Mutagenesis of p53 by reactive PAH and ROS

描述(由申请人提供)： 多环芳烃(PAH)是化石燃料燃烧和吸烟产生的一种普遍存在的环境空气污染物。PAH暴露是人类肺癌发生的主要危险因素。在多阶段癌变中的一个关键步骤是DNA加合物的形成所导致的突变事件。多环芳烃的代谢活化主要有三条途径，形成二元醇-环氧化物、自由基阳离子或反应性和氧化还原活性的邻苯二酚。这些反应性代谢物中的每一个都有可能形成DNA加合物，这些加合物可能导致突变。我们正在使用两种方法来模拟多环芳烃的致癌作用，一种是高度通用的酵母系统，另一种是人肺细胞系。在肺癌中，最常见的突变基因是p53，在肺癌中发现了三个明显的特征。(1)突变模式以G到T易位为主；(2)突变谱显示热点密码子，其中一些是肺癌所特有的；(3)突变模式和谱显示非转录链上的突变具有链偏向。我们的系统使用基因选择方法，因此可以很容易地检测到功能变化突变。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系统，导致突变菌落变红。我们的初步数据表明，当多环芳烃邻苯二酚被允许进行氧化还原循环时，产生8-oxo-dguo，主要导致G到T的转换，对热点有一定的偏好，但没有链偏见。我们最近设计了一种系统来检测人类肺细胞中的突变，使用依赖于p53的启动子来指导疱疹病毒TK基因的转录。在这个系统中，p53细胞通过暴露于更昔洛韦而被杀死，而p53细胞是耐药的。接下来，使用酵母系统从细胞中抢救出突变的p53，然后进行测序。利用这些方法模拟P53突变，我们将：(1)确定修复基因在P53突变中的作用，并比较不同的多环芳烃代谢产物；(2)定位多环芳烃诱导的DNA损伤在P53基因中的位置；(3)确定多环芳烃代谢产物是否能在肺细胞中突变P53。我们的假设是，多环芳烃邻苯二酚及其产生的ROS为肺癌中发现的p53突变提供了一条途径。与公共健康相关：多环芳烃(PAH)是化石燃料燃烧和吸烟产生的普遍存在的环境空气污染物。PAH暴露是人类肺癌发生的主要危险因素。这项建议将研究多环芳烃及其代谢产物对p53癌基因的诱变作用。

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.