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：
8052754
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 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8052754
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）是由化石燃料燃烧和吸烟引起的普遍环境空气污染物。 PAH暴露是人类肺癌发生的主要危险因素。多阶段致癌作用的关键步骤是诱变事件是由DNA加合物的形成而产生的。 PAH的代谢激活有三种主要途径，导致形成二醇环氧化物，自由基阳离子或反应性和氧化还原活性的O- Qu-金酮。这些反应性代谢产物中的每一个都有可能形成DNA添加剂，而这些加合物可能导致突变。我们正在使用两种方法来建模PAH致癌作用，一种用途广泛的酵母系统和人类肺细胞系。在肺癌中，最常见的基因是p53，其中在肺癌中发现了三个区别特征。（1）突变的模式由G到T横向占主导地位；（2）突变的光谱揭示了热点密码子，其中一些是肺癌独有的；（3）突变的模式和光谱显示出非转录链突变的链偏置。我们的系统使用遗传选择方法，以便可以轻松地检测到变化功能突变。 p53的酵母报道系统依赖于野生型p53与启动子结合，以驱动腺嘌呤记者，导致突变菌落变成红色。我们的初步数据表明，PAH O- Quinones允许进行氧化还原循环，会产生8-oxo-dguo，以使G转移到T横向t的t横向，但对热点的偏好却很大，但没有链偏置。我们最近设计了一个系统，使用p53依赖启动子来检测人肺细胞中的突变，以直接转录疱疹TK基因。在该系统中，p53+细胞通过暴露于ganciclovir杀死，而p53细胞具有抗性。接下来使用酵母系统从细胞中获救突变体p53，然后测序。使用这些测定法对p53诱变进行建模，我们将：（1）确定修复基因在p53诱变中的作用，并比较不同的PAH代谢产物，（2）绘制PAH诱导的p53基因（3）中PAH诱导的DNA病变的位置，确定PAH-Metabolites是否可以在肺细胞中突变P53。我们的假设是Pah o- Quinones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）是由化石燃料燃烧和吸烟引起的普遍环境空气污染物。 PAH暴露是人类肺癌发生的主要危险因素。该提案将研究PAH及其代谢产物对p53癌基因的诱变。