DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53 mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

Efeito da p53 sobre a expressão e atividade da enzima de reparo de DNA Timina-DNA Glicosilase / Effect of p53 on the expression and activity of DNA repair enzyme thymine-DNA glycosylase

O câncer de esôfago é uma malignidade altamente freqüente e letal. Uma característica específica das áreas de alta incidência de câncer de esôfago é a grande proporção de duplas mutações no gene TP53, sendo, ao menos uma delas, uma transição G para A em sítios CpG. Essas transições resultam de malpareamentos G.T causados pela desaminação espontânea da 5-metilcitosina em ilhotas CpG. A enzima de reparo de DNA Timina-DNA Glicosilase (TDG) é responsável pelo primeiro passo na remoção da timina de malpareamentos G.T em CpG. A alta proporção de mutações em sítios CpG em câncer de esôfago das áreas de alta incidência sugere que a via de reparo de DNA iniciada pela TDG pode estar prejudicada. A presença de duplas mutações, sendo ao menos uma delas em CpG, levantou a hipótese de que a primeira mutação no TP53 reduz a atividade da via de reparo iniciada pela TDG, que acarretaria a segunda mutação em sítios CpG. Dessa forma, o objetivo desse trabalho foi analisar o efeito da p53 sobre a expressão e atividade da TDG. Os resultados obtidos mostram que a expressão de TDG é regulada transcricionalmente pela p53 numa gama de linhagens celulares e é induzida pelo dano ao DNA, de forma p53-dependente. Além disto, os resultados apontam um possível papel da proteína p53 ativa na migração nuclear e atividade da TDG. Estes resultados ainda nos levam à conclusão de que o silenciamento de TDG aumenta a sensibilidade à morte celular induzida por MMS quando a p53 é encontrada na forma selvagem, mas não quando esta proteína é mutada, e de que o status mutacional de TP53 parece afetar a expressão de TDG em CEE primários. Juntos esses resultados sugerem que a p53 regula o reparo de DNA mediado pela TDG e que a inativação de p53 em células tumorais pode contribuir para a aquisição de um mutator phenotype

Esophageal squamous cell carcinoma (ESCC) is a highly frequent and fatal malignancy in the world. A peculiar characteristic of the high incidence areas of esophageal cancer is the large proportion of double mutations in TP53 gene, being, at least one of them, a G to A transition at CpG sites. These transitions result from G.T mismatches caused by the spontaneous deamination of 5-methylcytosine at CpG sites. The DNA repair enzyme Thymine-DNA Glycosylase (TDG) is responsible for the first step in the removal of the thymidine from the G.T mismatches at CpG sites. The high proportion of mutations at CpG sites in esophageal tumors in the high incidence areas suggests that the DNA repair pathway initiated by TDG might be impaired. The large number of double mutations, with one being at a CpG site, raised the possibility that the first mutation in TP53 reduces the activity of the TDG base excision repair pathway, increasing the chance of a second mutation event at a CpG site. In this way, the aim of this work was to analyze the effect of p53 on the expression and activity of TDG. The results achieved show that TDG expression is regulated by p53 in a variety of cells lines at the trancriptional level and induced by DNA–damage in a p53-dependent manner. Furthermore, these results point out a possible role of active p53 in the nuclear migration and activity of TDG. The results further support the notion that TDG silencing increases the sensitivity to cell death induced by Methylmethane sulphonate when p53 is found in a wild-type, but not in a mutant form, and that TP53 mutation seems to affect TDG expression in primary ESCC. Together, these results suggest that p53 regulates TDG-mediated repair and that p53 inactivation in cancer cells may contribute to a mutator phenotype through loss of TDG function