hMLH1 Promoter Methylation Status Research Articles

In vitro study of human mutL homolog 1 hypermethylation in inducing drug resistance of esophageal carcinoma.

Aberrant promoter methylation of tumor suppressor gene can inhibit corresponding protein expression and promote carcinogenesis. Many studies have demonstrated that human mutL homolog 1(hMLH1) promoter methylation is correlated with occurrence and progression of multiple types of tumors. However, its correlation with esophageal carcinoma drug resistance is still unknown. To confirm methylation status of hMLH1 promoter in drug-resistance cell line of esophageal carcinoma, further confirm whether hMLH1 promoter methylation is responsible for drug resistance. Two stable esophageal carcinoma drug-resistance cell lines were successfully established by Cisplatin (DDP) concentration increment method; methylation status of hMLH1 promoter, mRNA and protein expression of hMLH1 were detected by methylation-specific PCR (MSP), RT-PCR and western blot, respectively; Drug-resistance ability assay was used to detect drug-resistance ability. Stronger methylation status of hMLH1 promoter, lower hMLH1 mRNA and protein expression were found in both drug-resistance cell lines; after removing methylated bands using 5-aza-2'-deoxycytidine(5-Aza-CdR) in drug-resistance cell lines, hMLH1 mRNA and protein expression were restored and drug-resistance abilities declined nearly by half. hMLH1 promoter hypermethylation plays important roles in esophageal carcinoma drug-resistance and show us the prospect that combination of demethylation treatment with conventional chemotherapy drugs may bring better therapy effect.

Microsatellite instability and promoter hypermethylation in colorectal cancer in India

Microsatellite instability (MSI) is an important factor in tumor development and is a hypermutable phenotype caused by the loss of DNA mismatch repair activity. It is important to identify tumors with microsatellite instability as the patients have a better prognosis and differ with response to chemotherapy. Limited data are available on the incidence of MSI in Indian colorectal cancers (CRCs). The objectives of this study were to identify the extent of MSI in Indian CRC patients below 50 years and to determine promoter methylation status of hMLH1 and hMSH2 in relation to MSI. A total of 450 patients were diagnosed with CRC, out of which 91 individuals were recruited as per Bethesda guidelines and were tested for instability by the NCI-recommended Bethesda panel (BAT25, BAT26, D2S123, D5S346, and D17S2720) using labeled primers. The fragments were separated and analyzed on a Beckman GeXP sequencer. Promoter methylation status was determined by restriction enzyme digestion and PCR. MSI (high and low) was seen in 48.4% (44/91) of CRC patients, out of which microsatellite instability-high (MSI-H) was detected in 13.2% (12/91) and microsatellite instability-low (MSI-L) in 35.2% (32/91) and the rest were microsatellite stable (MSS), 51.6% (47/91). Majority of the MSI-H tumors were adenocarcinomas (10/12), in the rectum (8/12), and moderately or poorly differentiated (12/12). Promoter hypermethylation was seen in 75% of the MSI-H, 56.24% of MSI-L, and only 23.4% of MSS individuals. MSI (high and low) was associated with 48.4% of CRC patients, and a significantly higher proportion of promoter hypermethylation of hMLH1 and hMSH2 genes was associated with instable tumors.

Methylation of p16 and hMLH1 genes in esophageal squamous cell carcinoma and reflux esophagitis

To study the promoter methylation pattern of p16 and hMLH1 genes in esophageal squamous cell carcinoma and reflux esophagitis, and to correlate the results with clinical and pathologic findings. Twelve cases of normal esophagus, 13 cases of esophageal squamous cell carcinoma, 43 cases of reflux esophagitis with basal cell hyperplasia and 21 cases of reflux esophagitis with dysplasia, as confirmed by endoscopic and pathologic examination, were enrolled into the study. Genomic DNA was extracted. The promoter methylation status of p16 was measured by methylation-specific polymerase chain reaction. The promoter methylation status of hMLH1 was measured by sodium bisulfite-restriction enzyme digestion. Immunohistochemical study for p16 and hMLH1 proteins was also carried out. The rates of p16 methylation in normal esophageal epithelium, basal cell hyperplasia, dysplasia and esophageal squamous cell carcinoma were 0/12, 14.0% (6/43), 38.1% (8/21) and 6/13, respectively. The p16 methylation correlated with the progress of esophageal lesions. On the other hand, the hMLH1 methylation was not observed in the normal esophageal epithelium and reflux esophagitis. One case of esophageal squamous cell carcinoma showed the presence of hMLH1 methylation. The hMLH1 promoter hypermethylation did not correlate with the clinical and pathologic features. The p16 methylation may be one of the earliest events in the pathogenesis of esophageal squamous cell carcinoma and is also observed in reflux esophagitis. Reflux esophagitis may be related to the development of esophageal squamous cell carcinoma in Chinese population. In contrast, hMLH1 methylation may not be directly involved in the tumorigenesis of esophageal squamous cell carcinoma.

Promoter methylation status of hMLH1, hMSH2, and MGMT genes in colorectal cancer associated with adenoma–carcinoma sequence

Epigenetic silencing of the DNA mismatch repair genes has been poorly described in colorectal carcinomas showing the classic adenoma-carcinoma pathway of carcinogenesis. The aim of this study was to investigate the methylation status of MutL homolog 1 (hMLH1), MutS homolog 2 (hMSH2), and O-6-methylguanine-DNA methyltransferase (MGMT) in a series of colorectal carcinomas that contain both adenomas and carcinomas. Promoter methylation of hMLH1, hMSH2, and MGMT was evaluated in normal mucosa, adenoma, and carcinoma samples from 112 colorectal cancer patients. Methylation was assessed by bisulfite modification and methylation-specific PCR. Expression of the gene products was also examined by immunohistochemistry. Of the 112 adenomas, methylation was detected for hMLH1 (2, 1.8%), hMSH2 (9, 8.0%), and MGMT (38, 33.9%). In the carcinoma samples, methylation was seen in hMLH1 (2, 1.8%), hMSH2 (15, 13.4%), and MGMT (53, 47.3%). In normal mucosa, hMSH2 (6, 5.4%) and MGMT (12, 10.7%) were methylated, whereas hMLH1 was not. Immunohistochemical analysis revealed abnormal hMLH1 (14, 12.5%), hMSH2 (11, 9.8%), and MGMT (53, 47.3%) expression with a significant correlation between aberrant MGMT methylation and a loss of MGMT expression. These data suggest that CpG island methylation in hMSH2 and MGMT, but not hMLH1, is closely related to carcinogenesis in colorectal carcinomas presenting with a conventional adenoma-carcinoma sequence. Therefore, the detection of hMSH2 and MGMT methylation may have clinical significance in the evaluation of colon cancer patients and in tumor-specific management of the disease.

HMLH1 promoter methylation and JC virus T antigen presence in the tumor tissue of colorectal cancer Israeli patients of different ethnic groups

Hypermethylation of tumor suppressor genes' promoter and JC virus infection may be etiologic factors in the development of colorectal cancer (CRC). To look at both JC virus T antigen and hMLH1 promoter methylation in CRC tissue in Israeli ethnic groups with different incidence of CRC. Twenty-four consecutive patients with sporadic CRC were included in the study. Genomic DNA was isolated from paraffin-embedded microdomains removed from five slides of 7 mum by deparaffinizing in multiple xylene washes. Isolated DNA was used as a template for PCR to amplify DNA sequences coding the amino terminus of JC virus T antigen. Methylation-specific PCR was performed on bisulfite-modified DNA templates from CRC tissue materials to study methylation status of hMLH1 promoter, using two sets of primers specific for amplification of methylated and unmethylated alleles. hMLH1 promoter methylation was observed in five patients (20.8%) who were also positive for JC virus T antigen, with even distribution among the ethnic groups. JC virus T antigen DNA was found in cancer tissues of 20 of the 24 patients; 50, 90.9, and 100% of Asia-Africa-born Jews, Europe-America-born Jews, and Israeli Arabs, respectively (P = 0.036 between the first group to the other). Evidence for higher JC virus infection was shown among Europe-America-born Jews and Israeli Arabs. hMLH1 promoter methylation was evenly distributed between different ethnic groups in Israel.

Implications of mismatch repair genes hMLH1 and hMSH2 in patients with sporadic renal cell carcinoma

To analyse the implications of DNA mismatch repair genes hMLH1 and hMSH2 in sporadic renal cell carcinoma (RCC). Specimens of tumour and healthy renal tissue were collected from 89 patients treated for sporadic RCC. Another 95 blood samples taken from individuals with no history of cancer were also analysed. After DNA extraction and PCR amplification, microsatellite instability (MSI) was determined using the Bethesda microsatellite panel, two exonic microsatellites of the TGFbRII and BAX genes, and the microsatellite D3S1611. The promoter methylation status of hMLH1 was investigated using the HpaII and MspI restriction enzymes. In addition, a sequencing analysis of complete coding region of hMLH1 and hMSH2 genes was performed. MSI and promoter hypermethylation of hMLH1 were not detected. Interestingly, loss of heterozygosity (LOH) was common among patients with RCC, particularly in microsatellite D3S1611 (34.9%). Mutations were identified in eight patients: K618A and V716M in gene hMLH1; and I145V, G322D, and the novel mutation P349A, in gene hMSH2. The mutations also appeared in healthy renal tissue and therefore, were considered as germline DNA sequence variations. There were G322D and K618A changes in >1% of the healthy control subjects, suggesting that they are DNA polymorphisms. Our data show that loss of function of both hMLH1 and hMSH2 is not involved in sporadic RCC, either by promoter methylation or mutation in their exons. However, LOH indicated that chromosomal instability affecting large fragments of DNA was the main genetic alteration we detected associated with RCC.

HMLH1 promoter methylation and silencing in primary endometrial cancers are associated with specific alterations in MBDs occupancy and histone modifications

Objective. To investigate the relationship between hMLH1 promoter methylation and changes in chromatin composition. To study how the occupancy of methyl CpG binding domain proteins (MBDs) and histone acetylation/methylation in hMLH1 promoter may participate in hMLH1 silencing. Methods. 64 endometrial cancer samples were screened for hMLH1 mRNA expression. hMLH1 promoter methylation status was confirmed by methylation-specific PCR in cancers with high and low levels of hMLH1 expression. Chromatin immunoprecipitation was performed to compare the MBD occupancy and histone modifications between the methylated/silenced and unmethylated/active hMLH1 genes in multiple primary endometrial cancers. Results. We demonstrated that MeCP2, MBD1 and MBD2, but not MBD3 and MBD4, specifically bind to methylated hMLH1 promoters. Hyperacetylated histones H3 and H4 were found to be associated with the unmethylated and transcriptionally active hMLH1 promoters. While H3 lysine-4 methylation was present in unmethylated hMLH1 promoters, H3 lysine-9 methylation was found exclusively in methylated promoters. Western blot analysis showed that similar global levels of MBDs and histones were present in the two cancer groups with high and low hMLH1 expression. Conclusions. A distinct combination of MBDs and histone modification is associated with the silencing of the hMLH1 gene. The changes in hMLH1 chromatin composition are closely related to methylation status of hMLH1 promoters. These changes are not accounted by the global expression levels of MBDs and histones in endometrial cancers.

Promoter Methylation Precedes Chromosomal Alterations in Colorectal Cancer Development

Background: Colorectal cancers are characterized by genetic and epigenetic alterations. This study aimed to explore the timing of promoter methylation and relationship with mutations and chromosomal alterations in colorectal carcinogenesis. Methods: In a series of 47 nonprogressed adenomas, 41 progressed adenomas (malignant polyps), 38 colorectal carcinomas and 18 paired normal tissues, we evaluated promoter methylation status of hMLH1, O6MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5, and CHFR using methylation-specific PCR. Mutation status of TP53, APC and KRAS were studied by p53 immunohistochemistry and sequencing of the APC and KRAS mutation cluster regions. Chromosomal alterations were evaluated by comparative genomic hybridization. Results: Our data demonstrate that nonprogressed adenomas, progressed adenomas and carcinomas show similar frequencies of promoter methylation for the majority of the genes. Normal tissues showed significantly lower frequencies of promoter methylation of APC, p16INK4A, GATA-4, and GATA-5 (P-values: 0.02, 0.02, 1.1×10−5 and 0.008 respectively). P53 immunopositivity and chromosomal abnormalities occur predominantly in carcinomas (P values: 1.1×10−5 and 4.1×10−10). Conclusions: Since promoter methylation was already present in nonprogressed adenomas without chromosomal alterations, we conclude that promoter methylation can be regarded as an early event preceding TP53 mutation and chromosomal abnormalities in colorectal cancer development.

Microsatellite Instability Caused by hMLH1 Promoter Methylation Increases with Tumor Progression in Right-Sided Sporadic Colorectal Cancer

Objective: A subset of sporadic colorectal cancers (SCRCs) exhibits microsatellite instability (MSI). Most MSI in SCRCs is caused by hMLH1 inactivation due to promoter methylation. However, the role of MSI in the progression of SCRCs remains unclear. Methods: Thirty-two intramucosal cancers and 63 cancers with submucosal invasion were assigned to group 1 (early-stage cancer), and 30 Dukes’ B and 26 Dukes’ C cancers to group 2 (advanced-stage cancer). hMLH1 promoter methylation status was determined by methylation-specific PCR. MSI was determined using five markers. hMLH1 expression was determined immunohistochemically. Results: MSI was found in 1 of 95 (1.1%) tumors in group 1, compared with 4 of 56 (7.1%) tumors in group 2. In right-sided tumors, the overall frequency of hMLH1-methylation-positive tumors in group 1 was not significantly different from that in group 2 (17 of 43, 39.5%, vs. 9 of 23, 39.1%). In right-sided tumors with hMLH1 promoter methylation, the frequency of MSI-positive tumors in group 1 was significantly lower than that in group 2 (1 of 17, 5.9%, vs. 4 of 9, 44.4%, p = 0.0081). Conclusion: The frequency of MSI caused by hMLH1 promoter methylation increases with tumor progression in right-sided SCRCs.

Clinicopathological features and microsatellite instability (MSI) in colorectal cancers from African Americans

African Americans (AAs) have a 1.5 times higher risk of colorectal carcinoma (CRC) than Caucasians. Gene silencing through CpG island hypermethylation has been associated with the genesis or progression of microsatellite instability (MSI) largely due to 1 target for hypermethylation being the DNA mismatch repair gene hMLH1; there is anecdotal evidence of an increased incidence of MSI among AAs. P16 and hMLH1 can be inactivated by hypermethylation of their respective promoter regions, abrogating the ability to regulate cell proliferation and repair processes. We studied such methylation, as well as hMHS2 expression in colorectal cancers from AA patients to determine if MSI is associated with epigenetic silencing. Experiments were conducted on matched normal and colon cancer tissues from AA patients (n = 51). A total of 5 microsatellite markers (D2S123, D5S346, D17S250, BAT25 and BAT26) were used to evaluate MSI status. P16 and hMLH1 promoter methylation status was determined following bisulfite modification of DNA and using methylation specific PCR, while immunohistochemistry (IHC) was used to examine expression of hMLH1 and hMSH2. A total of 22 (43%) cancers demonstrated microsatellite instability-high (MSI-H), while 27 were microsatellite stable (MSS) and 2 were microsatellite instability-low (MSH-L). Most of the MSI-H tumors were proximal, well differentiated and highly mucinous. Most patients in the MSI-H group were females (68%). The p16 promoter was methylated in 19 of 47 (40%) tumors. A total of 7 of these CRCs demonstrated MSI-H (33%). The hMLH1 promoter was methylated in 29 of 34 (85%) tumors, of which 13 CRCs demonstrated MSI-H (87%). hMLH1 and hMSH2 staining was observed in 66% and 38% of MSI-H tumors, respectively. Overall, the prevalence of MSI-H colorectal tumor was 2-3-fold higher, while the defect in the percentage expression of mismatch repair (MMR) genes (hMLH1 and hMSH2) was similar in AA patients compared to the U.S. Caucasian population. Similar numbers of AA MSS tumors with p16 and hMLH1 methylation likely indicate hemimethylation of genes that might reflect environmental or genetic influences that might be more common in the AA population.

Low-level microsatellite instability phenotype in sporadic glioblastoma multiforme

Genetic instability is a hallmark of glioblastoma multiforme (GBM). Microsatellite instability (MSI) is a significant event in the tumorigenesis of many sporadic malignancies. The aim of our investigation was to study microsatellite instability in newly diagnosed glioblastomas. MSI was investigated in 109 GBMs with 15 microsatellite markers. Immunohistochemistry was performed for the mismatch repair (MMR) proteins hMLH1, hMSH2, hPMS2, and hMSH6 in cases showing MSI. Sequence and promoter methylation status of hMLH1 were analyzed in the case of a decreased hMLH1 protein expression as well. To further investigate MSI(+) GBMs we carried out studies of LOH at selected chromosome regions, EGFR amplification, and sequence of p53 and PTEN. MSI was observed in six GBMs (5.5%) and it was more frequent in GBMs with a previous lower grade astrocytoma (18.8% vs. 3.2%). MMR protein staining was positive in all MSI(+) GBMs except in one case, which showed an aberrant expression of hMLH1 and hPMS2 without hMLH1 inactivation. Among MSI(+) GBMs, one tumor corresponded to the GBM molecular type 1 (p53 mutation, no EGFR amplification), another tumor to type 2 (wild-type p53, EGFR amplification), and four tumors to neither type (wild-type p53, no EGFR amplification). None of the six tumors carried a PTEN mutation. MSI in GBM might be caused by inactivation of minor MMR genes rather than by a deficiency of hMLH1 or hMSH2 and it appears not to play a decisive role in the pathogenesis of these tumors. MSI(+) GBMs predominantly showed a profile which included wild-type of p53 and PTEN and absence of EGFR amplification but MSI occurred in all GBM molecular subtypes.

The contribution of genetic and epigenetic pathways in the molecular pathogenesis in neuroendocrine cancers of the gastro-entero-pancreatic system

Introduction: Very little is known about the mechanisms contributing to the molecular pathogenesis of neuroendocrine tumors of the gastro-entero-pancreatic system. Apart from tumors belonging to the MEN1-syndrome knowledge of genetic events in sporadic neuroendocrine tumors is scarce. We aimed to investigate the role of genetic and epigenetic events leading to the formation of these rare tumors. Methods: Allelic losses, microsatellite instability (MSI) and tumor specific promoter methylation were assessed in 127 tumors. MSI testing was performed by PCR using 5 microsatellite markers. The promoter methylation status of hMLH1, p16, APC, O6-MGMT, HIC1, RASSF1a, TIMP3 and MEN1 were investigated by methylation specific PCR. The loss of heterozygosity (LOH) status of chromosomal loci on 5q and 11q was analysed using markers linked to the corresponding loci. Results: Of the tumors investigated, 80% were microsatellite stable. Only one tumor was highly unstable, 13% of the tumors were low-grade unstable. Of all tumors, 39% showed LOH in at least one of the markers investigated. In specific, 24% showed evidence of LOH close to the MEN1 locus, and 20% had allelic losses at the APC site. We further showed that promoter hypermethylation was the most prominent alteration in these tumors. While the prevalence of hMLH1, p16 and TIMP3 promoter methylation was close to zero, APC was methylated in 51.3%, O6-MGMT in 13.3%, HIC1 in 82.2%, RASSF1a in 65.7% and two putative regulatory areas of the MEN1 promoter in 5.2% and 11%, respectively. Discussion: This is the first study demonstrating the significance of genetic and epigenetic alterations in tumors of the gastro-entero-pancreatic system. While MSI does not seem to be involved in the pathogenesis of these tumors, LOH can be frequently detected. We were able to define a unique epigenetic profile characterizing these tumors. Interestingly, silencing of the MEN1 gene by promoter methylation does not seem to be strongly involved in the pathogenesis of neuroendocrine tumors. Further studies are in progress to investigate the biological significance of our findings and to correlate tumor differentiation to genes and mechanisms of the molecular pathogenesis of neuroendocrine tumors of the gastro-entero-pancreatic system.

MSI and epigenetic changes in colorectal cancers from African Americans

3570 Background: Microsatellite instability (MSI) is observed in sporadic Colorectal carcinoma (CRC) reflecting promoter hypermethylation of the DNA mismatch repair gene hMLH1. Additionally, p16 and hMLH1 can be inactivated by hypermethylation of its promoter region, which would abrogate its ability to regulate cell proliferation and the repair process. This study was setup to determine if MSI is common in colorectal cancers from AA patients and if MSI is associated simultaneously with p16 and hMLH1 gene silencing by methylation in the CRC from AA patients. Methods: Five microsatellite markers were used to evaluate (normal and colon cancer tissues from the same subjects, N=34) MSI status, p16 and hMLH1 promoter methylation status were determined following bisulfite modification of DNA and using methylation specific PCR, while immunohistochemistry was used to examine expression of hMLH1. Results: Twenty three cancers demonstrated MSI-H, two cancer demonstrated MSI-L and the remaining twenty six tumors were microsatellite stable (MSS). Most of the MSI-H tumors were proximal, well differentiated and highly mucinous. Most patients in the MSI-H group were females (70%) while most of the MSI-L group (100) was male. The p16 promoter was methylated in five of the 34 tumors (14%). Seventeen CRCs demonstrated MSI-H, three of which showed p16 promoter methylation (16%). Most of the tumors were hMLH1 methylated (70%). Tumors were mostly right handed. In total, 37% tumors (that were assayed by IHC) showed loss of staining for hMLH1 and most of them were MSI-H (57%). In addition, loss of MSH2 staining was observed in 43% of MSI-H. Conclusions: The incidence of MSI-H lesions was 2–3 fold higher (45%) in our study group compared to the data reported for the general population. This difference might reflect dietary differences or genetic polymorphisms that may be common in the AA population. Odds ratio analysis indicates that the chance of female patients having MSI-H was 2.3 times more than male patients (P< 0.03). The reason for this gender difference is unknown. No significant financial relationships to disclose.

MSI and epigenetic changes in colorectal cancers from African Americans

3570 Background: Microsatellite instability (MSI) is observed in sporadic Colorectal carcinoma (CRC) reflecting promoter hypermethylation of the DNA mismatch repair gene hMLH1. Additionally, p16 and hMLH1 can be inactivated by hypermethylation of its promoter region, which would abrogate its ability to regulate cell proliferation and the repair process. This study was setup to determine if MSI is common in colorectal cancers from AA patients and if MSI is associated simultaneously with p16 and hMLH1 gene silencing by methylation in the CRC from AA patients. Methods: Five microsatellite markers were used to evaluate (normal and colon cancer tissues from the same subjects, N=34) MSI status, p16 and hMLH1 promoter methylation status were determined following bisulfite modification of DNA and using methylation specific PCR, while immunohistochemistry was used to examine expression of hMLH1. Results: Twenty three cancers demonstrated MSI-H, two cancer demonstrated MSI-L and the remaining twenty six tumors were microsatellite stable (MSS). Most of the MSI-H tumors were proximal, well differentiated and highly mucinous. Most patients in the MSI-H group were females (70%) while most of the MSI-L group (100) was male. The p16 promoter was methylated in five of the 34 tumors (14%). Seventeen CRCs demonstrated MSI-H, three of which showed p16 promoter methylation (16%). Most of the tumors were hMLH1 methylated (70%). Tumors were mostly right handed. In total, 37% tumors (that were assayed by IHC) showed loss of staining for hMLH1 and most of them were MSI-H (57%). In addition, loss of MSH2 staining was observed in 43% of MSI-H. Conclusions: The incidence of MSI-H lesions was 2–3 fold higher (45%) in our study group compared to the data reported for the general population. This difference might reflect dietary differences or genetic polymorphisms that may be common in the AA population. Odds ratio analysis indicates that the chance of female patients having MSI-H was 2.3 times more than male patients (P< 0.03). The reason for this gender difference is unknown. No significant financial relationships to disclose.

BRAF mutations and hMLH1 promoter methylation status in non-familial colorectal carcinoma with microsatellite instability

Peccent MSI status Location LN metastasis MSI status vadance H (12) vs. L(19) vs. R + ('12) vs. -(22); L (14) vs, Category L+S (29) (21); NA (1) NA (7) H+8 (27) % Component 3 0.0005 7.04 Component 5 0.0178 4.53 Component 6 0.0012 3,87 Component 10 0.0039 2.49 Table. The projections of the tumors on the principat components were identified, and correlations between groups of tumors and clinco-molecular features were per~rmed. Component 3 strongly correlated with MSI-H status, and component 10 correlated with MSI-L status. Anatomical location (fight vs. left colon) was associated with component 5 and lymph node metastasis was associated with component 6. Abbreviations: H MSI-H, L MSI-L, Snon-MSI, L left, Rright, + positive, negative, LN lymph node.

Profile of methylated CpG sites of hMLH1 promoter in primary gastric carcinoma with microsatellite instability.

A recent study using colorectal cancer cell lines has identified methylation on a small region of hMLH1 promoter (-248 to -178 relative to the transcription start site) to be critical for gene silencing, but shown that methylation on a more upstream region is frequent in cell lines with hMLH1 expression. Because cultured cell lines have a higher degree of CpG methylation than primary tumors, we attempted to examine methylation profiles of CpG sites of hMLH1 promoter in primary gastric carcinomas with or without microsatellite instability (MSI). Seven cases with MSI and six cases without MSI were assessed for the methylation status of hMLH1 promoter by bisulfite-sequencing. All of the MSI-positive cases previously showed loss of hMLH1 expression and six cases displayed methylated alleles in methylation-specific PCR (MSP) for hMLH1. Sequencing analysis revealed that: (i) CpG sites were overall methylated in MSI-positive tumors with positive MSP results; (ii) a small region (-248-178) was almost invariably methylated in MSI-positive tumors; and (iii) the vast majority of CpG sites were unmethylated in MSI-negative tumors, including a more upstream region (proximal to -248). Our study suggests that methylation of a more upstream region observed in colon cancer cell lines may be an acquired change during cell line establishment and it was not identified in primary gastric carcinomas without MSI.

DNA methylation status of hMLH1, p16(INK4a), and CDH1 is not associated with mRNA expression levels of DNA methyltransferase and DNA demethylase in gastric carcinomas.

DNA methyltransferase and DNA demethylase are enzymes potentially affecting promoter methylation status. We examined levels of DNA methyltransferase (DNMT1, DNMT3a, DNMT3b) and DNA demethylase (MBD2) mRNA expression by semi-quantitative RT-PCR. In addition, we examined promoter methylation status of hMLH1, p16(INK4a), and CDH1 by methylation-specific PCR since all three of these genes are reported to be hypermethylated in gastric carcinoma. MBD2 appeared to be down-regulated in neoplasms. The levels of DNMT1, DNMT3a, DNMT3b, and MBD2 mRNA expression were not associated with either tumor stage or histologic type. Promoter hypermethylation of hMLH1, p16(INK4a), and CDH1 was detected in 5/20 (25%), 8/20 (40%) and 8/20 (40%) of gastric carcinomas, respectively. There was no clear relation between DNA methylation status of hMLH1, p16(INK4a), and CDH1 and the mRNA expression levels of DNMT1, DNMT3a, DNMT3b or MBD2. We divided the examined cases into two groups according to the number of hypermethylated genes. Cases with more than two hypermethylated genes comprised a hypermethylation group, and cases with no hypermethylation comprised a non-hypermethylation group. We found no group association for levels of DNMT1, DNMT3a, DNMT3b, and MBD2 mRNA expression. Our results suggest that the mRNA expression levels for pro-methylating (DNMT1, DNMT3a, DNMT3b) and anti-methylating (MBD2) enzymes is not a critical determinate of tumor-specific promoter hypermethylation of hMLH1, p(16INK4a), or CDH1 in gastric carcinoma.

MSI-L Gastric Carcinomas Share the hMLH1 Methylation Status of MSI-H Carcinomas but Not Their Clinicopathological Profile

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Met+) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Met+ and MSI-H/Met+ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC.

Frequent Hypermethylation of the hMLH1 Gene Promoter in Differentiated-Type Tumors of the Stomach with the Gastric Foveolar Phenotype

Hypermethylation of the hMLH1 mismatch repair gene promoter has been revealed to lead to microsatellite instability (MSI). Previously, we demonstrated a high prevalence of MSI in differentiated-type gastric tumors showing distinct features of gastric foveolar epithelium (foveolar type). To clarify the significance of hMLH1 promoter hypermethylation in the development of this tumor type, we studied promoter methylation status and expression of hMLH1 in foveolar-type tumors and their surrounding non-neoplastic mucosae, as well as in tumors with other cellular phenotypes. The results were compared to MSI status. After phenotypical analyses using mucin histochemistry and immunohistochemistry, 41 differentiated-type tumors with distinct cellular phenotypes were classified into three categories: foveolar type, intestinal type (tumors with the distinct cellular phenotype of the intestine), and combined type (tumors with both foveolar and intestinal phenotypes). Methylation-specific polymerase chain reaction (MSP) was performed to determine the methylation status of hMLH1 promoter. hMLH1 protein expression was immunohistochemically examined. MSI was detected in 57% of the foveolar type, 8% of the intestinal type, and 67% of the combined-type tumors. Hypermethylation of hMLH1 promoter was found in 74% of the foveolar type, 33% of the intestinal type, and 83% of the combined-type tumors. Of 18 MSI-positive tumors, all but one were hypermethylated. Methylation status of hMLH1 promoter correlated well with protein expression in foveolar-type tumors. Moreover, hypermethylation was also detected frequently (71%) in the non-neoplastic surrounding mucosa of the hypermethylated tumors. Hypermethylation of hMLH1 promoter is an initial, vital event in the development of foveolar-type tumors of the stomach.