Frequent promoter hypermethylation and transcriptional downregulation of BTG4 gene in gastric cancer

The BTG4 gene belongs to the BTG family of genes endowed with antiproliferative properties. In this study, we have found that BTG4 undergoes promoter CpG island hypermethylation-associated inactivation in gastric cancer and 5′-aza-2′-deoxycytidine (DAC) treatment restores BTG4 expression. We also found BTG4 levels were significantly reduced in primary gastric cancer but not in normal gastric tissues. BTG4 reexpression in gastric cancer causes growth inhibition of colony assays and nude mice. Taken together, our data support BTG4 as a candidate tumor suppressor gene that is epigenetically silenced in the majority of gastric cancers.     

口腔白斑抑癌基因DAPK和TIG1高甲基化表达研究

摘要 目的：探讨抑癌基因DAPK、TIG1高甲基化在口腔白斑中表达状态及其对口腔癌发生发展中的作用。方法：取77例口腔白斑、32例口腔鳞癌、32份正常口腔黏膜组织,用实时定量甲基化特异性PCR技术检测组织中DAPK、TIG1高甲基化表达并进行统计学分析。结果：DAPK在口腔鳞癌组织中高甲基化表达率为46.9%,表达量为（0.0728±0.1617）,明显高于其在口腔白斑组织（19.5%,0.0070±0.0172）和口腔正常组织（18.8%,0.0021±0.0050）中的表达,差异有统计学意义（P<0.05）。DAPK高甲基化表达与口腔白斑组织上皮异常增生程度相关,上皮增生高风险组相对于低风险组DAPK高甲基化表达风险增加（OR,1.013;95% CI,1.004-1.023;P=0.004）。TIG1高甲基化在正常组织中未表达,在口腔鳞癌组织和口腔白斑组织表达为（28.1%,0.0174±0.0440）和（27.3%,0.0035±0.0076）,与正常组织相比具有统计学意义（P<0.05）。结论：抑癌基因 DAPK、TIG1高甲基化有望成为口腔黏膜癌变早期标志物。     

MGMT 甲基化影响替莫唑胺对胶质母细胞瘤疗效的研究

目的:探讨MGMT甲基化如何影响替莫唑胺对胶质母细胞瘤的治疗效果。方法:选取41个胶质母细胞瘤患者(根据相同替莫唑胺化疗方案治疗下临床结局的不同分为两组)的肿瘤组织,采用甲基化特异性聚合酶链反应分析胶质瘤组织中MGMT基因启动子区过甲基化状态,同时采用免疫组织化学法分析胶质瘤组织中MGMT蛋白表达情况。结果:临床结局不佳组中,以MGMT蛋白表达阳性的肿瘤为主(72.2%),而在结局相对良好组中,MGMT蛋白表达的阳性率仅为39.1%;在MGMT蛋白表达阳性的22例胶质母细胞瘤组织中,7例MGMT启动子甲基化,阳性率为31.8%,在MGMGT蛋白表达阴性的19例中,14例MGMT启动子甲基化,阳性率为73.7%(P<0.05)。结论:MGMT基因启动子区的甲基化状态与MGMT蛋白的表达相关。MGMT基因启动子过甲基化,MGMT蛋白表达较低;MGMT基因启动子去甲基化,MGMT蛋白表达较高。MGMT启动子过甲基化通过抑制MGMT基因的表达而增加替莫唑胺的疗效。     

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

Background

Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

Methods

CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

Results

Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2^′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O₂.

Conclusion

These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.     

Cisplatin-induced downregulation of SOX1 increases drug resistance by activating autophagy in non-small cell lung cancer cell

SOX1 was aberrant methylated in hepatocellular cancer and non-small cell lung cancer (NSCLC). Long-term cisplatin exposure promotes methylation of SOX1 in ovarian cancer cell, suggesting that SOX1 may be involved in cisplatin resistance. Our aim was to test the hypothesis that cisplatin resistance is associated with alteration of SOX1 expression in NSCLC. Expression of levels of SOX1 was examined using RT-PCR in cisplatin resistance cells and parental cells. The level of SOX1 mRNA in cisplatin resistance cells was markedly reduced when compared to parental cells. Promoter methylation of SOX1 was induced in cisplatin resistance cells. We also found that SOX1 silencing enhanced the cisplatin-mediated autophagy in NSCLC. This study shows that inactivation of SOX1 by promoter hypermethylation, at least in part, is responsible for cisplatin resistance in human NSCLC.     

Identification of differentially methylated sites within unmethylated DNA domains in normal and cancer cells

Altered DNA methylation has been linked to neoplastic cell transformation and is a hallmark of cancer progression. Therefore, the screening for differentially methylated sequences as tumor biomarkers has a significant implication in the clinical setting. To determine the cancer-linked alterations in DNA methylation pattern, we have applied an endonuclease, McrBC, to the existing methylation-sensitive arbitrarily primed polymerase chain reaction (msAP-PCR) method and developed McrBC-msAP-PCR. This modified approach allows detection of differentially methylated sites within unmethylated DNA domains enriched by regulatory sequences and CpG islands. In this method, we used digestion of DNA with the McrBC methylation-sensitive endonuclease to selectively exclude the methylated fraction of DNA, which comprises interspersed and tandem-repeated sequences and exons other than first exons, from analysis. The subsequent digestion of unmethylated DNA fragments with SmaI and HpaII methylation-sensitive restriction endonucleases followed by AP-PCR amplification resulted in the detection of unknown unique sequences associated with cancer-linked methylation changes in genomic DNA. Hypermethylation and hypomethylation are visualized by the increase or decrease in the band intensity of DNA fingerprints. By using this technique, we were able to differentiate clearly, identify, and characterize a number of novel unique DNA sequences with differentially methylated sites in normal and breast cancer cell lines and in normal and rat tumor liver tissues.     

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.