title: Non-CpG cytosine methylation of p53 exon 5 in non-small cell lung carcinoma.
abstract: Non-CpG methylation of cytosine residues, a mechanism associated with regulation of gene expression, has not been investigated in human cancer until now. Analysis of the p53 exon 5 mutation spectrum in mutation databases for lung cancer reveals frequent GC>AT transitions, several of which occur at non-CpG sequences. To investigate the involvement of cytosine methylation in this mutagenesis process, we analyzed the methylation profile of p53 exon 5, in lung carcinoma. In this report, we present evidence that extensive clustered non-CpG methylation is observed in three regions of this exon, namely the sequences spanning codons 156-159, 175-179 and the 3' splice site, as well as in scattered CpA sequences. This methylation pattern was verified using direct methylation sequencing, and a two-stage methylation-specific PCR assay (MSP), designed for the detection of methylation in a GC rich region (oligo C sequence, of codons 175-179) of exon 5. The results from this MSP assay reveal that DNA from cancerous specimens was more heavily methylated in non-CpG cytosines, compared to that from non-cancerous lung tissue of cancer patients (14/19 cancerous and 6/19 non-cancerous, respectively). DNA isolated from human leucocytes and some non-cancerous specimens (2/19) was free of non-CpG methylation. Careful analysis of the mutations reported in p53 mutation databases also provides corroborating evidence that the high incidence of GC>AT mutations in the p53 gene, observed in lung cancer, might also be related to non-CpG methylation, as well as to the overall increase of methylation sites in this locus.
authors.affiliation: Department of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. kouidou@auth.gr
authors.forename: Sofia
authors.initials: S
authors.lastname: Kouidou
authors.forename: Tania
authors.initials: T
authors.lastname: Agidou
authors.forename: Athena
authors.initials: A
authors.lastname: Kyrkou
authors.forename: Alexandra
authors.initials: A
authors.lastname: Andreou
authors.forename: Theodora
authors.initials: T
authors.lastname: Katopodi
authors.forename: Elisavet
authors.initials: E
authors.lastname: Georgiou
authors.forename: Dimitris
authors.initials: D
authors.lastname: Krikelis
authors.forename: Afroditi
authors.initials: A
authors.lastname: Dimitriadou
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Spanos
authors.forename: Christos
authors.initials: C
authors.lastname: Tsilikas
authors.forename: Hara
authors.initials: H
authors.lastname: Destouni
authors.forename: Georgios
authors.initials: G
authors.lastname: Tzimagiorgis
mesh_terms: D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics* / Q000502:physiology; D003596:Cytosine / Q000032:analysis*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030541:Databases, Genetic; D005091:Exons; D015972:Gene Expression Regulation, Neoplastic; D016158:Genes, p53; D006801:Humans; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D004273:DNA, Neoplasm; D003596:Cytosine