Sequence distribution of tobacco carcinogen-DNA adducts

描述(申请人提供)：吸烟诱发肺癌的特征是由于DNA序列的可遗传变化，例如突变、基因重排和DNA甲基化模式的变化，导致正常细胞基因的异常调控表达。内源性胞嘧啶甲基化通过介导特定蛋白(甲基CpG结合蛋白)与MeCG位点(MEC=5-甲基胞嘧啶)的结合和促进染色质重塑的组蛋白修饰酶的募集来控制基因的表达。然而，肺癌中甲基化模式改变的确切机制还不是很清楚。MEC的存在会导致DNA结构和动力学发生微小但明显的变化。以前的研究表明，MEC能够提高MeCG二核苷酸中鸟嘌呤碱基对致癌物的反应性。值得注意的是，在p53抑癌基因中观察到的大多数肺癌突变“热点”是在内源性甲基化的MeCG二核苷酸上发现的，例如p53密码子157、158、245、248和273。我们的长期目标是阐明吸烟诱导肺癌发生的分子机制。本应用的目的是检查烟草致癌物-DNA加合物在MeCG二核苷酸上形成的方法在肺癌中观察到的遗传和表观遗传学变化。我们的中心假设是，内源性胞嘧啶甲基化和局部DNA序列上下文控制着烟草致癌物-DNA加合物的形成和修复速度。在强大的初步数据的指导下，这一假说将通过追求以下四个具体目标来检验：1)确定5-甲基胞嘧啶(MEC)影响CG二核苷酸中B[a]P二环氧化物诱导的N-鸟嘌呤加合物产量的机制。2)考察MEC及其结构类似物对B[a]P二环氧化物诱导的N_2-鸟嘌呤加合物立体化学的影响。3)绘制DNA氧化损伤在P53和K-ras衍生DNA序列中的分布。4)在甲基化和未甲基化的CG二核苷酸上检测O6-烷基鸟嘌呤DNA烷基转移酶对NNK诱导的O6-鸟嘌呤损伤的修复作用。我们的方法是创新的，因为我们将使用我们实验室开发的一种基于质谱学的新方法和新的结构模型来确定内源性胞嘧啶甲基化对邻近鸟嘌呤对烟草致癌物的反应性的影响。公共卫生相关性：这项拟议的研究意义重大，因为人类广泛接触烟草产品，而且烟草产品在肺癌发病过程中起着核心作用。我们的研究将提供对肺癌相关遗传和表观遗传变化的结构起源的更多了解，这将促进针对高危个体的癌症治疗和化学预防策略的发展。据估计，总肺癌病例的80%-90%是吸烟造成的。烟草致癌物与基因组DNA的结合被认为是吸烟者肺癌发生的关键。本工作将采用一种新的、基于质谱学的方法来研究局部DNA序列和胞嘧啶的内源性甲基化影响烟草致癌物-DNA加合物的形成和修复速度的机制。这些研究的基本原理是，加深对肺癌相关遗传和表观遗传变化的结构起源的了解，将有助于为处于危险中的个人制定癌症治疗和化学预防策略。

DESCRIPTION (provided by applicant): Smoking-induced lung cancer is characterized by an abnormally regulated expression of normal cellular genes resulting from heritable alterations in DNA sequence, e.g. mutations, gene rearrangements, and changes in DNA methylation patterns. Endogenous cytosine methylation controls gene expression by mediating the binding of specific proteins (methyl-CpG binding proteins) to MeCG sites (MeC = 5-methylcytosine) and the recruitment of histone-modifying enzymes that promote chromatin remodeling. However, the exact mechanisms by which the altered methylation patterns arise in lung cancer are not well understood. The presence of MeC induces small, but noticeable changes in DNA structure and dynamics. Previous studies have revealed that MeC is capable of increasing the reactivity of guanine bases in MeCG dinucleotides towards carcinogens. Remarkably, the majority of lung cancer mutational "hot spots" observed within the p53 tumor suppressor gene are found at endogenously methylated MeCG dinucleotides, e.g. p53 codons 157, 158, 245, 248, and 273. Our long-range goal is to elucidate the molecular mechanisms of smoking-induced lung tumor initiation. The objective of this application is to examine the means by which tobacco carcinogen-DNA adduct formation at MeCG dinucleotides induces genetic and epigenetic changes observed in lung cancer. Our central hypothesis is that endogenous cytosine methylation and the local DNA sequence context control the rates of formation and repair of tobacco carcinogen-DNA adducts. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following four specific aims: 1) Identify the mechanisms by which 5-methylcytosine (MeC) influences the yields of B[a]P diolepoxide-induced N2-guanine adducts within CG dinucleotides. 2) Examine the effects of MeC and its structural analogs on the stereochemistry of N2-guanine adducts induced by B[a]P diolepoxides. 3) Map the distribution of oxidative DNA lesions within p53 and K-ras derived DNA sequences. 4) Examine O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-guanine lesions at methylated and unmethylated CG dinucleotides. Our approach is innovative, because we will be employing a novel, mass spectrometry based approach developed in our laboratory and novel structural models to identify the effects of endogenous cytosine methylation on reactivity of neighboring guanines towards tobacco carcinogens. PUBLIC HEALTH RELEVANCE: The proposed research is significant because of the widespread human exposure to tobacco products and because of their central role in lung cancer initiation. Our studies will provide an increased understanding of the structural origins of genetic and epigenetic changes associated with lung cancer, which will facilitate the development of cancer treatment and chemoprevention strategies for individuals at risk. An estimated 80-90% of total lung cancer cases are the result of cigarette smoking. Binding of tobacco carcinogens to genomic DNA is considered critical for lung cancer initiation in smokers. The present work will employ a novel, mass spectrometry based approach to investigate the mechanisms by which the local DNA sequence and endogenous methylation of cytosine influence the rates of formation and repair of tobacco carcinogen-DNA adducts. The rationale for these studies is that an increased understanding of the structural origins of genetic and epigenetic changes associated with lung cancer will facilitate the development of cancer treatment and chemoprevention strategies for individuals at risk.