Analysis of Inactivation of hMLH1 by Promoter Hypermethylation and Microsatellite Instability in Gastric Carcinogenesis

OBJECTIVE To study the microsatellite instability (MSI) and methylation state of the hMLH1 gene promoter and their mechanisms underlying the development of gastric cancer. METHODS Forty-one gastric cancer samples were obtained from patients undergoing surgery and 46 chronic atrophic gastritis tissues with dysplasia or intestinal metaplasia (IM) were obtained from patients undergoing gastro-endoscopy. Fourteen normal gastric mucosal samples were used as controls. Genomic DNA was extracted from the samples and 5 microsatellite markers were used to measure MSI. Methylation-specific PCR (MSP) was used to screen the methylation state of the samples. DNA sequencing and immunohistochemistry were performed to verify the results. RESULTS MSI was identified in 22 out of the 41 (53.7%) gastric cancers, of which 8 cases showed high-level MSI (2 or more loci altered) and 14 showed low-level MSI (1 locus altered). MSI was also detected in 12 out of 46 (26.1%) pre-cancerous lesions of the stomach, whereas it was not seen in the normal tissue. Moreover, hMLH1 hypermethylation was detected in 17 out of the 41 (41.5%) gastric cancers, 9 out of the 46 (19.6%) pre-cancerous lesions and 0 out of the 14 normal tissue. Significant differences in frequency of MSI and hMLH1 promoter methylation were observed among gastric cancers, precancerous lesions and normal gastric tissue. Gastric samples with MSI had a tendency to be hypermethylated in the hMLH1 promoter. DNA sequencing and immunohistochemistry results also confirmed that hMLH1 promoter methylation could lead to loss of the hMLH1 protein and gene silence which sequentely resulted in gene mismatch and MSI. CONCLUSION Accumulation of MSI and hMLH1 promoter methylation may be important early molecular events during gastric carcinogenesis and may contribute to the acquisition of a transformed cell phenotype and the development of gastric cancer.

Promoter hypermethylation of tissue specific tumor supressor genes and point mutation in K-ras, c-myc proto-oncogenes in urinary (transitional cell) bladder carcinoma

In a total of 83 UN specimens were investigated for proto-oncogene mutations, tumor supressor genes promoter methylation status and c-myc and Ki-67 expression. Point mutations in c-myc were detected in cases with high grade and proliferation index. Mutated K-ras proto-onco- gene profiles were detected in 17 (21%) tumoral spiecemens that examined. Tumor specimens were also showed hypermethylated promoter domain for the SFRP2, MGMT tumor supressor genes. These findings showed the combine effect of mutated c-myc and K-ras oncogene and epigenetic inactivation of tissue specific tumor supressor genes (TS) play a crucial role in tumor progression and recurrence in UN carcinogenesis.

Loss of disabled-2 expression is an early event in esophageal squamous tumorigenesis

AIM: Disabled-2 (DAB2) is a candidate tumor-suppressor gene identified in ovarian cancer that negatively influences mitogenic signal transduction of growth factors and blocks ras activity. In a recent study, we observed down-regulation of DAB2 transcripts in ESCCs using cDNA microarrays. In the present study, we aimed to determine the clinical significance of loss of DAB2 protein in esophageal tumorigenesis, hypothesizing that DAB2 promoter hypermethylation-mediated gene silencing may account for loss of the protein. METHODS: DAB2 expression was analyzed by immunohistochemistry in 50 primary esophageal squamous cell carcinomas (ESCCs), 30 distinct hyperplasia, 15 dysplasia and 10 non-malignant esophageal tissues. To determine whether promoter hypermethylation contributes to loss of DAB2 expression in ESCCs, methylation status of DAB2 promoter was analyzed in DAB2 immuno-negative tumors using methylation-specifi c PCR. RESULTS: Loss of DAB2 protein was observed in 5/30 (17%) hyperplasia, 10/15 (67%) dysplasia and 34/50 (68%) ESCCs. Significant loss of DAB2 protein was observed from esophageal normal mucosa to hyperplasia, dysplasia and invasive cancer (Ptrend < 0.001). Promoter hypermethylation of DAB2 was observed in 2 of 10 (20%) DAB2 immuno-negative ESCCs. CONCLUSION: Loss of DAB2 protein expression occurs in early pre-neoplastic stages of development of esophageal cancer and is sustained down the tumorigenic pathway. Infrequent DAB2 promoter methylation in ESCCs suggests that epigenetic genesilencing is only one of the mechanisms causing loss of DAB2 expression in ESCCs.

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients

Background Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine- DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas （pGBMs） and nine secondary glioblastomas （sGBMs） with the expectation to identify valuable prognostic and therapeutic targets. Methods We evaluated the methylation status of testis derived transcript （TES） gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines （U87 and U251） with 5-aza-2-deoxycytidines （5-aza-dC） and detected cell biological behaviors. Results Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs （P 〈0.05）. Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival （P 〈0.05）. This abnormality is also confirmed in glioblastoma cell lines （U87 and U251）. Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth （P=0.013）. Conclusions Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for gliobtastoma.

Aberrantly Methylated MGMT,hMLH1 and hMSH2 in Tumor and Serum DNA of Gliomas Patients

OBJECTIVE This study is to investigate the prevalence ofpromoter CpG island methylation of O^6-methylguananine-DNAmethyltransferase (MGMT), mismatch repair genes (hMLH1 andhMSH2) in both tumor and serum samples of gliomas.METHODS Methylation-specific PCR (MSP) was employed todetect promoter CpG island methylation of the MGMT, hMLH1and hMSH2 genes in 39 samples taken from surgery and 32samples of pretreatment serum all from the patients with gliomas.RESULTS Promoter CpG island methylation of MGMT, hMLH1and hMSH2 was detected and the results were 46.2%, 10.3% and20.5%, respectively in tumor DNA of the cases with gliomas,and 40.6%, 9.4% and 18.8%, respectively in serum DNA of thecases. The methylation pattern in primary tumor and serum wasfound to be concordant in matched tissue and serum samplesof 21 patients. In the cases with positive result of methylationfor MGMT, hMLH1 and hMSH2 in tumor tissues, the results ofdetection for those in the paired serum sample were 77.8% (7/9),66.7% (2/3) and 75.0 % (3/4), respectively. False positive resultswere not obtained in any of the patients who did not exhibitmethylation. No association was found between the promotermethylation of MGMT, hMLH1, and hMSH2 genes in primarygliomas and gender, age, localization, grade of malignant or tumorstage.CONCLUSION Promoter CpG island methylation is a frequentevent in gliomagenesis. Methylation analysis appears to bea promising predictive factor of the prognosis for the gliomapatients treated with alkylating drugs and a noninvasive tumormarker in serum DNA.