DAL-1表达下调与大肠腺癌发生发展的关系

目的探讨DAL-1表达下调与大肠腺癌发生发展的关系。方法RT-PCR检测6株大癌细胞系和51例组织标本DAL-1 mRNA表达;BSP和MSP法分别检测大肠癌细胞系和组织标本DAL-1基因启动子甲基化状态;5-aza-dC作用实验验证大 肠癌细胞系中DAL-1启动子甲基化情况。 结果6株大肠癌细胞系DAL-1均有表达,各细胞系间表达无明显差异;部分大肠腺癌组织中DAL-1表达下调,且在淋巴结转移患者和Dukes C、D期患者更常见（P＜0.05）。DAL-1基因启动子高甲基化是少见现象。结论DAL-1表达下调在大肠腺癌发生发展中发挥重要作用,特别是在进展期大肠腺癌;但该基因启动子高甲基化不是下调其表达的主要机制。     

Aberrant methylation of the ATM promoter correlates with increased radiosensitivity in a human colorectal tumor cell line

Recent findings suggest that DNA alkylating agents trigger cellular responses that overlap those activated after ionizing radiation. Moreover, activation of these responses is dependent upon a functional mismatch repair (MMR) system. These developments led us to test if MMR-deficient cells may be compromised in their ability to activate appropriate cellular signaling pathways after ionizing radiation. An initial experiment to address this notion was to determine the level of radiosensitivity of several MMR-deficient cell lines derived from patients with Hereditary Non-Polyposis Colorectal Cancer (HNPCC). While two of the three HNPCC lines investigated show levels of radiosensitivity consistent with that displayed by normal human fibroblasts, HCT-116 cells display moderate radiosensitivity compared to the other MMR-deficient lines. This increased sensitivity to ionizing radiation correlates with lowered levels of ATM expression in HCT-116. Analysis of genomic DNA from HCT-116 cells determined that these cells possess aberrant methylation of multiple CpG dinucleotides within the proximal promoter region of the ATM gene. The significance of this finding is underscored by our observations that co-culturing HCT-116 cells with the DNA demethylating agent 5-azacytidine reverses promoter methylation, promotes normal levels of ATM expression, and restores normal radiosensitivity. The proximal ATM promoter is a approximately 520 bp region shared with the NPAT gene, and current evidence suggests that this region functions as a bi-directional promoter. We found that, unlike ATM, the methylation status of this intergenic region does not effect the expression of the NPAT gene. In sum, these observations indicate that the ATM gene is a novel target for epigentic silencing through inappropriate methylation of its proximal promoter region.     

DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响

目的:探讨miR-133a在胶质瘤细胞中的表达及DNA甲基化对其的调控机制并初步探究miR-133a对胶质瘤细胞增殖及凋亡的影响。方法:通过RT-PCR检测胶质瘤组织与正常对照脑组织中miR-133a的表达差异;MSP实验检测胶质瘤组织与正常对照脑组织中miR-133a基因甲基化程度;BSP实验检测正常胶质细胞株HEB与胶质瘤细胞株A172、U87、U251中miR-133a基因的甲基化水平;去甲基化试剂AZA与胶质瘤细胞株A172、U87、U251共培养72 h后,RT-PCR检测上述3株胶质瘤细胞中的miR-133a表达变化;MTT及Annexin V-FITC凋亡实验分别检测AZA处理后的3株胶质瘤细胞的增殖与凋亡能力变化。结果:RT-PCR实验表明与正常对照脑组织相比,胶质瘤组织中miR-133a表达显著下降;MSP实验结果显示,与正常对照脑组织相比,胶质瘤组织中miR-133a基因的甲基化水平明显增高;BSP实验结果显示与正常对照胶质细胞株HEB相比,胶质瘤细胞A172、U87、U251中miR-133a基因的甲基化水平显著增加;RT-PCR实验显示与去甲基化试剂AZA共培养72 h后,胶质瘤细胞株A172、U87、U251细胞中miR-133a的表达显著增加。MTT与Annexin V-FITC凋亡实验结果表明,去甲基化试剂AZA处理后,胶质瘤细胞A172、U87、U251的增殖能力明显下降,细胞的凋亡发生率显著增加。结论:胶质瘤细胞中DNA甲基化通过调控miR-133a的表达而沉默后者发挥抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的功能。     

鼻咽癌细胞系中LTF基因的表达及遗传学与表观遗传学改变

目的 检测鼻咽癌(NPC)细胞系中LTF基因的表达水平,并分析遗传学和表观遗传学改变与其表达的关系.方法 采用逆转录聚合酶链反应(RT-PCR),检测NPC细胞系HNE1、HNE2、HNE3、CNE1、CNE2、5-8F、6-10B和15例慢性鼻咽炎组织中LTF mRNA表达水平,同时通过Western blot法分析6-10B细胞中LTF蛋白水平.运用微卫星分析技术、聚合酶链式反应-单链构象多态(PCR-SSCP)方法和甲基化特异性PCR(MSP)以及亚硫酸盐克隆测序方法,分别检测LTF的杂合性丢失、突变和启动子区甲基化情况.结果 在15例慢性鼻咽炎组织中,LTF基因稳定表达.在NPC细胞系中,除6-10B细胞有LTF基因弱表达外,其余均表达缺失,显著低于慢性鼻咽炎组织中的表达(Z=-3.738,P=0.000),且6-10B细胞中无LTF蛋白的表达.杂合性丢失分析显示,NPC细胞系中不存在LTF微卫星位点的缺失.突变检测显示,LTF在NPC细胞系中的突变率为14.3%(1/7),且突变存在于HNE1细胞系中,即启动子序列-305至-50 bp发生了缺失突变(-218delT).甲基化分析显示,LTF基因在慢性鼻咽炎组织中无甲基化,而在所有NPC细胞系中均有甲基化.用5-杂氮-2-脱氧包苷处理5-8F和6-10B细胞后,LTF mRNA表达上调.结论 LTF基因在NPC细胞系中表达失活,其分子机制与启动子高甲基化和缺失突变有关.     

人乳腺癌细胞系RUNX3基因启动子甲基化状态研究

目的:探讨人乳腺癌细胞系中抑癌基因RUNX3启动子甲基化状态,并分析其与RUNX3基因表达的相关性。方法:运用甲基化特异性PCR(MSP)检测5种乳腺癌细胞系和一种人类正常乳腺细胞系中RUNX3启动子甲基化状态,运用RT-PCR和Western印迹检测这些细胞系中RUNX3基因mRNA和蛋白的表达。结果:在6种细胞系中,有两种(T47D、MCF7)呈高甲基化状态,并且这两种细胞系的RUNX3 mRNA和蛋白表达阴性。SKBR3中没有检测出RUNX3启动子区的甲基化,但RUNX3 mRNA和蛋白表达阴性。结论:乳腺癌细胞系中RUNX3基因由于启动子区甲基化而失活,但尚有其它失活机制存在,需进一步研究探讨。     

人乳腺癌细胞系RUNX3基因启动子甲基化状态研究

目的：探讨人乳腺癌细胞系中抑癌基因RUNX3启动子甲基化状态,并分析其与RUNX3基因表达的相关性。方法：运用甲基化特异性PCR（MSP）检测5种乳腺癌细胞系和一种人类正常乳腺细胞系中RUNX3启动子甲基化状态,运用RT-PCR和Western印迹检测这些细胞系中RUNX3基因mRNA和蛋白的表达。结果：在6种细胞系中,有两种（T47D、MCF7）呈高甲基化状态,并且这两种细胞系的RUNX3 mRNA和蛋白表达阴性。SKBR3中没有检测出RUNX3启动子区的甲基化,但RUNX3 mRNA和蛋白表达阴性。结论：乳腺癌细胞系中RUNX3基因由于启动子区甲基化而失活,但尚有其它失活机制存在,需进一步研究探讨。     

Hypermethylation of the spleen tyrosine kinase promoter in T-lineage acute lymphoblastic leukemia

Sequence analysis of the noncoding first exon (exon 1) of the Syk gene demonstrated the presence of a previously cloned CpG island (GenBank #Z 65706). Transient transfection analysis in Daudi cells demonstrated promoter activity (18-fold increase over parental luciferase plasmid) for a 348 bp BstXI-BsrBI fragment containing this island. This region exhibits a high GC content (approximately 75%), contains several SP1 binding sites and a potential initiator sequence, but lacks a strong TATA consensus. Bisulfite sequencing and methylation-specific PCR (MSP) of this region demonstrated that the Syk promoter CpG island was largely unmethylated in B-lineage leukemia cell lines, control peripheral blood cells, human thymocytes and CD3(+) T lymphocytes. However, dense methylation was seen in four T-lineage leukemia cell lines, Jurkat, H9, Molt 3 and HUT 78. MSP screening of leukemia cells from six T-lineage acute lymphoblastic leukemia (ALL) patients demonstrated methylation of the Syk promoter CpG island in one T-lineage ALL patient. Promoter methylation was correlated with reduced to absent expression of Syk mRNA and SYK protein in the T-lineage leukemia cell lines. Treatment of the leukemia lines Ha and Molt 3, with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR) resulted in increased Syk mRNA expression. The presence of a methylated promoter sequence in these T-lineage leukemia cell lines and in one T-lineage patient suggests a potential role for SYK as a tumor suppressor in T-ALL.     

Aberrant methylation of p57KIP2 gene in lung and breast cancers and malignant mesotheliomas

The p57KIP2 gene belongs to the Cip/Kip family of CDK inhibitors and has been demonstrated to be a tumor suppressor gene, being inactivated in various types of human cancers. We analyzed the methylation and expression status of p57KIP2 in lung and breast cancers, and in malignant mesotheliomas (MMs). The promoter region of p57KIP2 was determined by methylation-specific PCR (MSP) in samples of lung and breast cancer, and of MM. The expression of the gene in the cell lines was determined by RT-PCR and correlated with the methylation status. Aberrant methylation was detected by MSP in 9 of 27 (33%) and 25 of 78 (32%) lung cancer cell lines and tumors, respectively, 11 of 18 (61%) and 17 of 38 (45%) breast cancer cell lines and tumors, respectively, and 1 of 25 (4%) MM tumors. DNA methylation was detected but rarely in the corresponding non-malignant tissues. In addition, the gene expression was restored in the methylated cell lines following 5-aza-2'-deoxycytidine treatment, confirming that the methylation was indeed responsible for the gene down-regulation. We also examined the relationship between the p57KIP2 methylation status and the clinicopathological features of the primary tumors, and found that there was no relationship between the p57KIP2 methylation status and any of the examined clinicopathological features. In summary, our results demonstrate that p57KIP2 methylation associated with the gene down-regulation is frequently present in lung and breast cancers and plays an important role at the molecular level in the pathogenesis of these cancers.     

Aberrant promoter methylation of human DAB2 interactive protein (hDAB2IP) gene in lung cancers

The human DOC-2/DAB2 interactive protein gene (hDAB2IP) is a novel member of the Ras GTPase-activating gene family that is known to act as a tumor suppressor gene and is inactivated by methylation in prostate and breast cancers. We established previously a methylation-specific PCR (MSP) for the promoter region (m2a and m2b regions) of hDAB2IP and examined hDAB2IP methylation status in breast cancers. We analyzed the methylation and expression status of hDAB2IP in lung cancers. The methylation status of hDAB2IP was examined in lung cancer cell lines using bisulfite sequencing and MSP. Expression was examined using conventional and real-time RT-PCR, and methylation was found to be inversely correlated with expression, confirming that the MSP can also be used to examine hDAB2IP methylation status in lung cancers. Aberrant methylation was detected at the m2a region in 19 of 47 lung cancer cell lines (40%) and 26 of 70 primary tumors (37%) and at the m2b in 16 lines (34%) and 25 of 70 tumors (36%). Gene expression was restored in methylated cell lines supplemented with 5-aza-2'-deoxycytidine, confirming that methylation was responsible for downregulation. We also examined the relationship between hDAB2IP methylation and clinico-pathological features of the lung cancers and found that hDAB2IP methylation was associated with advanced disease stage. Our results demonstrate that hDAB2IP methylation is frequently present in lung cancers and plays a key role in hDAB2IP silencing. hDAB2IP methylation could be used as a biomarker for disease stage, reflecting the degree of clinico-pathological malignancy of lung cancer.     

Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas

O6-Methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation has recently emerged as a powerful determinant of chemotherapy sensitivity in glioblastomas. To adapt such an important epigenetic biomarker to routine application in the clinical setting, we validated the conventionally used methylation-specific polymerase chain reaction (MSP) assay for its relevance in the determination of MGMT methylation status. MGMT promoter hypermethylation analysis employing MSP was performed on 25 primary glioblastoma samples and 7 cell lines, and compared with the more robust direct promoter sequencing that profiled the methylation status of 27 CpG sites within the MGMT promoter. In addition, the MGMT expression at the protein level was evaluated in the primary tumor samples using immunohistochemistry and in the cell lines using Western blotting analysis. Our MSP analyses yielded reproducible results, which were identical to the bisulfite sequencing data in all except one primary tumor that was negative on MSP. A poor correlation existed between the immunohistochemical staining results and the methylation status of the MGMT promoter in primary glioblastoma samples. Neither MSP-MGMT methylation nor immunohistochemical MGMT expression had prognostic implications in this small and non-uniform group of patients. In all of the cell lines with loss of MGMT expression, signals of methylated DNA were detected by MSP. Our data support the feasibility and reliability of MSP analysis, which could be routinely implemented in the diagnostic setting.     

CpG hypermethylation of promoter region and inactivation of E-cadherin gene in human bladder cancer

Several studies have shown that E-cadherin expression is lost during malignant transformation. We hypothesized that CpG methylation in the promoter region may inactivate the expression of the E-cadherin gene in human bladder cancer. Normal and bladder cancer samples from 51 patients were compared in terms of E-cadherin gene expression and methylation status by immunohistochemistry, methylation-specific polymerase chain reaction (MSP), and bisulfite genome-sequencing techniques. Ten different CpG sites (nt 863, 865, 873, 879, 887, 892, 901, 918, 920, and 940) in the promoter region were studied. Thirty-five of 51 (69%) bladder cancer samples lacked E-cadherin expression, whereas only six of 51 (12%) normal bladder samples lacked E-cadherin immunoreactivity. MSP analysis of bladder cancer samples suggested that 43 of 51 (84%) showed methylation of the promoter region, whereas only 12 of 51 (24%) normal bladder samples showed hypermethylation. Sodium bisulfite genome-sequencing analysis revealed that of 10 CpG sites, two sites (nt 892 and nt 940) showed 100% methylation in all the cancer samples analyzed. Other CpG sites were partially methylated (47-91%). Normal tissue showed only 12% methylation (range, 1-33%) on various CpG sites. Also supporting these data, E-cadherin-negative bladder cancer cell lines restored expression of the E-cadherin gene after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The present study showed that CpG hypermethylation was an important mechanism of E-cadherin gene inactivation in bladder cancer and also that specific CpG sites consistently presented higher methylation levels than others. These findings may provide a better strategy for the diagnosis and management of bladder cancer.     

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.     

Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene

Over 50% of human genes are associated with CpG islands and DNA methylation within such CpG islands has been clearly correlated with inhibition of expression. Whereas changes in DNA methylation play a key role in a number of human diseases, in particular cancer, in normal DNA CpG islands are nearly always methylation free, regardless of the expression status of the associated gene. Only limited evidence supports a role for DNA methylation in controlling tissue-specific expression in adult somatic tissue. Loss of expression of the MCJ gene has previously been linked to increased chemotherapeutic drug resistance in ovarian cancer. We report that loss of expression of MCJ in drug-resistant ovarian cancer cell lines depends on methylation of a CpG island within its first exon, but is independent of methylation within the promoter region. Furthermore, cell type-specific expression of the MCJ gene in normal cells also depends on the methylation status of the CpG island within its first exon. The MCJ CpG island is methylated and the gene is not expressed in cells of epithelial origin, but unmethylated and expressed in cells of lymphocyte or fibroblast origin. Chromatin immunoprecipitation assays determined that MCJ CpG island methylation was associated with loss of histone acetylation in ovarian epithelial cells compared with unmethylated fibroblast cells. Reduced acetylation was observed not only within the CpG island, but also within the promoter region, suggesting that CpG island methylation may direct alterations in chromatin structure within the promoter region, leading to gene inactivation.     

Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma

MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 MN/CA9 -positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at -74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at -74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at -74 bp. These findings suggest that hypomethylation of the CpG at -74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC.     

3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性

目的对3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性进行评估.方法通过甲基化特异性多聚酶链反应法(MSP)结合DNA测序来确定3株肺腺癌细胞株(A549,SH77和SPE-A-1)中9个基因的启动子CpG岛的甲基化状态:APC,CDH13,DAPK1,MGMT,MYOD1,p16INK4a,p73,RAR-β和WT1基因.同时采用半定量RT-PCR的方法来量度下列5个基因的表达:CDH13,MGMT,MYOD1,RAR-β和WT1基因.结果在9个受检基因之中,下列4个基因在三株细胞中均呈同样的甲基化谱式:仅有去甲基化:APC和DAPK1,以及兼有甲基化和去甲基化:p73和p16INK4a.而其余5个基因则表现为不同的甲基化模式.尽管,这5个基因的表达谱式大体上符合其启动子CpG岛甲基化的表达静息的原则,个别的例外亦是有的.这提示着与DNA甲基化状态无关的机制可能也会参与该基因的表达调控.结论肺癌细胞株中的肿瘤相关基因的启动子CpG岛的过甲基化可与该基因的表达状态有较高的相关性.     

宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响

背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31,STK31)基因在人类多种癌症中扮演重要角色,且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响;病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响,以及不同种类甲基转移酶(DNA methyltransferases,DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒,分别感染人乳头瘤病毒(human papillomavirus,HPV)阴性宫颈癌细胞系HT-3及C33A,获得稳定转染的细胞系;采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing,BGS)和甲基化特异性PCR (methylation-specific PCR,MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态;RT-PCR及蛋白［质］印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态,STK31 mRNA及蛋白质表达阳性;2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态,STK31 mRNA及蛋白质表达缺失;与未感染慢病毒HT-3和C33A细胞系比较,外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低,其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DNMT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达,差异有统计学意义(t=5.997,P<0.001;t=6.743,P<0.001;t=7.926,P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(t=7.451,P<0.001;t=2.451,P<0.05);DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134,P<0.001)。DNMT3LmRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低,低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。