Mutagenesis of p53 by reactive PAH and ROS

反应性 PAH 和 ROS 对 p53 的诱变

基本信息

批准号：
8235068
负责人：
JEFFREY M FIELD
金额：
$ 30.73万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-08 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8235068
关键词：
8-Oxo-2&apos -Deoxyguanosine A549 Adenine Aromatic Polycyclic Hydrocarbons Binding Biological Assay Carcinogens Cations Cell Culture Techniques Cell Line Cells Characteristics Code Codon Nucleotides Color DNA DNA Adduction DNA Adducts DNA Damage DNA lesion Data Environmental Air Pollutants Environmental Pollutants Epithelial Cells Epoxy Compounds Event Exposure to Fossil Fuels Frequencies Ganciclovir Genetic Transcription Glycols HSV-Tk Gene Health Human In Situ In Vitro Inherited Knock-out Lead Lesion Ligation Location Lung Lung Adenocarcinoma Malignant neoplasm of lung Mammalian Cell Maps Mediating Metabolic Activation Metabolic Pathway Methods Modeling Mutagenesis Mutagens Mutate Mutation Mutation Spectra OGG1 gene Oncogenes Oxidation-Reduction Pathway interactions Pattern Pharmaceutical Preparations Protein p53 Quinones Reactive Oxygen Species Relative (related person)Reporter Reporter Genes Resistance Risk Factors Role Route Site Staging System TK Gene TP53 gene Techniques Testing Tobacco Transcription-Coupled Repair Transcriptional Activation Yeasts adduct base carcinogenesis cell killing cigarette smoking design gene repair genetic selection lung carcinogenesis mutant novel preference promoter repaired research study

项目摘要

DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental air pollutants that result from fossil fuel combustion and cigarette smoking. PAH exposure is a major risk factor in human lung carcinogenesis. A critical step in multi-stage carcinogenesis is the mutagenic event that results from the formation of DNA adducts. There are three principle routes of metabolic activation of PAH resulting in the formation of diol- epoxides, radical cations, or reactive and redox-active o-quinones. Each of these reactive metabolites have the potential to form DNA-adducts and these adducts may lead to mutation. We are using two approaches to model PAH carcinogenesis, a highly versatile yeast system and human lung cell lines. In lung cancer, the gene most often mutated is p53 where three distinguishing characteristics are found in lung cancer. (1) The pattern of mutations is dominated by G to T transversions; (2) The spectrum of mutations reveals hotspot codons, a few of which are unique to lung cancer; (3) The pattern and spectrum of mutations show a strand bias for mutations on the non-transcribed strand. Our systems use genetic selection methods so that change-in- function mutations can be detected with ease. The yeast reporter system for p53 relies on wild-type p53 binding to a promoter to drive an adenine reporter causing mutant colonies turn red. Our preliminary data suggest that PAH o-quinones, when permitted to undergo redox-cycling, generate 8-oxo-dGuo to cause predominantly G to T transversions with a modest, but significant, preference for hotspots but with no strand bias. We recently devised a system to detect mutations in human lung cells using a p53 dependent promoter to direct transcription of the Herpes TK gene. In this system p53+ cells are killed by exposure to ganciclovir while p53 cells are resistant. The mutant p53 is next rescued from the cells using the yeast system and then sequenced. Using these assays to model p53 mutagenesis we will: (1) Determine the role of repair genes on p53 mutagenesis and compare different PAH metabolites, (2) Map the locations of PAH induced DNA lesions in the p53 gene(3) determine if PAH-metabolites can mutate p53 in lung cells. Our hypothesis is that PAH o- quinones and the ROS they generate provide a route to the p53 mutations found in lung cancer. PUBLIC HEALTH RELEVANCE: Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental air pollutants that result from fossil fuel combustion and cigarette smoking. PAH exposure is a major risk factor in human lung carcinogenesis. This proposal will study the mutagenesis of the p53 oncogene by PAH and their metabolites.

描述（由申请人提供）：多环芳烃 (PAH) 是普遍存在的环境空气污染物，由化石燃料燃烧和吸烟产生。 PAH 暴露是人类肺癌发生的主要危险因素。多阶段致癌过程中的一个关键步骤是 DNA 加合物形成导致的诱变事件。 PAH 的代谢活化有三种主要途径，导致二醇环氧化物、自由基阳离子或反应性和氧化还原活性邻醌的形成。这些反应性代谢物中的每一种都有可能形成 DNA 加合物，并且这些加合物可能导致突变。我们正在使用两种方法来模拟 PAH 致癌作用：高度通用的酵母系统和人类肺细胞系。在肺癌中，最常突变的基因是 p53，在肺癌中发现了三个显着特征。 (1)突变模式以G→T颠换为主； (2) 突变谱揭示了热点密码子，其中一些是肺癌特有的； (3) 突变的模式和谱显示非转录链上的突变具有链偏向性。我们的系统使用遗传选择方法，以便可以轻松检测功能变化突变。 p53 的酵母报告系统依赖于野生型 p53 与启动子的结合来驱动腺嘌呤报告基因，导致突变菌落变红。我们的初步数据表明，PAH邻醌当允许进行氧化还原循环时，会生成8-oxo-dGuo，主要引起G到T颠换，对热点有适度但显着的偏好，但没有链偏向。我们最近设计了一种系统，使用 p53 依赖性启动子来指导疱疹 TK 基因的转录，以检测人肺细胞中的突变。在该系统中，p53+细胞因暴露于更昔洛韦而被杀死，而p53细胞则具有抗药性。接下来使用酵母系统从细胞中拯救出突变体 p53，然后进行测序。使用这些测定来模拟 p53 突变，我们将：（1）确定修复基因对 p53 突变的作用并比较不同的 PAH 代谢物，（2）绘制 p53 基因中 PAH 诱导的 DNA 损伤的位置（3）确定 PAH 是否代谢物可使肺细胞中的 p53 发生突变。我们的假设是 PAH 邻醌及其产生的 ROS 为肺癌中发现的 p53 突变提供了途径。公共卫生相关性：多环芳烃 (PAH) 是普遍存在的环境空气污染物，由化石燃料燃烧和吸烟产生。 PAH 暴露是人类肺癌发生的主要危险因素。该提案将研究 PAH 及其代谢物对 p53 癌基因的诱变。