Stable Isotope Labeling−Mass Spectrometry Analysis of Methyl- and Pyridyloxobutyl-Guanine Adducts of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone inp53-Derived DNA Sequences

Abstract

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.

Mapping Structurally Defined Guanine Oxidation Products along DNA Duplexes: Influence of Local Sequence Context and Endogenous Cytosine Methylation

“…DNA quantification was based on HPLC–UV analysis of dG in enzymatic digests. 60 DNA oligomers were annealed to equimolar amounts of the corresponding complementary strands in the presence of 100 mM NaCl to obtain double-stranded DNA. DNA duplexes were characterized by UV melting and native gel electrophoresis to confirm their purity and stability.…”

Involvement of p53 Mutation and Mismatch Repair Proteins Dysregulation in NNK-Induced Malignant Transformation of Human Bronchial Epithelial Cells

“…Most mutational “hot spots” have been observed clustering in exon 5–8, within the DNA binding domain of p53 [7]. In the studies of NNK induced lung tumors, specific damage distribution patterns were found and factors other than NNK adduct formation may contribute to the mutagenesis of TP53 [8, 9]. …”

Multiplexed p53 Mutation Detection by Microchip Electro‐phoresis with Laser‐Induced Fluorescence Detector

“…2,3 One such approach involves isotopic 15 N or 13 C labeling of specific nucleobases within an oligonucleotide and determining the amount of damage at the site by mass spectrometry. 4,5 Although this method is rapid and also identifies the type of nucleobase adduct formed at the specific site, isotope-labeled oligonucleotides are up to 100-fold more expensive than fluorescent-labeled oligonucleotides. Also, with isotope labeling, it is only practical to characterize previously identified specific mutation sites, and the method is limited by the size of the labeled oligonucleotides.…”