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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• 作者：Mathur Rajesh ; Gang Wang ; Roger Jones ; and Natalia Tretyakova
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：February 15, 2005
• 年：2005
• 卷：44
• 期：6
• 页码：2197 - 2207
• 全文大小：213K
• 年卷期：v.44,no.6(February 15, 2005)
• ISSN：1520-4995

文摘

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 endogenouslymethylated ^MeCG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 (^MeC = 5-methylcytosine). Onepossible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolicallyactivated tobacco carcinogens to ^MeCG 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 duplexesrepresenting p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containingp53 codons 153-159, 243-250, and 269-275 were prepared, where ^MeC was incorporated at allphysiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH₂-¹⁵N₃-2-¹³C]-guanine, which served as an isotope "tag" to enable specific quantification of guaninelesions 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 atunlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine andN7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases withinpolypurine runs, while the formation of O⁶-methyl-2'-deoxyguanosine and O⁶-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGGsequences. In contrast, the presence of 5'-neighboring ^MeC inhibited O⁶-guanine adduct formation. Theseresults indicate that the N7- and O⁶-guanine adducts of NNK are not overproduced at the endogenouslymethylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other thanNNK adduct formation are responsible for mutagenesis at these sites.