Sequence distribution of tobacco carcinogen-DNA adducts

描述（由申请人提供）：吸烟诱导的肺癌的特征是由DNA序列的遗传性改变（例如突变、基因重排和DNA甲基化模式的变化）引起的正常细胞基因表达的异常调节。内源性胞嘧啶甲基化通过介导特异性蛋白（甲基-CpG结合蛋白）与MeCG位点（MeC = 5-甲基胞嘧啶）的结合以及促进染色质重塑的组蛋白修饰酶的募集来控制基因表达。然而，肺癌中甲基化模式改变的确切机制还不清楚。MeC的存在会引起DNA结构和动力学的微小但明显的变化。先前的研究表明，MeC能够增加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肿瘤抑制基因内观察到的大多数肺癌突变"热点"发现于内源性甲基化的MeCG二核苷酸，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肿瘤发生的分子机制。本申请的目的是检查烟草致癌物-DNA加合物在MeCG二核苷酸处形成诱导在肺癌中观察到的遗传和表观遗传变化的手段。我们的中心假设是，内源性胞嘧啶甲基化和当地的DNA序列上下文控制烟草致癌物DNA加合物的形成和修复的速度。在强有力的初步数据的指导下，这一假设将通过追求以下四个具体目标进行测试：1）确定5-甲基胞嘧啶（MeC）影响CG二核苷酸内B [a] P二环氧乙烷诱导的N2-鸟嘌呤加合物的产率的机制。2)检查MeC及其结构类似物对由B [a] P二醇环氧化物诱导的N2-鸟嘌呤加合物的立体化学的影响。3)绘制p53和K-ras衍生DNA序列内氧化DNA损伤的分布图。4)检查甲基化和未甲基化CG二核苷酸处NNK诱导的O6-鸟嘌呤损伤的O6-烷基鸟嘌呤DNA烷基转移酶催化修复。我们的方法是创新的，因为我们将采用一种新的，基于质谱的方法在我们的实验室和新的结构模型，以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：这项拟议中的研究意义重大，因为人类广泛接触烟草制品，而且烟草制品在肺癌发生中起着核心作用。我们的研究将增加对与肺癌相关的遗传和表观遗传变化的结构起源的理解，这将促进癌症治疗和化学预防策略的发展。据估计，80 - 90%的肺癌病例是吸烟的结果。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，质谱为基础的方法来调查的机制，其中的本地DNA序列和内源性甲基化的胞嘧啶影响烟草致癌物-DNA加合物的形成和修复的速度。这些研究的基本原理是，增加对与肺癌相关的遗传和表观遗传变化的结构起源的理解，将促进癌症治疗和风险个体的化学预防策略的发展。

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.