Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.     

p16基因启动子区甲基化在人嗜铬细胞瘤和副神经节瘤中的变化

目的 检测嗜铬细胞瘤(PHEO)和副神经节瘤(PGL)中p16基因突变和启动子区DNA甲基化改变,分析其与患者临床特征之间的关系.方法收集34例(PHEO 20例、PGL14例)组织标本及患者临床资料,通过甲基化特异性PCR(MSP)测定p16基因启动子区甲基化状态,DNA测序检测基因序列以及RT-PCR方法测定其mRNA表达.结果 (1)34例肿瘤组织中未发现p16基因纯合缺失及点突变;(2)35.3%(12/34)的患者存在p16基因高甲基化,p16基因甲基化阳性标本中,PHEO和PGL分别占25%和75%,两者差异有统计学意义(P=0.005);p16基因甲基化在恶性、单发肿瘤、发病年龄早的亚组中有增高趋势(P＞0.05);(3)甲基化与非甲基化肿瘤组织之间p16 mRNA表达无统计学差异;不同特点的肿瘤中其mRNA表达亦无统计学差异,但恶性肿瘤p16 mRNA表达与良性肿瘤相比有下降的趋势(0.83±0.65对1.12±0.81,P=0.278).结论人PHEO和PGL中,p16基因纯合缺失和突变并不常见,但p16基因启动子区甲基化是其失活的主要形式.     

Effects of von Hippel-Lindau gene mutation and methylation status on expression of transmembrane carbonic anhydrases in renal cell carcinoma

PURPOSE: Our primary goal was to determine whether increased expression of the transmembrane carbonic anhydrase (CA) isozymes in renal cell carcinoma (RCC) is regulated by the von Hippel-Lindau (VHL) gene. METHODS: We studied mRNA expression of all three transmembrane CAs - CA IX, XII, and XIV - in 17 RCC cell lines, transformants of the wild-type VHL gene, and normal kidney tissue cultures, and then compared them with the mutation status in the VHL gene. RESULTS: Northern blot analysis showed no detectable signal for CA XIV mRNA expression in normal and cancer cells. CA XII mRNA was ubiquitously expressed except in two cell lines. Although CA XII expression levels tended to be lower in RCC cell lines without the VHL mutation and in transformants of the wild-type VHL gene, the results were not conclusive. Significant expression of CA IX mRNA was seen in eight of 17 RCC cell lines. Among five cell lines which had no VHL mutation, four lines showed no detectable signal and one cell line showed a low amount of CA IX mRNA expression. In patients with RCC, VHL mutations and significant CA IX expression were seen in established tumor cell lines but not in primary tissue cultures from normal counterparts. Further study of methylation status showed that the 5' region in the CA9 gene was hypomethylated in all CA IX-positive cell lines and hypermethylated in all CA IX-negative cell lines. Especially, methylation status at -74 and -6 CpG sites perfectly correlated with CA IX expression. CONCLUSIONS: These findings indicate that VHL suppresses CA IX expression but has no conclusive effect on CA XII and XIV expressions in RCC. CA IX expression is also driven by the methylation status of the CA9 gene.     

Methylation status of p16 gene in colorectal carcinoma and normal colonic mucosa

INTRODUCTIONp16gene(als0knownasMTS-l,INK4a,CDKN2A),locatedonchromos0me9p21,isaG1-specificcell-cycleregulatorygene.Itiscomposedofthreeex0ns,whichenc0de156aminoacids[l].Thegeneisfrequentlyinactivatedinmanyhumancancers-Unlike0thertumor-suppress0rgenesthatarecommonlyinactivatedbypointmutations,smallhom0zygousdeletionsandmethylationoftheprom0terrepresentthemajormechanismofp16geneinactivati0n[2'3].IntheWesternc0untries,colorectalcarcinomaranksfirstamongmalignanttumors.Themortalityfromcolor…     

CpG island methylation in sporadic colorectal cancers and its relationship to microsatellite instability

BACKGROUND & AIMS: Methylation of CpG islands is increasingly recognized as an important event in colorectal carcinogenesis. We evaluated the extent of CpG island methylation in 426 sporadic colorectal cancers to define its relationship to microsatellite instability and to describe its clinicopathologic and genetic features. METHODS: Fresh cancer tissue was obtained from 417 consecutive individuals undergoing curative surgery for sporadic colorectal cancer. Methylation of p16 and hMLH1 promoters was determined by methylation-specific polymerase chain reaction (PCR), whereas methylation at MINT 1, 2, 12, and 31 loci was assessed by bisulfite PCR. Microsatellite instability and K-ras and p53 status were determined using microsatellite PCR, restriction enzyme-mediated PCR, and immunohistochemistry, respectively. RESULTS: Individual loci were commonly methylated, but locus-specific phenotypic changes were not seen. CpG island methylation was associated with right-sided location, female sex, and older age, as well as high tumor grade, mucinous type, wild-type P53, microsatellite instability, and K-ras mutations. More than half of tumors showing CpG island methylation were microsatellite stable. Compared with microsatellite unstable cancers, they were more commonly left-sided, had fewer intraepithelial lymphocytes, presented later, and had a worse outcome. CONCLUSIONS: Colorectal cancers with CpG island methylation have distinct clinicopathologic features and in some cases lead to sporadic microsatellite unstable cancers.     

Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for gamma interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5' CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell line, was treated with gamma interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma cell line, only did so when treated with 5-aza-2'-deoxycytidine followed by gamma interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of gamma interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell-lineage lymphomas.     

EMs患者在位子宫内膜hMLH1蛋白表达变化及其基因启动子CpG岛甲基化观察

目的观察子宫内膜异位症（EM s）患者在位子宫内膜组织中hMLH1蛋白的表达变化及hMLH1基因启动子CpG岛甲基化情况。方法采用免疫组化法分别检测52例（Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为9、16、12、15例）EM s患者及30例健康志原者（对照组）在位子宫内膜中的hMLH1,采用甲基化特异性聚合酶链反应（MSP）法检测两组在位子宫内膜hMLH1启动子CpG岛甲基化情况。分离hMLH1启动子CpG岛甲基化异常者在位内膜中的腺细胞和间质细胞,采用MSP志愿分别检测两种细胞hMLH1启动子CpG岛甲基化情况。结果 EM s组中hMLH1蛋白表达减弱共6例,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为0、0、1、5例,其余样本hMLH1蛋白表达均呈阳性;对照组30例无hMLH1蛋白表达减弱者,hMLH1蛋白表达均表现为阳性。EM s组中hMLH1启动子CpG岛部分甲基化3例,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为0、0、0、3例,余均表现为非甲基化;对照组30例均表现为hMLH1启动子CpG岛非甲基化。EM s组Ⅳ期患者与Ⅰ、Ⅱ、Ⅲ期患者及对照组相比,P均〈0.05。3例hMLH1启动子CpG岛表现为部分甲基化者子宫内膜组织中hMLH1蛋白表达均减弱,其腺细胞中hMLH1启动子区表现为半甲基化,间质细胞hMLH1启动子区未见明显甲基化条带。结论 EM s患者在位子宫内膜组织中存在hMLH1表达减弱及hMLH1启动子CpG岛部分甲基化,主要集中在腺细胞,hMLH1异常与严重EM s的发病有关。     

hMLH1基因高甲基化在胃癌发生、发展中的作用

基因启动子区CpG岛甲基化使许多基因失活，从而导致恶性肿瘤的发生和发展。目的：检测hMLHl基因启动子区CpG岛甲基化水平，探讨其胃癌发生、发展中的作用。方法：以甲基化特异性聚合酶链反应（MSP）检测41例胃癌、40例癌前病变和38例对照组织中hMLHl基因启动子区CpG岛甲基化状态，并分析其与胃癌患者临床病理特征的关系。结果：胃癌组织中hMLHl基因启动子区CpG岛甲基化阳性率为34．1％，显著高于癌前病变组的5．0％和对照组的0％（P〈0．05）。hMLHl基因甲基化阳性率与胃癌患者的年龄和肿瘤浸润深度有关（阳性率分别为46．4％对7．7％和55．0％对14.3％，P〈0．05），与性别、肿瘤分化程度和淋巴结转移无关（阳性率分别为34．8％对33．3％、28．0％对43．8％和38．1％对30．0％）。结论：胃癌组织中存在hMLHl基因启动子区CpG岛高甲基化，可能与胃癌的发生、发展有关．且可能在老年胃癌患者的肿瘤发生过程中起重要作用。