Carcinogenesis Advance Access originally published online on August 22, 2006
Carcinogenesis 2007 28(2):356-362; doi:10.1093/carcin/bgl150
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Potassium diazoacetate-induced p53 mutations in vitro in relation to formation of O6-carboxymethyl- and O6-methyl-2'-deoxyguanosine DNA adducts: relevance for gastrointestinal cancer
1 MRC Toxicology Unit, Hodgkin Building, University of Leicester P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK
2 Leeds Institute of Molecular Medicine, St James's University Hospital Leeds LS9 7TF, UK
3 Department of Chemistry, The Open University Walton Hall, Milton Keynes MK7 6AA UK
4 Present address: School of Biomedical Sciences, University of Nottingham Queen's Medical Centre, Nottingham NG7 2UH, UK
*To whom requests for reprints and correspondence should be addressed at: Department of Chemistry, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK. Tel: +44 0 1908 653199; Fax: +44 0 1908 858327; Email: d.e.g.shuker{at}open.ac.uk
Nitrosated glycine derivatives react with DNA to form O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) and O6-methyl-2'-deoxyguanosine (O6-MedG) adducts concurrently. O6-CMdG is not repaired by O6-alkylguanine alkyltransferases and might be expected to lead to mutations via a similar mechanism to O6-MedG. Potassium diazoacetate (KDA) is a stable form of nitrosated glycine and its ability to induce mutations in the p53 gene in a functional yeast assay was studied. Treatment of a plasmid containing the human p53 cDNA sequence with KDA afforded readily detectable levels of O6-CMdG and O6-MedG. The treated plasmid was used to transform yeast cells and coloured colonies harbouring a p53 sequence with functional mutations were detected. Recovery of the mutated plasmids followed by DNA sequencing enabled the mutation spectrum of KDA to be characterised. The most common mutations induced by KDA were substitutions with >50% occurring at GC base pairs. In contrast to the methylating agent methylnitrosourea which gives predominantly (>80%) GC
AT transitions, KDA produced almost equal amounts of transitions (GCAT) and transversions (GCTA and ATTA). This difference is probably due to a different mode of base mispairing for O6-CMdG compared with O6-MedG. The pattern of mutations induced by KDA was very similar to the patterns observed in mutated p53 in human gastrointestinal tract tumours. These results are consistent with the hypothesis that nitrosation of glycine (or glycine derivatives) may contribute to characteristic human p53 mutation profiles. This conclusion is borne out by recent observations that O6-CMdG is present in human DNA both from blood and exfoliated colorectal cells and is consistent with recent epidemiological studies that have concluded that endogenous nitrosation arising from red meat consumption is related to an increased risk of colorectal cancer.
Abbreviations: O6-CMG, O6-carboxymethylguanine; O6-CMdG, O6-carboxymethyl-2'-deoxyguanosine; O6-MeG, O6-methylguanine; O6-MedG, O6-methyl-2'-deoxyguanosine; KDA, potassium diazoacetate; GI, gastrointestinal; MNU, N-methyl-N-nitrosourea
Both the authors have contributed equally to this paper. Received March 15, 2006; revised July 18, 2006; accepted August 8, 2006.
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