Densely methylated <i>MLH1</i> promoter correlates with decreased mRNA expression in sporadic colorectal cancers

Furukawa, Taiji; Konishi, Fumio; Masubuchi, Shigehiko; Shitoh, Kazuhisa; Nagai, Hideo; Tsukamoto, Tatsuro

doi:10.1002/gcc.10100

Cited by 35 publications

(31 citation statements)

References 22 publications

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“…Methylation at both the A and C regions of the promoter was strongly related to loss of hMLH1 expression. This is consistent with recent reports that only dense methylation of the hMLH1 promoter is associated with gene silencing and loss of protein expression (Furukawa et al, 2002). The presence of partial methylation at both the A and C regions was also seen in cell lines lacking hMLH1 expression (LS411).…”

Section: Discussionsupporting

confidence: 92%

CpG island methylation is a common finding in colorectal cancer cell lines

Suter

Norrie

et al. 2003

Br J Cancer

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Tumour cell lines are commonly used in colorectal cancer (CRC) research, including studies designed to assess methylation defects. Although many of the known genetic aberrations in CRC cell lines have been comprehensively described, no studies have been performed on their methylation status. In this study, 30 commonly used CRC cell lines as well as seven primary tumours from individuals with hereditary nonpolyposis colorectal cancer (HNPCC) were assessed for methylation at six CpG islands known to be hypermethylated in colorectal cancer: hMLH1, p16, methylated in tumour (MINT-)-1, -2, -12 and -31. The cell lines were also assessed for microsatellite instability (MSI), ploidy status, hMLH1 expression, and mutations in APC and Ki-ras. Methylation was frequently observed at all examined loci in most cell lines, and no differences were observed between germline-derived and sporadic cell lines. Methylation was found at MINT 1 in 63%, MINT 2 in 57%, MINT 12 in 71%, MINT 31 in 53%, p16 in 71%, and hMLH1 in 30% of cell lines. Overall only one cell line, SW1417, did not show methylation at any locus. Methylation was found with equal frequency in MSI and chromosomally unstable lines. MSI was over-represented in the cell lines relative to sporadic CRC, being detected in 47% of cell lines. The rate of codon 13 Ki-ras mutations was also over three times that expected from in vivo studies. We conclude that CpG island hypermethylation, whether acquired in vivo or in culture, is a ubiquitous phenomenon in CRC cell lines. We suggest that CRC cell lines may be only representative of a small subset of real tumours, and this should be taken into account in the use of CRC cell lines for epigenetic studies.

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Section: Discussionsupporting

confidence: 92%

CpG island methylation is a common finding in colorectal cancer cell lines

Suter

Norrie

et al. 2003

Br J Cancer

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“…48 CpG islands of some genes, among them MLH1, are more prone to methylation in several types of cancers. 7,9,11,49 In this respect it is noteworthy that the methylated alleles traced back were of maternal origin in three cases in the literature 24,27 and in one case published here. An increased maternal age at pregnancy can be excluded, as all pregnancies came prior to a maternal age of 34 years, as well as methylation errors due to assisted reproductive techniques.…”

Section: Discussionsupporting

confidence: 51%

“…42 CpG islands D and B/C, both downstream of c.1-500 bp are unmethylated in normal cells. 7,43 Abnormal methylation of these CpG dinucleotides causes transcriptional silencing, whereas in the upstream CpG island A/B (c.1 -755 to c.1 -574 bp), methylation is not relevant for MLH1 transcription regulation. Figure 2a) but was still heterozygous in two cases (Table 1, patients 9 and 12, Figure 2b).…”

Section: Cpg Island Predictionmentioning

confidence: 99%

“…Methylation of cytosines in single CpG dinucleotides located in the promoter region of the MLH1 gene can epigenetically modify gene expression. 5,6 The MLH1 CpG islands D and B/C (Figure 1b) are normally unmethylated 7 while biallelic hypermethylation in the tumour tissue was found to be a causative mechanism for 15% of sporadic endometrial, 8,9 gastric 10,11 and colorectal 10,12 -14 cancers showing MSI-H and immunohistochemical (IHC) MLH1-deficiency. The transcriptional activity of the MLH1 promoter is strongly dependent on two CAAT boxes at c.1-282 and c.1-145 bp 15 and on transcriptional enhancers in a region from c.1-250 to c.1-151 bp 16 ( Figure 1a).…”

Section: Introductionmentioning

confidence: 98%

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Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC

et al. 2008

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Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCRscreening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.

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“…8,9 Nevertheless, while several lines of evidence implicate promoter methylation as a causal factor in hMLH1 silencing, in some MSI-H CRCs, hMLH1 silencing has been seen in the absence of hMLH1 promoter methylation. [10][11][12][13] Conversely, in some MSS/MSI-L CRCs, despite the fact that hMLH1 promoter sequences show increased methylation, hMLH1 gene expression is retained. 9,10-13 Some histopathological and clinical features have been associated with the MSI-H phenotype in sporadic CRCs.…”

Section: B I O S C I E N C E N O T F O R D I S T R I B U T I O Nmentioning

confidence: 99%