Sequence distribution of tobacco carcinogen-DNA adducts

描述（由申请人提供）：吸烟引起的肺癌的特征是正常细胞基因的异常表达，这是由DNA序列的遗传性改变引起的，例如突变、基因重排和DNA甲基化模式的改变。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，甲基化模式改变在肺癌中产生的确切机制尚不清楚。MeC的存在引起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meg二核苷酸中鸟嘌呤碱基对致癌物质的反应性。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”都位于内源性甲基化的MeCG二核苷酸上，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起始的分子机制。本应用程序的目的是检查烟草致癌物质- dna加合物在MeCG二核苷酸上形成的方式诱导肺癌中观察到的遗传和表观遗传变化。我们的中心假设是内源性胞嘧啶甲基化和局部DNA序列背景控制烟草致癌物质-DNA加合物的形成和修复速率。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来验证：1)确定5-甲基胞嘧啶（MeC）影响CG二核苷酸中B[a]P二环氧化合物诱导的n2 -鸟嘌呤加合物产率的机制。2)研究MeC及其结构类似物对B[a]P二聚环氧化合物诱导的n2 -鸟嘌呤加合物立体化学的影响。3)绘制p53和K-ras衍生DNA序列中氧化性DNA损伤的分布。4)检测甲基化和未甲基化CG二核苷酸的o6 -烷基鸟嘌呤DNA烷基转移酶催化修复nnk诱导的o6 -鸟嘌呤损伤。我们的方法是创新的，因为我们将采用我们实验室开发的一种新的基于质谱的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：拟议的研究具有重要意义，因为人类广泛接触烟草制品，并且烟草制品在肺癌发病中起核心作用。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高危个体化学预防策略的发展。据估计，肺癌病例总数的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开始肺癌的关键。目前的工作将采用一种新的、基于质谱的方法来研究局部DNA序列和内源性胞嘧啶甲基化影响烟草致癌物质-DNA加合物形成和修复速率的机制。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，将促进癌症治疗和对高危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.