Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.
Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.
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DOI：
10.1021/bi0480032
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发表时间：
2005-02
期刊：
影响因子：
2.9
通讯作者：
M. Rajesh;Gang Wang;Roger A. Jones;N. Tretyakova
中科院分区：
文献类型：
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作者：
M. Rajesh;Gang Wang;Roger A. Jones;N. Tretyakova
p53肿瘤抑制基因是吸烟诱发肺癌的主要靶点。有趣的是，在吸烟者肺肿瘤中观察到的p53突变集中在内源性甲基化（Me）CG二核苷酸中的鸟嘌呤碱基上，例如密码子157、158、245、248和273 （(Me)C = 5-甲基胞嘧啶）。这些位点突变增加的一个可能机制涉及代谢激活的烟草致癌物质与（Me）CG序列的靶向结合。本研究采用稳定同位素标记的HPLC-ESI(+)-MS/MS方法分析了烟草特异性肺癌致癌物4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）在代表p53突变“热点”的DNA双链及其周围序列中诱导的鸟嘌呤病变的形成。制备了含有p53密码子153-159、243-250和269-275的合成DNA双链，其中（Me）C结合在所有生理甲基化的CG位点上。在每个双链中，其中一个鸟嘌呤碱基被[1,7，NH(2)-(15)N(3)-2-(13)C]-鸟嘌呤取代，作为同位素“标签”，能够对源自该位置的鸟嘌呤病变进行特异性定量。与NNK重氮氢氧化物孵育后，采用HPLC-ESI(+)-MS/MS分析测定序列中同位素标记的鸟嘌呤和其他未标记的鸟嘌呤碱基上NNK加合物的产率。我们发现N7-甲基-2'-脱氧鸟苷和N7-[4-氧-4-（3-吡啶基）- 1-基]鸟嘌呤病变在多嘌呤序列的3'-鸟嘌呤碱基上过量产生，而在5'- gg和5'- ggg序列的3'-鸟嘌呤碱基上特别倾向于形成O(6)-甲基-2'-脱氧鸟嘌呤和O(6)-[4-氧-4-（3-吡啶基）- 1-基]-2'-脱氧鸟嘌呤加合物。相反，5'-邻（Me）C的存在抑制了O(6)-鸟嘌呤加合物的形成。这些结果表明，NNK的N7-和O(6)-鸟嘌呤加合物在p53肿瘤抑制基因内的内源性甲基化CG二核苷酸上不会过量产生，这表明NNK加合物形成以外的因素在这些位点上负责突变。
The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.