摘要
胃癌是常见的恶性肿瘤之一,在我国的死亡率居各种癌症的首位,全国每年死于胃癌的人数约为15万,我国胃癌的死亡率在世界范围内属于较高的水平。随着现代分子生物学理论和技术的发展,认为胃癌的发生是涉及到多基因、多分子水平变化、多阶段的一个复杂过程,而癌基因的激活和抑癌基因的失活是该过程中的重要事件之一。近年来提出的表观遗传学说,使人们注意到DNA甲基化在肿瘤发生、发展中起着重要的作用。DNA甲基化是具有可逆性与遗传性的一种基因修饰方式,是指DNA双螺旋中,胞嘧啶核苷的嘧啶环第5位碳原子在DNA甲基转移酶(DNMT)介导下以S-腺苷蛋氨酸(SAM)为甲基供体发生甲基化。甲基化状态的改变是引起肿瘤的一个重要因素,这种变化包括基因组整体甲基化水平降低和CpG岛局部甲基化水平的异常升高,从而导致癌基因的激活和抑癌基因的沉默,以及基因组的不稳定。
S-腺苷蛋氨酸,又名S-腺苷甲硫氨酸,是人体内所有甲基化反应的甲基供体,由S-腺苷蛋氨酸合成酶催化合成。S-腺苷蛋氨酸被认为是一种去甲基化抑制剂,能够逆转基因的低甲基化和诱导基因高甲基化。近年来许多研究显示S-腺苷蛋氨酸有预防和治疗肿瘤的作用。通过对肝癌、结肠癌、乳腺癌等体内外研究发现,S-腺苷蛋氨酸可以抑制肿瘤细胞的生长、促进细胞凋亡,并可使存在低甲基化的基因发生甲基化,达到抑制肿瘤的作用。但是在目前众多的文献中,尚无S-腺苷蛋氨酸对胃癌细胞体内外作用的报导,因此我们对S-腺苷蛋氨酸对体内外胃癌细胞的影响进行了研究,探讨S-腺苷蛋氨酸对胃癌的抑制作用。癌基因c-myc的异常表达与肿瘤的发生、发展密切相关,并且研究发现其在肝癌、胃癌和肺癌等肿瘤的发生过程中出现基因甲基化水平的降低。uPA(尿激酶型纤溶酶原激活剂)由于具有促进肿瘤细胞生长、侵袭、转移及肿瘤新生血管形成的能力,被看作是一个与转移有关的肿瘤促进基因,近年来发现结肠癌、乳腺癌和前列腺癌等肿瘤中uPA基因启动子区呈低甲基化。因此我们对胃癌中c-myc和uPA基因的表达和启动子区甲基化状态进行了研究,以期探讨S-腺苷蛋氨酸作用可能的机制。
实验方法
1.S-腺苷蛋氨酸对体外胃癌细胞系增殖能力的影响
使用MTT方法检测最终浓度为0.5mmol/L、1mmol/L、2mmol/L、4mmol/L、8mmol/L、16mmol/L和32mmol/L的S-腺苷蛋氨酸对胃癌细胞系MKN-28、SGC-7901、BGC-823和MKN-45作用24h、48h和72h的影响,检测细胞吸光度值(A492值),计算细胞的生长抑制率和IC5。并绘制细胞增殖抑制曲线。
2.S-腺苷蛋氨酸对体外胃癌细胞系细胞周期和凋亡率的影响
使用流式细胞仪检测不同浓度的S-腺苷蛋氨酸(0mmol/L、2mmol/L和4mmol/L)对4种胃癌细胞系作用72h后细胞周期和凋亡率的影响。
3. S-腺苷蛋氨酸对体外胃癌细胞系侵袭力和迁移力的影响
收集不同浓度的S-腺苷蛋氨酸(0mmol/L、2mmol/L和4mmol/L)作用72h后的4种胃癌细胞,在聚碳酯膜覆盖有Matrigel的Transwell小室内加入各组细胞(1×105个/孔)孵育24h,观察侵袭力。或在Transwell小室内直接加入各组细胞(1×106个/孔)孵育20h,观察迁移力。检测穿膜细胞数,计算侵袭抑制率和迁移抑制率。
4.S-腺苷蛋氨酸对体外胃癌细胞中c-myc和uPA基因mRNA和蛋白表达及启动子区甲基化状态的影响
收集不同浓度的S-腺苷蛋氨酸(0mmol/L、2mmol/L和4mmol/L)作用72h后的4种胃癌细胞,使用半定量RT-PCR法和Western Blot方法检测各组细胞中c-myc和uPA基因mRNA和蛋白的表达,使用甲基化特异性PCR(MSP)方法检测c-myc和uPA基因启动子区的甲基化状态。
5.建立人胃癌裸鼠移植瘤模型及药物处理
将SGC-7901细胞(1×107个/只)接种于裸鼠左侧腋窝皮下,建立移植瘤模型。随机分为低浓度SAM组[192μmol/(kg·d)]、高浓度SAM组[768μmol/(kg·d)]和对照组(NS),每组5只,腹腔给药15天。
6.S-腺苷蛋氨酸对体内胃癌生长及c-myc和uPA基因的影响
每3天测量一次裸鼠体质量及移植瘤体积,绘制移植瘤生长曲线。HE染色观察移植瘤组织病理学改变和肿瘤细胞凋亡指数,观察肝脏和肺部的转移灶。使用免疫组化SP法和Western Blot方法检测各组移植瘤中c-myc基因和uPA基因蛋白的表达,使用半定量RT-PCR法检测基因mRNA的表达,使用甲基化特异性PCR方法检测基因启动子区的甲基化状态。
统计学分析
采用SPSS 11.1软件进行统计学分析。计量资料数据以均数±标准差(x±5)表示。细胞体外侵袭结果、细胞体外迁移结果和移植瘤免疫组化结果使用多个独立样本的秩和检验,移植瘤MSP结果使用列联表x2检验,其余结果使用单因素方差分析和成组t检验,以α=0.05为检验水准。
结果
1.随着S-腺苷蛋氨酸浓度和作用时间的增加,MKN-28、SGC-7901、BGC-823和MKN-45这4种细胞均逐渐出现生长抑制,凋亡细胞增多。MTT结果显示这4种细胞随S-腺苷蛋氨酸浓度和作用时间的增加,细胞的增殖活性逐渐受到抑制(P均<0.05),细胞的生长抑制率也逐渐加大。根据生长抑制率得到72h的半数抑制率(IC50)分别为：MKN-28细胞IC50 6.37mmol/L、SGC-7901细胞IC50 5.40mmol/L、BGC-823细胞IC50 4.01mmol/L和MKN-45细胞IC504.77mmol/L.
2.随SAM浓度增加,SGC-7901和BGC-823细胞G1期细胞比例逐渐增加(P分别<0.05和<0.01)；细胞增殖指数PI明显降低(P分别<0.05和<0.01)；而MKN-28和MKN-45细胞无明显变化(P均>0.05)；4种细胞凋亡率均显著升高(P均<0.01)
3.4种细胞的侵袭细胞数和迁移细胞数均随S-腺苷蛋氨酸浓度的增加而逐渐减少(P均<0.01),侵袭抑制率和迁移抑制率逐渐增大。
4.SGC-7901和BGC-823细胞中c-myc基因,4种细胞中uPA基因mRNA和蛋白的表达随S-腺苷蛋氨酸浓度增加而逐渐减弱(P分别<0.05和<0.01),基因启动子区的低甲基化逐渐改善；而MKN-28和MKN-45细胞中c-myc基因mRNA和蛋白的表达无明显变化(P均>0.05),基因启动子区的低甲基化状态也无改善。
5.S-腺苷蛋氨酸对裸鼠体质量无影响,低浓度组和高浓度组的移植瘤体积分别为(618.51±149.27)mm3和(444.32±118.51)mm3,与对照组[(1018.22±223.07)mm3]相比,体积明显减小(P均<0.01),S-腺昔蛋氨酸对移植瘤的生长有明显的抑制作用,低浓度SAM组和高浓度SAM组抑瘤率分别为39.26%和56.36%。
6.3组移植瘤组织均呈人中分化胃腺癌的表现。实验组移植瘤组织中有较多的凝固性坏死灶和凋亡细胞,肿瘤细胞凋亡指数随S-腺苷蛋氨酸浓度增大而逐渐增加(P均<0.01)。3组裸鼠均发现未有肝脏和肺部的转移灶。
7.免疫组化、Western Blot方法及半定量RT-PCR方法检测均显示裸鼠移植瘤中c-myc和uPA基因蛋白和mRNA的表达强度随S-腺苷蛋氨酸浓度增大而明显减弱(P分别<0.05和<0.01)；对照组中c-myc和uPA基因启动子区未甲基化,随着S-腺苷蛋氨酸浓度增高,逐渐恢复甲基化(c-mycx2值=10.179,P<0.01；uPA x2值=11.667,P<0.01)。
结论
1、S-腺苷蛋氨酸能够抑制体外胃癌细胞的增殖、侵袭力和迁移力,诱导凋亡,使细胞阻滞在G1期,且作用与浓度和时间成正相关。S-腺苷蛋氨酸可逆转体外胃癌细胞中c-myc和uPA基因启动子区的低甲基化,抑制其表达。胃癌细胞的增殖、周期、凋亡、侵袭力和迁移力的改变可能与S-腺苷蛋氨酸改善c-myc和uPA基因启动子区的低甲基化状态,抑制其表达有关。
2、S-腺苷蛋氨酸可抑制人胃癌裸鼠移植瘤的生长,促进细胞凋亡,并能逆转移植瘤中c-myc和uPA基因启动子区的低甲基化状态,使其表达受到抑制,正是这种作用可能导致了S-腺苷蛋氨酸对体内胃癌生长的抑制。
3、我们研究并证实了S-腺苷蛋氨酸对体内外胃癌的增殖及其肿瘤特性有抑制作用；其作用机制可能是SAM通过提供甲基基团,逆转了癌基因的低甲基化状态,使其失活。
Gastric cancer is one of the most prevalent malignant tumors, and the number of death from it is more than 150 thousand per year in China. Gastric cancer continue to be the most common fatal cancer recently in China. The carcinogenesis is known as a complicated process involved in polygene, poly-molecular change and multistage. And the most important events are the activation of oncogene and inactivation of tumor suppressor gene in the process. Recently, there are more and more attention to epigenetics, such as DNA methylation. DNA methylation, the covalent addition of a methyl group to the C-5 position of cytosine in the context of the CpG dinucleotide, is catalysised by the DNA methyltransferase enzyme, and has the significant role in the process of carcinogenesis and growth. DNA methylation is a process modified by genes, which is reversible and hereditary. The changes of methylation status, including the decrease of global genome methylation level and the abnomaly raise of CpG island partly methylation level, are important factors in carcinogenesis. These changes can lead to the activation of oncogene and inactivation of tumor suppressor gene, as well as the instability of genome.
S-adenosylmethionine (SAM) is synthetized through S-adenosylmethionine synthetase, and is the primary methyl group donor for most biological methylation reactions. SAM is known as a demethylation suppressor and can reverse gene hypomethylation, induce gene hypermethylation. In recent studies, SAM has been found having the effect on chemopreventing and treating on cancers of liver, colon and breast, because it could promote apoptosis and suppress the growth of cancer cells. So far there were no reports on gastric cancer. For the reason, we investigated the potential benefits of SAM in inhibiting the growth of gastric cancer in vivo and in vitro. Abnomal expression of c-myc is closely correlated with carcinogenesis. And the methylation level of c-myc gene is decreased in the process of carcinogenesis such as liver cancer, gastric cancer, lung cancer. Urokinase-type plasminogen activator (uPA) is related with tumor metastasis. uPA is regard as a tumor trigger gene, for promoting the growth, invasion, transfer, and neovascularity of tumor. We studied the methylation status of gene promoter region and the expression of c-myc and uPA genes, trying to identify the anti-tumor mechanisms of SAM in gastric cancer.
Methods
1 Effects of SAM on proliferation of gastric cancer cell lines
For dose-response experiments, MKN-28, SGC-7901, BGC-823 and MKN-45 cells were incubated with increasing concentrations (0.5-32mmol/L) of SAM for 24h,48h and 72h. Cell growth was detected by MTT colorimetric method. The cell growth inhibition ratio and IC50 were calculated, and the restrain curves were ploted.
2 Effects of SAM on cell cycle and apoptosis of gastric cancer cell lines
The four cell lines were incubated with different concentrations(0,2 and 4 mmol/L) of SAM for 72h. Cell cycle distribution and cell apoptosis were calculated by using of flow cytometry system.
3 Effects of SAM on invasiveness and migration of gastric cancer cell lines
The four cell lines were incubated with different concentrations(0,2 and 4mmol/L) of SAM for 72h. Cells were incubated in the transwell caves at a density of 1×105/well for 24h, to investigate the invasiveness, after the polycarbonate membrane of transwell cave was covered by Matrigel. Or, cells were incubated at a density of 1×106/well for 20h, to investigate the migration. The number of cell which going through the membrane was counted.
4 Effects of SAM on expression and methylation status of c-myc and uPA of gastric cancer cell lines
The four cell lines were incubated with different concentrations(0,2 and 4mmol/L) of SAM for 72h. RT-PCR analysis of c-myc and uPA mRNA expression in cells. Western blot analysis of c-myc and uPA protein expression in cells. MSP analysis of c-myc and uPA gene promoter region methylation status in cells.
5 Establishment of nude mice model of tumor xenografts and treatment by SAM
Tumor xenografts were established by inoculation of SGC-7901 cells subcutaneously in BALB/c nude mice. The mice were randomized separated into three groups:low concentration group(192μmol/(kg-d) SAM), high concentration group(768μmol/(kg-d) SAM) and control group(NS), and treated by peritoneal injection for 15 days.
6 Effects of SAM on growth and c-myc,uPA genes of xenografts
Tumor volume, the weight of nude mice were recorded every three days. After gotten the samples,the changes of histopathology and apoptotic index were observed by HE. The metastasis of liver and lung of nude mice were observed. Immunohistochemistry, Western Blot and RT-PCR were used to analysis c-myc and uPA protein and mRNA expression. MSP analysis of methylation status of gene promoter regions in xenografts.
Statistical Analysis
Data were expressed as mean±SD. The results of the invasiveness and migration of cells, the immunohistochemistry of xenografts were evaluated by rank-sum test, The results of MSP of xenografts were evaluated by contingency table x2 test. The results of the rest were evaluated by ANOVA with SPSS 11.1. The statistical level of significance was set at P<0.05.
Results
1 The growths of MKN-28,SGC-7901,BGC-823 and MKN-45 cell were inhibited and the apoptosis cells were increasing with increase of SAM concentration and action time. The MTT assay showed that the proliferation of the four cell lines were restrained gradually (P<0.05) and the growth inhibition ratio were increasing after treated with SAM. The IC50 of cells of 72h were: MKN-28 IC50 6.37 mmol/L, SGC-7901 IC50 5.40 mmol/L, BGC-823 IC50 4.01mmol/L and MKN-45 IC50 4.77 mmol/L.
2 The apoptosis cells were all significantly increased (P<0,01) after treated with SAM. In SGC-7901 and BGC-823, the cell percentages of G0/G1 phase were significantly increased (P<0.05 and P<0.01), whereas the cell proliferation indexes (PI) were significantly decreased (P<0.05 and P<0.01). But in MKN-28 and MKN-45, the cell cycle and the PI did not show any difference (P>0.05).
3 The number of invasion and migration cell of the four cell lines were decreasing(P<0.01) and the invasion and migration inhibition ratio were all increasing, with increase of SAM concentration.
4 The expressions of c-myc mRNA and protein in SGC-7901 and BGC-823, and the expressions of uPA mRNA and protein in the four cell lines were all significantly decreasing(P<0.05 and P<0.01) with increase of SAM concentration, and the hypomethylation of gene promoter regions were reversed. But the the expressions of c-myc mRNA and protein in MKN-28 and MKN-45, and the hypomethylation of gene promoter regions were no marked change(P>0.05).
5 When nude mice were puting to death, the body weights of three groups were no difference (P>0.05). Compared with control group[(1018.22±223.07)mm3], the tumor volume of low concentration group[(618.51±149.27)mm3] and high concentration group[(444.32±118.51)mm3] were obviousily diminished(P<0.01), and the inhibitory rate of tumor growth of low and high concentration group were 39.26% and 56.36%, respectively.
6 The xenografts pathology conformed with the characteristics of moderately differentiated human gastric carcinoma. The xenografts had more coagulative necrosis and apoptosis after treated with SAM. Apoptotic index had positive correlation with SAM concentration (P<0.01). The metastases of liver and lung of three groups were no found.
7 In the xenografts, the expressions of c-myc and uPA mRNA and protein were all significantly weakening(P<0.05 and P<0.01), and the hypomethylation of gene promoter regions were improving (c-mycχ2=10.179, uPAχ2=11.667, all P<0.01), with increase of SAM concentration.
Conclusions
1 SAM can suppress the proliferation, invasion and migration, induce apoptosis, blocke at G1 phase of the gastric cancer cell lines, in a dose-and time-dependent manner in vitro. And SAM can reverse the hypomethylation of c-myc, uPA gene promoter regions, reduce their expression. The changes of proliferation, cell cycle, apoptosis, invasion and migration of gastric cancer cells may correlate with the effects of SAM on hypomethylation of c-myc and uPA gene promoter region and their expression.
2 SAM can inhibit the growth, promote apoptosis, reverse the hypomethylation of c-myc and uPA gene promoter region, and reduce genes'expression of human gastric carcinoma xenografts. May be these effects result the inhibition of human gastric carcinoma in vivo.
3 Our data demonstrate the inhibition of SAM on human gastric carcinoma in vivo and in vitro. The mechanisms of the effects may be SAM can reverse the hypomethylation of oncogenes and re-inactivate them by providing methyl groups.
引文
[1]Wolffe A P, Matzke M A. Epigenetics:regulation through repression[J]. Science,1999, 286:481-486
[2]Holliday R. Aging and the biochemistry of life[J].Trends Biochem Sci,2001,26:68-71
[3]Laird P W, Rudolf Jaenisch R. The role of DNA methylation in cancer genetics and epigenetics[J]. Annu Rev Genet,1996,30:441-464
[4]Nuovo G J, Plaia T W, Belinsky S A, et al. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis[J]. Proc Natl Acad Sci USA,1999,96:12754-12759
[5]Ehrlich M. DNA methylation in cancer:too much, but also too little [J]. Oncogene,2002, 21:5400-5413
[6]Gaudet F, Hodgson J G, Edcn A, et al. Induction of tumors in mice by genomic hypomethylation [J]. Science,2003,300:489-492
[7]Lengauer C. Cancer:An unstable liaison[J]. Science,2003,300:442-443
[8]Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability and tumors promoted by DNA hypomethylation[J]. Science,2003,455
[9]Tao L,Yang S,Xie M, et al. Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors [J]. Cancer Lett,2000,158:185-193
[10]Fang J Y, Zhu S S, Xiao S D, et al. Studies on the hypomethylation of c-myc, c-Ha-ras oncogenes and histopathological changes in human gastric carcinoma[J]. J Gastroenterol Hepatol,1996,11:1079-1082
[11]Cameron E E, Bachman K E, Myohanen S, et al. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer[J]. Nat Genet, 1999,21:103-107
[12]Li Q L, Ito K, Sakakura C, et al. Causal relationship between the loss of RUNX3 expression and gastric cancer [J]. Cell,2002,109:113-124
[13]Koen D, Eleonora B, Igor K, et al. The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorrigenesis [J]. Proc Natl Acad Sci USA,2001,98:7504-7509
[14]Harada K, Toyooka S, Maitra A, et al. Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines [J]. Oncogene,2002,21:4345-4349
[15]Kresge N, Tabor H, Simoni RD, et al. An escape from Italy, the discovery of S-adenosylmethionine, and the biosynthesis of creatine by Giulio L. Cantoni.1953[J]. J Biol Chem,2005,280:e35
[16]Ahmad I, Rao D N. Chemistry and biology of DNA methyltransferases [J]. Crit Rev Biochem Mol Biol,1996,31:361-380
[17]白坚石,马晴,王萍,等.S-腺苷酰L-甲硫氨酸(SAM)对HL-60细胞DNA甲基化酶活力和甲基化水平的影响[J].中国生物化学与分子生物学报,2001,Vol.17(2)：155-159
[18]Pascale R M, Simile M M, De Miglio M R, et al. Chemoprevention of hepatocarcinogenesis:S-adenosyl-L-methionine [J]. Alcohol,2002,27:193-198
[19]Guruswamy S, Swamy MV, Choi CI, et al. S-adenosyl L-methionine inhibits azoxymethane-induced colonic aberrant crypt foci in F344 rats and suppresses human colon cancer Caco-2 cell growth in 3D culture [J]. Int J Cancer,2008,122:25-30
[20]Pakneshan P, Szyf M, Farias-Eisner R, et al. Reversal of the hypomethylation status of urokinase (uPA) promoter blocks breast cancer growth and metastasis [J]. J Biol Chem, 2004,279:31735-31744
[21]Shen L, Fang J, Qiu D, Zhang T, et al. Correlation between DNA methylation and pathological changes in human hepatocellular carcinoma[J]. Hepatogastroenterology, 1998,45:1753-1759
[22]Sharrard R M, Royds J A, Rogers S, et al. Patterns of methylation of the c-myc gene in human colorectal cancer progression [J]. Br J Cancer,1992,65:667-672
[23]Xing R H, Rabbani S A. Transcriptional regulation of urokinase (uPA) gene expression in breast cancer cell:role of DNA methylation [J]. Int J Cancer,1999,81:443-450
[24]Pulukuri S M, Estes N, Patel J, et al. Demethylation-linked activation of urokinase plasminogen activator is involved in progression of prostate cancer[J]. Cancer Res, 2007,67:930-939
[25]Shukeir N, Pakneshan P, Chen G, et al. Alteration of the methylation status of tumor-promoting genes decreases prostate cancer cell invasiveness and tumorigensis in vitro and in vivo [J]. Cancer Res,2006,66:9202-9210
[26]Pascale R M, Marras V, Simile M M, et al. Chemoprevention of rat liver carcinogenesis by S-adenosyl-L-methionine:a long-term study [J]. Cancer Res,1992,52:4979-4986
[27]Lu SC, Ramani K, Ou X, et al. S-Adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model [J]. Hepatology,2009,50:462-471
[28]Li T W, Zhang Q, Oh P, et al. S-Adenosylmethionine and methylthioadenosine inhibit cellular FLICE inhibitory protein expression and induce apoptosis in colon cancer cells[J]. Mol Pharmacol,2009,76:192-200
[29]董志伟, 乔友林, 李连弟,等.中国癌症控制策略研究报告[J].中国肿瘤,2002,Vol.11(5)：250-260
[30]Eskelinen M J, Haglund UH. Prognosis of human pancreatic adenocarcinoma:review of clinical and histopathological variables and possible use of new molecular methods[J]. Eur J Surg,1999,165:292-306
[31]Wainfan E, Pooroer L A. Methyl groups in carcinogenesis:effects on DNA methylation and gene expression[J]. Cancer Res,1992,152:2071s-2077s
[32]Simile M M, Pascale R M, De Miglio M R, et al. Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis [J]. Cancer Lett,1994,79:9-16
[33]Schmutte C, Yang A S, Nguyen T T, et al. Mechanisms for the involvement of DNA methylation in colon carcinogenesis[J]. Cancer Res,1996,56:2375-2381
[34]Mosmann T. Rapid colorimetric assay of cellular groth and suvrival:application to proliferation and cytotoxiciyt assays[J]. J Immunol Methods,1983,65:55-63
[35]King K L, Cidlowwski J A. Cell cycle regulation and apoptosis[J]. Annu Rev Physiol, 1998,60:601-617
[36]Houtpraaf J H, Versmissen J, van der Giessen W J. A concise review of DNA damage checkpoints and repair in mammalian cells[J]. Cardiovase Revase Med,2006,7:165-172
[37]Lobrich M, Jeggo PA. The impact of a negligent G2/M checkpoint on genomic instability and cancer induction[J]. Nat Rev Cancer,2007,7:861-869
[38]Hartwell L H, Kastan M B. Cell cycle control and cancer[J]. Science,1994,266: 1821-1828
[39]Hino N, Higashi T, Nouso K, et al. Apoptosis and proliferation of human hepatocellular carcinoma[J]. Liver,1996,16:123-129
[40]Shoji Y, Saegusa M, Takano Y, et al. Correlation of apoptosis with tumor cell differentiation, progression, and HPV infection in cervical cancinoma[J]. J Clin Pathol, 1996,49:134-138
[41]潘銮凤.分子生物学技术[M].上海：复旦大学出版社,2008.158-160.
[42]Kleiner D E, Stetler-Stevenson W G. Matrix metalloproteinases and metastasis[J]. Cancer Chemother Pharmacol,1999,43:S42-51
[43]Liotta L A, Kohn E C. The microenvironment of the tumor-host interface[J]. Nature, 2001,411:375-379
[44]Nabil I R, Watanabe H, Raz A. Autocrine motility factor and its receptor:role in cell locomotion and metastasis[J]. Cancer Metastasis Rev,1992,11:5-20
[45]朱静,万远廉,高嵩.细胞体外侵袭实验常见问题及对策[J].实用医技杂志,2006,Vol.13(15)：2583-2584
[46]Soares J, Pinto A E, Cunha C V, et al. Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor progression [J]. Cancer,1999,85:112-118
[47]Cravo M, Pinto R, Fidalgo P, et al. Global DNA hypomethylation occurs in the early stages of intestinal type gastric carcinoma [J]. Gut,1996,39:434-438
[48]Feinberg A P, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts[J]. Nature,1983,301:89-92
[49]Kim Y I, Giuliano A, Hatch K D, et al. Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma[J]. Cancer,1994,74:893-899
[50]Fang J Y, Cheng Z H, Chen Y X, et al. Expression of Dnmtl, demethylase, MeCP2 and methylation of tumor-related genes in human gastric cancer [J]. World J Gastroenterol, 2004,10:3394-3398
[51]Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands[J]. Proc Natl Acad Sci USA,1996,93:9821-9826
[52]Ahmad I, Rao D N. Chemistry and biology of DNA methyltransferases[J]. Crit Rev Biochem Mol Biol,1996,31:361-380
[53]Vertino P M, Yen R W, Gao J, et al. De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5)-methyltransferase[J]. Mol Cell Biol, 1996,16:4555-4565
[54]Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells[J]. Nat Rev Cancer,2005,5:223-231
[55]Almeida A, Kokalj-Vokac N, Lefrancois D, et al. Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines [J]. Hum Genet,1993,91: 538-546
[56]Jones P A. DNA methylation errors and cancer[J]. Cancer Res,1996,56:2463-2467
[57]Wong I H, Lo Y M, Zhang J, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients[J]. Cancer Res,1999,59:71-73
[58]Sun L,Hui A M, Kanai Y, et al. Increased DNA methyltransferase expression is associated with an early stage of human hepatocarcinogenesis[J]. Jpn J Cancer Res,1997, 88:1165-1170
[59]Gonzalez-Zulueta M, Bender C M, Yang A S, et al. Methylation of the 5'CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing[J]. Cancer Res,1995,55:4531-4535
[60]Herman J G, Jen J, Merlo A, et al. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B[J]. Cancer Res,1996,56:722-727
[61]Kang G H, Lee S, Kim W H, et al. Epstein-barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma[J]. Am J Pathol,2002,160:787-794
[62]Tsuchiya T, Tamura G, Sato K, et al. Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia [J]. Oncogene,2000,19:3642-3646
[63]Kane M F, Loda M, Gaida G M, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines[J]. Cancer Res 1997,57:808-811
[64]Teitz T, Wei T, Valentine M B, et al. Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN[J]. Nat Med,2000,6:529-535
[65]Dammann R, Li C, Yoon J H, et al. Epigenetic inactivation of a RAS association domain family protein fonn the lung tumour suppressor locus 3p21.3.[J]. Nat Genet,2000,25: 315-319
[66]Kang G H, Lee S, Kim J S, et al. Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis [J]. Lab Invest,2003,83:635-641
[67]Limnander A, Rothman P B. Abl oncogene bypasses normal regulation of Jak/STAT activation[J]. Cell Cycle,2004,3:1486-1488
[68]Crossen P E, Morrison M J. Methylation status of 3rd exon of the c-MYC oncogene in B-cell malignancies. Leuk Res,1999,23:251-253
[69]Nishigaki M, Aoyagi K, Danjoh I, et al. Discovery of aberrant expression of R-RAS by cancer-linked DNA hypomethylation in gastric cancer using microarrys[J]. Cancer Res, 2005,65:2115-2124
[70]Jang S J, Soria J C, Wang L, et al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis[J]. Cancer Res,2001, 61:7959-7963
[71]Pakneshan P, Xing R H, Rabbani S A. Methylation status of uPA promoter as a molecular mechanism regulating prostate cancer invasion and growth in vitro and in vivo[J]. FASEB J,2003,17:1081-1088
[72]Gaudet F,Hodgson J G,Edcn A,ct al. Induction of tumors in mice by genomic hypomethylation[J]. Science,2003,300:489-492
[73]Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability and tumors promoted by DNA hypomethylation[J]. Science,2003,300:455.
[74]Lengauer C, Kinzler K W, Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells [J]. Proc Natl Acad Sci USA,1997,94:2545-2450
[75]Riggs A D, Jones P A.5-methylcytosine, gene regulation, and cancer [J]. Adv Cancer Res, 1984,40:1-30.
[76]Nuovo G J, Plaia T W, Belinsky S A, et al. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis [J]. Proc Natl Acad Sci USA,1999,96:12754-12759
[77]Momparler R L, Ayoub J. Potential of 5-aza-2'-deoxycytidine (Decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer [J]. Lung Cancer,2001,34:S111-115
[78]Fang J Y, Yang L, Zhu HY, et al.5-Aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16INK4A gene in human gastric cancer cell lines[J]. Chin Med J,2004,117:99-103
[79]Kang G H, Lee S, Kim J S, et al. Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis [J]. Lab Invest,2003,83:635-641
[80]Tsuchiya T, Tamura G, Sato K, et al. Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia [J]. Oncogene,2000,19:3642-3646
[81]Dahl C, Guldberg P. DNA methylation analysis techniques [J]. Biogerontology,2003,4: 233-250
[82]黄琼晓,金帆,黄荷凤.DNA甲基化的研究方法学[J].国外医学遗传学分册,2004,Vol.27(6)：354-358
[83]Kuo K C, McCune R A, Gehrke C W, et al. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA [J]. Nucleic Acids Res,1980,8:4763-4776
[84]Wu J, Issa J P, Hermen J, et al. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells [J]. Proc Natl Acad Sei USA,1993,90:8891-8895
[85]Oakeley E J, Schmitt F, Jost J P. Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction [J]. Biotechniques,1999,27:744-746,748-750,752
[86]Costello J F, Fruhwald M C, Smiraglia D J, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns [J]. Nat Genet,2000,24:132-138
[87]Yegnasubramanian S, Lin X, Haffner M C, et al. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation [J]. Nucleic Acids Res, 2006,34:e19
[88]de Nigris F, Sica V, Hermann J, et al. c-myc oncoprptein:cell cycle-related evevts and new therapeutic challenges in cancer and cardiovascular diseases[J]. Cell Cycle,2003,2: 325-328
[89]Cheah M S, Wallace C D, Hoffman R M. Hypomethylation of DNA in human cancer cells:a site-specific change in the c-myc oncogene [J]. J Natl Cancer Inst,1984,73: 1057-1065
[90]Rabbani S A, Xing R H. Role of urokinase (uPA) and its receptor (uPAR)in invasion and metastasis of hormone-dependent malignancies [J]. Int J Oncol,1998,12:911-920
[91]Liotta L A, Kohn E C. The microenvironment of the tumour-host interface. Nature,2001, 411:375-379
[92]施新猷.医用实验动物学[M].西安：陕西科学技术出版社,1989.300-303
[93]Ostrand-Rosenberg S. Animal models of tumor immunity, immunotherapy and cancer vaccines[J]. Curr Opin Immunol,2004,16:143-150
[94]施新猷.医用实验动物学[M].西安：陕西科学技术出版社,1989.128-150
[95]Hoffman R M. Orthotopic metastatic mouse models for anticancer drug discovery and evaluation:a bridge to the clinic[J].Invest New Drugs,1999,17:343-359
[96]朱新江,戴冬秋.表遗传学与胃肠道肿瘤[J].世界华人消化杂志,2006,Vol.14(34)：3251-3256
[97]Chiang P K, Gordon P K, Tal J, et al. S-adenosylmethionine and methylation [J]. FASEB J,1996,10:471-480
[98]Detich N, Hamm S, Just G, et al. The methyl donor S-adenosylmethionine inhibits active demethylation of DNA:a candidate novel mechanism for the pharmacological effects of S-adenosylmethionine [J]. J Biol Chem,2003,278:20812-20820
[99]Fang J Y, Xiao S D. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis[J]. J Gastroenterol,2003,38:821-829
[100]Harnack L,Jacobs D R Jr,Nicodemus K, et al. Relationship of folate, vitamin B-6, vitamin B-12, and methionine intake to incidence of colorectal cancers[J]. Nutr Cancer,2002,43: 152-158
[101]Davis C D, Uthus E O. DNA methylation, cancer susceptibility, and nutrient interactions. Exp Biol Med (Maywood),2004,229:988-995
[102]Lowrey C H, Nienhuls A W. Brief report:treatment with azacitidine of patients with end-stage beta-thalassemia[J]. N Engl J Med,1993,329:845-848
[103]Charache S, Dover G, Smith K, et al. Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the gamma-delta-beta-globin gene complex[J]. Proc Natl Acad Sci USA,1983,80:4842-4846
[104]Maslak P, Chanel S, Camacho L H, et al. Pilot study of combination transcriptional modulation therapy with sodium phenylbutyrate and 5-azacytidine in patients with acute myeloid leukemia or myelodysplastic syndrome[J]. Leukemia、2006,20:212-217
[105]Hackanson B, Robbel C, Wijermans P, et al. In vivo effects of decitabine in myelodysplasia and acute myeloid leukemia:review of cytogenetic and molecular studies [J]. Ann Hematol,2005,84 Supp 1:32-38
[106]Ateeq B, Unterberger A, Szyf M, et al. Pharmacological inhibition of DNA methylation induces proinvasive and prometastatic genes in vitro and in vivo[J]. Neoplasia,2008,10: 266-278
[107]Shukeir N, Pakneshan P, Chen G, et al. Alteration of the methylation status of tumor-promoting genes decreases prostate cancer cell invasiveness and tumorigenesis in vitro and in ivivo[J]. Cancer Res,2006,66:9202-9210
[108]Pakneshan P, Szyf M, Rabbani S A. Hypomethylation of urokinase(uPA) promoter in breast and prostate cancer:prognostic and therapeutic implications[J]. Curr Cancer Drug Targets,2005,5:471-488
[109]Van Wart H E, Birkedal-Hansen H. The cysteine switch:a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family [J]. Proc Natl Acad Sci USA,1990,87:5578-5582
[110]Duffy M J. The urokinase plasminogen activator system:role in malignancy[J]. Curr Pharm Des,2004,10:39-49
[111]Rabbani S A, Mazar A P. The role of the plasminogen activation system in angiogenesis and metastasis[J]. Surg Oncol Clin N Am.2001,10:393-415
[112]Blasi F, Carmeliet P. uPAR:a versatile signalling or chestrator[J]. Nat Rev Mol Cell Biol. 2002,3:932-943
[1]Singal R, Ginder G D. DNA methylation[J]. Blood,1999,93:4059-4070
[2]Baylin S B. Tying it all together:Epigenetics, genetics, cell cycle, and cancer. Science, 1997,277:1948-1949
[3]Laird P W. The power and the promise of DNA methylation markers. Nat Rev Cancer, 2003,3:253-266
[4]Amir R E, Van den Veyver I B, Wan M, et al:Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet,1999,23:185-188
[5]Ramsahoye B H, Biniszkiewicz D, Lyko F, et al:Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci U S A,2000,97:5237-5242
[6]Antequera F, Bird A. CpG islands. Exs,1993,64:169-185
[7]Bogdanovic O, Veenstra GJ. DNA methylation and methyl-CpG binding proteins: developmental requirements and function. Chromosoma,2009,118:549-565
[8]Ushijima T, Asada K. Aberrant DNA methylation in contrast with mutations. Cancer Sci, 2010,101:300-305
[9]Robertson K D. DNA methylation and chromatin- unraveling the tangled web. Oncogene,2002,21:5361-5379
[10]Costello J F, Plass C. Methylation matters.J Med Genet,2001,38:285-303
[11]Szyf M. Targeting DNA methylation in cancer. Ageing Res Rev,2003,2:299-328
[12]Fremont M, Siegmann M, Gaulis S, et al. Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA. Nucleic Acids Res,1997,25:2375-2380
[13]Bhattacharya S K, Ramchandani S, Cervoni N, et al. A mammalian protein with specific demethylase activity for mCpG DNA. Nature,1999,397:579-583
[14]Ng HH, Zhang Y, Hendrich B, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet,1999,23:58-61
[15]Wade P A, Gegonne A, Jones P L, et al. Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat Genet,1999,23:62-66
[16]Jones P A, Laird P W. Cancer epigenetics comes of age. Nat Genet,1999,21:163-167
[17]Trinh B N, Long T I, Nickel A E, et al. DNA methyltransferase deficiency modifies cancer susceptibility in mice lacking DNA mismatch repair. Mol Cell Biol,2002,22: 2906-2917
[18]Gaudet F, Hodgson J G, Eden A, el al. Induction of tumors in mice by genomic hypomethylation. Science,2003,300:489-492
[19]Jones P A. The DNA methylation paradox.Trends Genet,1999,15:34-37
[20]Tate P H, Bird A P. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev,1993,3:226-231
[21]Prokhortchouk E, Hendrich B. Methyl-CpG binding proteins and cancer:Are MeCpGs more important than MBDs? Oncogene,2002,21:5394-5399
[22]Prokhortchouk A, Hendrich B, Jorgensen H, et al. The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor. Genes Dev,2001,15:1613-1618
[23]Unoki M, Brunet J, Mousli M. Drug discovery targeting epigenetic codes:the great potential of UHRF1, which links DNA methylation and histone modifications, as a drug target in cancers and toxoplasmosis. Biochem Pharmacol,2009,78:1279-1288
[24]Tamaru H, Selker EU. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature,2001,414:277-283
[25]Fahrner J A, Eguchi S, Herman J G, et al. Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res,2002,62:7213-7218
[26]Baylin S B, Herman J G. DNA hypermethylation in tumorigenesis:epigenetics joins genetics. Trends Genet,2000,16:168-174
[27]Ehrlich M. DNA methylation in cancer:Too much, but also too little. Oncogene,2002,21: 5400-5413
[28]Clark S J, Melki J. DNA methylation and gene silencing in cancer:Which is the guilty party? Oncogene,2002,21:5380-5387
[29]Lee W H, Morton R A, Epstein J I, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci U S A,1994,91:11733-11737
[30]Melki J R, Vincent P C, Clark S J. Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res,1999,59:3730-3740
[31]Di Croce L, Raker V A, Corsaro M, et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science,2002, 295:1079-1082
[32]Cheung H H, Lee T L, Rennert O M, et al. DNA methylation of cancer genome. Birth Defects Res C Embryo Today,2009,87:335-350
[33]Martens JW, Margossian AL, Schmitt M, et al. DNA methylation as a biomarker in breast cancer. Future Oncol,2009,5:1245-1256
[34]Catteau A, Morris J R. BRCA1 methylation:A significant role in tumour development? Semin Cancer Biol,2002,12:359-371
[35]Leone G, Teofili L, Voso M T, et al:DNA methylation and demethylating drugs in myelodysplastic syndromes and secondary leukemias. Haematologica,2002,87: 1324-1341
[36]Toyota M, Kopecky K J, Toyota M 0, et al. Methylation profiling in acute myeloid leukemia. Blood,2001,97:2823-2829
[37]Feinberg A P, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature,1983,301:89-92
[38]Kerr K M, Galler J S, Hagen J A, et al. The role of DNA methylation in the development and progression of lung adenocarcinoma. Dis Markers,2007,23:5-30
[39]Lin C H, Hsieh S Y, Sheen I S, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res,2001,61:4238-4243
[40]Okano M, Bell D W, Haber D A, et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell,1999,99:247-257
[41]Tuck-Muller C M, Narayan A, Tsien F, et al. DNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients. Cytogenet Cell Genet,2000,89:121-128
[42]Gershoni-Baruch R, Dagan E, Israeli D, et al. Association of the C677T polymorphism in the MTHFR gene with breast and/or ovarian cancer risk in Jewish women. Eur J Cancer,2000,36:2313-2316
[43]Song C, Xing D, Tan W, et al. Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population. Cancer Res,2001,61:3272-3275
[44]Miao X, Xing D, Tan W, et al. Susceptibility to gastric cardia adenocarcinoma and genetic polymorphisms in methylenetetrahydrofolate reductase in an at-risk chinese population. Cancer Epidemiol Biomarkers Prev,2002,11:1454-1458
[45]Wiemels J L, Smith R N, Taylor G M, et al. Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia. Proc Natl Acad Sci U S A,2001,98:4004-4009
[46]Chen J, Giovannucci E, Kelsey K, et al. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res,1996,56:4862-4864
[47]Kim Y I, Pogribny I P, Salomon R N, et al. Exon-specific DNA hypomethylation of the p53 gene of rat colon induced by dimethylhydrazine:Modulation by dietary folate. Am J Pathol,1996,149:1129-1137
[48]Christman J K, Sheikhnejad G, Dizik M, et al. Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency. Carcinogenesis,1993,14:551-557
[49]Pogribny I P, Miller B J, James S J. Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat. Cancer Lett,1997,115: 31-38
[50]Fraga M F, Esteller M. DNA methylation:A profile of methods and applications. Biotechniques,2002,33:632,634,636-649
[51]Dominguez G, Carballido J, Silva J, et al. P14ARF promoter hypermethylation in plasma DNA as an indicator of disease recurrence in bladder cancer patients. Clin Cancer Res, 2002,8:980-985
[52]Evron E, Dooley W C, Umbricht C B, et al. Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. Lancet,2001,357:1335-1336
[53]Zou H Z, Yu B M, Wang Z W, et al. Detection of aberrant p16 methylation in the serum of colorectal cancer patients. Clin Cancer Res,2002,8:188-191
[54]Kersting M, Friedl C, Kraus A, et al. Differential frequencies of p16(INK4a) promoter hypermethylation, p53 mutation, and K-ras mutation in exfoliative material mark the development of lung cancer in symptomatic chronic smokers. J Clin Oncol,2000,18: 3221-3229
[55]Kawakami K, Brabender J, Lord R V, et al. Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma.J Natl Cancer Inst,2000,92: 1805-1811
[56]Sanchez-Cespedes M, Esteller M, Wu L,et al. Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res,2000,60:892-895
[57]Wong I H, Lo Y M, Yeo W, et al. Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients. Clin Cancer Res,2000,6: 3516-3521
[58]Maeda K, Kawakami K, Ishida Y, et al. Hypermethylation of the CDKN2A gene in colorectal cancer is associated with shorter survival. Oncol Rep,2003,10:935-938
[59]Yi J, Wang Z W, Cang H, et al. P16 gene methylation in colorectal cancers associated with Duke's staging. World J Gastroenterol,2001,7:722-725
[60]Tomizawa Y, Kohno T, Kondo H, et al. Clinicopathological significance of epigenetic inactivation of RASSF1A at 3p21.3 in stage I lung adenocarcinoma. Clin Cancer Res, 2002,8:2362-2368
[61]Ng C S, Zhang J, Wan S, et al. Tumor p16M is a possible marker of advanced stage in non-small cell lung cancer. J Surg Oncol,2002,79:101-106
[62]Hayashi M, Tokuchi Y, Hashimoto T, et al. Reduced HIC-1 gene expression in non-small cell lung cancer and its clinical significance. Anticancer Res,2001,21:535-540
[63]Maruyama R, Toyooka S, Toyooka K O, et al. Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features. Clin Cancer Res, 2002,8:514-519
[64]Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med,2000, 343:1350-1354
[65]Arnold C N, Goel A, Boland C R. Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines. Int J Cancer,2003,106:66-73
[66]Strathdee G, MacKean M J, Illand M, et al. A role for methylation of the hMLH1 promoter in loss of hMLH1 expression and drug resistance in ovarian cancer. Oncogene, 1999,18:2335-2341
[67]Lubbert M. DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies:Clinical results and possible mechanisms of action. Curr Top Microbiol Immunol,2000,249:135-164
[68]Strathdee G, Brown R. Epigenetic cancer therapies:DNA methyltransferase inhibitors. Expert Opin Investig Drugs,2002,11:747-754
[69]Goffin J, Eisenhauer E. DNA methyltransferase inhibitors-state of the art. Ann Oncol, 2002,13:1699-1716
[70]Thiagalingam S, Cheng K H, Lee H J, et al. Histone deacetylases:Unique players in shaping the epigenetic histone code. Ann N Y Acad Sci,2003,983:84-100
[71]Santini V, Kantarjian H M, Issa J P. Changes in DNA methylation in neoplasia: Pathophysiology and therapeutic implications. Ann Intern Med,2001,134:573-586
[72]Momparler R L. Epigenetic therapy of cancer with 5-aza-2'-deoxycytidine (decitabine). Semin Oncol,2005,32:443-451
[73]Momparler R L, Bouchard J, Onetto N, et al.5-aza-2'-deoxycytidine therapy in patients with acute leukemia inhibits DNA methylation. Leuk Res,1984,8:181-185
[74]Steuber C P, Krischer J, Holbrook T, et al. Therapy of refractory or recurrent childhood acute myeloid leukemia using amsacrine and etoposide with or without azacitidine:A Pediatric Oncology Group randomized phase Ⅱ study. J Clin Oncol,1996,14:1521-1525
[75]Kantarjian H M, O'Brien S, Cortes J, et al. Results of decitabine (5-aza-2_deoxycytidine) therapy in 130 patients with chronic myelogenous leukemia. Cancer,2003,98:522-528
[76]Daskalakis M, Nguyen T T, Nguyen C, et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidirie (decitabine) treatment. Blood,2002,100:2957-2964
[77]Sato N, Maehara N, Su GH, et al. Effects of 5-aza-2'-deoxycytidine on matrix metalloproteinase expression and pancreatic cancer cell invasiveness. J Natl Cancer Inst, 2003,95:327-330