hMLH1 Promoter Hypermethylation Research Articles

Three independent genetic profiles based on mucin expression in early differentiated-type gastric cancers—a new concept of genetic carcinogenesis of early differentiated-type adenocarcinomas

Recent molecular studies have shown that the genetic profiles of differentiated-type adenocarcinomas of the stomach are associated with distinct cellular mucin phenotypes (gastric- intestinal- and mixed-phenotypes). Therefore, we examined whether these cellular mucin phenotypes reflect specific molecular genetic alterations, and whether the phenotypes can be used to help categorize the intramucosal neoplasias of gastric tumors. We subclassified tumors into four cellular phenotypes using immunohistochemical mucin analysis. In all, 62 early gastric carcinomas (gastric-phenotype, 13; intestinal-phenotype, 17; mixed-phenotype, 31; unclassified-phenotype, 1) were examined using a combination of polymerase chain reaction microsatellite assays and immunohistochemical analysis in order to detect chromosomal allelic losses of multiple cancer-related chromosomal loci (1p, 3p. 4p, 5q, 8p, 9p, 13p, 17p, 18q and 22q), microsatellite instability (MSI), and overexpression of the p53 protein. In addition, we analyzed the relationship between MSI status and hMLH1 promoter hypermethylation, which is thought to be a cause of high MSI status. For gastric phenotype cancers, the frequency of 3p allelic loss was higher than that of other microsatellite markers, whereas 5q allelic loss was frequently found in intestinal phenotype cancers. The genetic profile of mixed phenotype cancers is comprised of two distinct genetic types: LOH and MSI types. In the former, 5q, 3p and 18q allelic losses are seen frequently in intramucosal carcinomas. On the other hand, 17p, 1p and 9p allelic losses are associated with the development of submucosal carcinomas. MSI was observed only in mixed phenotype cancers (six of 31 mixed phenotype cancers). Overexpression of the p53 protein is common in differentiated-type gastric cancers. In addition, the MSI status of the tumor cells was correlated with the extent of hypermethylation of the hMLH1 promoter. We suggest that the cellular mucin phenotypes of the differentiated-type adenocarcinomas result from distinct genetic alterations.

Hypermethylation of hMLH1 promoter in sporadic colorectal cancer

To identify CpG island hypermethylation of 5'region of hMLH1 promotor and to explore its relationship to microsatellite instability(MSI)in sporadic colorectal carcinoma. Forty-one pairs of tissue specimens (normal and cancer) were collected from 41 patients with colorectal cancer. Hypermethylation of hMLH1 promoter was detected by methylation specific PCR; the relationship between methylation and clinicopathological features was analyzed. Combined with BAT25 and BAT26, the MSI status was detected using an automated fluorescent DNA sequencer. Hypermethylation of hMLH1 promoter was detected in 75.6 % (31/41) of samples. Mean age of unmethylation cases (49.2 y) was significantly younger than that of methylation cases (63.6 y) (P<0.05), but there were no differences between two groups in other clinicopathological features. MSI was detected in 43.9 % samples (18/41); hypermethylation of hMLH1 promoter was detected in 94.4 % (17/18) of MSI(+) samples, which was higher than that in MSI(-) samples (60.9 %,14/23, P<0.05). Age-related hypermethylation is generally found in patients with sporadic colorectal cancers, which may cause MSI and might be the mechanism in the development of colorectal cancer of elderly people.

Patrón de metilación génico en el cáncer de mama

Genomic DNA methylation, mutations and allelic deletions explain the inactivation of genes involved in cell proliferation and cell cycle control mechanisms. To analyze the methylation pattern of important genes related to different carcinogenic mechanisms in patients with breast cancer and the relationship with its biological behavior. Seventy fresh-frozen breast cancer samples were selected. The methylation specific PCR (MSP) test was used to analyze promoter methylation status for genes CDKN2A (p16), hMLH1, APC, CDH1 (Cadherin E) and FHIT. We found methylation in at least one of the genes studied in 88% of cases and in 3 or more genes in 40.5% of cases. The frequencies of promoter hypermethylation of CDKN2A, hMLH1, APC, CDH1 and FHT were 41.4%, 11.4%, 52.9%, 70% and 42.9%, respectively. We found a relationship between CDKN2A methylatlon and better survival (p=0.002). CDH1 methylation and poor histological differentiation (p=0.007), hMLH1 methylation and non-Mapuche ethnicity (p=-0.03), APC methylation and larger tumor size (p<0.05), FHIT methylatton and lack of estrogen rectptor IHC expression (p<0.05). The high frequency of promoter methylation in patients with breast cancer confirms its role in breast carcinogenesis. The finding of alterations in the methylation pattern of genes studied and its association with prognostic factors is a helpful tool in the search of new criteria for clinical and therapeutic decision making.

Promoter hypermethylation is a major event of hMLH1 gene inactivation in liver fluke related cholangiocarcinoma

Cholangiocarcinoma is a crucial health problem in Northeast Thailand where liver fluke infection is endemic. However, molecular genetic and epigenetic mechanisms involved in carcinogenesis of this cancer remain unclear. We attempted to study genetic and epigenetic alterations of hMLH1 gene in 65 intrahepatic cholangiocarcinoma using polymerase chain reaction (PCR) based microsatellite marker D3S1611 and methylation-specific PCR, respectively. Of 65 cases, 29 (44.6%) showed hypermethylation of hMLH1 promoter. Loss of heterozygosity (LOH) of hMLH1 was detected in 12 of 51 informative cases (23.5%). Five out of 29 (17.2%) methylated cases demonstrated LOH. Aberrant methylation of hMLH1 promoter was significantly associated with poorly differentiated type ( P=0.013). Our study suggests that both genetic and epigenetic mechanisms cause the inactivation of hMLH1 where epigenetic is a major event resulting in mismatch repair deficiency and contributing to carcinogenesis of liver fluke related cholangiocarcinoma. Since, gene silencing by methylation is an early event in carcinogenesis, promoter hypermethylation of hMLH1 may be a molecular targeted therapy and prevention of liver fluke related cholangiocarcinoma.

Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status.

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.

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.

Methylation profiling of the DNA repair genes in glioblastoma multiforme and its clinical relevance

1572 Background: Alterations of DNA repair protein have been associated with sensitivity to anticancer drugs, and transcriptional repression by promoter hypermethylation is an essential role in the protein inactivation. Recently methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter is an expected predictor not only of responsiveness to chemotherapy but also of prognosis of patient with glioma. The aim of this study was to investigate the methylation profile in DNA repair genes, MGMT, FANCF and hMLH1, of glioblastoma multiforme and correlation between the methylation status and outcome of the patients. Methods: In 40 adult patients with glioblastoma multiforme treated with surgery, irradiation, and/or chemotherapy (ACNU: n=19, cisplatin: n=11), median age at diagnosis was 55 years old, and median overall survival time was 14 months. Promoter hypermethylation of MGMT, hMLH1 and FANCF genes in tumor DNA was examined using the methylation-specific PCR assay and COBRA (combined bisulfite restriction analysis) method. We analyzed relation between methylation status of the MGMT promoter and survival in these patients. Results: The hypermethylation in MGMT, FANCF and hMLH1 promoter were detected 24 of 40 (60%), 1 of 40 (2.5%), and 0 of 40, respectively. Kaplan-Meier analysis and log-rank test revealed no significant difference in overall survival between patient subgroups divided by methylation status of MGMT promoter. Cox proportional hazard regression analysis adjusted for age of onset and extent of surgical resection showed the MGMT promoter hypermethylation had no significant association with overall survival time. Conclusions: In our series, promoter hypermethylation of MGMT was not a predictive factor for outcome, however, more controlled study would be required to validate this result. And it is still suggested epigenetic alteration of MGMT might influence nature of glioblastoma compared with other DNA repair genes. No significant financial relationships to disclose.

Methylation profiling of the DNA repair genes in glioblastoma multiforme and its clinical relevance

1572 Background: Alterations of DNA repair protein have been associated with sensitivity to anticancer drugs, and transcriptional repression by promoter hypermethylation is an essential role in the protein inactivation. Recently methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter is an expected predictor not only of responsiveness to chemotherapy but also of prognosis of patient with glioma. The aim of this study was to investigate the methylation profile in DNA repair genes, MGMT, FANCF and hMLH1, of glioblastoma multiforme and correlation between the methylation status and outcome of the patients. Methods: In 40 adult patients with glioblastoma multiforme treated with surgery, irradiation, and/or chemotherapy (ACNU: n=19, cisplatin: n=11), median age at diagnosis was 55 years old, and median overall survival time was 14 months. Promoter hypermethylation of MGMT, hMLH1 and FANCF genes in tumor DNA was examined using the methylation-specific PCR assay and COBRA (combined bisulfite re...

Colorectal adenomas: a prototype for the use of surrogate end points in the development of cancer prevention drugs.

Carcinogenesis is a multiyear, multistep, multipath disease of progressive genetic alterations and associated tissue damage. Chemoprevention is the use of drugs or other agents to inhibit, delay, or reverse this process. Despite the enormous population at risk of cancer who would benefit, drug

Expressions of O (6)-methylguanine-DNA methyl transferase MGMT and p53 in breast cancer

The existence of genetic alterations affecting genes involved in cellular proliferation and death, such as TP53 and K-ras, is one of the most common features of tumour cells. Recently, gene inactivation by promoter hypermethylation has been demonstrated. Methylation is the main epigenetic modification in mammals and abnormal methylation of the CpG islands located in the promoter region of the genes leads to transcriptional silencing. Examples include the p16INK4a, p15INK4B, p14ARF, Von Hippel-Lindau (VHL), the oestrogen and progesterone receptors, E-cadherin, death associated protein (DAP) kinase and the first tumour suppressor gene described, retinoblastoma (Rb) gene. In most cases, methylation involves loss of expression, absence of a coding mutation and restoration of transcription by the use of demethylating agents. However, is there a linkage between genetic and epigenetic alterations? Our results show one side of this puzzle demonstrating that epigenetic lesions drive genetic lesions in cancer. Four specific epigenetic lesions, promoter hypermethylation of the DNA mismatch repair gene hMLH1, the DNA alkyl-repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), the detoxifier glutathione S-transferase P1 (GSTP1) and the familial breast cancer gene BRCA1 may lead to four specific genetic lesions, microsatellite instability, G to A transitions, steroid-related adducts and double-strand breaks in DNA. This is probably only the beginning of an extensive list of epigenetic events that change and make the genetic environment of the transformed cell unstable.

Hypermethylation of the hMLH1 promoter with absent hMLH1 expression in medullary-type poorly differentiated colorectal adenocarcinoma in the elderly

To clarify the significance of hMLH1 promoter hypermethylation in the development of medullary-type poorly differentiated colorectal adenocarcinoma, we studied the status of promoter methylation and hMLH1 expression in 23 medullary-type and 12 pleomorphic-type carcinomas, as well as the pathology and microsatellite status. In medullary-type carcinomas, the percentages of cases with promoter methylation (83%) and an absence of hMLH1 expression (91%) were significantly higher than in pleomorphic-type carcinomas (14 and 17%), respectively. The rate of microsatellite instability in the medullary type was significantly higher than that of the pleomorphic type (87 vs 40%, P<0.01). Compared with pleomorphic-type carcinomas, medullary-type carcinomas were significantly associated with hMLH1 promoter methylation, absent expression of hMLH1 protein, microsatellite instability, as well as a proximal location, a Crohn's-like lymphoid reaction, a low incidence of lymph node metastasis, and a favorable outcome. Medullary-type carcinomas accumulated with advancing age, especially in the female. These results indicated that hMLH1 hypermethylation, concurrent with a lack of its protein expression, may play an important role in the development of medullary-type poorly differentiated colorectal adenocarcinomas in the elderly.

Evaluation of Microsatellite Instability, hMLH1 Expression and hMLH1 Promoter Hypermethylation in Defining the MSI Phenotype of Colorectal Cancer

About 10-15% of all colorectal cancers (CRCs) demonstrate high levels of microsatellite instability (MSI-H) and are currently best identified by molecular analysis of microsatellite markers. Most sporadic CRCs with MSI-H are known to be associated with the methylation of the hMLH1 promoter. Promoter methylation coincides with lack of the hMLH1 expression. We aimed to investigate the association between MSI status, hMLH1 protein expression and methylation status of the hMLH1 promoter, and to determine the usefulness of each method in defining the MSI phenotype in sporadic CRCs. Methods: CRCs from 173 patients from the Cancer and Leukemia Group B (CALGB) were assessed for their MSI status. An additional cohort of 18 MSI-H tumors from the University of California San Diego (UCSD) was included in the analysis of the MSI-H subgroup. MSI testing was performed by PCR using five standard MSI markers. hMLH1 promoter analysis was investigated by methylation specific PCR (MSP), and expression of the MMR genes hMLH1 and hMSH2 was examined by immunohistochemistry (IHC). Results: Of the 173 CALGB tumors, 111 (64%) were MSS, 35 (20%) were MSI-L and 27 (16%) MSI-H, respectively. Data on hMLH1 protein expression, MSH2 protein expression and hMLH1 methylation are available on 128, 173 and 81 of these tumors, respectively. Presence of hMLH1 and hMSH2 protein expression was significantly associated with MSI status. Four of 45 (8.9%) MSI-H tumors and 0 of 146 (0%) MSS/MSI-L tumors did not express hMSH2 (p=0.0028). hMLH1 protein expression was present in 107 of 108 (99%) MSS and MSI-L tumors versus 11 of 20 (55%) MSI-H tumors (p

HMLH1 and hMSH2 somatic inactivation mechanisms in sporadic colorectal cancer patients.

Much is known about the role of germline inactivation in mismatch repair (MMR) genes in hereditary non-polyposis colorectal cancer (HNPCC), but the impact of somatic MMR gene changes on sporadic colorectal cancer remains to be elucidated. In hereditary cases the hMLH1 and hMSH2 genes were shown to have a great importance, and in order to examine the somatic inactivation mechanisms of the two MMR genes hMLH1 and hMSH2 we screened 37 Hungarian sporadic colorectal cancer patients for allelic imbalance (AI), microsatellite instability (MSI), hMLH1 promoter hypermethylation and somatic mutations. Thirteen of the examined tumours (35%) were characterized by low-level MSI and none of the cases belonged to the high MSI group. Nine (24%) and seven (19%) cases had AI at the hMLH1 and hMSH2 genes, respectively. Seven tumours (19%) showed dense promoter hypermethylation of hMLH1, but only two patients had somatic mutations, one for each MMR gene. According to our study on this limited set of cases the most prominent mismatch repair inactivation mechanism in sporadic colorectal cancer patients is the hMLH1 promoter hypermethylation which may have a role in the carcinogenesis of sporadic colorectal cancer.

Downregulation of ID4 by promoter hypermethylation in gastric adenocarcinoma.

Promoter hypermethylation has become apparent as a common mechanism of gene silencing in cancer. Based on our published microarray expression data, we noticed a prominent downregulation of ID4 in gastric adenocarcinoma. The dense 5' CpG island covering the previously mapped upstream promoter of ID4 has prompted us to relate its downregulation to promoter hypermethylation. ID proteins are distinct members in the helix-loop-helix family of transcriptional regulators, which modulate various key developmental processes. Emerging data have suggested the involvement of ID genes in tumorigenesis. In this study using bisulfite genomic sequencing, we have found hypermethylation of ID4 promoter in most gastric cancer cell lines and 30% of primary tumors. This correlated with decreased level of ID4 expression. Restoration of ID4 expression in various gastric cancer cell lines was achieved by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which at times required the synergistic action of the histone deacetylase inhibitor trichostatin A, but not with trichostatin A alone. Re-expression was accompanied by the corresponding ID4 promoter demethylation. Furthermore, we have found significant association of ID4 promoter methylation with hMLH1 promoter methylation (P=0.008) and microsatellite instability (P=0.006). Overall, our results have shown that transcriptional silencing of ID4 is related to the aberrant methylation of its promoter in gastric cancer. The significant association of ID4 and hMLH1 promoter hypermethylation suggested that ID4 may also be among the genes being targeted in the CpG island methylator phenotype tumorigenic pathway.

Microsatellite Instability Is Associated with Hypermethylation of the hMLH1 Gene and Reduced Gene Expression in Mycosis Fungoides

Fifty-one mycosis fungoides samples were analyzed for microsatellite instability (MSI) using the panel of markers recommended for hereditary nonpolyposis colorectal cancer kindred and a panel we designed for cutaneous T cell lymphoma in order to compare detection rates and determine if MSI is a genome-wide phenomenon. Samples demonstrating MSI were analyzed for abnormalities of the hMLH1 gene including loss of heterozygosity, mutations, and promoter hypermethylation. MSI was detected in 16% using the hereditary nonpolyposis colorectal cancer panel and 22% with the cutaneous T cell lymphoma panel. Overall, 27% demonstrated MSI and 73% had a stable phenotype. hMLH1 gene studies did not detect loss of heterozygosity or reveal any mutations. Promoter hypermethylation was detected in nine of 14 patients with MSI, however (64%). In addition hMLH1 and hMSH2 protein expression was studied using immunohistochemical techniques. Five of nine patients with MSI and hMLH1 promoter methylation showed abnormal hMLH1 protein expression with normal hMSH2 gene expression. All other patients tested demonstrated normal hMLH1 and hMSH2 protein expression. MSI was found to be more prevalent in tumor stage mycosis fungoides (47%) than early stage disease (20%) and was associated with an older age of onset of mycosis fungoides. MSI may be a consequence of hMLH1 promoter hypermethylation in mycosis fungoides patients and may prevent transcription in a subset of patients. This suggests that the development of a mutator phenotype may contribute to disease progression in mycosis fungoides.

Mismatch repair gene expression defects contribute to microsatellite instability in ovarian carcinoma.

hMLH1, the human MutL homologue, has been linked to microsatellite instability (MSI) in gastrointestinal tumors. However, to the authors' knowledge, the role of hMLH1, the other mismatch repair genes (MMR), and MSI in ovarian carcinoma has not been well defined. The purpose of the current study was to determine the relation between MSI of ovarian carcinoma and MMR gene expression, hMLH1 and hMSH2 hypermethylation, and hMLH1 and hMSH2 null mutations. hMLH1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and amplification of cDNA using a housekeeping gene (glycerol 3-phosphate dehydrogenase) as a control for mRNA quality and quantity. Methylation-specific PCR (MS-PCR) was used to correlate methylation of the hMLH1 and hMSH2 CpG islands with mRNA expression status. Similar techniques were used to evaluate the concomitant expression of five other MMR: hMSH2, hMSH3, hMSH6, PMS1, and PMS2. Microsatellite instability was studied using the National Cancer Institute consensus markers (D2S123, D5S346, D17S250, BAT25, and BAT26) and NM23 as described previously. One hundred twenty-five primary tumors were analyzed. High-frequency MSI (MSI-H) was found in 21 tumors (16.8%). hMLH1 mRNA was absent in 10 of these 21 tumors (47.6%). In each case, coordinated hypermethylation of both regions A and C of the promoter was identified. Microsatellite stable and low-frequency MSI tumors all were found to express not only hMLH1 but the other MMR genes as well (P < 0.001). Absence of expression of hMSH2 and the four other MMRs occurred in tumors with absent hMLH1 mRNA expression because of CpG island hypermethylation. No absence of expression of hMSH2, hMSH3, hMSH6, PMS1, or PMS2 was found to occur in tumors expressing hMLH1. None of the 11 MSI-H tumors without promoter hypermethylation demonstrated a null mutation in hMLH1 or hMSH2. A molecular mechanism to explain > 50% of the MSI-H phenotype in ovarian carcinoma cases was demonstrated. MSI-H may occur because of MMR defects, especially hMLH1 promoter hypermethylation. Additional mechanisms are required to explain the balance between the cases of MSI-H as well as the phenomenon of MSI-L tumors.

Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines.

Loss of DNA mismatch repair (MMR) occurs in 10-15% of sporadic colorectal cancer, is usually caused by hMLH1 hypermethylation, and has been shown to confer resistance to various chemotherapeutic reagents, including 5-fluorouracil (5-FU). We tested the hypothesis that demethylation of the hMLH1 promoter in hypermethylated colorectal cancer cells would restore MMR proficiency and drug sensitivity to 5-FU. We used the MMR-deficient cell lines SW48, HCT116, HCT116+chr2 and the -proficient cell line HCT116+chr3. After treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5 aza-dC), hMLH1 mRNA and protein expression were determined by RT-PCR and immunoblots. The methylation status for hMLH1 was investigated by methylation-specific PCR. Cells were subsequently treated with 5-FU and the growth characteristics ascertained by clonogenic assays. hMLH1 hypermethylation was reverted in SW48 cells 24 hr after treatment with 5 aza-dC and was accompanied by hMLH1 mRNA and protein reexpression. While 5 aza-dC alone did not affect the growth of SW48 cells, all other cell lines responded with a pronounced growth inhibition. 5-FU treatment strongly reduced the colony formation of HCT116+chr3 cells. These effects were significantly less in the MMR-deficient cells. Combined treatment of SW48 cells resulted in a similar growth pattern as seen in 5-FU only treated HCT116+chr3 cells. We demonstrate that in vitro resistance to 5-FU can be overcome by reexpression of hMLH1 protein through 5 aza-dC-induced demethylation in hypermethylated cell lines. Induction of the expression of methylated tumor suppressor or MMR genes could have a significant impact on the development of future chemotherapy strategies.

Microsatellite instability in esophageal squamous cell carcinoma is not associated with hMLH1 promoter hypermethylation.

To test whether a subset of esophageal squamous cell carcinomas (SCC) develop through a deficiency in DNA mismatch repair, we examined microsatellite instability (MSI) using 11 microsatellite markers including BAT-26, hMLH1 protein expression by immunohistochemistry, and methylation status of the hMLH1 promoter by methylation-specific polymerase chain reaction (MSP). p53 mutations were also investigated. Microsatellite instability at one or more loci was observed in 40% (12/30) of esophageal SCC tumor samples, although only one of these tumors was categorized as high-frequency MSI (MSI-H) and none showed BAT-26 instability. While immunohistochemistry revealed decreased hMLH1 protein expression in 27% (8/30) of the tumors, hMLH1 promoter hypermethylation was not observed. Absence of hMLH1 protein expression was relatively common in well-differentiated (keratinizing-type) esophageal SCC, but was not associated with hMLH1 promoter hypermethylation. p53 mutation was detected in 37% (11/30) and loss of heterozygosity (LOH) in 90% (27/30) of esophageal SCC samples. Our results suggested that most esophageal SCC develop through defects in tumor suppressor genes (i.e. the suppressor pathway), and that MSI in esophageal SCC probably represent random replication errors rather than being associated with DNA mismatch repair deficiency.

Evaluation of microsatellite instability, hMLH1 expression and hMLH1 promoter hypermethylation in defining the phenotype of colorectal cancer: a study from CALGB 9865

Evaluation of microsatellite instability, hMLH1 expression and hMLH1 promoter hypermethylation in defining the phenotype of colorectal cancer: a study from CALGB 9865