采用蛋白质组学技术筛选鼻咽癌细胞的甲基化基因
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摘要
鼻咽癌(NPC)是我国南方地区常见的一种恶性肿瘤,其发病机制还不清楚。DNA甲基化修饰是基因表达调控的重要表观遗传学机制之一,DNA甲基化修饰导致抑瘤基因等的沉默在癌变过程中发挥重要的作用。因此,筛选鼻咽癌的甲基化失活基因将有助于揭示鼻咽癌的发病机制,具有重要的理论和应用价值。
本研究以高转移的鼻咽癌细胞系5-8F为对象,采用蛋白质组学技术筛选鼻咽癌细胞的甲基化失活基因。首先用MTT比色法和流式细胞仪分析确定去甲基化药物5-杂氮-2'-脱氧胞苷(5-aza-2-dC)处理5-8F细胞的最适浓度。然后应用双向凝胶电泳(2-DE)技术分离5-aza-2-dC处理与未处理5-8F细胞的蛋白质;利用PDQuest图像分析软件比较两者2-DE图谱的异同,识别差异表达的蛋白质点;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达的蛋白质;采用Western blot和RT-PCR检测差异蛋白质nm23-H1的表达水平;再采用甲基化特异性PCR(MSP)检测5-8F细胞中nm23-H1基因的甲基化。主要结果如下:
1.采用蛋白质组学技术建立了5-aza-2-dC处理与未处理鼻咽癌细胞株5-8F细胞蛋白质的2-DE图谱,识别了49个差异表达的蛋白质点,鉴定了33个差异表达的蛋白质,其中包括nm23-H1在内的15个蛋白质在5-aza-2-dC处理后表达上调,而18个蛋白质表达下调。15个5-aza-2-dC处理后表达上调的基因可能是5-8F细胞中的甲基化沉默基因。
2.Western blot结果显示:在5-aza-2-dC处理5-8F细胞后,nm23-H1蛋白质的表达上调,这与蛋白质组学的研究结果一致。
3.MSP结果显示,5-aza-2-dC处理5-8F细胞后,nm23-H1基因启动子甲基化水平降低,而非甲基化水平增加。RT-PCR结果显示,5-aza-2-dC处理后,nm23-H1基因mRNA的表达明显上调。结果说明,nm23-H1基因是5-8F细胞中的甲基化沉默基因。
本研究首次采用蛋白质组学技术,从蛋白质组水平筛选鼻咽癌细胞的甲基化失活基因,鉴定了15个候选的鼻咽癌细胞的甲基化失活基因,证实鼻咽癌细胞的nm23-H1基因存在甲基化沉默,可能在鼻咽癌发病中具有重要作用。本文结果为研究鼻咽癌的甲基化失活基因提供了实验依据,为筛选与肿瘤发生发展相关的甲基化失活基因提供了新思路。
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China. However, the mechanism underlying the pathogenesis of NPC remains unclear. DNA methylation is one of the epigenetics mechanisms of gene expression regulation. And promoter methylation of tumor suppression genes plays a critical role in carcinogenesis. Therefore, screening methylation silenced genes may help to reveal the pathogenesis of NPC, which has important theoretical and clinical value.
Proteomic analysis was performed to screen for methylation silenced genes in NPC cell line 5-8F with high metastatic potential. MTT assay and flow cytometry were used to determine the optimum concentration of demethylating agent 5-aza-2-dC, which was used to treat 5-8F cells. Two-dimensional gel electrophoresis (2-DE) was performed to separate the proteins of treated and untreated 5-8F cells with 5-aza-2-dC. PDQuest software was used to analyze 2-DE images to determine the differential expression proteins between the treated and untreated 5-8F cells. MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot and RT-PCR analysis were used to examine the expression levels of the differential expression protein nm23-H1. Methylation-specific PCR (MSP) was performed to examine the methylation status of nm23-H1 gene in 5-8F cells. The results were as following: (1) 2-DE reference patterns of 5-8F cells treated and untreated with 5-aza-2-dC were established. Forty-nine differential protein spots were found between treated and untreated 5-8F cells, and thirty-three non-redundant proteins were identified. Among them, 15 proteins including nm23-H1 were up-regulated, and 18 proteins were down-regulated after treated with 5-aza-2-dC. Encoding genes of 15 up-regulated proteins may be methylation silenced genes in 5-8F cells; (2) Western blot analysis showed that the expression of nm23-H1 was up-regulated in 5-8F treated with 5-aza-2-dC, which was consistent with the result of proteomic analysis; (3) MSP showed that methylation level of nm23-H1 promoter was decreased, while unmethylation level of that was increased after treated with 5-aza-2-dC, and RT-PCR showed that nm23-H1 mRNA expression was increased obviously after treated with 5-aza-2-dC, which suggested that nm23-H1 is methylation silenced gene in 5-8F.
In this study, it is the first time using proteomic analysis to screen for methylation silenced gene in NPC. 15 candidate methylation silenced genes in NPC cells were identified, and our data suggested that nm23-H1 is a methylation silenced gene in NPC cells, which may play a critical role in the pathogenesis of NPC. The results will be helpful for further investigating methylation silenced genes in NPC, and provide a new idea for screening methylation silenced genes involved in the pathogenesis of tumor.
本研究以高转移的鼻咽癌细胞系5-8F为对象,采用蛋白质组学技术筛选鼻咽癌细胞的甲基化失活基因。首先用MTT比色法和流式细胞仪分析确定去甲基化药物5-杂氮-2'-脱氧胞苷(5-aza-2-dC)处理5-8F细胞的最适浓度。然后应用双向凝胶电泳(2-DE)技术分离5-aza-2-dC处理与未处理5-8F细胞的蛋白质;利用PDQuest图像分析软件比较两者2-DE图谱的异同,识别差异表达的蛋白质点;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达的蛋白质;采用Western blot和RT-PCR检测差异蛋白质nm23-H1的表达水平;再采用甲基化特异性PCR(MSP)检测5-8F细胞中nm23-H1基因的甲基化。主要结果如下:
1.采用蛋白质组学技术建立了5-aza-2-dC处理与未处理鼻咽癌细胞株5-8F细胞蛋白质的2-DE图谱,识别了49个差异表达的蛋白质点,鉴定了33个差异表达的蛋白质,其中包括nm23-H1在内的15个蛋白质在5-aza-2-dC处理后表达上调,而18个蛋白质表达下调。15个5-aza-2-dC处理后表达上调的基因可能是5-8F细胞中的甲基化沉默基因。
2.Western blot结果显示:在5-aza-2-dC处理5-8F细胞后,nm23-H1蛋白质的表达上调,这与蛋白质组学的研究结果一致。
3.MSP结果显示,5-aza-2-dC处理5-8F细胞后,nm23-H1基因启动子甲基化水平降低,而非甲基化水平增加。RT-PCR结果显示,5-aza-2-dC处理后,nm23-H1基因mRNA的表达明显上调。结果说明,nm23-H1基因是5-8F细胞中的甲基化沉默基因。
本研究首次采用蛋白质组学技术,从蛋白质组水平筛选鼻咽癌细胞的甲基化失活基因,鉴定了15个候选的鼻咽癌细胞的甲基化失活基因,证实鼻咽癌细胞的nm23-H1基因存在甲基化沉默,可能在鼻咽癌发病中具有重要作用。本文结果为研究鼻咽癌的甲基化失活基因提供了实验依据,为筛选与肿瘤发生发展相关的甲基化失活基因提供了新思路。
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China. However, the mechanism underlying the pathogenesis of NPC remains unclear. DNA methylation is one of the epigenetics mechanisms of gene expression regulation. And promoter methylation of tumor suppression genes plays a critical role in carcinogenesis. Therefore, screening methylation silenced genes may help to reveal the pathogenesis of NPC, which has important theoretical and clinical value.
Proteomic analysis was performed to screen for methylation silenced genes in NPC cell line 5-8F with high metastatic potential. MTT assay and flow cytometry were used to determine the optimum concentration of demethylating agent 5-aza-2-dC, which was used to treat 5-8F cells. Two-dimensional gel electrophoresis (2-DE) was performed to separate the proteins of treated and untreated 5-8F cells with 5-aza-2-dC. PDQuest software was used to analyze 2-DE images to determine the differential expression proteins between the treated and untreated 5-8F cells. MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot and RT-PCR analysis were used to examine the expression levels of the differential expression protein nm23-H1. Methylation-specific PCR (MSP) was performed to examine the methylation status of nm23-H1 gene in 5-8F cells. The results were as following: (1) 2-DE reference patterns of 5-8F cells treated and untreated with 5-aza-2-dC were established. Forty-nine differential protein spots were found between treated and untreated 5-8F cells, and thirty-three non-redundant proteins were identified. Among them, 15 proteins including nm23-H1 were up-regulated, and 18 proteins were down-regulated after treated with 5-aza-2-dC. Encoding genes of 15 up-regulated proteins may be methylation silenced genes in 5-8F cells; (2) Western blot analysis showed that the expression of nm23-H1 was up-regulated in 5-8F treated with 5-aza-2-dC, which was consistent with the result of proteomic analysis; (3) MSP showed that methylation level of nm23-H1 promoter was decreased, while unmethylation level of that was increased after treated with 5-aza-2-dC, and RT-PCR showed that nm23-H1 mRNA expression was increased obviously after treated with 5-aza-2-dC, which suggested that nm23-H1 is methylation silenced gene in 5-8F.
In this study, it is the first time using proteomic analysis to screen for methylation silenced gene in NPC. 15 candidate methylation silenced genes in NPC cells were identified, and our data suggested that nm23-H1 is a methylation silenced gene in NPC cells, which may play a critical role in the pathogenesis of NPC. The results will be helpful for further investigating methylation silenced genes in NPC, and provide a new idea for screening methylation silenced genes involved in the pathogenesis of tumor.
引文
[1] Jones P.A, Laird P.W. Cancer epigenetics comes of age. Nat Genet, 1999, 21(2): 163-167
[2] Jones PA. Epigenetics in carcinogenesis and cancer prevention. Ann N Y Acad Sci, 2003, 983: 213-219
[3] Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med, 2003, 349(21):2042-2054
[4] Costello JF, Fruhwald MC, Smiraglia DJ,et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000, 24(2): 132- 138
[5] Costello JF, Plass C. Methylation matters. J Med Genet, 2001, 38(5):285-303
[6] Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science, 2001, 293(5532):1068-1070
[7] Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci U S A, 1993, 90(24): 11995-11999
[8] Karpf AR, Jones DA. Reactivating the expression of methylation silenced genes in human cancer.Oncogene, 2002,21(35):5496-5503
[9] Baylin SB, Herman JG, Graff JR, et al. Alternations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res, 1988, 72: 141-196
[10] Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Letter, 2003, 190: 125-133
[1 l]Merlo A, Herman JG, Mao L, et al. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTSl in human cancers. Nat Med, 1995, 1(7):686-692
[12]Hsu HS, Wen CK, Tang YA,et al. Promoter hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in nonsmoking lung cancer. Clin Cancer Res, 2005, 11(15):5410-5416
[13]Schildhaus HU, Krockel I, Lippert H, et al. Promoter hypermethylation of p16INK4a, E-cadherin, 06-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach. Int J Oncol, 2005, 26(6):1493-1500
[14]Laird PW,Jackson-Grusby L,Fazeli A,et al.Suppression of intestinal neoplasia by DNA hypomethylation.Cell,1995,81(2):197-205
[15]Jeronimo C,Henrique R,Hoque MO,et al.Quantitative RARbeta2 hyperme-thylation:a promising prostate cancer marker.Clin Cancer Res,2004,10(12 Pt 1):4010-4014
[16]Belinsky SA,Nikula KJ,Palmisano WA,et al.Aberrant methylation of p16(INK4a)is an early event in lung cancer and a potential biomarker for early diagnosis.Proc Natl Acad Sci U S A,1998,95(20):11891-11896
[17]Balch C,Montgomery JS,Paik HI,et al.New anti-cancer strategies:epigenetic therapies and biomarkers.Front Biosci,2005,10:1897-1931
[18]Fraga MF,Esteller M.DNA methylation:a profile of methods and applications.Biotechniques,2002,33(3):632,634,636-649
[19]Rush LJ,Raval A,Funchain P,et al.Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets.Cancer Res,2004,64(7):2424-2433
[20]Pompeia C,Hodge DR,Plass C,et al.Microarray analysis of epigenetic silencing of gene expression in the KAS-6/1 multiple myeloma cell line.Cancer Res,2004,64(10):3465-3473
[21]Shi H,Yan PS,Chen CM,et al.Expressed CpG island sequence tag microarray for dual screening of DNA hypermethylation and gene silencing in cancer cells.Cancer Res,2002,62(11):3214-3220
[22]Wilkins MR,Williams KL,Hohstrasser DF.(ed)Proteome Research:New Frontiers in Fuctional Genomics.New York,Spring,1997
[23]陈主初,梁宋平,肖志强.肿瘤蛋白质组学.长沙:湖南科学技术出版社,2002
[24]Kwong J,Lo KW,To KF,et al.Promoter hypermethylation of multiple gene in nasopharyngeal carcinoma.Clin Cancer Res,2002,8(1):131-137
[25]Lo KW,Kwong J,Hui AB,et al.High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma.Cancer Res,2001,61(10):3877-3881
[26]Zhou L,Jiang W,Ren C,et al.Frequent hyperrnethylation of RASSF1A and TSLC1,and high viral load of Epstein-Barr virus DNA in nasopharyngeal carcinoma and matched minor-adjacent tissues.Neoplasia,2005,7(9):809-815
[27]宋立兵,鄢践,简少文,等.鼻咽癌细胞亚株不同成瘤与转移潜能的分子机制.癌症,2002,21(2):158-162
[28]宋立兵,汪慧民,曾木圣.鼻咽癌细胞SUNE-1的异质性研究.癌症,1998,17(5):324-327
[29]姚开泰.鼻咽癌研究的回顾与展望.湖南医科大学学报,1995,20(2):99-102
[30]Shao JY,Zeng WF,Zeng YX,et al.Molecular genetic progression on nasopharyngeal carcinoma.Ai Zheng,2002,21(1):1-10
[31]Sidagis J,Ueno K,Tokunaga M,et al.Molecular epidemiology of Epstein-Barr virus(EBV)in EBV-related malignancies.Int J Cancer,1997,72(1):72-76
[32]Niedobitek G.Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma.Mol Pathol,2000,53(5):248-254
[33]Choi PH,Suen MW,Huang DP,et al.Nasopharyngeal carcinoma:genetic changes,Epstein-Barr virus infection,or both.A clinical and molecular study of 36 patients.Cancer,1993,72(10):2873-2878
[34]Lo k,Huang PW,Lee CK.Genetic changes in nasopharyngeal carcinoma.Clin Med J(Engl),1997(7),110:548-549
[35]Vogelstein B,Kinzler KW.The multistep nature of cancer.Trends Genet,1993,9(4):138-141
[36]Lo KW,Huang DP.Genetic and epigenetic changes in nasopharyngeal carcinoma.Semin Cancer Biol,2002,12(6):451-462
[37]Steeg PS,Bevilacqua G,Kopper L,et al.Evidence for a novel gene associated with low tumor metastatic p rotential.J Natl Cancer Inst,1988,80(3):200-204
[38]Freije JM,MacDonald NJ,Steeg PS.Differential gene expression in tumor metastasis:nm23.In:Gunthert U,Shlag PM,Birchmeier W,eds.Attempts to understand metastasis formation Ⅱ:regulatory factors.Berlin:Springer-Verlag,1996:215-232
[39]Qian M,Feng Y,Xu L,et al.Expression of antimetastatic gene nm23-H1 in epithelial ovarian cancer.Chin Med J(Engl),1997,110(2):142-144
[40]Busmanis I.Biomarkers in carcinoma of the cervix:emphasis on tissue-related factors and their potential prognostic factors.Ann Acad Med Singapore,1998,27(5):671-675
[41]Freije JM,MacDonald NJ,Steeg PS,et al.Nm23 and tumour metastasis:basic and translational advances.Biochem Soc Symp,1998,63:261-271
[42]Wang CS,Lin KH,Hsu YC,et al.Distant metastasis of gastric cancer is associated with elevated expression of the antimetastatic nm23 gene.Cancer Lett, 1998,128(1):23-29
[43]Boissan M, Lacombe ML. Nm23/NDP kinases in hepatocellular carcinoma. J Bioenerg Biomembr,2006 ,38(3-4):169-175
[44]Gazzeri S, Brambilla E, Negoescu A,et al. Overexpression of nucleoside diphosphate/kinase A/nm23-H1 protein in human lung tumors: association with tumor progression in squamous carcinoma. Lab Invest, 1996,74(1):158-167
[45]Niitsu N, Nakamine H, OkamotoM, et al. Expression of nm23-Hl is associated with poor prognosis in peripheral T-cell lymphoma. Br J Haematol, 2003, 123(4):621-630
[1]Feinberg AP,Gehrke CW,Kuo KC,et al.Reduced genomic 5-methylcytosine content in human colonic neoplasia.Cancer Res,1998,48(5):1159-1161
[2]De Smet C,De Backer O,Faraoni I,et al.The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.Proc Natl Acad Sci USA,1996,93(14):7149-7153
[3]Fang JY,Zhu SS,Xiao SD,et al.Studies on the hypomethylation of c-myc,c-Ha-ras oncogenes and histopathological changes in human gastric carcinoma.J Gastroenterol Hepatol,1996,11(11):1079-1082
[4]Jackson-Grusby L,Laird PW,Magge SN,et al.Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase.Proc Natl Acad Sci USA,1997,94(9):4681-4685
[5]Baylin SB,Herman JG.DNA hypermethylation in tumorigenesis:epigenetics joins genetics.Trends Genet,2000,16(4):168-174
[6]Clark SJ,Harrison J,Molloy PL.Spl binding is inhibited by(m)Cp(m)CpG methylation.Gene,1997,195(1):67-71
[7]Gaston K,Fried M.CpG methylation has differential effects on the binding of YY1 and ETS proteins to the bi-directional promoter of the Surf-1 and Surf-2genes.Nucl Acids Res,1995,23(6):901-909
[8]Umezawa A,Yamamoto H,Rhodes K,et al.Methylation of an ETS site in the intron enhancer of the keratin 18 gene participates in tissue-specific repression. Mol Cell Biol, 1997,17(9): 4885-4894
[9] Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol, 1998,18(11): 6538 -6547
[10] Nan X, Campoy FJ, Bird A. MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell,1997, 88(4): 471- 481
[11]Ng HH, Zhang Y, Hendrich B,et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet,1999, 23(1):58- 61
[12]Ng HH, Jeppesen P, Bird A. Active repression of methylated genes by the chromosomal protein MBD1 . Mol Cell Biol, 2000, 20(4): 1394-1406
[13]Jones PL, Veenstra GJ, Wade PA, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet, 1998 ,19(2): 187-191
[14]Bachman KE, Rountree MR, Baylin SB. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. J Biol Chem, 2001, 276(34): 32282 - 32287
[15]Fuks F, Hurd PJ, Wolf D,et al. The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem,2003, 278(6): 4035-4040
[16]Fuks F, Burgers WA, Godin N,et al. Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription. EMBO J,2001, 20(10): 2536-2544
[17] Robertson KD, Ait-Si-Ali S, Yokochi T, et al. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet, 2000,25(3): 338-342
[18] Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet, 2000, 25(3):269-277
[19]Schena M, Shalon D, Davis RW, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995,270(5235): 467-470
[20]Jackson-Grusby L, Beard C, Possemato R,et al. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet, 2001, 27(1): 31-39
[21]Kawai J, Hirotsune S, Hirose K,et al. Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method. Nucl Acids Res, 1993,21(24): 5604-5608
[22]Costello JF, Fruhward MC, Smiraglia D,et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000,24(2): 132-138
[23]Yan PS, Perry MR, Laux DE, et al. CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer. Clin Cancer Res, 2000,6(4): 1432-1438
[24]Yan PS, Chen CM, Shi H,et al. Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res, 2001, 61(23):8375-8380
[25]Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature, 2000,406(6797): 747-752
[26] Bender CM, Zingg JM, Jones PA. DNA methylation as a target for drug design. Pharm Res, 1998,15(2): 175-187
[27]Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation,Cell, 1980, 20(1): 85-93
[28]Ferguson AT, Vertino PM, Spitzner JR, et al. Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells. J Biol Chem 1997,272:32260-32266
[29]Bigey P, Knox JD, Croteau S, et al. Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase. J Biol Chem, 1999, 274(8):4594-4606
[30]Fournel M, Sapieha P, Beaulieu N, et al. Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16(ink4A) and p21(WAF/Cip1) by distinct mechanisms. J Biol Chem, 1999, 274(34): 24250-24256
[31] Cameron EE, Bachman KE, Myohanen S, et al . Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet,1999,21(1):103-107
[32]Leshin M. 5-Azacytidine and sodium butyrate induce expression of aromatase in fibroblasts from chickens carrying the henny feathering trait but not from wild-type chickens. Proc Natl Acad Sci USA, 1985, 82(9): 3005-3009
[33]Magdinier F, Wolffe AP. Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia. Proc Natl Acad Sci USA, 2001, 98(9): 4990-4995
[34]Bender CM, Gonzalgo ML, Gonzales FA,et al. Roles of cell division and gene transcription in the methylation of CpG islands.Mol Cell Biol,1999, 19(10):6690-6698
[35]Liang G, Gonzales FA, Jones PA,et al. Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine. Cancer Res, 2002, 62(4): 961-966
[36]Albanesi T, Polani S, Cozzi R, et al. DNA strand methylation and sister chromatid exchanges in mammalian cells in vitro. Mutat Res 1999,429(2) :23 9-48
[37] Chen RZ, Pettersson U, Beard C, et al. DNA hypomethylation leads to elevated mutation rates. Nature, 1998, 395(6697): 89-93
[38]Neidhart M, Rethage J, Kuchen S, et al. Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression. Arthritis Rheum, 2000, 43(12): 2634 -2647
[39] Wade PA. Methyl CpG binding proteins: coupling chromatin architecture to gene regulation. Oncogene, 2001, 20(24):3166-3173
[40]Liu PQ, Rebar EJ, Zhang L, et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem, 2001, 276(14): 11323-11334
[41] Plumb JA, Strathdee G, Sludden J, et al. Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLHl gene promoter. Cancer Res, 2000, 60(21): 6039-6044
[42] Strathdee G, MacKean MJ, Illand M et al. A role for methylation of the hMLH1 promoter in loss of hMLHl expression and drug resistance in ovarian cancer. Oncogene, 1999,18(14):2335-2341
[43]Abril E, Real LM, Serrano A,et al, Unresponsiveness to interferon associated with STAT1 protein deficiency in a gastric adenocarcinoma cell line. Cancer Immunol Immunother, 1998,47(2): 113-120
[44] Sun WH, Pabon C, Alsayed Y,et al. Interferon-alpha resistance in a cutaneous T-cell lymphoma cell line is associated with lack of STAT1 expression.Blood,1998,91(2):570-576
[45]Wong LH,Krauer KG,Hatzinisiriou I,et al.Interferon-resistant human melanoma cells are deficient in ISGF3 components,STAT1,STAT2,and p48-ISGF3gamma.J Biol Chem,1997,272(45):28779-28785
[46]Bovenzi V,Le NL,Cote S,et al.DNA methylation of retinoic acid receptor beta in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine.Anticancer Drugs,1999,10(5):471-476
[47]Altucci L and Gronemeyer H.The promise of retinoids to fight against cancer.Nat Rev Cancer,2001,1(3):181-193
[48]Gillespie AM,Coleman RE.The potential of melanoma antigen expression in cancer therapy.Cancer Treat Rev,1999,25(4):219-227
[49]Weber J,Salgaller M,Samid D,et al.Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2'-deoxycytidine.Cancer Res,1994,54(7):1766-1771
[2] Jones PA. Epigenetics in carcinogenesis and cancer prevention. Ann N Y Acad Sci, 2003, 983: 213-219
[3] Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med, 2003, 349(21):2042-2054
[4] Costello JF, Fruhwald MC, Smiraglia DJ,et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000, 24(2): 132- 138
[5] Costello JF, Plass C. Methylation matters. J Med Genet, 2001, 38(5):285-303
[6] Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science, 2001, 293(5532):1068-1070
[7] Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci U S A, 1993, 90(24): 11995-11999
[8] Karpf AR, Jones DA. Reactivating the expression of methylation silenced genes in human cancer.Oncogene, 2002,21(35):5496-5503
[9] Baylin SB, Herman JG, Graff JR, et al. Alternations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res, 1988, 72: 141-196
[10] Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Letter, 2003, 190: 125-133
[1 l]Merlo A, Herman JG, Mao L, et al. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTSl in human cancers. Nat Med, 1995, 1(7):686-692
[12]Hsu HS, Wen CK, Tang YA,et al. Promoter hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in nonsmoking lung cancer. Clin Cancer Res, 2005, 11(15):5410-5416
[13]Schildhaus HU, Krockel I, Lippert H, et al. Promoter hypermethylation of p16INK4a, E-cadherin, 06-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach. Int J Oncol, 2005, 26(6):1493-1500
[14]Laird PW,Jackson-Grusby L,Fazeli A,et al.Suppression of intestinal neoplasia by DNA hypomethylation.Cell,1995,81(2):197-205
[15]Jeronimo C,Henrique R,Hoque MO,et al.Quantitative RARbeta2 hyperme-thylation:a promising prostate cancer marker.Clin Cancer Res,2004,10(12 Pt 1):4010-4014
[16]Belinsky SA,Nikula KJ,Palmisano WA,et al.Aberrant methylation of p16(INK4a)is an early event in lung cancer and a potential biomarker for early diagnosis.Proc Natl Acad Sci U S A,1998,95(20):11891-11896
[17]Balch C,Montgomery JS,Paik HI,et al.New anti-cancer strategies:epigenetic therapies and biomarkers.Front Biosci,2005,10:1897-1931
[18]Fraga MF,Esteller M.DNA methylation:a profile of methods and applications.Biotechniques,2002,33(3):632,634,636-649
[19]Rush LJ,Raval A,Funchain P,et al.Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets.Cancer Res,2004,64(7):2424-2433
[20]Pompeia C,Hodge DR,Plass C,et al.Microarray analysis of epigenetic silencing of gene expression in the KAS-6/1 multiple myeloma cell line.Cancer Res,2004,64(10):3465-3473
[21]Shi H,Yan PS,Chen CM,et al.Expressed CpG island sequence tag microarray for dual screening of DNA hypermethylation and gene silencing in cancer cells.Cancer Res,2002,62(11):3214-3220
[22]Wilkins MR,Williams KL,Hohstrasser DF.(ed)Proteome Research:New Frontiers in Fuctional Genomics.New York,Spring,1997
[23]陈主初,梁宋平,肖志强.肿瘤蛋白质组学.长沙:湖南科学技术出版社,2002
[24]Kwong J,Lo KW,To KF,et al.Promoter hypermethylation of multiple gene in nasopharyngeal carcinoma.Clin Cancer Res,2002,8(1):131-137
[25]Lo KW,Kwong J,Hui AB,et al.High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma.Cancer Res,2001,61(10):3877-3881
[26]Zhou L,Jiang W,Ren C,et al.Frequent hyperrnethylation of RASSF1A and TSLC1,and high viral load of Epstein-Barr virus DNA in nasopharyngeal carcinoma and matched minor-adjacent tissues.Neoplasia,2005,7(9):809-815
[27]宋立兵,鄢践,简少文,等.鼻咽癌细胞亚株不同成瘤与转移潜能的分子机制.癌症,2002,21(2):158-162
[28]宋立兵,汪慧民,曾木圣.鼻咽癌细胞SUNE-1的异质性研究.癌症,1998,17(5):324-327
[29]姚开泰.鼻咽癌研究的回顾与展望.湖南医科大学学报,1995,20(2):99-102
[30]Shao JY,Zeng WF,Zeng YX,et al.Molecular genetic progression on nasopharyngeal carcinoma.Ai Zheng,2002,21(1):1-10
[31]Sidagis J,Ueno K,Tokunaga M,et al.Molecular epidemiology of Epstein-Barr virus(EBV)in EBV-related malignancies.Int J Cancer,1997,72(1):72-76
[32]Niedobitek G.Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma.Mol Pathol,2000,53(5):248-254
[33]Choi PH,Suen MW,Huang DP,et al.Nasopharyngeal carcinoma:genetic changes,Epstein-Barr virus infection,or both.A clinical and molecular study of 36 patients.Cancer,1993,72(10):2873-2878
[34]Lo k,Huang PW,Lee CK.Genetic changes in nasopharyngeal carcinoma.Clin Med J(Engl),1997(7),110:548-549
[35]Vogelstein B,Kinzler KW.The multistep nature of cancer.Trends Genet,1993,9(4):138-141
[36]Lo KW,Huang DP.Genetic and epigenetic changes in nasopharyngeal carcinoma.Semin Cancer Biol,2002,12(6):451-462
[37]Steeg PS,Bevilacqua G,Kopper L,et al.Evidence for a novel gene associated with low tumor metastatic p rotential.J Natl Cancer Inst,1988,80(3):200-204
[38]Freije JM,MacDonald NJ,Steeg PS.Differential gene expression in tumor metastasis:nm23.In:Gunthert U,Shlag PM,Birchmeier W,eds.Attempts to understand metastasis formation Ⅱ:regulatory factors.Berlin:Springer-Verlag,1996:215-232
[39]Qian M,Feng Y,Xu L,et al.Expression of antimetastatic gene nm23-H1 in epithelial ovarian cancer.Chin Med J(Engl),1997,110(2):142-144
[40]Busmanis I.Biomarkers in carcinoma of the cervix:emphasis on tissue-related factors and their potential prognostic factors.Ann Acad Med Singapore,1998,27(5):671-675
[41]Freije JM,MacDonald NJ,Steeg PS,et al.Nm23 and tumour metastasis:basic and translational advances.Biochem Soc Symp,1998,63:261-271
[42]Wang CS,Lin KH,Hsu YC,et al.Distant metastasis of gastric cancer is associated with elevated expression of the antimetastatic nm23 gene.Cancer Lett, 1998,128(1):23-29
[43]Boissan M, Lacombe ML. Nm23/NDP kinases in hepatocellular carcinoma. J Bioenerg Biomembr,2006 ,38(3-4):169-175
[44]Gazzeri S, Brambilla E, Negoescu A,et al. Overexpression of nucleoside diphosphate/kinase A/nm23-H1 protein in human lung tumors: association with tumor progression in squamous carcinoma. Lab Invest, 1996,74(1):158-167
[45]Niitsu N, Nakamine H, OkamotoM, et al. Expression of nm23-Hl is associated with poor prognosis in peripheral T-cell lymphoma. Br J Haematol, 2003, 123(4):621-630
[1]Feinberg AP,Gehrke CW,Kuo KC,et al.Reduced genomic 5-methylcytosine content in human colonic neoplasia.Cancer Res,1998,48(5):1159-1161
[2]De Smet C,De Backer O,Faraoni I,et al.The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.Proc Natl Acad Sci USA,1996,93(14):7149-7153
[3]Fang JY,Zhu SS,Xiao SD,et al.Studies on the hypomethylation of c-myc,c-Ha-ras oncogenes and histopathological changes in human gastric carcinoma.J Gastroenterol Hepatol,1996,11(11):1079-1082
[4]Jackson-Grusby L,Laird PW,Magge SN,et al.Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase.Proc Natl Acad Sci USA,1997,94(9):4681-4685
[5]Baylin SB,Herman JG.DNA hypermethylation in tumorigenesis:epigenetics joins genetics.Trends Genet,2000,16(4):168-174
[6]Clark SJ,Harrison J,Molloy PL.Spl binding is inhibited by(m)Cp(m)CpG methylation.Gene,1997,195(1):67-71
[7]Gaston K,Fried M.CpG methylation has differential effects on the binding of YY1 and ETS proteins to the bi-directional promoter of the Surf-1 and Surf-2genes.Nucl Acids Res,1995,23(6):901-909
[8]Umezawa A,Yamamoto H,Rhodes K,et al.Methylation of an ETS site in the intron enhancer of the keratin 18 gene participates in tissue-specific repression. Mol Cell Biol, 1997,17(9): 4885-4894
[9] Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol, 1998,18(11): 6538 -6547
[10] Nan X, Campoy FJ, Bird A. MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell,1997, 88(4): 471- 481
[11]Ng HH, Zhang Y, Hendrich B,et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet,1999, 23(1):58- 61
[12]Ng HH, Jeppesen P, Bird A. Active repression of methylated genes by the chromosomal protein MBD1 . Mol Cell Biol, 2000, 20(4): 1394-1406
[13]Jones PL, Veenstra GJ, Wade PA, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet, 1998 ,19(2): 187-191
[14]Bachman KE, Rountree MR, Baylin SB. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. J Biol Chem, 2001, 276(34): 32282 - 32287
[15]Fuks F, Hurd PJ, Wolf D,et al. The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem,2003, 278(6): 4035-4040
[16]Fuks F, Burgers WA, Godin N,et al. Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription. EMBO J,2001, 20(10): 2536-2544
[17] Robertson KD, Ait-Si-Ali S, Yokochi T, et al. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet, 2000,25(3): 338-342
[18] Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet, 2000, 25(3):269-277
[19]Schena M, Shalon D, Davis RW, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995,270(5235): 467-470
[20]Jackson-Grusby L, Beard C, Possemato R,et al. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet, 2001, 27(1): 31-39
[21]Kawai J, Hirotsune S, Hirose K,et al. Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method. Nucl Acids Res, 1993,21(24): 5604-5608
[22]Costello JF, Fruhward MC, Smiraglia D,et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000,24(2): 132-138
[23]Yan PS, Perry MR, Laux DE, et al. CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer. Clin Cancer Res, 2000,6(4): 1432-1438
[24]Yan PS, Chen CM, Shi H,et al. Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res, 2001, 61(23):8375-8380
[25]Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature, 2000,406(6797): 747-752
[26] Bender CM, Zingg JM, Jones PA. DNA methylation as a target for drug design. Pharm Res, 1998,15(2): 175-187
[27]Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation,Cell, 1980, 20(1): 85-93
[28]Ferguson AT, Vertino PM, Spitzner JR, et al. Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells. J Biol Chem 1997,272:32260-32266
[29]Bigey P, Knox JD, Croteau S, et al. Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase. J Biol Chem, 1999, 274(8):4594-4606
[30]Fournel M, Sapieha P, Beaulieu N, et al. Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16(ink4A) and p21(WAF/Cip1) by distinct mechanisms. J Biol Chem, 1999, 274(34): 24250-24256
[31] Cameron EE, Bachman KE, Myohanen S, et al . Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet,1999,21(1):103-107
[32]Leshin M. 5-Azacytidine and sodium butyrate induce expression of aromatase in fibroblasts from chickens carrying the henny feathering trait but not from wild-type chickens. Proc Natl Acad Sci USA, 1985, 82(9): 3005-3009
[33]Magdinier F, Wolffe AP. Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia. Proc Natl Acad Sci USA, 2001, 98(9): 4990-4995
[34]Bender CM, Gonzalgo ML, Gonzales FA,et al. Roles of cell division and gene transcription in the methylation of CpG islands.Mol Cell Biol,1999, 19(10):6690-6698
[35]Liang G, Gonzales FA, Jones PA,et al. Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine. Cancer Res, 2002, 62(4): 961-966
[36]Albanesi T, Polani S, Cozzi R, et al. DNA strand methylation and sister chromatid exchanges in mammalian cells in vitro. Mutat Res 1999,429(2) :23 9-48
[37] Chen RZ, Pettersson U, Beard C, et al. DNA hypomethylation leads to elevated mutation rates. Nature, 1998, 395(6697): 89-93
[38]Neidhart M, Rethage J, Kuchen S, et al. Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression. Arthritis Rheum, 2000, 43(12): 2634 -2647
[39] Wade PA. Methyl CpG binding proteins: coupling chromatin architecture to gene regulation. Oncogene, 2001, 20(24):3166-3173
[40]Liu PQ, Rebar EJ, Zhang L, et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem, 2001, 276(14): 11323-11334
[41] Plumb JA, Strathdee G, Sludden J, et al. Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLHl gene promoter. Cancer Res, 2000, 60(21): 6039-6044
[42] Strathdee G, MacKean MJ, Illand M et al. A role for methylation of the hMLH1 promoter in loss of hMLHl expression and drug resistance in ovarian cancer. Oncogene, 1999,18(14):2335-2341
[43]Abril E, Real LM, Serrano A,et al, Unresponsiveness to interferon associated with STAT1 protein deficiency in a gastric adenocarcinoma cell line. Cancer Immunol Immunother, 1998,47(2): 113-120
[44] Sun WH, Pabon C, Alsayed Y,et al. Interferon-alpha resistance in a cutaneous T-cell lymphoma cell line is associated with lack of STAT1 expression.Blood,1998,91(2):570-576
[45]Wong LH,Krauer KG,Hatzinisiriou I,et al.Interferon-resistant human melanoma cells are deficient in ISGF3 components,STAT1,STAT2,and p48-ISGF3gamma.J Biol Chem,1997,272(45):28779-28785
[46]Bovenzi V,Le NL,Cote S,et al.DNA methylation of retinoic acid receptor beta in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine.Anticancer Drugs,1999,10(5):471-476
[47]Altucci L and Gronemeyer H.The promise of retinoids to fight against cancer.Nat Rev Cancer,2001,1(3):181-193
[48]Gillespie AM,Coleman RE.The potential of melanoma antigen expression in cancer therapy.Cancer Treat Rev,1999,25(4):219-227
[49]Weber J,Salgaller M,Samid D,et al.Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2'-deoxycytidine.Cancer Res,1994,54(7):1766-1771