanI polymorphic variation by a reporter gene assay. 2. To determine the effects of polymorphic alterations on AGT function and expression in humans by measuring AGT activity, protein and mRNA levels in human peripheral lymphocytes and esophageal mucosa (a target tissue of alkylating carcinogens). Samples with extremely high or low AGT activity and expression levels will also be screened for new AGT genetic polymorphisms. The current proposal will contribute to our understanding of AGT, an important environmentally relevant enzyme, and help assess the role of AGT genetic polymorphism in human carcinogenesis and toxicity related to environmental alkylating agents. It could have a significant impact on cancer prevention by identifying susceptible subpopulations in future epidemiological studies.

大量的环境致癌物和有毒物都在发生烷化反应 剂. 人O 6-烷基鸟嘌呤-DNA烷基转移酶（AGT，EC 2.1.1.63） 是一种重要的DNA修复蛋白，特异性修复O 6- DNA中的烷基鸟嘌呤，是由 环境烷基化剂。 AGT在以下方面发挥着关键作用： 保护细胞免受突变和毒性反应诱导 烷基化剂，包括烷基化化疗药物。 人类中显著的个体间变异（大于10倍） AGT活性水平已得到报告。 然而，人们对 这些变化的分子基础及其在一个 个人对环境致癌作用和毒性的易感性 与烷基化试剂有关。 最近，我们发现了两个新的 人类AGT的遗传多态性形式。 一个错误的改变 发生在密码子143（异亮氨酸到缬氨酸）。 密码子143（Ile 143） 与AGT活性位点的烷基受体Cys 145相邻， 在哺乳动物AGT中保守。另一种多态性变化是 位于AGT基因的启动子区，这创造了一个新的 BanI限制性位点。 我们的假设是， AGT的多态性，特别是那些发生在酶活性 网站或在一个重要的监管区域，是一个主要的原因， 人类AGT活性的个体间差异较大。 因此，AGT基因多态性可能发挥重要作用 在确定一个人对环境的敏感性时， 烷化剂致癌物和对烷化剂化疗的反应 毒品 目前的建议将通过集中于 人AGT的遗传多态性的功能表征（已知 或待确定），具体目标如下：1. 阐明 AGT基因多态性的功能意义。 初步工作 将集中在密码子143和BanI多态性，我们最近 鉴定 这将通过以下方式实现：（a）生物化学研究， AGT错义多态性变体;（B）毒性反应的比较 涉及表达野生型的人细胞之间的烷化剂， 错义变体AGT;和（c）BanI的转录分析 通过报告基因测定的多态性变异。 2. 确定 多态性改变对AGT功能和表达的影响 通过测量人类中AGT活性、蛋白质和mRNA水平， 外周淋巴细胞和食管粘膜（ 烷基化致癌物）。 AGT极高或极低的样本 活性和表达水平也将筛选新的AGT基因， 多态性 目前的建议将有助于我们了解AGT， 重要的环境相关酶，并帮助评估的作用， AGT基因多态性与人类肿瘤发生及毒性的关系 涉及环境烷基化剂。 这可能会对 未来通过识别易感亚群预防癌症 流行病学研究。