Detection of p16 Hypermethylation in Gastric Carcinomas Using a Seminested Methylation-Specific PCR

Aberrant DNA methylation of a CpG site is among the earliest and most frequent alterations in various tumors including gastric carcinoma. The aim of this study is to detect tumor-associated aberrant hypermethylation of the p16 gene from 60 gastric tumor and corresponding normal tissues using a seminested methylation-specific PCR (MSP). The results indicated that hypermethylation of the p16 gene could be detected in 80% (48/60) of the gastric tumor samples from the first PCR. However, the frequency increased significantly to 86.7% (52/60) of the gastric tumor samples after the second PCR. These results show that this technique increases the sensitivity of detecting p16 hypermethylation from tumor samples. Furthermore, the aberrant methylation of p16 was observed in all of the stages, confirming that this epigenetic alteration is an early event during gastric carcinogenesis. Clinicopathologic parameters such as age, sex, and histological differentiation of GC were not significantly associated with the methylation status.     

Transmembrane protein 106A is silenced by promoter region hypermethylation and suppresses gastric cancer growth by inducing apoptosis

Inactivation of tumour suppressor genes by promoter methylation plays an important role in the initiation and progression of gastric cancer (GC). Transmembrane 106A gene (TMEM106A) encodes a novel protein of previously unknown function. This study analysed the biological functions, epigenetic changes and the clinical significance of TMEM106A in GC. Data from experiments indicate that TMEM106A is a type II membrane protein, which is localized to mitochondria and the plasma membrane. TMEM106A was down‐regulated or silenced by promoter region hypermethylation in GC cell lines, but expressed in normal gastric tissues. Overexpression of TMEM106A suppressed cell growth and induced apoptosis in GC cell lines, and retarded the growth of xenografts in nude mice. These effects were associated with the activation of caspase‐2, caspase‐9, and caspase‐3, cleavage of BID and inactivation of poly (ADP‐ribose) polymerase (PARP). In primary GC samples, loss or reduction of TMEM106A expression was associated with promoter region hypermethylation. TMEM106A was methylated in 88.6% (93/105) of primary GC and 18.1% (2/11) in cancer adjacent normal tissue samples. Further analysis suggested that TMEM106A methylation in primary GCs was significantly correlated with smoking and tumour metastasis. In conclusion, TMEM106A is frequently methylated in human GC. The expression of TMEM106A is regulated by promoter hypermethylation. TMEM106A is a novel functional tumour suppressor in gastric carcinogenesis.     

p16 hypermethylation during gastric carcinogenesis of Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine

Inactivation of the tumor suppressor gene, p16 by CpG hypermethylation is a common event in various tumors including gastric carcinoma. The aim of this study is to investigate if p16 hypermethylation is an early and frequent event in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The frequency and timing of p16 hypermethylation during the multistep gastric carcinogenesis in Wistar rats were analyzed in various microdissected gastric lesions. The p16 methylation status and the presence of p16 protein were analyzed by methylation-specific PCR and immunohistochemistry, respectively. Results showed that p16 methylation frequency was correlated with the severity of gastric pathologic lesions, positively. For instance, p16 methylation was found in 2.7% of normal gastric epithelium (n = 36), 16.7% of chronic atrophy gastritis (n = 24), 37.5% of dysplasia (n = 24), 67.4% of gastric adenoma (n = 43), and 85.2% of gastric carcinoma (n = 27). The p16 methylation in the distal stomach epithelium was higher than that in the proximal stomach. p16 protein was expressed in all of 15 p16 unmethylated gastric epithelial samples, but not expressed in all of 12 p16 methylated samples. These results suggest that CpG island hypermethylation may account for the silencing of p16 in rat stomach and is an early event whose accumulation will finally lead to gastric carcinogenesis.     

ZIC1 is downregulated through promoter hypermethylation in gastric cancer

As one of major epigenetic changes responsible for tumor suppressor gene inactivation in the development of cancer, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes. In the current study we identified ZIC1 (Zic family member 1, odd-paired Drosophila homolog) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. In all of gastric cancer cells lines examined, ZIC1 expression was downregulated and such downregulation was accompanied with the hypermethylation of ZIC1 promoter. Demethylation treatment with 5-aza-2′-deoxycytidine (Aza) reversed ZIC1 downregulation, highlighting the importance of promoter methylation to ZIC1 downregulation in gastric cancer cells. Notably, ZIC1 expression was significantly downregulated in primary gastric carcinoma tissues in comparison with non-tumor adjacent gastric tissues (p < 0.01). Accordingly, promoter methylation of ZIC1 was frequently detected in primary gastric carcinoma tissues (94.6%, 35/37) but not normal gastric tissues, indicating that promoter hypermethylation mediated ZIC1 downregulation may play an important role in gastric carcinogenesis. Indeed, ectopic expression of ZIC1 led to the growth inhibition of gastric cancer cells through the induction of S-phase cell cycle arrest (p < 0.01). Our results revealed ZIC1 as a novel candidate tumor suppressor gene downregulated through promoter hypermethylation in gastric cancer.     

CpG island hypermethylation of tumor-suppressor genes in H. pylori-infected non-neoplastic gastric mucosa is linked with gastric cancer risk

Background and aim: Gastric carcinogenesis involves CpG island hypermethylation (CIHM) of tumor‐suppressor genes. Although the CIHM of these genes occurs in non‐neoplastic gastric cells, it is unclear whether this epigenetic alteration is linked with aging and/or gastric cancer risk. We investigated this linkage in noncancerous gastric mucosa infected with H. pylori. Subjects and methods: Noncancerous corpus mucosa was endoscopically obtained from H. pylori‐positive gastric cancer patients (n = 34), and age‐matched H. pylori‐positive noncancerous controls (n = 68). Genomic DNA retrieved from the mucosa was subjected to methylation‐specific polymerase chain reaction for p16, Ecad, and DAPK genes. Linkage between CIHM and clinicopathologic factors was evaluated. Results: CIHM rates of DAPK, Ecad, and p16 promoters were significantly higher in noncancerous gastric mucosa of gastric cancer patients (91, 88, and 68%, respectively) than in noncancerous controls (71, 53, and 25%, respectively). Multivariate regression analysis showed a significant linkage between CIHM in noncancerous mucosa and coexistence of gastric cancer. Significant linkage between polymorphoneutrophil infiltration and CIHM was observed except for CIHM of p16. No linkage was observed between CIHM and other parameters, including age. High CIHM status (all three tested genes methylated) was associated with an increased risk of gastric cancer, with an odds ratio of 9.8 (95% confidence interval, 3.8–25.3). Conclusions: In a subset of the H. pylori‐infected population, CIHM of tumor‐suppressor genes in noncancerous gastric mucosa is linked with the risk of gastric cancer and polymorphoneutrophil infiltration, but not aging. CIHM is a potential marker of gastric cancer risk.     

Hypermethylation-mediated partial transcriptional silencing of DAP-kinase gene in bladder cancer

Death-associated protein kinase (DAP-kinase) is a novel serine/threonine kinase whose expression is required for interferon-γ-induced apoptosis. This study evaluated the methylation pattern and its impact on the expression of the DAP-kinase gene in transitional cell carcinoma of the bladder as hypermethylation is one of the earliest and most frequent alterations leading to cancer. The frequency of hypermethylation of the gene promoter was 37.8%. On correlation with clinicopathological features, methylation was seen mostly in superficial tumours in the group aged?>?60 years (42.9 vs 33.3% of those?≤?60 years) and in smokers (48.1 vs 27.4% of non-smokers). The increased risk of bladder cancer was 6.70-fold (95% confidence interval (CI) 2.09–23.87; p?=?0.000) in those carrying methylated DAP-kinase and it was elevated in patients who smoked (odds ratio 7.87; 95% CI 1.50–54.96; p?=?0.007). This study demonstrated that methylation in the gene promoter on its own could significantly decrease the mRNA expression level of DAP-kinase by 27.68%. Interestingly, patients within the group aged?>?60 years and with a smoking habit showed increased downregulation of mRNA compared with non-smokers of this age group (similar pattern of methylation). Hypermethylation can decrease the expression of DAP-kinase and may be one of the reasons for conversion of normal cells to malignant cells, as the frequency of methylation at the early stage (superficial) of tumours was elevated. Methylation of DAP-kinase can be considered as one of the prognosis indicators for progression and development of bladder cancer.     

Noninvasive diagnostics of bladder cancer based on analysis of telomerase activity and expression hTERT and hTR coding its subunits

In order to develop noninvasive diagnostics of bladder cancer (BC), telomerase activity has been examined by means the TRAP method (telomerase repeat amplification protocol) in tumor tissue and urine pellet samples taken from patients with bladder cancer. The levels of relative expression of genes encoding telomerase catalytic subunit (hTERT) and its RNA subunit (hTR) were evaluated by RT-PCR. Telomerase activity and expression of genes encoding its subunits were detected in both tumor tissues and in the urine cell pellet from each BC patient. Results of our study demonstrate possibility of noninvasive BC diagnostics using combination of these methods with sensitivity of 96% and specificity of 100% in the case of telomerase detection and with sensitivity of 80% and specificity of 100% in the case of hTERT detection in urine pellet samples.