Promoter methylation status of the DNA repair genes hMLH1 and MGMT in gastric carcinoma and metaplastic mucosa.

Hypermethylation of CpG islands in the promoter region is associated with the silencing of a variety of tumor suppressor genes. DNA repair genes human Mut L homologue 1 (hMLH1) and O(6)-methylguanine-DNA methyltransferase (MGMT) have been shown to be hypermethylated in certain carcinomas. We studied DNA methylation of CpG islands in hMLH1 and MGMT in 50 gastric carcinomas and 10 intestinal metaplastic mucosa samples. We analyzed the methylation status of hMLH1 and MGMT using methylation-specific polymerase chain reaction and DNA sequencing analysis. We measured protein levels of hMLH1 using Western blot and immunohistochemical analysis. CpG island hypermethylation of hMLH1 and MGMT was detected in 11 (22%) and 8 (16%) of the 50 gastric tumors, respectively. Hypermethylation of the promoter was more common in intestinal-type gastric carcinomas than in poorly diffuse-type gastric carcinomas (p = 0.016 and 0.021, respectively; Fisher's exact test). However, hMLH1 promoter hypermethylation did not coincide with MGMT promoter hypermethylation except in 1 patient. Hypermethylation of the hMLH1 promoter but not the MGMT promoter occurred in intestinal metaplastic mucosae. Immunohistochemical analysis revealed a corresponding reduction in hMLH1 protein expression in some of the intestinal metaplastic mucosae. Our results suggest that at least two types of promoter methylation participate in the development of gastric carcinoma. Tumor-specific promoter hypermethylation of hMLH1 may be an early event in carcinogenesis in the stomach.     

Multipoint methylation analysis indicates a distinctive epigenetic phenotype among testicular germ cell tumors and testicular malignant lymphomas

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. Although a growing number of genes showing hypermethylation is being reported in human cancer, methylation profiles of tumor-related genes in testicular neoplasms have not been well elucidated. This study was designed to show the methylation profiles of multiple CpG islands in testicular germ cell tumors (TGCTs) in comparison with those in testicular malignant lymphomas. We studied the methylation status of E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 by use of TGCT tissues and testicular malignant lymphoma tissues (25 primary TGCT tissues and three primary testicular lymphoma tissues). Methylation was not observed in E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 in any of the TGCT tissues. In contrast, all three (100%) of the testicular lymphoma tissues demonstrated hypermethylation of E-cadherin, RASSF1A, and RARB, but not CDKN2B, CDKN2A, BRCA1, RB1, VHL, and GSTP1. These data demonstrate that a distinctive epigenetic phenotype underlies the TGCTs and testicular lymphomas at the CpG sites of E-cadherin, RASSF1A, and RARB; a distinctive epigenetic phenotype was not observed among seminomatous TGCTs and non-seminomatous TGCTs at the CpG sites examined.     

Hypermethylation of the MYOD1 gene is a novel prognostic factor in patients with colorectal cancer

MYOD1 promoter methylation occurs in various malignancies including colorectal cancer. We analyzed MYOD1 methylation in 80 colorectal cancer and 74 adjacent normal tissues using MethyLight, which enabled quantitative DNA methylation analysis. The measured methylation value was expressed as a percentage of methylated reference (PMR). The results were compared with clinicopathological features and patient prognosis in order to investigate whether MYOD1 methylation may serve as an independent prognostic factor of colorectal cancer. MYOD1 promoter methylation was detectable in all samples and was significantly higher in tumor compared to normal mucosa, where the median level of methylation was 5.3 PMR (range 0.03-133.4) in normal mucosa and 42.3 PMR (range 0.44-742.9) in tumor. The methylation value of MYOD1 was higher with elder patients both in normal colonic mucosa (p=0.034) and in cancer tissues (p=0.0012). Patients without MYOD1 hypermethylation showed significantly longer survival than those with hypermethylation (p=0.0077). In multivariate analysis of prognostic factors, MYOD1 hypermethylation was a significant prognostic factor (p=0.015) independent to patients' age. These results suggest that MYOD1 hypermethylation plays an important role in colorectal cancer and may be a novel prognostic factor. In addition, quantitative methylation analysis by MethyLight is encouraged for other genes showing age-related and non-cancer-specific methylation.     

Promoter hypermethylation of the 14-3-3 sigma, SYK and CAGE-1 genes is related to the various phenotypes of urinary bladder carcinomas and associated with progression of transitional cell carcinomas

To explore the significance of epigenetic mechanisms in urinary bladder carcinogenesis mediated by methylation of cytosine in CpG dinucleotides at 5' promoter regions, we analysed the methylation status of a broad panel of different genes in transitional cell carcinomas (TCC) and nonurothelial cancers, among which the 14-3-3 sigma, SYK and CAGE-1 genes were recognised as promising target genes. Using methylation-specific PCR, the rate of DNA hypermethylation proved to be related to the various histopathological cancer subtypes. The higher frequency of promoter methylation of the 14-3-3 sigma (57.1%) and SYK (64.3%) genes in high-grade, high-stage TCC in association with a reduced or even lacking immunohistochemical protein expression than in low-grade, low-stage TCC (28.6% and 42.9%, respectively), indicates that aberrant methylation of these genes plays an essential role in the progression of TCC. The importance of DNA hypermethylation in the conversion of TCC from a low to a high malignant potential was strongly supported by the finding that, unlike superficial low-grade TCC, advanced muscle invasive TCC showed a concurrent promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes. Squamous cell carcinomas revealed a peak incidence of hypermethylation of the 14-3-3 sigma gene (80%), and conversely, the lowest methylation frequency of the SYK gene (13.3%). Undifferentiated small cell carcinomas disclosed a promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in only a quarter each for the cases. Although a correlation between the methylation status and gene activity in squamous cell and undifferentiated small cell carcinomas was not observed, the underexpression of the SYK protein products in both cancer types and additionally of the 14-3-3 sigma protein in small cell carcinomas appeared to be related to the aggressive clinical behaviour of both these nonurothelial bladder carcinomas. The relevance of the high frequency of DNA hypermethylation of the CAGE-1 antigen in TCC and squamous cell carcinomas merits further study, particularly in relation to anticancer immunotherapy. The methylation status of the PTEN, COX-2, RUNX-3 and HIC-1 genes was found to be unaltered. In conclusion, the different patterns of aberrant methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in the various histopathological cancer types of the urinary bladder point to a role in tumor cell differentiation, resulting in the phenotypical conversion of TCC into nonurothelial carcinomas and in the progression of TCC to a more malignant potential.     

DNA hypermethylation of tumor-related genes in gastric carcinoma

The hypermethylation of the CpG islands is a common mechanism for the inactivation of tumor-related genes. In the present study, we analyzed the methylation status of genes for cell repair such as hMLH1, MGMT, and GSTP1, and a gastric cancer-specifically methylated DNA fragment, MINT 25 in gastric cancer cases and control groups. The study population consisted of 100 gastric cancer patients (50 distal and 50 proximal carcinomas), and 238 healthy controls. All genes showed more frequent hypermethylation in the cases than in the control group (p<0.0001). We investigated the association between promoter hypermethylation and relevant parameters including age, gender, alcohol consumption, smoking, and family history. There was a common hypermethylation of hMLH1 (p=0.008), MGMT (p= 0.0001), and GSTP1 (p=0.0003) in females. This study also demonstrates that hypermethylation was strongly associated with non-drinkers (MGMT, p=0.046 and MINT 25, p=0.049) and non-smokers (hMLH1, p=0.044; MGMT, p=0.0003; MINT 25, p=0.029). Moreover, the frequency of MINT 25 hypermethylation increased with age (p=0.037), and MGMT methylation was frequently detected in distal gastric cancer than in proximal type (p=0.038). Our study suggested that promoter hypermethylation of the genes involved in cell repair system and MINT 25 is associated strongly with some subgroups of primary gastric carcinoma.     

肾细胞癌RASSF1A基因启动子区甲基化状况与表达水平研究

目的检测19例肾细胞癌患者癌组织及癌旁组织Ras相关区域家族蛋白1A（RASSFlA）基因启动子区甲基化情况并检测其mRNA表达水平，探索两者之间的联系。方法收集肾细胞癌患者癌组织及相应癌旁组织19份，分别提取其基因组DNA，以甲基化特异性PCR（Methylation special PCR，MSP）法检测RASSFlA基因启动子区甲基化情况。并以QPCR法检测了RASSFlA基因的mRNA表达水平。结果19例中有lO例肾细胞癌患者癌组织存在高甲基化，mRNA表达水平表达降低，二者之间存在显著负相关性（r=-0．8734，P〈0．01）。结论肾细胞癌中RASSFlA基因启动子区存在高甲基化，并抑制该基因的表达。     

Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLC1 promoter hypermethylation

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.     

Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.     

Epigenetic markers for molecular detection of prostate cancer

Prostate cancer is a highly prevalent malignancy, which is clinically silent but curable while organ-confined. Because available screening methods show poor sensitivity and specificity, the development of new molecular markers is warranted. Epigenetic alterations, mainly promoter hypermethylation of cancer-related genes, are common events in prostate cancer and might be used as cancer biomarkers. Moreover, the development of quantitative, high-throughput techniques to assess promoter methylation enabled the simultaneous screening of multiple clinical samples. From the numerous cancer-related genes hypermethylated in prostate cancer only a few proved to be strong candidates to become routine biomarkers. This small set of genes includes GSTP1, APC, RARbeta2, Cyclin D2, MDR1, and PTGS2. Single and/or multigene analyses demonstrated the feasibility of detecting early prostate cancer, with high sensitivity and specificity, in body fluids (serum, plasma, urine, and ejaculates) and tissue samples. In addition, quantitative hypermethylation of several genes has been associated with clinicopathologic features of tumor aggressiveness, and also reported as independent prognostic factor for relapse. The identification of age-related methylation at specific loci and the differential frequency of methylation among ethnical groups, also provided interesting data linking methylation and prostate cancer risk. Although large trials are needed to validate these findings, the clinical use of these markers might be envisaged for the near future.     

Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.     

Loss of heterozygosity of selected tumor suppressor genes in human testicular germ cell tumors

Human testicular germ cell tumors (TGCTs) are histologically heterogenous neoplasms with a variable malignant potential. Two main groups of germ cell tumors occur in men: seminomas and nonseminomas. In the present study, a set of four tumor suppressor genes was investigated in testicular cancers. CDH1, APC, p53, and nm23-H1 genes were tested for loss of heterozygosity (LOH). Thirty-eight testicular germ cell tumors (17 seminomas and 21 nonseminomas) were analyzed by PCR using restriction fragment length polymorphism or the dinucleotide/tetranucleotide repeat polymorphism method. An allelic loss of p53 at exon 4 was detected in five nonseminomas, whereas LOH of p53 at intron 6 occurred in one of the seminoma and two of the nonseminoma samples. Allelic losses of the APC gene were present in three seminomas and one nonseminoma, whereas one seminoma and three nonseminomas showed LOH of CDH1. The analysis of allelic losses showed no common structural genetic alterations in tumor tissues, although a different pattern of LOH was observed between the two main histological groups of TGCTs.     

FOXA1 positively regulates gene expression by changing gene methylation status in human breast cancer MCF-7 cells

Objective: DNA methylation is an important epigenetic modification with tumor suppressor gene silencing in cancer. The mechanisms underlying DNA methylation patterns are still poorly understood. This study aims to evaluate the potential value of FOXA1 for controlling gene CpG island methylation in breast cancer. Methods: FOXA1 was down-regulated by transfection with siRNA and up-regulated by transfection with plasmid in MCF-7 cell lines. The DNA methylation and mRNA levels were examined by qMSP and qRT-PCR. The cell proliferation and apoptosis was detected by MTT and Flow cytometry. Results: Suppression of FOXA1 enhanced the methylation status of DAPK, MGMT, RASSF1A, p53, and depressed mRNA levels of these tumor suppressor genes, whereas over-expression of FOXA1 showed the opposite effects. DNMT1, DNMT3A and DNMT3B mRNA were up-regulated by siRNA knock-down of FOXA1. At the same time, FOXA1 suppression promoted cell growth and inhibited apoptosis. Conclusions: FOXA1 may be associated with methylation of the tumor suppressor genes promoter through changing DNMTs expression. FOXA1 could be a potential demethylation target for prevention and treatment of breast cancer.     

DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis.

Cancer cells have aberrant patterns of DNA methylation including hypermethylation of gene promoter CpG islands and global demethylation of the genome. Genes that cause familial cancer, as well as other genes, can be silenced by promoter hypermethylation in sporadic tumors, but the methylation of these genes in tumors from kindreds with inherited cancer syndromes has not been well characterized. Here, we examine CpG island methylation of 10 genes (hMLH1, BRCA1, APC, LKB1, CDH1, p16(INK4a), p14(ARF), MGMT, GSTP1 and RARbeta2) and 5-methylcytosine DNA content, in inherited (n = 342) and non-inherited (n = 215) breast and colorectal cancers. Our results show that singly retained alleles of germline mutated genes are never hypermethylated in inherited tumors. However, this epigenetic change is a frequent second "hit", associated with the wild-type copy of these genes in inherited tumors where both alleles are retained. Global hypomethylation was similar between sporadic and hereditary cases, but distinct differences existed in patterns of methylation at non-familial genes. This study demonstrates that hereditary cancers "mimic" the DNA methylation patterns present in the sporadic tumors.