Sequence Distribution of Tabacco Carcinogen-DNA Adducts

烟草致癌物-DNA 加合物的序列分布

• 批准号: 7484005
• 负责人: NATALIA Y TRETYAKOVA
• 金额: $ 20.69万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7484005
• 关键词: Alkylation; Aromatic Polycyclic Hydrocarbons; Benzo(a)pyrene; Binding; Biological Assay; Butanones; Carcinogens; Chemicals; Chromosomal Stability; Codon Nucleotides; Controlled Environment; CpG dinucleotide; Cytosine; DNA; DNA Adduction; DNA Adducts; DNA Sequence; DNA lesion; DNA-Protein Interaction; Dinucleoside Phosphates; Environment; Enzymes; Epigenetic Process; Exposure to; Gene Expression; Genes; Genetic; Genomic Imprinting; Goals; Guanine; Host Defense; Hot Spot; Human; Human Genome; Investigation; Kinetics; Laboratories; Lesion; Life; Link; Lung Neoplasms; Malignant neoplasm of lung; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Methylation; Methyltransferase; Minnesota; Minor; Modification; Molecular; Monitor; Mutagenesis; Mutation; Nitrosamines; O(6)-Methylguanine-DNA Methyltransferase; Oncogene Activation; Pattern; Physiological Processes; Play; Positioning Attribute; Promoter Regions; Protein p53; Proto-Oncogenes; Range; Rate; Reactive Oxygen Species; Research; Resources; Role; Series; Site; Smoke; Smoker; Smoking; Stable Isotope Labeling; Stereoisomer; Structure; TP53 gene; Testing; Tobacco; Tobacco smoke; Tobacco-Associated Carcinogen; Tumor Suppressor Genes; Universities; X Inactivation; adduct; alkyltransferase; analog; base; chromatin remodeling; exposed human population; innovation; insight; mammalian genome; methyl group; novel; nucleobase; oxidation; repaired; stereochemistry; success; tool; tumor; tumor initiation

Endogenous cytosine methylation at 5'-CG-3' sites within the human genome is involved in many physiological processes, including gene expression, host defense, genomic imprinting, and X chromosome inactivation. Interestingly, many tumor types, including smoking-induced lung cancer, are characterized by aberrant DMA methylation patterns. The presence of 5-methylcytosine (MeC) induces small, but noticeable changes in DMA structure and dynamics, leading to altered DNA-protein interactions and chromatin remodeling. Furthermore, 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 previous investigations have revealed that the presence of eC at these sites modulates the reactivity of neighboring guanine bases towards tobacco carcinogens, leading to targeted binding of benzo[a]pyrene diolepoxides (BPDE) to MeCG sequences. In contrast, alkylation of MeCG dinucleotides by reactive metabolites of tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), is inhibited. We now propose to determine the structural basis for these effects by conducting stable isotope labeling studies with a series of MeC analogs. We will also test the hypothesis that cytosine methylation status and local DMA sequence context influence guanine adduct formation by reactive oxygen species generated as a result of exposure to tobacco smoke. Finally, any effects of MeC on O6- alkylguanine DMA alkyltransferase-mediated repair of NNK-induced O6-alkylguanine adducts will also be examined. These studies will: 1. Determine the mechanisms by which 5-methylcytosine (MeC) influences the reactivity of neighboring guanine bases towards tobacco carcinogens. 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 DMA 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. The results of this research will identify the mechanisms by which endogenous MeC modulates the reactivity of CG sites towards tobacco carcinogens, providing an insight into the origins of genetic and epigenetic changes observed in lung cancer. Our approach is innovative because our laboratory is employing a novel, mass spectrometry based approach to probe the reactivity of specific bases within DNA duplexes towards alkylating and oxidative agents. The proposed research is significant because of the widespread human exposure to tobacco products and because of their central role in the initiation of lung cancer in humans.

人类基因组内5 '-CG-3'位点的内源性胞嘧啶甲基化参与了许多 生理过程，包括基因表达，宿主防御，基因组印记和X染色体 失活有趣的是，许多肿瘤类型，包括吸烟诱发的肺癌，其特征是 异常的DNA甲基化模式5-甲基胞嘧啶（MeC）的存在诱导了小的，但值得注意的 DMA结构和动力学的变化，导致DNA-蛋白质相互作用和染色质的改变 重塑此外，MeC能够增加MeCG二核苷酸中鸟嘌呤碱基的反应性 致癌物质的研究值得注意的是，大多数肺癌突变“热点”在p53内观察到， 肿瘤抑制基因存在于内源性甲基化的MeCG二核苷酸，例如p53密码子157， 158、245、248和273。我们之前的调查显示，在这些地点存在的电子C 调节邻近鸟嘌呤碱基对烟草致癌物的反应性， 苯并[a]芘二醇环氧化物（BPDE）与MeCG序列的结合。相比之下，MeCG的烷基化 二核苷酸通过烟草特异性亚硝胺，4-（甲基亚硝胺基）-1-（3-吡啶基）-1- 丁酮（NNK）被抑制。我们现在建议通过以下方式确定这些影响的结构基础： 用一系列MeC类似物进行稳定同位素标记研究。我们还将检验以下假设： 胞嘧啶甲基化状态和局部DNA序列环境通过反应性DNA序列影响鸟嘌呤加合物的形成。 由于暴露于烟草烟雾而产生的氧物质。最后，MEC对O 6- 烷基鸟嘌呤DMA烷基转移酶介导的NNK诱导的O 6-烷基鸟嘌呤加合物的修复也将被 考察这些研究将： 1.确定5-甲基胞嘧啶（MeC）影响相邻细胞反应性的机制。 鸟嘌呤碱基对烟草致癌物。 2.检查MeC及其结构类似物对N2-鸟嘌呤加合物立体化学的影响 由B[a]P二醇环氧化物诱导。 3.绘制p53和K-ras衍生DNA序列中氧化性DNA损伤的分布图。 4.检查O 6-烷基鸟嘌呤DNA烷基转移酶催化的NNK诱导的O 6-鸟嘌呤损伤的修复， 甲基化和未甲基化的CG二核苷酸。 本研究的结果将确定内源性MeC调节反应性的机制 CG网站对烟草致癌物，提供了一个深入了解的起源，遗传和表观遗传 在肺癌中观察到的变化我们的方法是创新的，因为我们的实验室采用了一种新颖的， 基于质谱的方法来探测DNA双链体内特定碱基对 烷基化剂和氧化剂。这项研究之所以重要，是因为人类的广泛存在。 烟草制品是导致肺癌的主要原因，也是因为烟草制品在人类肺癌的发生中起着重要作用。