DNA甲基化与结肠癌

DNA异常甲基化是肿瘤常见的表观遗传学改变.广泛的低甲基化和区域性高甲基化是基因异常表达的常见机制.某些基因异常甲基化与结肠癌发生密切相关,且常见于结肠癌发病早期.     

胃癌DNA甲基化研究进展

胃癌的发生发展是一个涉及多基因多步骤的复杂过程。随着研究的不断深入,人们开始关注DNA 甲基化这种表遗传学修饰方式在胃癌发生过程中的作用。DNA的异常甲基化通过影响基因转录,促基因突变,增加染色体结构的不稳定性等多方面促进胃癌的发生发展。随着DNA甲基化研究的不断深入,甲基化的检测技术也不断革新。基因启动子甲基化已经作为一种重要的肿瘤生物学标志被应用于胃癌的临床诊断,甲基化制剂作为抗肿瘤药物也逐步应用于临床。     

The emergence of DNA methylation as a key modulator of aberrant cell death in prostate cancer

It is now well established that cancer cells exhibit a number of genetic defects in the machinery that governs programmed cell death and that sabotage of apoptosis is one of the principal factors aiding in the evolution of the carcinogenic phenotype. A number of studies have implicated aberrant DNA methylation as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many processes including apoptosis. To date, studies on the methylation profile of apoptotic genes have largely focused on cancers of the breast, colon and stomach, with only limited data available on prostate cancer. Here we discuss the major developments in the field of DNA methylation and its role in the regulation of aberrant apoptosis in prostate cancer. The most significant advances have involved the discovery of apoptotic gene targets of methylation, including XAF1, (fragile histidine triad (FHIT ), cellular retinol binding protein 1 (CRBP1), decoy receptor 1(DCR1), decoy receptor 2 (DCR2 ), target of methylation-induced silenceing 1 (TMS1), TNF receptor superfamily, member 6 (FAS), Reprimo (RPRM) and GLI pathogenesis-related 1 (GLIPR1). These genes are reported to be hypermethylated in prostate cancer and some offer potential as diagnostic and prognostic markers. We also introduce the concept of an 'apoptotic methylation signature' for prostate cancer and evaluate its potential in a diagnostic, prognostic and therapeutic setting.     

Neonatal exposure to estradiol/bisphenol A alters promoter methylation and expression of Nsbp1 and Hpcal1 genes and transcriptional programs of Dnmt3a/b and Mbd2/4 in the rat prostate gland throughout life

Evidence supporting an early origin of prostate cancer is growing. We demonstrated previously that brief exposure of neonatal rats to estradiol or bisphenol A elevated their risk of developing precancerous lesions in the prostate upon androgen-supported treatment with estradiol as adults. Epigenetic reprogramming may be a mechanism underlying this inductive event in early life, because we observed overexpression of phosphodiesterase 4D variant 4 (Pde4d4) through induction of hypomethylation of its promoter. This epigenetic mark was invisible in early life (postnatal d 10), becoming apparent only after sexual maturation. Here, we asked whether other estrogen-reprogrammable epigenetic marks have similar or different patterns in gene methylation changes throughout life. We found that hypomethylation of the promoter of nucleosome binding protein-1 (Nsbp1), unlike Pde4d4, is an early and permanent epigenetic mark of neonatal exposure to estradiol/bisphenol A that persists throughout life, unaffected by events during adulthood. In contrast, hippocalcin-like 1 (Hpcal1) is a highly plastic epigenetic mark whose hypermethylation depends on both type of early-life exposure and adult-life events. Four of the eight genes involved in DNA methylation/demethylation showed early and persistent overexpression that was not a function of DNA methylation at their promoters, including genes encoding de novo DNA methyltransferases (Dnmt3a/b) and methyl-CpG binding domain proteins (Mbd2/4) that have demethylating activities. Their lifelong aberrant expression implicates them in early-life reprogramming and prostate carcinogenesis during adulthood. We speculate that the distinctly different fate of early-life epigenetic marks during adulthood reflects the complex nature of lifelong editing of early-life epigenetic reprogramming.     

胰腺癌细胞系BxPC3及胰腺癌组织Ras相关区域家族1A启动子CpG岛的甲基化状态

目的 检测Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)在胰腺癌细胞株BxPC3及胰腺癌组织中的甲基化状态,探讨其启动子异常甲基化在胰腺癌发病机制中的可能作用.方法 采用结合重亚硫酸盐的限制性内切酶法(combined bisulfite restriction analysis,COBRA)检测胰腺癌细胞株BxPC3、5例正常胰腺组织、13对胰腺癌及相应癌旁正常胰腺组织中RASSF1A启动子CpG岛的甲基化状态,计算其甲基化率.以甲基化酶抑制剂5-Aza-dC(5-Aza-2-deoxycitydine)处理BxPC3,观察处理前后甲基化率变化情况及RASSF1A mRNA表达变化.结果 在BxPC3细胞株中,RASSF1A启动子的CpG岛甲基化率为62.90%;正常胰腺、癌旁及癌组织中平均分别为9.14%、53.79%和55.82%.与正常胰腺组织相比,胰腺癌旁及癌组织的RASSF1A启动子甲基化率明显增高(P值<0.01),而癌旁及癌组织之间无明显差异(P>0.05).BxPC3经5-Aza-dC处理后,RASSF1A的CpG岛甲基化率显著下降至42.50%(P<0.05),同时RASSF1A mRNA表达增强.结论 RASSF1A启动子CpG岛异常甲基化是胰腺癌发生发展中的早期事件,可能参与胰腺癌的发病过程.     

The emerging role of epigenetics in urological cancers

DNA methylation and histone modifications constitute the common epigenetic modifications in vertebrate genomes. The epigenetic changes are early event in the cancer development and are reversible. Over the last decade, the field of epigenetics has made considerable progress both in the diagnosis and treatment of variety of malignancies. Novel epigenetic markers are being studied, which have the potential as sensitive diagnostic and prognostic markers. DNA methylation has been identified as a powerful diagnostic tool in classification, detection and risk assessment of cancers. As DNA methylation is reversible, inhibitors of DNA methyl transferases and histone deacteylases have been designed for use in treatment of a variety of urological malignancies. Variety of drugs targeting epigenetic changes are being studied, which can be effective individually or in combination with other conventional drugs used in cancer therapy. The emerging area of epigenetic therapy holds great promise for novel chemotherapeutic and chemoprevention approaches against cancer.     

Clinical application of methylation specific-polymerase chain reaction in serum of patients with gastric cancer

BACKGROUND/AIMS: We previously reported that aberrant methylation of p16 and/or E-cadherin genes in serum DNA could serve together as a tumor marker in gastric cancer. We presently investigated whether sensitivity could be increased by consideration of a third gene, which encodes retinoic acid receptor-1 (RARbeta). METHODOLOGY: We performed a methylation-specific polymerase chain reaction (MSP) in serum DNA to detect aberrant methylation of RARbeta from 109 preoperative gastric cancer patients, in which the first two genes had been characterized. We also examined all three genes in sera from 10 outpatients during postgastrectomy follow-up. RESULTS: Aberrant methylation of RARbeta was demonstrated in 26 preoperative patients (24%). Considering this with previous results, 52 patients (48%) of the 109 preoperative showed hypermethylation of at least one gene (p16, E-cadherin, and/or RARbeta). No aberrant methylation was detected in control sera. In the follow-up group, aberrant methylation was demonstrated in 2 of the 3 patients who had definite radiologic evidence of recurrences. One of the patients showing promoter hypermethylation without definite findings of recurrence at the time of analysis developed peritoneal recurrence 6 months later. CONCLUSIONS: Including the MSP assay in conventional follow-up could facilitate early detection of recurrent disease in gastric cancer patients.     

外周血SARP2基因甲基化检测对胰腺癌的诊断价值

目的 探讨胰腺癌患者外周血DNA中SARP2基因甲基化对胰腺癌的诊断价值.方法 收集12例原发性胰腺癌、10例慢性胰腺炎和6例健康志愿者外周静脉血,提取血清游离DNA,经亚硫酸氢盐修饰后,行SARP2基因第一外显子区域的BSP扩增和测序.结果 12例胰腺癌外周血DNA、10例慢性胰腺炎外周血DNA中分别有10例(83%)和4例(40%)检测出明显的SARP2基因甲基化改变.胰腺癌患者外周血中SARP2基因第一外显子区CpG位点甲基化率为16.8%;慢性胰腺炎患者的甲基化率为10.4%,健康志愿者为2.2%.3组间的SARP2 CpG岛甲基化率差别非常显著(P<0.01或P<0.05).结论 胰腺癌患者外周血DNA中可以检测到SARP2甲基化改变,SARP2甲基化的检测对胰腺癌的诊断可能有潜在的临床价值.     

Microarray-based method for detecting methylation changes of p16^Ink4a gene 5＇-CpG islands in gastric carcinomas

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.     

Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement

Cellular DNA damage triggers the DNA damage response pathway and leads to enforcement of cell cycle checkpoints, which are essential for the maintenance of genomic integrity and are activated in early stages of tumorigenesis. A special feature of prostate cancer is its high incidence and multifocality. To address the functionality of DNA damage checkpoints in the prostate, we analyzed the responses of human primary prostate epithelial cells (HPECs) and freshly isolated human prostate tissues to gamma-irradiation. We find that gamma-irradiation activates the ataxia telangiectasia mutated-associated DNA damage response pathway in the HPECs but that the clearance of phosphorylated histone H2AX (gammaH2AX) foci is delayed. Surprisingly, gamma-irradiated HPECs were unable to enforce cell cycle checkpoint arrest and had sustained cyclin-dependent kinase 2 (Cdk2)-associated kinase activity because of a lack of inhibitory Cdk phosphorylation by Wee1A tyrosine kinase. We further show that HPECs express low levels of Wee1A and that ectopic Wee1A efficiently rescues the checkpoints. We recapitulate the absence of checkpoint responses in epithelium of ex vivo irradiated human prostate tissue despite robust induction of gammaH2AX. The findings show that prostate epithelium has a surprising inability to control checkpoint arrest, the lack of which may predispose to accrual of DNA lesions.     

特异性PCR法检测胰腺癌p16基因甲基化改变

目的：探讨胰腺癌p16基因启动子区5'CpG岛甲基化改变的特点及其与临床病理特征的关系。方法：应用甲基化特异性PCR法进行甲基化检测。结果：14／36例胰腺癌标本的p16基因启动子区5'CpG岛检测到甲基化，甲基化频率为38．9％，5例癌旁组织、1例正常胰腺组织、7例慢性胰腺炎及2例胰腺粘液性囊腺瘤未发现相应区域的甲基化。结论：p16基因启动子区5'CpG岛甲基化是胰腺癌p16基因失活的机制之一，是胰腺癌细胞区别与正常细胞的分子事件。检则p16基因甲基化可能有助于胰腺癌的诊断及鉴别诊断。     

Methylation-mediated gene silencing as biomarkers of gastric cancer:a review

Despite a decline in the overall incidence of gastric cancer(GC),the disease remains the second most common cause of cancer-related death worldwide and is thus a significant global health problem.The best means of improving the survival of GC patients is to screen for and treat early lesions.However,GC is often diagnosed at an advanced stage and is associated with a poor prognosis.Current diagnostic and therapeutic strategies have not been successful in decreasing the global burden of the disease;therefore,the identification of reliable biomarkers for an early diagnosis,predictive markers of recurrence and survival and markers of drug sensitivity and/or resistance is urgently needed.The initiation and progression of GC depends not only on genetic alterations but also epigenetic changes,such as DNA methylation and histone modification.Aberrant DNA methylation is the most well-defined epigenetic change in human cancers and is associated with inappropriate gene silencing.Therefore,an increasing number of genes methylated at the promoter region have been targeted as possible biomarkers for different purposes,including early detection,classification,the assessment of the tumor prognosis,the development of therapeutic strategies and patient follow-up.This review article summarizes the current understanding and recent evidence regarding DNA methylation markers in GC with a focus on the clinical potential of these markers.     

DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results

The prognosis of lung cancer is very much limited by the difficulties of diagnosing early stage disease amenable to surgery. Thus, novel diagnostic and therapeutic approaches are urgently needed for this common type of cancer. Recently, epigenetic alterations of tumor cells have been defined for a multitude of tissues and genes. Thus, promoter hypermethylation of tumor suppressor genes, and other targets of neoplasia-associated methylation disturbances, have become the most frequent recurrent alteration in solid tumors and hematologic neoplasia. In lung cancer, several sets of genes including the tumor suppressor gene p16, the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), E-cadherin and retinoic acid receptor beta have been shown to be frequently methylated and inactivated. Distinct methylation patterns can provide molecular distinctions between different histologic subtypes of lung cancer. Gene hypermethylation in lung cancer is an early event associated with exposure to tobacco-specific carcinogens. Highly sensitive detection of hypermethylated DNA in sputum and peripheral blood offers a powerful tool for detecting lung cancer at an early stage. Epigenetic alterations in cancer, as opposed to genetic lesions, are potentially reversible. Thus, hypermethylation has been studied as a therapeutic target for agents which revert this epigenotype. The most advanced drugs to inhibit methylation are two azanucleosides, decitabine and its ribonucleoside analogue 5-azacytidine. In vitro, demethylating agents given at low doses reactivate tumor suppressor genes, and in mouse models, the development of lung cancer can be retarded. This effect is more powerful when histone acetylation, as a second epigenetic silencing mechanism, is also inhibited pharmacologically (HDAC inhibitors). Clinical trials of both groups of agents have been performed, and novel demethylating agents which are not incorporated into DNA offer further perspectives for epigenetic therapy of lung cancer and other malignancies.     

DNA甲基化与恶性肿瘤

DNA甲基化是脊椎动物DNA唯一的自然共价修饰方式.DNA的异常甲基化可发生在多数人类恶性肿瘤及其癌前病变,其在转录水平抑制基因的表达,与肿瘤的发生、发展关系密切.DNA甲基化可能在肿瘤的早期诊断、预后判断及癌前病变的逆转方面发挥一定作用.     

Global DNA hypomethylation occurs in the early stages of intestinal type gastric carcinoma.

BACKGROUND: Global DNA hypomethylation has been found in the premalignant stages of some neoplasms and has been implicated as an important factor for tumour progression. AIMS: The aim of this study was to evaluate whether DNA hypomethylation occurs during the process of gastric carcinogenesis. METHODS: Gastric specimens were obtained from 49 patients and histologically classified as: normal 10, superficial gastritis 14, chronic atrophic gastritis with intestinal metaplasia 15, and intestinal type of gastric carcinoma 10. Global DNA methylation was assessed by incubating DNA with (3H)-S-adenosylmethionine and Sss1 methylase. A higher incorporation of (3H) methyl groups reflects a lower degree of intrinsic methylation. RESULTS: A graduated increase in (3H) methyl group incorporation into DNA was found over the range extending from normal gastric mucosa, to superficial gastritis and to chronic atrophic gastritis (136,556 (24,085) v 235,725 (38,636) v 400,998 (26,747 dpm/micrograms/DNA respectively; p = 0.0002). No further increase was found in specimens from patients with carcinoma. No differences were found between extent of DNA methylation in neoplastic or non-neoplastic mucosa from patients with gastric carcinoma. Hypomethylation of DNA increased substantially with severe atrophy (p = 0.01) or with type III intestinal metaplasia (p = 0.15). CONCLUSIONS: Global DNA hypomethylation occurs in the early stages of gastric carcinogenesis, and it may be a novel biomarker of gastric neoplasia, useful in monitoring the response to chemopreventive agents.     

Role of DNMT3A, TET2, and IDH1/2 mutations in pre-leukemic stem cells in acute myeloid leukemia

Aberrant changes in the epigenome are now recognized to be important in driving the development of multiple human cancers including acute myeloid leukemia. Recent advances in sequencing technologies have led to the identification of recurrent mutations in genes that regulate DNA methylation including DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 2 (TET2), and isocitrate dehydrogenase 1 (IDH1) and IDH2. These mutations have been shown to promote self-renewal and block differentiation of hematopoietic stem/progenitor cells. Acquisition of these mutations in hematopoietic stem cells can lead to their clonal expansion resulting in a pre-leukemic stem cell (pre-LSC) population. Pre-LSCs retain the ability to differentiate into the full spectrum of mature daughter cells but can become fully transformed with the acquisition of additional driver mutations. Here, we review the effects of mutations in DNMT3A, TET2, and IDH1/2 on mouse and human hematopoiesis, the current understanding of their role in pre-LSCs, and therapeutic strategies to eliminate this population which may serve as a cellular reservoir for relapse.     

Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma

AIM: Detection of methylation in the p16 gene, an inhibitor of cyclin D-dependent protein kinase, as a new tumor marker for early detection of esophageal squamous cell carcinoma (ESCC) in DNA derived from blood and serum. METHOD: A large family with clustering of ESCC was assessed in Khorasan province in northeastern Iran. The family had three histologically proven cases of ESCC in two consecutive generations and several other deceased cases with histories of ESCC. DNA from blood of 28 living family members in three consecutive generations, 30 sporadic ESCC cases (from serum, blood, and tumor tissues), and 30 healthy volunteers (from blood) were examined for the methylation status of p16 promoter using methylation-specific PCR (MSP). RESULTS: Aberrant p16 promoter methylation was found in 64.3% (n = 28) of ESCC family members and none (n = 30) of our normal volunteers. Five of the 28 family members with esophageal cancer symptoms had negative endoscopy results for ESCC, while four of these members had p16 hypermethylation in their blood. The family members with negative endoscopy and positive p16 promoter methylation are being monitored closely for signs of ESCC development through regular check-ups and chromoendoscopies. In sporadic ESCC in northeastern Iran, 73.3% (n = 30) of tumor tissue samples had p16 hypermethylation. Serum and blood samples from the same patients showed p16 hypermethylation in 26.6% and 43.3% of the samples, respectively. CONCLUSION: Aberrant p16 methylation may be a valuable diagnostic tool as a tumor marker for the early identification of individuals in high risk ESCC families.     

食管鳞癌组织p16基因调控区甲基化及其蛋白表达研究

目的探讨p16基因在食管癌变过程中表达缺失与其启动子区甲基化的关系。方法采用MSP免疫组化方法,检测环太行山地区45例食管鳞癌患者癌组织p16基因启动子区甲基化状态及蛋白表达情况。结果p16基因在癌组织中表达异常41例（91．1％）,间变组织中表达异常38例（84．4％）,发生纯合型甲基化的组织分别为33例（73．3％）（癌组织）和32例（71．1％）（间变组织）,而其周围正常组织26例（57．8％）均发生了p16启动子区的杂合型甲基化。p16基因纯合型甲基化与癌组织、间变组织、p16蛋白表达缺失相关（P〈0．05）。结论该地区食管癌组织p16基因在癌前病变中p16启动子区即发生了纯合型甲基化、食管癌变的早期事件。p16基因启动子区甲基化可单独影响p16蛋白的正常表达。