Sequence Distribution of Tobacco Carcinogen-DNA Adducts

项目描述（申请人提供）：本项目主要研究烟草致癌物- dna加合物在吸烟诱导肺癌中常见的两个基因中的分布：p53抑癌基因和K-ras原癌基因。吸烟者的肺肿瘤通常在K-ras密码子12和p53密码子157,158,248和273中存在特发性点突变“热点”。这些突变被认为是DNA聚合酶在被代谢激活的烟草致癌物化学修饰的DNA复制过程中出错的结果。K-ras和p53基因特定位点的大量突变可能源于它们对烟草致癌物的高反应性、DNA损伤修复缺陷或特定序列背景下病变错配率升高。虽然在特定的序列环境中引入的DNA损伤的错配率可以通过位点特异性诱变准确地建立，但缺乏能够绘制基因序列中特定DNA损伤形成和修复的分析方法。本研究将使用该实验室最近开发的基于质谱的方法来量化p53和k -ras衍生DNA序列中特定位置的DNA加合物的形成。该研究将重点关注两种主要的烟草致癌物苯并[a]芘（B[a]P）和4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）引起的DNA损伤。代表p53和K-ras基因突变易发区域的双链合成寡脱氧核苷酸将用B[a]P和NNK的反应性代谢物处理，每个位置的加合物形成程度将通过液相色谱-电喷雾串联质谱结合稳定同位素标记来确定。同样的方法将用于分析内源性胞嘧啶甲基化对烟草致癌物质诱导的鸟嘌呤病变形成的影响，并确定K-ras和p53序列背景对nnk诱导的o6 -烷基鸟嘌呤加合物修复的影响。这些研究将1)在含有已知突变热点的K-ras和p53来源的DNA序列中建立B[a]P二聚环氧化合物诱导的核碱基损伤的分布。2)沿着K-ras和p53衍生的DNA序列绘制nnk诱导的甲基化和吡啶氧基丁基化病变的形成图谱。3)分析内源性胞嘧啶甲基化对邻近鸟嘌呤上B[a]P和NNK加合物形成的影响。4)研究K-ras序列背景对nnk诱导的o6 -烷基鸟嘌呤DNA烷基转移酶催化的o6 -烷基鸟嘌呤损伤修复的影响。这项工作的结果将为吸烟引起的肺癌中观察到的遗传变化的分子基础提供新的见解，并将有助于为风险个体制定合理的预防策略和基于机制的生物标志物。本研究也将为今后致癌物质修饰核碱基在单核苷酸分辨率下的体内研究奠定基础。

DESCRIPTION (provided by applicant): This project focuses on the distribution of specific tobacco carcinogen-DNA adducts within the two genes frequently targeted in smoking induced lung cancer: the p53 tumor suppressor gene and the K-ras proto-oncogene. Lung tumors of smokers often contain characteristic "hotspots" for point mutations within K-ras codon12 and p53 codons 157,158, 248, and 273. These mutations are believed to be a result of DNA polymerase errors during the replication of DNA chemically modified by metabolically activated tobacco carcinogens. The large numbers of mutations at specific sites of the K-ras and p53 genes may originate from their high reactivity towards tobacco carcinogens, deficient repair of DNA lesions, or elevated mispairing rates for the lesions within specific sequence context. While the mispairing rates of DNA lesions introduced in a defined sequence environment can be accurately established by site specific mutagenesis, analytical methods capable of mapping the formation and repair of specific DNA lesions within gene sequences are lacking. The present study will use a mass spectrometry based approach recently developed in this laboratory to quantify the formation of DNA adducts at specific positions within p53 and K-ras-derived DNA sequences. The research will focus on DNA damage induced by two prominent tobacco carcinogens, benzo[a]pyrene(B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Double-stranded synthetic oligodeoxynucleotides representing mutation-prone regions of the p53 and K-ras genes will be treated with reactive metabolites of B[a]P and NNK, and the extent of adduct formation at each position will be determined by liquid chromatography-electrospray ionization tandem mass spectrometry in combination with stable isotope labeling. The same approach will be used to analyze the effect of endogenous cytosine methylation on the formation of tobacco carcinogen-induced guanine lesions and to determine the effect of K-ras and p53 sequence context on the repair of NNK-induced O6-alkylguanine adducts. These studies will 1) Establish the distribution of B[a]P diolepoxide-induced nucleobasel esions within K-ras and p53-derived DNA sequences containing known mutational hotspots. 2) Map the formation of NNK-induced methylated and pyridyloxobutylated lesions along K-ras and p53-derived DNA sequences. 3) Analyze the effects of endogenous cytosine methylation on the formation of B[a]P and NNK adducts at neighboring guanines. 4) Examine the effects of K-ras sequence context on O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-alkylguanine lesions. The results of this work will afford new insights into the molecular basis of genetic changes observed in smoking-induced lung cancer and will aid in the development of rational prevention strategies and mechanism-based biomarkers for individuals at risk. This research will also lay a foundation for future in vivo studies of carcinogen-modified nucleobases at single nucleotide resolution.