结肠癌细胞系中E-cadherin甲基化状况的研究
|
摘要
目的：探讨E-cadherin在结肠癌HT-29细胞系中的蛋白表达及其甲基化状况，分析二者的关系以及甲基化抑制剂对其影响，了解DNA甲基化在结肠癌发生发展中的作用。
方法：利用人结肠癌细胞系HT-29；用免疫细胞化学染色检测甲基化转移酶抑制剂5-氮杂脱氧胞苷(5-Aza-CdR)干预前后的HT-29细胞系E-cadherin的表达变化；采用甲基化特异性聚合酶链式反应(MSP)检测HT-29细胞中E-cadherin基因启动区域甲基化状态及去甲基化作用后甲基化的变化。
结果：(一)*5-Aza-CdR处理前后HT-29细胞形态学出现明显改变：未经5-Aza-CdR处理的HT-29细胞呈梭形或多边形，不规则，有时成团生长；经5-Aza-CdR处理后，部分细胞于出现细胞体积缩小、核固缩，染色质边集，核质比例减少；呈分裂相的细胞数目减少，部分核细胞出现核仁消失，提示HT-29细胞系在5-Aza-CdR干预作用下生长情况同对照组比较有显著性差异(P＜0.01)；且随着药物浓度的增加，细胞生长速度呈现减缓的趋势。*免疫细胞化学染色检测结果示，在5-Aza-CdR干预作用下，24h后E-cadherin表达较干预前增强，干预前后相比具有明显的差异(P＜0.01)；细胞E-cadherin阳性表达率由1μM药物处理时的21％±7％提高到5μM药物处理时的39％±13％；积分光密度值检测结果示5-Aza-CdR对HT-29细胞株E-cadherin的表达有明显的上调作用。(二) MSP检测结果示，未经5-Aza-CdR处理的HT-29细胞中E-cadherin基因启动子区域甲基化阳性；而经1μM和5μM 5-Aza-CdR各干预24h后HT-29细胞中E-cadherin基因启动子区域均为甲基化阴性。
结论：1)结肠癌中E-cadherin的蛋白表达下调，且E-cadherin低表达与其启动区域异常甲基化相关；2)去甲化作用对HT-29细胞株E-cadherin的表达有明显的上调作用，提示寻找特异性抑制甲基化酶活性的药物是肿瘤治疗的一个方向；3) E-cadherin基因启动区域异常甲基化可能是与结肠癌发生有密切关系的重要分子事件，为结肠癌的临床早期诊断、检测和治疗提供一定的分子理论与实验依据。
Objective To explore the expression and the methylation status ofE-cadherin in colon carcinoma cell line HT-29, to analyze the relationbetween the expression and DNA methylation of E-cadherin, and tounderstand the role of DNA methylation during the development of coloncarcinoma.
Materials and method Colon carcinoma cell line HT-29 fromhomo were examined; Immunocytochemistry dicho-step method wasused to detect the expression change of E-cadherin protein in HT-29 cellswith 5-Aza-CdR and without 5-Aza-CdR; Methylation specific-PCR(MSP) was used to detect the methylation status at promoter ofE-cadherin gene and the change of this status after demethylation in cellline HT-29.
Results (一) * There were much morphological change in HT-29cells: the shape of HT-29 cells without 5-Aza-CdR was fusiform orpolygon, irregular, sometimes agglomerate growth; to some of HT-29cells with 5-Aza-CdR, whose change included cell shrinkage, nucleuspycnosis, chromatin marhgination and so on. These change showed therewere significant differences between the treated group and the controlgroup(P＜0.01), and the cell growth velocity slowed down gradually with the increasing drug concentration. * Immunocytochemistry stainingshowed the expression of E-cadherin protein, enhanced obviously withthe role of 5-Aza-CdR contrast to the control after 24 hours, there wereobvious differences between the treated group and the control group(P＜0.01); the positiveexpression ratio of E-cadherin rised from 21%±7%(1μM) to 39%±13% (5μM); 5-Aza-CdR had made the expression ofE-cadherin protein in HT-29 cells up-regulated significantly. (二) MSPshowed that the promoter methylation of E-cadherin gene in the controlgroup was positive, and it was negative in the treated.
Conclusion 1) The expression of E-cadherin protein was downregulation in colon carcinoma; there was significant correlation betweenthe low expression of E-cadherin protein and the aberrant promotermethylation of E-cadherin gene; 2) Demethylation had an obviousup-regulation role to the expression of E-cadherin in HT-29 cells, whichshowed that drugs with controlling DMT were a direction of tumortreatment; 3) Aberrant promoter methylation of E-cadherin gene might bean inchoate molecular event; which offered some molecular theory andexperiment evidence for clinical early diagnosis, detection and treatmentof colon carcinoma.
方法：利用人结肠癌细胞系HT-29；用免疫细胞化学染色检测甲基化转移酶抑制剂5-氮杂脱氧胞苷(5-Aza-CdR)干预前后的HT-29细胞系E-cadherin的表达变化；采用甲基化特异性聚合酶链式反应(MSP)检测HT-29细胞中E-cadherin基因启动区域甲基化状态及去甲基化作用后甲基化的变化。
结果：(一)*5-Aza-CdR处理前后HT-29细胞形态学出现明显改变：未经5-Aza-CdR处理的HT-29细胞呈梭形或多边形，不规则，有时成团生长；经5-Aza-CdR处理后，部分细胞于出现细胞体积缩小、核固缩，染色质边集，核质比例减少；呈分裂相的细胞数目减少，部分核细胞出现核仁消失，提示HT-29细胞系在5-Aza-CdR干预作用下生长情况同对照组比较有显著性差异(P＜0.01)；且随着药物浓度的增加，细胞生长速度呈现减缓的趋势。*免疫细胞化学染色检测结果示，在5-Aza-CdR干预作用下，24h后E-cadherin表达较干预前增强，干预前后相比具有明显的差异(P＜0.01)；细胞E-cadherin阳性表达率由1μM药物处理时的21％±7％提高到5μM药物处理时的39％±13％；积分光密度值检测结果示5-Aza-CdR对HT-29细胞株E-cadherin的表达有明显的上调作用。(二) MSP检测结果示，未经5-Aza-CdR处理的HT-29细胞中E-cadherin基因启动子区域甲基化阳性；而经1μM和5μM 5-Aza-CdR各干预24h后HT-29细胞中E-cadherin基因启动子区域均为甲基化阴性。
结论：1)结肠癌中E-cadherin的蛋白表达下调，且E-cadherin低表达与其启动区域异常甲基化相关；2)去甲化作用对HT-29细胞株E-cadherin的表达有明显的上调作用，提示寻找特异性抑制甲基化酶活性的药物是肿瘤治疗的一个方向；3) E-cadherin基因启动区域异常甲基化可能是与结肠癌发生有密切关系的重要分子事件，为结肠癌的临床早期诊断、检测和治疗提供一定的分子理论与实验依据。
Objective To explore the expression and the methylation status ofE-cadherin in colon carcinoma cell line HT-29, to analyze the relationbetween the expression and DNA methylation of E-cadherin, and tounderstand the role of DNA methylation during the development of coloncarcinoma.
Materials and method Colon carcinoma cell line HT-29 fromhomo were examined; Immunocytochemistry dicho-step method wasused to detect the expression change of E-cadherin protein in HT-29 cellswith 5-Aza-CdR and without 5-Aza-CdR; Methylation specific-PCR(MSP) was used to detect the methylation status at promoter ofE-cadherin gene and the change of this status after demethylation in cellline HT-29.
Results (一) * There were much morphological change in HT-29cells: the shape of HT-29 cells without 5-Aza-CdR was fusiform orpolygon, irregular, sometimes agglomerate growth; to some of HT-29cells with 5-Aza-CdR, whose change included cell shrinkage, nucleuspycnosis, chromatin marhgination and so on. These change showed therewere significant differences between the treated group and the controlgroup(P＜0.01), and the cell growth velocity slowed down gradually with the increasing drug concentration. * Immunocytochemistry stainingshowed the expression of E-cadherin protein, enhanced obviously withthe role of 5-Aza-CdR contrast to the control after 24 hours, there wereobvious differences between the treated group and the control group(P＜0.01); the positiveexpression ratio of E-cadherin rised from 21%±7%(1μM) to 39%±13% (5μM); 5-Aza-CdR had made the expression ofE-cadherin protein in HT-29 cells up-regulated significantly. (二) MSPshowed that the promoter methylation of E-cadherin gene in the controlgroup was positive, and it was negative in the treated.
Conclusion 1) The expression of E-cadherin protein was downregulation in colon carcinoma; there was significant correlation betweenthe low expression of E-cadherin protein and the aberrant promotermethylation of E-cadherin gene; 2) Demethylation had an obviousup-regulation role to the expression of E-cadherin in HT-29 cells, whichshowed that drugs with controlling DMT were a direction of tumortreatment; 3) Aberrant promoter methylation of E-cadherin gene might bean inchoate molecular event; which offered some molecular theory andexperiment evidence for clinical early diagnosis, detection and treatmentof colon carcinoma.
引文
[1] Frigola Jordi, Ribas Maria, Risques Rosa-Ana. Methylome profiling of cancer cells by amplification of inter-methlated sites (AIMS). Nucleic Acids Research. 30(7):28, April 1, 2002
[2] 来茂德主编，医学分子生物学，人民卫生出版社，2001，213
[3] Roman Jose, Castillejo Juan Antonio, Jimenez Antonio. Hypermethylation of the calcitonin gene in acute lymphoblastic leukaemia is associated with unfavourable clinical outcome. British Journal of Haematology. 113(2):329-338, May 2001
[4] Chan A O-O, Lam S-K, Wong B C-Y. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut, 2003 April; 52(4): 502-506
[5] Hayashi Ken, Yokozaki Hiroshi, Goodison Steve. Inactivation of retinoic acid receptor [beta] by promoter CpG hypermethylation in gastric cancer. Differentiation. 2001 August; 68(1): 13-21
[6] Clarke LE, Leitzel K, Smith J, Ali SM, Lipton A. Epidermal growth factor receptor mRNA in peripheral blood of patients with pancreatic, lung, and colon carcinomas detected by RTPCR. Int J Oncol 2003;22:425-430
[7] 李存玺，章静波．癌细胞粘附与癌细胞侵袭和转移．癌的侵袭及转移高进主编北京：北京医科大学协和医科大学联合出版社，1996，145
[8] Anastasiadis PZ,Reynolds AB. The p120 catenin family: complex roles in adhesion, signaling and cancer. J Cell Sci,2000,113(Pt8): 1319-1334
[9] 李东印，马玉泉．胃癌中E钙粘蛋白的表达及意义．世界华人消化杂志，2001，9(8)．974-975
[10] Rimm DL, Morrow JS, et al. Molecular cloning of human H-CD suggest a novel subdivision of the CD subdivision of the CD superfamily biochem. Biophys Res Commun, 1994; 200(3): 1745
[11] Bussemakers MJG; Bokhoven AV, Mees SGM, et al. Molecular cloning and characterization of the human E-CD cDNA. Mol Biol Rep, 1993; 17; 123
[12] Si HX, Tsao SW, Lam KY, et al. E-cadherin expression is commonly downregulated by CpG island hypermethylation in esophageal cancer cells. Cancer Lett, 2001 Nov 8; 173(1): 71-8
[13] 司徒镇强，吴军正主编．《细胞培养》，西安：世界图书出版公司，2004．3
[14] 赵京，孙玉鄂，张连斌．CD44的表达与人类非小细胞肺癌淋巴转移相 关性的初步研究．中国肺癌杂志，2000，3(2)：115
[15] Herman JG,Graff J R, Myohanen S et al. Methylation specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93: 9821-9826
[16] Yamura G, Yin J, Wang S, et al. E-cadherin gene promoter hypermethylation in primary human gastric cancers [J]. J Natl Cancer Inst, 2000; 92(7): 569-573
[17] 李士谔．DNA甲基化、基因表达与肿瘤．基础医学与临床，1996，5：321-340
[18] Laird W P, Jaenisch R. The role of DNA methylation in cancer genetics and epigenetics [J]. Annu Rev Genet, 1996, 30:441-464
[19] Thiery JP. Epithelial-mesenchymal transitions in tumour Progression. Nat Rev Cancer. 2002(2): 442-454
[20] Perl AK, Wilgenbus P, Dahl U, et al. A causal role for E-eadherin in the transition from adenoma to carcinoma [J]. Nature, 1998, 392: 190-193.
[21] Sommers CL, Thompson EW, Torri JA, et al. Cell adhesion molecule uvomorulin expression in human breast cancer cell lines: relationship to morphology and invasive capacities. Cell Growth Differ. 1991 Aug; 2(8): 365-72.
[22] Frixen U, Behren J, Sachs Met al. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol, 1991: 113: 173-85
[23] Beavon, IR. Regulation of E-cadherin: does hypoxia initiate the metastatic cascade? Mol Pathol 1999, 52: 179-188.
[24] St.Croix B, Sheehan C W, Rak J, et al. E-cadherin-dependent growth suppression is mediated by the Cycline-dependent Kinse inhibitor p~(27kipl). K Cell Biol, 1998, 142(2): 557-571.
[25] Kinsella AR, Iets GC, Hill CL, et al. Reduced E-cadherin expression correlates with increased invasivenessin colorectal carcinoma cell lines. Clin Exp Metastasis, 1994, 12 (4): 335-342.
[26] Melki JR, Vincent PC, Brown RD, et al. Hypermethylation of E-cadherin in leukemia [J]. Melki Blood, 2000; 95(10): 3208-3213
[27] Suzuki H, Itoh F, Toyota M, et al. Distinct methylation pattern and microsatellite instability in sporadic gastric cancer [J]. Int J Cancer, 1999; 83(3): 309-313.
[28] SzyfM. DNA methylation machinery as a target for anticancer therapy. Pharmacol Ther, 1996; 76(7): 501
[29] Yoshiura K, Kanai Y, Ochiai A, et al. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human cancers[J]. Proc Natl Acad Sci USA, 1995; 92(16): 7416
[30] Nojima D, Nakajima K, Li LC, et al. CpG methylation of promoter region inactivates E-cadherin gene in renal cell carcinoma. Mol Carcinog, 2001, 32 (1): 19-27.
[31] Darwanto A,Kitazawa R,Maeda S, Kitazawa S. MeCP2 and promoter methylation cooperatively regulate E-cadherin gene expression in colorectal carcinoma.Cancer Sci, 2003, 94 (5): 442-447.
[32] Garinis GA,Menounos PG, Spanakis NE, et al. Hypermethylation-associated transcriptional silencing of E-cadherin in primary sporadic colorectal carcinomas. J Pathol, 2002, 198 (4): 442-449.
[33] Wheeler JMD, Kim HC, Efstathiou J A, et al. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut, 2001, 48(3): 367-371
[34] Corn Paul G, Summers Matthew K, Fogt Franz. Frequent hypermethylation of the 5'CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer. Carcinogenesis. 24(1): 47-51, January 2003
[35] Nakayama T, Watanabe M, Yamanaka M, et al. The role of epigenetic modifications in retinoic acid receptor beta2 geneexpression in human prostate cancers[J]. Lab Invest, 2001, 81(7): 1049-1057
[36] Lubomierski N, Kersting M. Tumor suppressor genes in the 9p21 gene cluster are selective targets of inactivation in gastroenteropancreatic tumor[J]. Cancer Res, 2001, 61(15): 5905-5910
[37] Gagnon Jacynthe, Shaker Sepideh, Primeau Melanie. Interaction of 5-aza-2'-deoxycytidine and depsipeptide on antineoplastic activity and activation of 14-3-3[sigma], E-cadherin and tissue inhibitor of metalloproteinase 3 expression in human breast carcinoma cells. Anti-Cancer Drugs. 14(3): 193-202, March 2003
[1] Byun DS, Lee MG, Chae KS, et al. Frequent epigenetic inactivation of RASSFIA by aberrant promoter hypermethylation in human gastric adenocarcinoma[J]. Cancer Res, 2001, 61 (19): 7034-7038.
[2] Wajed SA, Laird PW, Demeester TR. DNA methylation: an alternative pathyway to cancer. Ann Surg, 2001, 234(1): 10-20.
[3] Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methylatransferases DNM T1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179.
[4] Tamura G, Yin J, Wang S, et al. E-cadherin gene promoter hypermethylation in primary human gastric cancers [J]. J Natl Cancer Inst, 2000; 92(7): 569-573.
[5] Clark SJ, Molloy PL. Spl binding is inhibited by (m) Cp (m)CpG methylation. Gene, 1997, 195(1): 67-71.
[6] Mancini DN, Rodenhiser DI, Ainsworth PJ, et al. CpG methylation within the 5'regulatory region of BRCA1 gene is tumor specific and includes a putative CREB binding site [J]. Oncogene, 1998, 15: 1161-1169.
[7] Suske G. The Sp-family of transcription factors[J].Gene,1999, 238(2):291-300
[8] Momparler RL, Bovenzi V. DNA methylation and cancer[J]. 2000, J Cell Phsiology, 2000, 183: 145-154.
[9] Nan X, NG HH, Johnson CA, et al. transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex[J]. Nature, 1998, 393:386-389.
[10] Magewu WN, Jones PA. Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer[J]. Mol Cell Biol, 1994, 14: 4225.
[11] Dehissenko M, Chen JX, Tang MS, et al. Cytosine methylation determines hot spots of DNA damage in the human p53 gene [J]. Proc Natl Acad Sci USA, 1997, 94: 3893-3898.
[12] Garcia-Moll X, Cole D, Zouridakis E, et al. Increased serum neopterin: a marker of coronary artery disease activity in women. Heart, 2000, 83(3): 346-350.
[13] Ze-jun liu, Masato Maekawa. Polymerase chain reaction based methods of DNA methlation analysis[J]. Analytical Biochemistry, 2003; 317:259-65.
[14] Thomas GA, Williams ED. Production of thyroid tumous in mice by demethylating agents[J]. Carcinogenesis, 1992, 3(6): 1039-1042.
[15] Jones PA. DNA methylation and cancer. Oncogene, 2002, 21(35): 5358-5360.
[16] 程中华，房静远，杨丽，等．hMSH2在胃癌组织中的表达及其与DNA甲基化之间的关系，世界华人消化杂志，2004，12(7)：1731-1733．
[17] Sardi I, Dal-Canto M, Bartoletti R, et al. Molecular genetic alterations of c-myc oncogene in superficial and locally advanced bladder cancer. Eur Urol, 1998, 33(4): 424-430.
[18] Robertson KD, Uzvolgyi E, Liang G, et al. The human DNA methytransferases(DNMTs)1, 3a, and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res, 1999, 27(11): 2291-2298.
[19] Tao L,Yang S, Xie M, et al. Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity indichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Cancer Lett, 2000, 58(2):185-193.
[20] Jong S J, Soria JC, Wang Let al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res,2001,61 (21): 7959-7963.
[21] Feng Z, Hu W, RomWN, et al. N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation. Biochemistry, 2002,41 (20):6414-6421.
[22] Florl AR, Steinhoff C, Muller M, et al. Coordinate hypermethylation at specific genes in prostate carcinoma precedes L 1NE-1 hypomethylation. Br J Cancer, 2004,91 (5):985-994.
[23] ia4. Sciandrello G, Caradonna F, Mauro M, et al. Arsenic-induced DNA hypomethylation affects chromosomal instability in mammalian cells. Carcinogenesis, 2004,25 (3):413-417.
[24] Ehrlich M. The ICF syndrome, a DNA methyltransferase 3B deficiency and immunodeficiency disease. Clin Immunol, 2003,109(1): 17-28.
[25] Gaudet F, Hodgson JG, Eden A et al. Induction of Tumors in Mice by Genomic Hypomethylation. Science, 2003,300 (5618):489-492.
[26] Butcher DT, Mancini-DiNardo DN, Archer TK, et al. DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter. Int J Cancer, 2004,111 (5): 669-678.
[27] Etoh T, Kanai Y, Ushijima S, et al. Increased DNA methyltransferase 1 (DNMTI) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpC islands in gastric cancers. AmJ Pathol, 2004,164(2): 689-699.
[28] Nagai M, Nakamura A, Makino R, et al. Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas. Hepatol Res, 2003, 26(3): 186-191.
[29] Herman J G, Merlo A, Mao L, et al. Inactivation of the CDKN2/P 16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers[J]. Cancer Res,1995,55:4525-4530.
[30] Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis. Trend Genet, 2000,16(4): 168-174.
[31] Otterson GA, Khleif SN, Chen W, et al. CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16/INK4 protein induction by 52Aza-2-doexycytidine[J]. Oncogene, 1995, 11: 1211.
[32] Wheeler JM, Kim HC, Efstathiou JA, et al. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer[J]. Gut, 2001, 48(3): 367.
[33] C^oteS , Sinnett D , Momparler RL, et al. Demethylation by 52Aza-2-doexycytidine of specific 5-methylcytosine sites in the promoter region of the retinoic acid receptorβgene in human colon carcinoma cells[J]. Anti Cancer Drugs,1998,9: 743-750.
[34] Bovenzi V, Le NL, Cote S, et al. DNA methylation of the retinoic acid receptorβgene in breast cancer and possible therapeutic role of 5-Aza-2-doexycytidine[J]. Anti Cancer Drugs, 1999, 10: 471-476.
[35] Esteller M. DNA methylation and cancer therapy: new developments and expectations. Curr Opin Oncol, 2005, 17(1): 55-60.
[36] Das PM, Singal R. DNA methylation and cancer. J Clin Oncol, 2004, 22(22): 4632-4642.
[37] Laird PW. The power and the promise of DNA methylation markers. Nat Rev Cancer, 2003, 3(4): 253-266.
[38] Robert MF, Morin S, Beaulieu N, et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells, Nat Genet, 2003, 33(1): 61-65.
[39] Xiong Y, Dowdy SC, Podratz KC, et al. Histone deacetylase DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells. Cancer Res, 2005, 65(7): 2684-2689.
[40] EstellerM. Ep igenetic lesions causing genetic lesions in human cancer: promoter hypermethylation of DNA repar genes [J]. Euro J Cancer, 2000, 36(18): 2294-2300.
[2] 来茂德主编，医学分子生物学，人民卫生出版社，2001，213
[3] Roman Jose, Castillejo Juan Antonio, Jimenez Antonio. Hypermethylation of the calcitonin gene in acute lymphoblastic leukaemia is associated with unfavourable clinical outcome. British Journal of Haematology. 113(2):329-338, May 2001
[4] Chan A O-O, Lam S-K, Wong B C-Y. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut, 2003 April; 52(4): 502-506
[5] Hayashi Ken, Yokozaki Hiroshi, Goodison Steve. Inactivation of retinoic acid receptor [beta] by promoter CpG hypermethylation in gastric cancer. Differentiation. 2001 August; 68(1): 13-21
[6] Clarke LE, Leitzel K, Smith J, Ali SM, Lipton A. Epidermal growth factor receptor mRNA in peripheral blood of patients with pancreatic, lung, and colon carcinomas detected by RTPCR. Int J Oncol 2003;22:425-430
[7] 李存玺，章静波．癌细胞粘附与癌细胞侵袭和转移．癌的侵袭及转移高进主编北京：北京医科大学协和医科大学联合出版社，1996，145
[8] Anastasiadis PZ,Reynolds AB. The p120 catenin family: complex roles in adhesion, signaling and cancer. J Cell Sci,2000,113(Pt8): 1319-1334
[9] 李东印，马玉泉．胃癌中E钙粘蛋白的表达及意义．世界华人消化杂志，2001，9(8)．974-975
[10] Rimm DL, Morrow JS, et al. Molecular cloning of human H-CD suggest a novel subdivision of the CD subdivision of the CD superfamily biochem. Biophys Res Commun, 1994; 200(3): 1745
[11] Bussemakers MJG; Bokhoven AV, Mees SGM, et al. Molecular cloning and characterization of the human E-CD cDNA. Mol Biol Rep, 1993; 17; 123
[12] Si HX, Tsao SW, Lam KY, et al. E-cadherin expression is commonly downregulated by CpG island hypermethylation in esophageal cancer cells. Cancer Lett, 2001 Nov 8; 173(1): 71-8
[13] 司徒镇强，吴军正主编．《细胞培养》，西安：世界图书出版公司，2004．3
[14] 赵京，孙玉鄂，张连斌．CD44的表达与人类非小细胞肺癌淋巴转移相 关性的初步研究．中国肺癌杂志，2000，3(2)：115
[15] Herman JG,Graff J R, Myohanen S et al. Methylation specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93: 9821-9826
[16] Yamura G, Yin J, Wang S, et al. E-cadherin gene promoter hypermethylation in primary human gastric cancers [J]. J Natl Cancer Inst, 2000; 92(7): 569-573
[17] 李士谔．DNA甲基化、基因表达与肿瘤．基础医学与临床，1996，5：321-340
[18] Laird W P, Jaenisch R. The role of DNA methylation in cancer genetics and epigenetics [J]. Annu Rev Genet, 1996, 30:441-464
[19] Thiery JP. Epithelial-mesenchymal transitions in tumour Progression. Nat Rev Cancer. 2002(2): 442-454
[20] Perl AK, Wilgenbus P, Dahl U, et al. A causal role for E-eadherin in the transition from adenoma to carcinoma [J]. Nature, 1998, 392: 190-193.
[21] Sommers CL, Thompson EW, Torri JA, et al. Cell adhesion molecule uvomorulin expression in human breast cancer cell lines: relationship to morphology and invasive capacities. Cell Growth Differ. 1991 Aug; 2(8): 365-72.
[22] Frixen U, Behren J, Sachs Met al. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol, 1991: 113: 173-85
[23] Beavon, IR. Regulation of E-cadherin: does hypoxia initiate the metastatic cascade? Mol Pathol 1999, 52: 179-188.
[24] St.Croix B, Sheehan C W, Rak J, et al. E-cadherin-dependent growth suppression is mediated by the Cycline-dependent Kinse inhibitor p~(27kipl). K Cell Biol, 1998, 142(2): 557-571.
[25] Kinsella AR, Iets GC, Hill CL, et al. Reduced E-cadherin expression correlates with increased invasivenessin colorectal carcinoma cell lines. Clin Exp Metastasis, 1994, 12 (4): 335-342.
[26] Melki JR, Vincent PC, Brown RD, et al. Hypermethylation of E-cadherin in leukemia [J]. Melki Blood, 2000; 95(10): 3208-3213
[27] Suzuki H, Itoh F, Toyota M, et al. Distinct methylation pattern and microsatellite instability in sporadic gastric cancer [J]. Int J Cancer, 1999; 83(3): 309-313.
[28] SzyfM. DNA methylation machinery as a target for anticancer therapy. Pharmacol Ther, 1996; 76(7): 501
[29] Yoshiura K, Kanai Y, Ochiai A, et al. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human cancers[J]. Proc Natl Acad Sci USA, 1995; 92(16): 7416
[30] Nojima D, Nakajima K, Li LC, et al. CpG methylation of promoter region inactivates E-cadherin gene in renal cell carcinoma. Mol Carcinog, 2001, 32 (1): 19-27.
[31] Darwanto A,Kitazawa R,Maeda S, Kitazawa S. MeCP2 and promoter methylation cooperatively regulate E-cadherin gene expression in colorectal carcinoma.Cancer Sci, 2003, 94 (5): 442-447.
[32] Garinis GA,Menounos PG, Spanakis NE, et al. Hypermethylation-associated transcriptional silencing of E-cadherin in primary sporadic colorectal carcinomas. J Pathol, 2002, 198 (4): 442-449.
[33] Wheeler JMD, Kim HC, Efstathiou J A, et al. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut, 2001, 48(3): 367-371
[34] Corn Paul G, Summers Matthew K, Fogt Franz. Frequent hypermethylation of the 5'CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer. Carcinogenesis. 24(1): 47-51, January 2003
[35] Nakayama T, Watanabe M, Yamanaka M, et al. The role of epigenetic modifications in retinoic acid receptor beta2 geneexpression in human prostate cancers[J]. Lab Invest, 2001, 81(7): 1049-1057
[36] Lubomierski N, Kersting M. Tumor suppressor genes in the 9p21 gene cluster are selective targets of inactivation in gastroenteropancreatic tumor[J]. Cancer Res, 2001, 61(15): 5905-5910
[37] Gagnon Jacynthe, Shaker Sepideh, Primeau Melanie. Interaction of 5-aza-2'-deoxycytidine and depsipeptide on antineoplastic activity and activation of 14-3-3[sigma], E-cadherin and tissue inhibitor of metalloproteinase 3 expression in human breast carcinoma cells. Anti-Cancer Drugs. 14(3): 193-202, March 2003
[1] Byun DS, Lee MG, Chae KS, et al. Frequent epigenetic inactivation of RASSFIA by aberrant promoter hypermethylation in human gastric adenocarcinoma[J]. Cancer Res, 2001, 61 (19): 7034-7038.
[2] Wajed SA, Laird PW, Demeester TR. DNA methylation: an alternative pathyway to cancer. Ann Surg, 2001, 234(1): 10-20.
[3] Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methylatransferases DNM T1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179.
[4] Tamura G, Yin J, Wang S, et al. E-cadherin gene promoter hypermethylation in primary human gastric cancers [J]. J Natl Cancer Inst, 2000; 92(7): 569-573.
[5] Clark SJ, Molloy PL. Spl binding is inhibited by (m) Cp (m)CpG methylation. Gene, 1997, 195(1): 67-71.
[6] Mancini DN, Rodenhiser DI, Ainsworth PJ, et al. CpG methylation within the 5'regulatory region of BRCA1 gene is tumor specific and includes a putative CREB binding site [J]. Oncogene, 1998, 15: 1161-1169.
[7] Suske G. The Sp-family of transcription factors[J].Gene,1999, 238(2):291-300
[8] Momparler RL, Bovenzi V. DNA methylation and cancer[J]. 2000, J Cell Phsiology, 2000, 183: 145-154.
[9] Nan X, NG HH, Johnson CA, et al. transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex[J]. Nature, 1998, 393:386-389.
[10] Magewu WN, Jones PA. Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer[J]. Mol Cell Biol, 1994, 14: 4225.
[11] Dehissenko M, Chen JX, Tang MS, et al. Cytosine methylation determines hot spots of DNA damage in the human p53 gene [J]. Proc Natl Acad Sci USA, 1997, 94: 3893-3898.
[12] Garcia-Moll X, Cole D, Zouridakis E, et al. Increased serum neopterin: a marker of coronary artery disease activity in women. Heart, 2000, 83(3): 346-350.
[13] Ze-jun liu, Masato Maekawa. Polymerase chain reaction based methods of DNA methlation analysis[J]. Analytical Biochemistry, 2003; 317:259-65.
[14] Thomas GA, Williams ED. Production of thyroid tumous in mice by demethylating agents[J]. Carcinogenesis, 1992, 3(6): 1039-1042.
[15] Jones PA. DNA methylation and cancer. Oncogene, 2002, 21(35): 5358-5360.
[16] 程中华，房静远，杨丽，等．hMSH2在胃癌组织中的表达及其与DNA甲基化之间的关系，世界华人消化杂志，2004，12(7)：1731-1733．
[17] Sardi I, Dal-Canto M, Bartoletti R, et al. Molecular genetic alterations of c-myc oncogene in superficial and locally advanced bladder cancer. Eur Urol, 1998, 33(4): 424-430.
[18] Robertson KD, Uzvolgyi E, Liang G, et al. The human DNA methytransferases(DNMTs)1, 3a, and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res, 1999, 27(11): 2291-2298.
[19] Tao L,Yang S, Xie M, et al. Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity indichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Cancer Lett, 2000, 58(2):185-193.
[20] Jong S J, Soria JC, Wang Let al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res,2001,61 (21): 7959-7963.
[21] Feng Z, Hu W, RomWN, et al. N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation. Biochemistry, 2002,41 (20):6414-6421.
[22] Florl AR, Steinhoff C, Muller M, et al. Coordinate hypermethylation at specific genes in prostate carcinoma precedes L 1NE-1 hypomethylation. Br J Cancer, 2004,91 (5):985-994.
[23] ia4. Sciandrello G, Caradonna F, Mauro M, et al. Arsenic-induced DNA hypomethylation affects chromosomal instability in mammalian cells. Carcinogenesis, 2004,25 (3):413-417.
[24] Ehrlich M. The ICF syndrome, a DNA methyltransferase 3B deficiency and immunodeficiency disease. Clin Immunol, 2003,109(1): 17-28.
[25] Gaudet F, Hodgson JG, Eden A et al. Induction of Tumors in Mice by Genomic Hypomethylation. Science, 2003,300 (5618):489-492.
[26] Butcher DT, Mancini-DiNardo DN, Archer TK, et al. DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter. Int J Cancer, 2004,111 (5): 669-678.
[27] Etoh T, Kanai Y, Ushijima S, et al. Increased DNA methyltransferase 1 (DNMTI) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpC islands in gastric cancers. AmJ Pathol, 2004,164(2): 689-699.
[28] Nagai M, Nakamura A, Makino R, et al. Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas. Hepatol Res, 2003, 26(3): 186-191.
[29] Herman J G, Merlo A, Mao L, et al. Inactivation of the CDKN2/P 16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers[J]. Cancer Res,1995,55:4525-4530.
[30] Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis. Trend Genet, 2000,16(4): 168-174.
[31] Otterson GA, Khleif SN, Chen W, et al. CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16/INK4 protein induction by 52Aza-2-doexycytidine[J]. Oncogene, 1995, 11: 1211.
[32] Wheeler JM, Kim HC, Efstathiou JA, et al. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer[J]. Gut, 2001, 48(3): 367.
[33] C^oteS , Sinnett D , Momparler RL, et al. Demethylation by 52Aza-2-doexycytidine of specific 5-methylcytosine sites in the promoter region of the retinoic acid receptorβgene in human colon carcinoma cells[J]. Anti Cancer Drugs,1998,9: 743-750.
[34] Bovenzi V, Le NL, Cote S, et al. DNA methylation of the retinoic acid receptorβgene in breast cancer and possible therapeutic role of 5-Aza-2-doexycytidine[J]. Anti Cancer Drugs, 1999, 10: 471-476.
[35] Esteller M. DNA methylation and cancer therapy: new developments and expectations. Curr Opin Oncol, 2005, 17(1): 55-60.
[36] Das PM, Singal R. DNA methylation and cancer. J Clin Oncol, 2004, 22(22): 4632-4642.
[37] Laird PW. The power and the promise of DNA methylation markers. Nat Rev Cancer, 2003, 3(4): 253-266.
[38] Robert MF, Morin S, Beaulieu N, et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells, Nat Genet, 2003, 33(1): 61-65.
[39] Xiong Y, Dowdy SC, Podratz KC, et al. Histone deacetylase DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells. Cancer Res, 2005, 65(7): 2684-2689.
[40] EstellerM. Ep igenetic lesions causing genetic lesions in human cancer: promoter hypermethylation of DNA repar genes [J]. Euro J Cancer, 2000, 36(18): 2294-2300.