Hypermethylation of CpG island in O^6-methylguanine-DNA methyltransferase gene was associated with K-rasG to A mutation in colorectal tumor

AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas,62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutantallele-specific amplification was used to detect K-rasG to A point mutation in codon 12.RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation,respectively (P = 0.027, P＜0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P = 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group to a cysteine residue in its active site and became inactive.The chemical carcinogen N-nitroso compounds(NOCs)can directly bind to the DNA and induce the O_(6)-methylguanine adducts,which is an important cause of gene mutation and tumorigenesis.However,the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis,especially in the initiation phase,remains largely unclear.AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODS We established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)or N-methyl-N-nitroso-urea(MNU)treatment.Cell proliferation,colony formation,soft agar,cell migration,and xenograft assays were used to verify the malignant phenotype.By using quantitative real-time polymerase chain reaction(qPCR)and Western blot analysis,we detected the MGMT expression in malignant transformed cells.We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry.MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR.The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.RESULTS We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment.Moreover,we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells.Inhibition of the MGMT expression by O_(6)-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes.Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU.Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues,but downregulated in the gastric cancer tissues.CONCLUSION Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation.The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis,which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.

O^6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY AND SENSITIVITY OF 20 CHINESE TUMOR CELL STRAINS TO 1-(4-AMINO-2-METHYL-5-PYRIMIDINYL) METHYL-3-(2-CHLOROETHYL)-3-NITROSOUREA

O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl-5-pyrimidinyl) methyl-3- ( 2-chloroethyl)-3-nitrosourea (ACNU) of 20 Chinese tumor cell strains were assayed. A linear response between MGMT activity and ACNU sensitivity (D10) was observed. The lower the MGMT activity In the cells, the more the sensitivity to ACNU killing. It suggested that assay of MGMT activity in tumor biopsy could be used as a guide to predict the effectiveness of ACNU treatment in chemotherapy of human cancer.

Expression of O⁶-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.

Temozolomide and radiotherapy in newly diagnosed glioblastoma patients:O^6-methylguanine-DNA methyltransferase (MGMT) promotor methylation status and Ki-67 as biomarkers for survival and response to treatment

Objective:This phase II study aimed at investigating the correlation between O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and protein expression,together with Ki-67 labeling index (LI),to response,time to progression (TTP),and overall survival (OS) in newly diagnosed glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ) concomitant with and adjuvant to radiotherapy (RT).Methods:From June 2005 to August 2008,34 adult patients (18-65 years),PS ≥70,with newly diagnosed GBM received TMZ 75 mg/m2 plus RT up to 60 Gy,followed by TMZ 175 mg/m2 5 days every 4 weeks for 12 doses.MGMT Methylation-specific PCR assay,MGMT protein expression,and Ki-67 expression using immunohistochemistry (IHC) were performed on the tissue blocks.The patients were followed by MRI while MR spectroscopy (MRS) was performed for the stable cases or to confirm progression and accordingly Bevacizumab 10 mg/kg every 2 weeks was added to 7 patients till further progression was proved.Results:31 cases were evaluable,12 (38.7%) had unmethylated MGMT,while 19 (61.3%) were methylated.Seventeen cases (55%) were MGMT immunonegative while 14 cases (45%) were immunopositive.The cut off value of Ki-67 LI in relation to survival was 17%,where 15 were < 17% (48.4%),and 16 were ≥ 17% (51.6%).Response evaluation started after the second dose of the adjuvant TMZ and was repeated every 2 months.The overall disease control rate (ODC) was 74.2%,where 2 patients had complete response (CR),14 had partial response (PR),and 7 had stable disease (SD),while 8 (25.8%) had progressive disease (PD).The ODC was significantly higher among methylated patients and in those with Ki-67 < 17% (P=0.0003).The median overall TTP was 12 months and the median OS was 20 months for all the patients including those who received Bevacizumab for some stable cases or as a salvage treatment in patients with good PS,the MGMT-methylated patients had a higher median TTP of 13 months (range 8 to 18 months,95% CI of 9.36 to 12.9),and OS of 24 months (range 12 to 31 months,95% CI of 16.1 to 21.32),while the unmethylated patients had a median TTP of 6.5 months and a median OS of 12 months,such correlations were highly significant (P=0.0001).MGMT immunoexpression failed to show significant correlation with MGMT promotor methylation or the outcome of the patients.Patients with Ki-67 < 17% had a median TTP of 16 months and median OS of 24 months compared to 7 and 12.5 months respectively for the patients with Ki-67 ≥17%.Significant correlation was found between the ODC,TTP,and OS with age < 52,near total excision,and TMZ doses received ≥ 10.The commonest grade 3 and 4 toxicities was neutropenia recorded in 3 patients (9.67%),thrombocytopenia in 4 patients (12.9%),and one patient with G3 nausea,vomiting,and constipations (3%),all were medically manageable.Conclusion:MGMT promotor methylation status and Ki-67 LI (but not the MGMT protein expression),could serve as prognostic markers for survival,also MGMT could identify the newly diagnosed GBM patients who will have better response to TMZ.

O^6-甲基鸟嘌呤-DNA-甲基转移酶基因表达与胃癌发生的关系

目的研究人胃癌组织O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)基因的表达特点及其与胃癌发生的关系。方法应用加强型原位杂交方法及寡核苷酸探针高效标记技术对76例胃癌组织、73例癌旁组织、20例正常胃组织进行MGMT基因标记检测,同时应用自动图像分析系统进行定量分析。结果胃癌组织MGMT基因阳性表达率为40.8%(31/76),低于正常胃组织的60.0%(12/20),MGMT表达强阳性率为32.3%(10/31),也明显低于正常组织的83.3%(10/12),差异均有显著性意义(P<0.01或P<0.05);癌旁组织MGMT基因阳性表达率为46.6%(34/73),MG-MT表达强阳性率为41.1%(14/34),与胃癌组织差异无显著性意义。定量分析示,胃癌组织MGMT阳性表达的积分吸光度、阳性单位值明显低于正常组织(P<0.05)。胃癌组织与癌旁组织、早期胃癌与进展期胃癌组织、癌旁不同程度不典型增生之间MGMT表达无明显差异。结论人体胃组织存在高度表达的MGMT基因,胃癌发生与MGMT基因表达水平降低有关,MGMT基因异常表达是胃癌形态学改变出现之前的早期行为。

O^6-甲基鸟嘌呤-DNA甲基转移酶基因多态性与胃癌遗传易感性的关系

目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因第3外显子上第84和第53密码子上的多态性改变与胃癌易感性的关系以及与环境因素在胃癌发生中的交互作用。方法应用以人群为基础的病例对照研究,采用PCR-RFLP技术检测191例胃癌病例组与251例健康对照组MGMT-Leu53Leu和MGMT-Leu84Phe多态改变的频率。结果MGMT-Leu84Phe各基因型在胃癌病例组(CC 74.3%,CT 23.6%,TT 2.1%)和对照组(CC 74.1%,CT 23.5%,TT 2.4%)的分布情况与MGMT-Leu53Leu基因型相似,差异无显著性;MGMT-Leu53Leu各基因型在胃癌病例组中的分布分别为CC型(75.4%)、CT型(21.5%)、TT型(3.1%),对照组分布为CC型(72.1%)、CT型(24.7%)、TT型(3.2%),两组比较差异无显著性;分别按年龄、性别、吸烟、饮酒、H.pylori感染以及胃癌家族史等因素分层分析也没有发现两个位点的突变基因型与胃癌遗传易感性的相关性。结论结果显示MGMT-Leu53Leu、Leu84Phe与胃癌的发生没有显著关联。

O^6-methylguanine-DNA methyltransferase gene expression confers alkylation resistance to tumor cells

The emergence of drug resistance is a major obstacle limiting the successful chemotherapy of malignant tumors. Our previous studies have demonstrated that O^6-methylguanine-DNA methyltransferase (MGMT, EC 2.1.1.63) is an important contributor to tumor cellular resistance toward mono- and bifunctional alkylating agents, such as 1- (4-amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl) -3-nitrosourea (ACNU) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Mechanistically, MGMT can specifically remove the induced alkyl groups at the O^6-position of guanine which finally would lead to a G→ A transition or a lethal DNA interstrand cross-link unless repaired. Cells

Promoter methylation status of hMLH1,MGMT,and CDKN2A/p16 in colorectal adenomas

AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.

Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

Protective effect of O^6-methylguanine-DNA-methyltransferase on mammalian cells

Background O^6-methylguanine-DNA-methyltransferase （MGMT） is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells. Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells （PBMCs） via liposome, then assayed for gene expression at RNA and protein levels. MIF assay was used to check the drug resistance of cells transfected with MGMT gene. Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC. Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

G156A六氧甲基鸟嘌呤-DNA-甲基转移酶原核表达载体的构建和表达

目的 :构建原核表达载体pPROEXHTb G15 6AMGMT ,观察突变型G15 6AMGMT在大肠杆菌中的表达情况。方法 :通过基因重组技术将G15 6AMGMT亚克隆至pPROEXHTb原核表达载体 ,在大肠杆菌DH5α中经异丙基硫代 β D半乳糖苷 (IPTG)诱导表达。 结果 :经PCR及酶切分析证实 ,成功构建了pPROEXHTb G15 6AMGMT原核表达载体 ,经SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)显示重组克隆化基因pPROEXHTb G15 6AMGMT在大肠杆菌DH5α中有表达。结论 :G15 6AMGMT在大肠杆菌DH5α中有表达 ,为获取突变型MGMT工程蛋白奠定了基础。

MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acidinduced spina bifida aperta

O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.

Temozolomide resistance in high grade gliomas

High grade gliomas are always the research focus in the field of neurosurgery due to their poor prognosis despite the current standard therapeutic regimen of surgical resection followed by radiation therapy and chemotherapy. Alkylating agent temozolomide has been established as the standard chemotherapy while its resistance inevitable during treatment. This phenomenon seriously influences the prognosis of patients suffering from high grade gliomas. This review aims to elucidate temozolomide chemoresistance mechanisms through three chapters including O^6-methylguanine-DNA methyltransferase(MGMT) methylation, mismatch repair mutation and epigenetic regulation consisting of p21, chromatin and histone, Y-box binding protein-1 and micro RNAs.

MGMT、hMLH1和hMSH2基因启动子甲基化状态对脑胶质瘤预后的影响

目的探讨脑胶质瘤DNA修复基因O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)和错配修复基因(MMR)(hMLH1、hMSH2)启动子甲基化状态及其对患者预后和烷化剂化疗敏感性的影响。方法采用甲基化特异性PCR(MSP)方法检测39例脑胶质瘤和6例正常脑组织MGMT、hMLH1和hMSH2基因启动子区的甲基化状态,免疫组化方法测定其蛋白表达。绘制Kaplan-merier生存曲线。结果脑胶质瘤组织MGMT、hMLH1和hMSH2基因启动子区甲基化发生率分别为46.2%、10.3%和20.5%,而正常脑组织相应基因启动子区未发生甲基化;三种基因启动子未甲基化模式与其对应蛋白表达模式相似。MGMT基因甲基化的脑胶质瘤患者存活率显著高于未甲基化者(P<0.05);MMR基因甲基化患者中MGMT基因甲基化与未甲基化者的生存期无统计学差异(P>0.05)。结论hMLH1、hMSH2及MGMT甲基化是脑胶质瘤发生过程中常见的分子事件;联合检测MGMT、hMLH1和hM-SH2基因启动子甲基化状态可判断脑胶质瘤患者的预后及其对烷化剂化疗的敏感性。

胃癌组织中MDR和MGMT基因表达及其相关意义

目的:检测胃癌组织多药耐药(multidrugresistance,MDR)基因和O6-甲基鸟嘌呤-DNA-甲基转移酶(O6-methylguanin-e-DNA-meyhyltransferase,MGMT)基因的表达,探讨MDR、MGMT基因表达的相关性及其在胃癌组织中的临床病理学意义。方法:应用加强型原位杂交技术通过核酸探针对76例胃癌组织进行MDR、MGMT基因检测,同时应用自动图像分析系统进行定量分析。结果:76例胃癌组织中MDR基因阳性表达率为68·4%,积分吸光度为351·5±121·4,分别高于正常组织(40·0%,168·4±68·8);MGMT阳性表达率为40·8%,积分吸光度为161·4±78·84,分别低于正常组织(60·4%,332·9±118·2)。生存时间5年以下组MDR和MGMT基因表达分别高于5年以上组,rs分别为0·250和0·287,P值分别为0·010和0·008;白细胞计数>3×109L-1组MGMT基因表达高于<3×109L-1组,rs=0·354,P=0·001;有淋巴结转移组MDR基因表达高于无淋巴结转移组,rs=0·280,P=0·004;术前化疗组MDR表达明显高于未化疗组,rs=0·300,P=0·002。结论:胃癌组织存在原发性和获得性MDR基因耐药的双重性,而MGMT基因主要是原发性,两种基因表达可能是2个相互独立的事件,在胃癌预后和骨髓抑制的判断上有一定意义。

燃煤污染型地方性氟中毒患者血细胞hMLH1与MGMT mRNA表达改变

目的探讨燃煤污染型地方性氟中毒(简称燃煤型地氟病)对血液中错配修复基因(hMLH1)及O(6)-甲基鸟嘌呤DNA甲基转移酶基因(MGMT)mRNA表达水平的影响。方法在贵州省毕节地区燃煤型地氟病区抽取45例患者,按其氟斑牙和氟骨症严重程度分为轻、中、重度组各15例。在毕节地区非病区抽取15名村民作为对照组。实时荧光定量PCR技术检测各组血样本MGMT、hMLH1mRNA水平。结果燃煤型氟中毒轻、中、重度3个组中hMLH1及MGMT mRNA水平与对照组相比明显降低,差异有统计学意义(P<0.05),且随着氟中毒程度的加重,其降低程度更加明显。组间两两比较,随着中毒程度加重,表达水平明显降低(P<0.05)。结论 MGMT与hMLH1mRNA水平在燃煤型氟中毒所造成的DNA损伤及修复机制中可能有负面作用。

反义RNA调节MGMT基因表达逆转肿瘤细胞对嘧啶亚硝脲的耐药性

目的：抑制细胞内的Ｏ６－甲基鸟嘌呤－ＤＮＡ甲基转移酶（ＭＧＭＴ）基因表达水平，逆转肿瘤细胞对嘧啶亚硝脲的耐药性。方法：本实验构建了三个表达ＭＧＭＴ反义ＲＮＡ的逆转录病毒载体（ｐＬＭＴＳＮＡＳ，ｐＬＭ５ＳＮＡＳ和ｐＬＭ３ＳＮＡＳ），并用它们转导ＨｅＬａＳ３肿瘤细胞，观察细胞在转导前后ＭＧＭＴ基因表达水平及其对嘧啶亚硝脲（ＡＣＮＵ）抗药性的变化。结果：研究发现针对ＭＧＭＴｍＲＮＡ５端的反义ＲＮＡ能够有效地降低ＨｅＬａＳ３细胞的ＭＧＭＴ基因表达水平（为对照细胞的３０％～４０％），并使细胞对ＡＣＮＵ的敏感性提高４．６倍；针对ＭＧＭＴｍＲＮＡ全长的反义ＲＮＡ，也能在一定程度上调节细胞的ＭＧＭＴ基因表达水平并增加细胞对ＡＣＮＵ的敏感性，而针对３端序列的反义ＲＮＡ对ＭＧＭＴ基因无调节作用。转导细胞对ＡＣＮＵ耐药性的变化与ＭＧＭＴ基因表达水平的抑制程度密切相关。结论：合适的反义ＲＮＡ能够有效地调节ＭＧＭＴ基因表达水平。

Chromosome 1p/19q status combined with expression of protein improves the diagnostic and prognostic evaluation of oligodendrogliomas

Background Our previous study confirmed that oligodendrogliomas had higher frequency of chromosome 1p/19q deletion. In order to improve the diagnostic criteria and to predict the prognosis of oligodendroglioma patients, the status of chromosome 1 p/19q deletion, the methylation of O6-methylguanine-DNA methyltransferase （MGMT）, and the expression of p53 protein were evaluated and investigated in relation to patients＇ outcomes.Methods Methylation of MGMT in 73 cases was analyzed by nested methylation-specific PCR （MSP）. The levels of MGMT and p53 protein were tested with immunohistochemistry. Pearson＇s chi-square test and Fisher＇s exact test were used. Multivariate and Kaplan-Meier analysis were performed to determine patients＇ outcomes.Results Both oligodendrogliomas and astrocytic gliomas exhibited frequent methylation of MGMT. However, the results of MSP did not completely correspond to that of the immunohistochemical staining for MGMT. The expression of p53 protein was more frequently observed in patients without a 1 p or 19q deletion in anaplastic oligodendrogliomas （=0.032,0.025）. In low-grade oligodendrogliomas, methylation of MGMT was more frequent in patients with 1 p/19q deletion than in patients with 1p/19q intact （P=0.038）. Patients with oligodendrogliomas with 1p/19q loss of heterozygosity and p53-negative showed a longer progression-free survival.Conclusion Detection of chromosome 1p/19q status combined with p53 protein immunohistochemistry might be beneficial to improve the pathological diagnosis and to determine the prognosis of patients with oligodendrogliomas.

燃煤污染型砷中毒人群MGMT基因甲基化、转录及表达的研究

目的了解O6-甲基鸟嘌呤DNA甲基转移酶基因(MGMT基因)启动子区甲基化、转录及表达以及DNA甲基转移酶1(DNMT1)的转录及表达与砷中毒的关系。方法采集68例燃煤污染型砷中毒(以下简称砷中毒)患者(轻度24例、中度28例、重度16例)及23例非病区居民外周血。用甲基化特异性PCR法(MSP)检测MGMT基因启动子区甲基化,实时荧光定量PCR(FQ-PCR)法检测MGMT及DNMT1 mRNA的水平。利用自愿手术治疗的砷中毒患者皮肤组织标本(61例,其中34例为一般病变,21例为癌前病变,6例为癌变)和对照皮肤组织标本(15例),以免疫组织化学(IHC)法检测砷中毒患者及对照皮肤组织中MGMT及DNMT1蛋白的表达。结果 MGMT基因启动子区甲基化阳性率与砷中毒程度有关(χ2=13.739,P<0.01)。砷中毒组MGMT mRNA表达水平与对照组比较,差异无统计学意义(P>0.05);MGMT基因启动子区甲基化患者和非甲基化患者之间MGMT mRNA和DNMT1 mRNA表达差异无统计学意义(P>0.05)。但轻、中度患者DNMT1 mRNA表达明显低于对照组(P<0.01);癌前病变组和癌变组皮肤组织中MGMT蛋白表达明显低于对照组(P<0.01),与皮肤损害程度有关(rs=-0.446,P<0.01);MGMT基因启动子区甲基化患者MGMT蛋白表达明显低于非甲基化组(P<0.05)。砷中毒组皮肤组织中DNMT1蛋白表达强于对照组(P<0.01),并与皮肤损害程度有关(rs=0.740,P<0.01);且MGMT基因启动子区甲基化患者组织中DNMT1蛋白表达明显高于非甲基化组(P<0.05)。结论 MGMT基因启动子区高甲基化抑制了燃煤污染型砷中毒患者皮肤组织中MGMT蛋白的表达,且DNMT1蛋白的高表达参与了此抑制过程。