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Spreading of Alu methylation to the promoter of the MLH1 gene in gastrointestinal cancer.

ABSTRACT: The highly repetitive Alu retroelements are regarded as methylation centres in the genome. Methylation in the gene promoters could be spreading from them. Promoter methylation of MLH1 is frequently detected in cancers, but the underlying mechanism is unclear. The aim of this study is to understand whether the methylation in the Alu elements is associated with promoter methylation in the MLH1 gene. Bisulfite genomic sequencing was used to analyse the CpG sites of the 5' end (promoter, exon 1 and Alu-containing intron 1) of the MLH1 gene in colorectal cancer cells and tissues, and gastric cancer tissues. Hypomethylation in the Alu elements and hypermethylation in the promoters and the regions between the promoters and the Alu elements were detected in two cancer cell lines and seven cancer tissues. However, demethylation or hypomethylation of the MLH1 promoter and regions between promoter and the Alu elements, and hypermethylation in the Alu elements, were identified in the normal tissues. MLH1 promoter methylation may spread from Alu elements that are located in intron 1 of the MLH1 gene. The trans-acting elements binding to the mutation sites could play a role in the methylation spreading.

SUBMITTER: Wang X

PROVIDER: S-EPMC3192117 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Spreading of Alu methylation to the promoter of the MLH1 gene in gastrointestinal cancer.

Wang Xiyin X Fan Jing J Liu Dong D Fu Siqing S Ingvarsson Sigurdur S Chen Huiping H

PloS one 20111012 10

The highly repetitive Alu retroelements are regarded as methylation centres in the genome. Methylation in the gene promoters could be spreading from them. Promoter methylation of MLH1 is frequently detected in cancers, but the underlying mechanism is unclear. The aim of this study is to understand whether the methylation in the Alu elements is associated with promoter methylation in the MLH1 gene. Bisulfite genomic sequencing was used to analyse the CpG sites of the 5' end (promoter, exon 1 and ...[more]

PMID: 22022465

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Project description:BackgroundTo investigate the etiology of MLH1 promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high ages, young patients bearing this type of malignancy are rare and could provide additional insight into the etiology of sporadic MSI-H colon cancer.MethodsWe studied a set of 46 MSI-H colon tumors cases with MLH1 promoter methylation which was enriched for patients with an age of onset below 50 years (n=13). Tumors were tested for CIMP marker methylation and mutations linked to methylation: BRAF, KRAS, GADD45A and the MLH1 -93G>A polymorphism. When available, normal colon and leukocyte DNA was tested for GADD45A mutations and germline MLH1 methylation. SNP array analysis was performed on a subset of tumors.ResultsWe identified two cases (33 and 60 years) with MLH1 germline promoter methylation. BRAF mutations were less frequent in colon cancer patients below 50 years relative to patients above 50 years (p-value: 0.044). CIMP-high was infrequent and related to BRAF mutations in patients below 50 years. In comparison with published controls the G>A polymorphism was associated with our cohort. Although similar distribution of the pathogenic A allele was observed in the patients with an age of onset above and below 50 years, the significance for the association was lost for the group under 50 years. GADD45A sequencing yielded an unclassified variant. Tumors from both age groups showed infrequent copy number changes and loss-of-heterozygosity.ConclusionSomatic or germline GADD45A mutations did not explain sporadic MSI-H colon cancer. Although germline MLH1 methylation was found in two individuals, locus-specific somatic MLH1 hypermethylation explained the majority of sporadic early onset MSI-H colon cancer cases. Our data do not suggest an intrinsic tendency for CpG island hypermethylation in these early onset MSI-H tumors other than through somatic mutation of BRAF.

Project description:The human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumour-suppressor gene inactivated by methylation in several cancers. In this study, we analysed the methylation and expression status of hDAB2IP in gastrointestinal tumours. The promoter region of hDAB2IP was divided into two regions (m2a and m2b) based on our previous report, and the methylation status was determined by bisulphite DNA sequencing in gastric cancer cell lines. The gene expression was semiquantified by real-time RT-PCR, and the results indicated that the m2b promoter region might be an authentic methylation-mediated key regulator of the gene expression. Based on the sequence data, we developed a methylation-specific PCR (MSP) for the m2a and m2b regions and applied it to the samples. Methylation-specific PCR revealed aberrant methylation in the m2a region in eight of 12 gastric cancer cell lines (67%), 16 of 35 gastric cancer tissues (46%) and 29 of 60 colorectal cancer tissues (48%), and in the m2b region in eight of 12 cell lines (67%), 15 of 35 gastric cancer tissues (43%) and 28 of 60 colorectal cancer tissues (47%). On the other hand, seven (12%) and 11 (19%) of 59 gastrointestinal nonmalignant mucosal specimens showed methylation in the m2a and m2b regions, respectively, suggesting that hDAB2IP methylation might play a causative role in carcinogenesis. The 5-aza-2'-deoxycytidine treatment restored the gene expression in the m2b-methylated cell lines, confirming that the methylation caused gene downregulation. We also examined the relationship between hDAB2IP methylation and the clinicopathological features in patients with primary tumours, and determined that methylation in the m2b region was associated with location of the tumour in the stomach. In summary, our results demonstrated that hDAB2IP methylation is frequently present in gastrointestinal tumours and that the resulting gene silencing plays an important role in gastrointestinal carcinogenesis.

Project description:UnlabelledBackgroundOxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG).MethodsIn this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student's two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount.ResultsThe intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate.ConclusionThis vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.

Dataset Information

Spreading of Alu methylation to the promoter of the MLH1 gene in gastrointestinal cancer.

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Spreading of Alu methylation to the promoter of the MLH1 gene in gastrointestinal cancer.

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