5-氮杂-2’-脱氧胞苷对人子宫颈癌细胞系HeLa及SiHa细胞Syk基因异常甲基化和表达的影响
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摘要
目的研究不同浓度的去甲基化药物5-氮杂-2’-脱氧胞苷对人子宫颈癌细胞系HeLa及SiHa细胞Syk基因启动子区异常甲基化和表达的影响。
方法MSP方法检测宫颈癌细胞系HeLa及SiHa细胞启动子区Syk基因甲基化情况；MTT检测不同浓度的5-氮杂-2’-脱氧胞苷(5-Aza-CdR)干预后HeLa细胞的增殖情况；MSP法检测不同浓度的5-氮杂-2’-脱氧胞苷(5-Aza-CdR)干预前后HeLa及SiHa细胞的Syk基因启动子区的甲基化状态的改变；RT-PCR法检测用药前后HeLa及SiHa细胞中SykmRNA表达水平的变化。
结果HeLa细胞Syk基因启动子区存在甲基化,SiHa细胞Syk基因启动子区无甲基化；5-氮杂-2'-脱氧胞苷对HeLa细胞的生长有抑制作用,5-氮杂-2'-脱氧胞苷浓度为1μM时对HeLa细胞生长的抑制作用最佳；5-氮杂-2'-脱氧胞苷对HeLa细胞启动子区甲基化有逆转作用,药物浓度为1μM时去甲基化效果最好：可使HeLa细胞SykmRNA的表达水平明显增高。
结论HeLa细胞Syk基因启动子区存在甲基化,5-氮杂-2’-脱氧胞苷干预能使其Syk甲基化的基因重新表达并抑制细胞生长。
Objective To investigate the effect of different concentrations of demethylating agent 5-Aza-2',-deoxycytidine (5-Aza-CdR) on the expression and its methylation of Syk gene promoter region in cervical cancer cell lines of HeLa and SiHa.
Methods Methylation-specific PCR (MSP) was utilized to detect the methylation state of Syk gene promoter in cervical cancer cell lines of Hela and SiHa; Activation in HeLa cells proliferation was monitored by MTT assay pre/post treatment at different concentrations of 5-Aza-2'-deoxycytidine; MSP was also applied to detect the changes of methylation on Syk gene promoter in cervical cell lines of HeLa and SiHa pre/post treatment with different concentrations of 5-Aza-2'-deoxycytidine; in the meantime, the expression level of Syk mRNA was detected using RT-PCR pre/post treatment with 5-Aza-CdR.
Results The methylation of Syk gene promoter was found in HeLa cells and which was not present in SiHa cells;the proliferation of Hela cells could be inhibited by 5-Aza-2'-deoxycytidine, its optimal concentration to inhibit the proliferation of HeLa cells was 1μM. It was also shown that treatment with 5-Aza-CdR could induce the demethylation of the Syk gene promoter, its optimal concentration was luM; further investigation found that the demethylation with 5-Aza-CdR restored Syk gene expression in HeLa cells.
Conclusion It was found there was methylation in the Syk gene promoter in Hela cells, its methhylation could be demethylated by 5-Aza-2'-deoxycytidine, which resulted in restored the expression of Syk gene.5-Aza-2'-deoxycytidine could inhibit the proliferation of Hela cells.
方法MSP方法检测宫颈癌细胞系HeLa及SiHa细胞启动子区Syk基因甲基化情况；MTT检测不同浓度的5-氮杂-2’-脱氧胞苷(5-Aza-CdR)干预后HeLa细胞的增殖情况；MSP法检测不同浓度的5-氮杂-2’-脱氧胞苷(5-Aza-CdR)干预前后HeLa及SiHa细胞的Syk基因启动子区的甲基化状态的改变；RT-PCR法检测用药前后HeLa及SiHa细胞中SykmRNA表达水平的变化。
结果HeLa细胞Syk基因启动子区存在甲基化,SiHa细胞Syk基因启动子区无甲基化；5-氮杂-2'-脱氧胞苷对HeLa细胞的生长有抑制作用,5-氮杂-2'-脱氧胞苷浓度为1μM时对HeLa细胞生长的抑制作用最佳；5-氮杂-2'-脱氧胞苷对HeLa细胞启动子区甲基化有逆转作用,药物浓度为1μM时去甲基化效果最好：可使HeLa细胞SykmRNA的表达水平明显增高。
结论HeLa细胞Syk基因启动子区存在甲基化,5-氮杂-2’-脱氧胞苷干预能使其Syk甲基化的基因重新表达并抑制细胞生长。
Objective To investigate the effect of different concentrations of demethylating agent 5-Aza-2',-deoxycytidine (5-Aza-CdR) on the expression and its methylation of Syk gene promoter region in cervical cancer cell lines of HeLa and SiHa.
Methods Methylation-specific PCR (MSP) was utilized to detect the methylation state of Syk gene promoter in cervical cancer cell lines of Hela and SiHa; Activation in HeLa cells proliferation was monitored by MTT assay pre/post treatment at different concentrations of 5-Aza-2'-deoxycytidine; MSP was also applied to detect the changes of methylation on Syk gene promoter in cervical cell lines of HeLa and SiHa pre/post treatment with different concentrations of 5-Aza-2'-deoxycytidine; in the meantime, the expression level of Syk mRNA was detected using RT-PCR pre/post treatment with 5-Aza-CdR.
Results The methylation of Syk gene promoter was found in HeLa cells and which was not present in SiHa cells;the proliferation of Hela cells could be inhibited by 5-Aza-2'-deoxycytidine, its optimal concentration to inhibit the proliferation of HeLa cells was 1μM. It was also shown that treatment with 5-Aza-CdR could induce the demethylation of the Syk gene promoter, its optimal concentration was luM; further investigation found that the demethylation with 5-Aza-CdR restored Syk gene expression in HeLa cells.
Conclusion It was found there was methylation in the Syk gene promoter in Hela cells, its methhylation could be demethylated by 5-Aza-2'-deoxycytidine, which resulted in restored the expression of Syk gene.5-Aza-2'-deoxycytidine could inhibit the proliferation of Hela cells.
引文
[1]Greenspan DI, Connolly DC, Wu r, et al.loss of fhit expression in cervical carcinoma cell lines and primary tumors[J].cancer res,1997,57:4692-4698.
[2]Taniguchi T,Kobayashi T,Kondo J,et al.Molecular cloning of a porcine gene Syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to prteolysis[J].J Biol Chem,1991,266 (24);15790-15796.
[3]Turner M, Schweighoffer E, Colucci F, et al. Tyrosine kinase SYK:essential functions for immunoreceptor signaling[J].Immunol Today,2000,21(3):148-154.
[4]Coopman P J,Do M T,Barth M et a.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells[J].Nature,2000,406(8):742-747.
[5]Takano T,Sada K, Yamamura H.Role of protein-tyrosine kinase syk in oxidative stress signaling in B cells [J].Antioxid Redox Signal,2002,4 (3):533-541.
[6]Murphy MA,Schnall RG, Venter DJ,et al.Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficientmice [J].Mol Cell Biol,1998,18 (8):4872-4882.
[7]陈明霞,丁永斌,戴存才,等.MSP法检测胰腺癌Syk基因甲基化改变的研究[J].河北医学,2003,9(7)：577-579.
[8]Ulanova M, Puttagunta L, Marcet-Palacios M, et al. Syk tyrosine kinaseparticipates in betal-integrin signaling and inflammatory responses in airwayepithelial cells [J].Am J PhysiolLung Cell Mol Physiol,2005,288(3):497-507.
[9]Yamada T, Fujieda S, Yanagi S, etal. Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production[J].J Immunol,2001,166(1):538-543.
[10]Inatome R, Yanagi S, Takano T, etal. A critical role for Syk in endothelial cell proliferation and migration [J].Biochem Biophys Res Commun,2001,286(1):195-199.
[11]Tsujimura T, Yanagi S,Inatome R,et al. Syk protein-tyrosine kinase isinvolved in neuron-like differentiation of embryonal carcinoma P19 cells[J].FEBS Lett,2001,489 (2-3):129-133.
[12]Tsuchida S, Yanagi S, Inatome R,et al.Purification of a 72-kDaprotein-tyrosine kinase from rat liver and its identification as Syk:involvement of Syk in signaling events of hepatocytes[J].J Biochem(Tokyo),2000,127(2):321-327.
[13]Hoeller C, Thallinger C, PratscherB,et al. The non-receptor-associatedtyrosine kinase Syk is a regulator of metastatic behavior in humanmelanoma cells[J].J Invest Dermatol,2005,124(6): 1293-1299.
[14]Wang S, Ding YB,Chen GY,et al.Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma. World J Gastroenterol.2004 Jun 15；10(12)：1815-1818.
[15]孙桂霞,马德花,陈荣辉,等.候选抑癌基因Syk与宫颈癌发生发展相关性研究[J].中华肿瘤防治杂志2009,13(1)814-816+824
[16]邢学森,黄利呜,等.改良的甲基化特异PCR法在人宫颈癌Hela细胞检测中的应用[J].中国现代医学杂志,2006,16(2)：191-194.
[17]Yuan Y,Liu H,Sahin A,et al.Reactivation of Syk expression by inhibition of DNA methylation suppresses breast cancer cell invisiveness.Int J Cancer,2005,113:654-659.
[18]Bender CM,PaoMM,Jones PA.Inhibition of DNA by 5-Aza-2'-deoxycytidine suppresses the growth of human tumor cell lines[J].Cancer Res,1998,58(1):95-101.
[19]Wijermans P,Lubbert M,Vverhoef G,et al.Low dose 5-Aza-2'-deoxycytidine,a DNA hypomethylation agent,for the treatment of high risk myelodyspastic syndrome:a multicenter phase II studying elderly patients[J].J Clin Oncol,2000,18(5):956-960.
[20]刘特,刘志学,等.5-杂氮-2’-脱氧胞营对宫颈癌细胞生长及其甲基化转移酶和甲基结合蛋白DNA转录的影响[J].中国临床医学,2008,15(4)：524-527.
[21]郑伟,丁永斌,武正炎.SYK基因表达对乳腺癌生长、转移影响的动物实验[J].中华普通外科杂志,2005，20：5
[22]Heman J G, Jen J,Merlo A et al.Hypermethylation associated inactivation indicates a turmor suppressor role for p151NK4B[J].Cancer Res,1996;56:722-727.
[23]Corn P G, Kuerbitz S J,Noesel M M et al. Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5'CpG island methylation[J]. Cancer Res,1999;59:3352-3356.
[24]Yunfei Y, Richard M, Aysegul S et al. Hypermethylation leads to silencing of the SYK gene in human breast cancer[J].Cancer Res,2001; 61 (14):5558-55561.
[25]Feng Q, Stern JE, Hawes SE, et al. DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection.[J]. Exp Mol Pathol.2010 Jan 14.
[1]Mike F J,Hervy E A. Cervical cancer:prevention, diagnosis and therapeutics [J].CA Cancer J Clin, 2001,51(2):92-114.
[2]Slack A,Cervoni N,Pinard M,et al.Feedback regulation of DNA methyltransferase gene expression by methylation.Eur J Biochem,1999,264(1):191
[3]Lehmann U,Brakensiek K, Kreipe H.Role of epigenetic changes in hematological malignancies[J].Ann Hematol,2004,83(3):137-152.
[4]Yu J, Ni M, Xu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis[J].BMC Cancer,2002,2(1):29.
[5]Lee S, Lee HJ, Kim JH,et al.Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis[J].Am J Pathol,2003,163:1371-1378.
[6]Robertson KD,Jones PA. DNA methylation:past,present and future directions[J]. Carcinogenesis,2000,21(3):461-467.
[7]Garinis GA,Patrinos GP, Spanakis NE, et al. DNA hypermethylation:when tumour suppressor genes go silent[J].Hum Genet,2002,111(2):115-127. [8]Jones PA, Laird PW. Cancer epigenetics comes of age[J].Nat Genet,1999,21(2):163-167.
[9]Esteller M,Cirn PG,Baylin SB,et al.A gene hypermethylation profile of human cancer.Cancer Res,2001,61:3225
[10]Belinsky SA,Nikula Kj,Palmisano WA,et al.Aberrant methylation ofp16 is an early event in lung cancer and a potential biomarker forearly diagnosis.Proc NaT1 scad Sci USA,1998,95:11891
[11]Taniguchi T, Kobayashi T, Kondo J, et al. Molecular cloning of a porcine gene Syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis[J].J Biol Chem,1991,266(24):15790-15796.
[12]Turner M, Schweighoffer E, Colucci F, et al. Tyrosine kinase SYK:essential functions for immunoreceptor signaling[J].Immunol Today,2000,21(3):148-154.
[13]David Frommhold,Ingrid Mannigel,Jurgen Schymeinsky,et al.Spleen tyrosine kinase SYK is critical for sustained leukocyte adhesion during inflammation in vivo.BMC Immunology,2007,8:31
[14]Murali Gururajan,Trivikram Dasu,Seif Shahidain C.et al.Spleen Tyrosine Kinase(SYK),a Novel Target of Curcumin, Is Required for B Lymphoma Growth.The Journal of Immunology,2007,178:111
[15]Sada K, Takano T,Yanagi S,et al. Structure and function of Syk protein-tyrosine kinase [J].J Biochem,2001,130(2):177-186.
[16]Carter WB,Hoying JB,Boswell C,et al.HER2/neu over expression induces endothelial cell retraction[J].Int J Cancer,2001,91 (3):295-299.
[17]Murphy MA,Schnall RG, Venter DJ,et al.Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficientmice [J].Mol Cell Biol,1998,18(8):4872-4882.
[18]Coopman PJ,Do MT,Bart h M,et al.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells [J].Nature,2000,406 (6 797):742-747.
[19]Mahabeleshwar G H,Kundu G C.Syk, a protein-tyrosine kinase,suppresses the cell motility andnuclear factor kappa B-mediatedsecretion of urokinase typeplasminogen activator by inhibiting thephosphatidylinositol 3'-kinase activityin breast cancer cells[J].J BiolChem,2003,278(8):6209-6221.
[20]Takada Y, Aggarwal B B.TNF activates Syk protein tyrosine kinaseleading to TNF-induced MAPKactivation, NF-kappaB activation,and apoptosis[J].J Immunol,2004,173(2):1066-1077.
[21]Takada Y, Mukhopadhyay A, KunduG C,et al.Hydrogen peroxideactivates NF-kappa B through tyrosinephosphorylation of I kappa B alphaand serine phosphorylation of p65:evidence for the involvement of Ikappa B alpha kinase and Sykprotein-tyrosine kinase[J].J BiolChem,2003, 278(26):24233-24241.
[22]Moroni M, Soldatenkov V, Zhang L,et al.Progressive loss of Syk andabnormal proliferation in breastcancer cells[J].Cancer Res,2004,64(20):7346-7354.
[23]Ruschel A, Ullrich A. Proteintyrosine kinase Syk modulates EGFRsignalling in human mammaryepithelial cells [J].Cell Signal,2004,16(11):1249-1261.
[24]Jabril-Cuenod B,Zhang C,Scharenberg A M, et al.Sykdependentphosphorylation of She. Apotential link between FcepsilonRIand the ras/mitogen-activated proteinkinase signaling pathway through SOSand Grb2[J].J Biol Chem,1996,271(27):16268-16272.
[25]Kawakami Y, Kitaura J, Yao L, etal. A Ras activation pathwaydependent on Syk phosphorylation ofprotein kinase C[J].Proc Natl AcadSci USA,2003,100(16):9470-9475.
[26]Craxton A, Jiang A, Kurosaki T, etal.Syk and Brutons tyrosine kinaseare required for B cell antigenreceptor-mediated activation of thekinase Akt[J].J Biol Chem,1999,274(43):30644-30650.
[27]Moon K D, Post C B,Durden D L,et al.Molecular basis for a directinteraction between the Syk proteintyrosinekinase and phosphoinositide3-kinase[J].J Biol Chem,2005,280(2):1543-1551.
[28]Li B,Allendorf D J, Hansen R, etal.Yeast beta-glucan amplifiesphagocyte killing of iC3b-opsonizedtumor cells via complementreceptor3-Syk-phosphatidylinositol 3-kinasepathway [J]. J Immunol,2006,177(3):1661-1669.
[29]Peters J D, Furlong M T, Asai D J,et al.Syk, activated by cross-linkingthe B-cell antigen receptor, localizesto the cytosol where it interacts withand phosphorylates alpha-tubulin ontyrosine[J].J Biol Chem,1996,271(9):4755-4762.
[30]Nan X, Ng HH, Johnson CA, et al.Tanscriptional repression by the methyl-CpG2binding p roteinMeCP2 invovles a histone deacetylase comp lex[J].Nature,1998,393 (6683):3862389.
[31]Bachman KE,Rountree MR, Baylin SB.Dnmt3a and Dnmt3b are transcriptional repressor that exhibit unique localization properties to heterochromation[J].J Biol Chem,2001,276(34): 32282232287.
[32]Heman J G, Jen J,Merlo A et al.Hypermethylation associated inactivation indicates a turmor suppressor role for p151NK4B[J].Cancer Res,1996;56:722-727.
[33]Corn P G, Kuerbitz S J, Noesel M M et al. Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5'CpG island methylation[J]. Cancer Res,1999;59:3352-3356.
[34]Yunfei Y, Richard M, Aysegul S et al. Hypermethylation leads to silencing of the SYK gene in human breast cancer[J].Cancer Res,2001;61 (14):5558-55561.
[35]Toyama T, Iwase H,Yamashita H,et al.Reduced expression of the Syk gene is correlated with poor prognosis in human breast cancer [J].Cancer Lett,2003,189(1):97-102.
[36]Yuan Y, Mendez R,Sahin A,et al.Hypermethylation leads to silencing of the Syk gene in human breast cancer [J].Cancer Res,2001,61(14):5558-5561.[8]
[37]Wang S,Ding YB,Chen GY,et al.Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma[J].World J Gastroenterol,2004,10(12):1 815-1818.
[38]Nakashima H, Natsugoe S,IshigamiS,et al. Clinical significance ofnuclear expression of spleen tyrosinekinase(Syk) in gastric cancer[J].Cancer Lett,2006,236 (1):89-94.
[39]Goodman PA,Wood CM,Vassilev A,et al.Spleen tyrosine kinase(Syk) deficiency in childhood pro2B cell acute lympho2blastic leukemia[J] Oncogene,2001,20 (30):3969-3978.
[40]Goodman PA,Burkhardt N,Juran B,et al.Hypermethylation of t he spleen tyrosine kinase promoter in T-lineage acute lymphoblastic leukemia[J]. Oncogene,2003,22(16):2504-2 514.
[41]Watanabe Y.Expression of tyrosine kinase Syk in pancreatic cancers [J].Hokkaido Igaku Zasshi,2001,76(3):173-181.
[42]陈明霞,丁永斌,戴存才,等.MSP法检测胰腺癌Syk基因甲基化改变的研究[J].河北医学,2003,9(7)：577-579.
[43]Ulanova M, Puttagunta L, Marcet-Palacios M, et al.Syk tyrosine kinaseparticipates in betal-integrin signaling and inflammatory responses in airwayepithelial cells[J].Am J PhysiolLung Cell Mol Physiol,2005,288(3):497-507.
[44]Yamada T, Fujieda S,Yanagi S, etal.Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production[J].J Immunol,2001,166(1):538-543.
[45]Inatome R, Yanagi S,Takano T, etal. A critical role for Syk in endothelial cell proliferation and migration[J].Biochem Biophys Res Commun,2001,286(1):195-199.
[46]Tsujimura T, Yanagi S,Inatome R,et al.Syk protein-tyrosine kinase isinvolved in neuron-like differentiation of embryonal carcinoma P19 cells[J].FEBS Lett,2001,489(2-3):129-133.
[47]Tsuchida S, Yanagi S, Inatome R,et al.Purification of a 72-kDaprotein-tyrosine kinase from rat liver and its identification as Syk:involvement of Syk in signaling events of hepatocytes[J].J Biochem(Tokyo),2000,127(2):321-327.
[48]Hoeller C, Thallinger C, PratscherB, et al. The non-receptor-associatedtyrosine kinase Syk is a regulator of metastatic behavior in humanmelanoma cells[J].J Invest Dermatol,2005,124(6): 1293-1299.
[49]Yuan Y, Mendez R, Sahin A, et al.Hypermethylation leads to silencing ofthe SYK gene in human breast cancer[J].Cancer Res,2001,61(14):5558-5561.
[50]丁永斌,武正炎,王水,等.候选抑癌基因Syk启动子甲基化与乳腺癌发生转移的关[J].中华医学杂志,2004,84(4)：290-294.
[51]杨祖立,汪建平,元云飞,等.结直肠癌侵袭性与脾酪氨酸激酶基因Syk启动子甲基化的关系[J].中华实验外科杂志,2005,22(1)：68-70.
[52]陈明霞,丁永斌,蒋奎荣,等.酪氨酸激酶syk在胰腺癌中的表达检测及其临床意义[J].南京医科大学学报(自然科学版),2003,23(5)：492-493.
[53]元云飞,汪建平,李锦清,等.DNA甲基化导致肝细胞癌SYK基因失表达[J].中国肿瘤临床,2006,33(6)：307-309.
[54]Dhillon V S, Young A R, Husain SA, et al.Promoter hypermethylation of MGMT, CDH1, RAR-beta andSYK tumour suppressor genes ingranulosa cell tumours(GCTs) ofovarian origin[J]. Br J Cancer,2004,90(4):874-881.
[55]Skomedal H, Kristensen GH, Lie AK et aL Aberrant expression of the cell cycle associated proteins TP53,MDM7,P21,P27,CDK4,cuclinDl,Rb and EGFR in cervical carcinoma. Gyneonl Oncol, 1999,73(2):223-228
[56]Jones PA, Takai D. The role of DNA methylation in mamma lion epigenetics[J].Science,2001, 293 (5532):106821070.
[57]Graff JR, Gabrielson, E, Fujii H, et al. Methylation patterns of the E-cadherin 5'-CpG island are unstable and reflect the dynam2ic, heterogeneous loss of E-cadherin expression during metastatic progression[J].J Biol chem,2000,275 (4):272722732.
[58]Bender CM, PaoMM, Jones PA. Inhibition of DNA by 5-Aza-2'deoxycytidine suppresses the growth of human tumor cell lines[J].Cancer Res,1998,58 (1):952101.
[59]Wijermans P, LubbertM, Verhoef G, et al. Low2dose 5-Aza-2'-deoxycytidine, a DNA hypomethylation agent, for the treatment ofhighrisk myelodyspastic syndrome:a multicenter phase Ⅱ study in elderly patients[J].J Clin Oncol,2000,18 (5):9562960.
[2]Taniguchi T,Kobayashi T,Kondo J,et al.Molecular cloning of a porcine gene Syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to prteolysis[J].J Biol Chem,1991,266 (24);15790-15796.
[3]Turner M, Schweighoffer E, Colucci F, et al. Tyrosine kinase SYK:essential functions for immunoreceptor signaling[J].Immunol Today,2000,21(3):148-154.
[4]Coopman P J,Do M T,Barth M et a.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells[J].Nature,2000,406(8):742-747.
[5]Takano T,Sada K, Yamamura H.Role of protein-tyrosine kinase syk in oxidative stress signaling in B cells [J].Antioxid Redox Signal,2002,4 (3):533-541.
[6]Murphy MA,Schnall RG, Venter DJ,et al.Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficientmice [J].Mol Cell Biol,1998,18 (8):4872-4882.
[7]陈明霞,丁永斌,戴存才,等.MSP法检测胰腺癌Syk基因甲基化改变的研究[J].河北医学,2003,9(7)：577-579.
[8]Ulanova M, Puttagunta L, Marcet-Palacios M, et al. Syk tyrosine kinaseparticipates in betal-integrin signaling and inflammatory responses in airwayepithelial cells [J].Am J PhysiolLung Cell Mol Physiol,2005,288(3):497-507.
[9]Yamada T, Fujieda S, Yanagi S, etal. Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production[J].J Immunol,2001,166(1):538-543.
[10]Inatome R, Yanagi S, Takano T, etal. A critical role for Syk in endothelial cell proliferation and migration [J].Biochem Biophys Res Commun,2001,286(1):195-199.
[11]Tsujimura T, Yanagi S,Inatome R,et al. Syk protein-tyrosine kinase isinvolved in neuron-like differentiation of embryonal carcinoma P19 cells[J].FEBS Lett,2001,489 (2-3):129-133.
[12]Tsuchida S, Yanagi S, Inatome R,et al.Purification of a 72-kDaprotein-tyrosine kinase from rat liver and its identification as Syk:involvement of Syk in signaling events of hepatocytes[J].J Biochem(Tokyo),2000,127(2):321-327.
[13]Hoeller C, Thallinger C, PratscherB,et al. The non-receptor-associatedtyrosine kinase Syk is a regulator of metastatic behavior in humanmelanoma cells[J].J Invest Dermatol,2005,124(6): 1293-1299.
[14]Wang S, Ding YB,Chen GY,et al.Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma. World J Gastroenterol.2004 Jun 15；10(12)：1815-1818.
[15]孙桂霞,马德花,陈荣辉,等.候选抑癌基因Syk与宫颈癌发生发展相关性研究[J].中华肿瘤防治杂志2009,13(1)814-816+824
[16]邢学森,黄利呜,等.改良的甲基化特异PCR法在人宫颈癌Hela细胞检测中的应用[J].中国现代医学杂志,2006,16(2)：191-194.
[17]Yuan Y,Liu H,Sahin A,et al.Reactivation of Syk expression by inhibition of DNA methylation suppresses breast cancer cell invisiveness.Int J Cancer,2005,113:654-659.
[18]Bender CM,PaoMM,Jones PA.Inhibition of DNA by 5-Aza-2'-deoxycytidine suppresses the growth of human tumor cell lines[J].Cancer Res,1998,58(1):95-101.
[19]Wijermans P,Lubbert M,Vverhoef G,et al.Low dose 5-Aza-2'-deoxycytidine,a DNA hypomethylation agent,for the treatment of high risk myelodyspastic syndrome:a multicenter phase II studying elderly patients[J].J Clin Oncol,2000,18(5):956-960.
[20]刘特,刘志学,等.5-杂氮-2’-脱氧胞营对宫颈癌细胞生长及其甲基化转移酶和甲基结合蛋白DNA转录的影响[J].中国临床医学,2008,15(4)：524-527.
[21]郑伟,丁永斌,武正炎.SYK基因表达对乳腺癌生长、转移影响的动物实验[J].中华普通外科杂志,2005，20：5
[22]Heman J G, Jen J,Merlo A et al.Hypermethylation associated inactivation indicates a turmor suppressor role for p151NK4B[J].Cancer Res,1996;56:722-727.
[23]Corn P G, Kuerbitz S J,Noesel M M et al. Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5'CpG island methylation[J]. Cancer Res,1999;59:3352-3356.
[24]Yunfei Y, Richard M, Aysegul S et al. Hypermethylation leads to silencing of the SYK gene in human breast cancer[J].Cancer Res,2001; 61 (14):5558-55561.
[25]Feng Q, Stern JE, Hawes SE, et al. DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection.[J]. Exp Mol Pathol.2010 Jan 14.
[1]Mike F J,Hervy E A. Cervical cancer:prevention, diagnosis and therapeutics [J].CA Cancer J Clin, 2001,51(2):92-114.
[2]Slack A,Cervoni N,Pinard M,et al.Feedback regulation of DNA methyltransferase gene expression by methylation.Eur J Biochem,1999,264(1):191
[3]Lehmann U,Brakensiek K, Kreipe H.Role of epigenetic changes in hematological malignancies[J].Ann Hematol,2004,83(3):137-152.
[4]Yu J, Ni M, Xu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis[J].BMC Cancer,2002,2(1):29.
[5]Lee S, Lee HJ, Kim JH,et al.Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis[J].Am J Pathol,2003,163:1371-1378.
[6]Robertson KD,Jones PA. DNA methylation:past,present and future directions[J]. Carcinogenesis,2000,21(3):461-467.
[7]Garinis GA,Patrinos GP, Spanakis NE, et al. DNA hypermethylation:when tumour suppressor genes go silent[J].Hum Genet,2002,111(2):115-127. [8]Jones PA, Laird PW. Cancer epigenetics comes of age[J].Nat Genet,1999,21(2):163-167.
[9]Esteller M,Cirn PG,Baylin SB,et al.A gene hypermethylation profile of human cancer.Cancer Res,2001,61:3225
[10]Belinsky SA,Nikula Kj,Palmisano WA,et al.Aberrant methylation ofp16 is an early event in lung cancer and a potential biomarker forearly diagnosis.Proc NaT1 scad Sci USA,1998,95:11891
[11]Taniguchi T, Kobayashi T, Kondo J, et al. Molecular cloning of a porcine gene Syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis[J].J Biol Chem,1991,266(24):15790-15796.
[12]Turner M, Schweighoffer E, Colucci F, et al. Tyrosine kinase SYK:essential functions for immunoreceptor signaling[J].Immunol Today,2000,21(3):148-154.
[13]David Frommhold,Ingrid Mannigel,Jurgen Schymeinsky,et al.Spleen tyrosine kinase SYK is critical for sustained leukocyte adhesion during inflammation in vivo.BMC Immunology,2007,8:31
[14]Murali Gururajan,Trivikram Dasu,Seif Shahidain C.et al.Spleen Tyrosine Kinase(SYK),a Novel Target of Curcumin, Is Required for B Lymphoma Growth.The Journal of Immunology,2007,178:111
[15]Sada K, Takano T,Yanagi S,et al. Structure and function of Syk protein-tyrosine kinase [J].J Biochem,2001,130(2):177-186.
[16]Carter WB,Hoying JB,Boswell C,et al.HER2/neu over expression induces endothelial cell retraction[J].Int J Cancer,2001,91 (3):295-299.
[17]Murphy MA,Schnall RG, Venter DJ,et al.Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficientmice [J].Mol Cell Biol,1998,18(8):4872-4882.
[18]Coopman PJ,Do MT,Bart h M,et al.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells [J].Nature,2000,406 (6 797):742-747.
[19]Mahabeleshwar G H,Kundu G C.Syk, a protein-tyrosine kinase,suppresses the cell motility andnuclear factor kappa B-mediatedsecretion of urokinase typeplasminogen activator by inhibiting thephosphatidylinositol 3'-kinase activityin breast cancer cells[J].J BiolChem,2003,278(8):6209-6221.
[20]Takada Y, Aggarwal B B.TNF activates Syk protein tyrosine kinaseleading to TNF-induced MAPKactivation, NF-kappaB activation,and apoptosis[J].J Immunol,2004,173(2):1066-1077.
[21]Takada Y, Mukhopadhyay A, KunduG C,et al.Hydrogen peroxideactivates NF-kappa B through tyrosinephosphorylation of I kappa B alphaand serine phosphorylation of p65:evidence for the involvement of Ikappa B alpha kinase and Sykprotein-tyrosine kinase[J].J BiolChem,2003, 278(26):24233-24241.
[22]Moroni M, Soldatenkov V, Zhang L,et al.Progressive loss of Syk andabnormal proliferation in breastcancer cells[J].Cancer Res,2004,64(20):7346-7354.
[23]Ruschel A, Ullrich A. Proteintyrosine kinase Syk modulates EGFRsignalling in human mammaryepithelial cells [J].Cell Signal,2004,16(11):1249-1261.
[24]Jabril-Cuenod B,Zhang C,Scharenberg A M, et al.Sykdependentphosphorylation of She. Apotential link between FcepsilonRIand the ras/mitogen-activated proteinkinase signaling pathway through SOSand Grb2[J].J Biol Chem,1996,271(27):16268-16272.
[25]Kawakami Y, Kitaura J, Yao L, etal. A Ras activation pathwaydependent on Syk phosphorylation ofprotein kinase C[J].Proc Natl AcadSci USA,2003,100(16):9470-9475.
[26]Craxton A, Jiang A, Kurosaki T, etal.Syk and Brutons tyrosine kinaseare required for B cell antigenreceptor-mediated activation of thekinase Akt[J].J Biol Chem,1999,274(43):30644-30650.
[27]Moon K D, Post C B,Durden D L,et al.Molecular basis for a directinteraction between the Syk proteintyrosinekinase and phosphoinositide3-kinase[J].J Biol Chem,2005,280(2):1543-1551.
[28]Li B,Allendorf D J, Hansen R, etal.Yeast beta-glucan amplifiesphagocyte killing of iC3b-opsonizedtumor cells via complementreceptor3-Syk-phosphatidylinositol 3-kinasepathway [J]. J Immunol,2006,177(3):1661-1669.
[29]Peters J D, Furlong M T, Asai D J,et al.Syk, activated by cross-linkingthe B-cell antigen receptor, localizesto the cytosol where it interacts withand phosphorylates alpha-tubulin ontyrosine[J].J Biol Chem,1996,271(9):4755-4762.
[30]Nan X, Ng HH, Johnson CA, et al.Tanscriptional repression by the methyl-CpG2binding p roteinMeCP2 invovles a histone deacetylase comp lex[J].Nature,1998,393 (6683):3862389.
[31]Bachman KE,Rountree MR, Baylin SB.Dnmt3a and Dnmt3b are transcriptional repressor that exhibit unique localization properties to heterochromation[J].J Biol Chem,2001,276(34): 32282232287.
[32]Heman J G, Jen J,Merlo A et al.Hypermethylation associated inactivation indicates a turmor suppressor role for p151NK4B[J].Cancer Res,1996;56:722-727.
[33]Corn P G, Kuerbitz S J, Noesel M M et al. Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5'CpG island methylation[J]. Cancer Res,1999;59:3352-3356.
[34]Yunfei Y, Richard M, Aysegul S et al. Hypermethylation leads to silencing of the SYK gene in human breast cancer[J].Cancer Res,2001;61 (14):5558-55561.
[35]Toyama T, Iwase H,Yamashita H,et al.Reduced expression of the Syk gene is correlated with poor prognosis in human breast cancer [J].Cancer Lett,2003,189(1):97-102.
[36]Yuan Y, Mendez R,Sahin A,et al.Hypermethylation leads to silencing of the Syk gene in human breast cancer [J].Cancer Res,2001,61(14):5558-5561.[8]
[37]Wang S,Ding YB,Chen GY,et al.Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma[J].World J Gastroenterol,2004,10(12):1 815-1818.
[38]Nakashima H, Natsugoe S,IshigamiS,et al. Clinical significance ofnuclear expression of spleen tyrosinekinase(Syk) in gastric cancer[J].Cancer Lett,2006,236 (1):89-94.
[39]Goodman PA,Wood CM,Vassilev A,et al.Spleen tyrosine kinase(Syk) deficiency in childhood pro2B cell acute lympho2blastic leukemia[J] Oncogene,2001,20 (30):3969-3978.
[40]Goodman PA,Burkhardt N,Juran B,et al.Hypermethylation of t he spleen tyrosine kinase promoter in T-lineage acute lymphoblastic leukemia[J]. Oncogene,2003,22(16):2504-2 514.
[41]Watanabe Y.Expression of tyrosine kinase Syk in pancreatic cancers [J].Hokkaido Igaku Zasshi,2001,76(3):173-181.
[42]陈明霞,丁永斌,戴存才,等.MSP法检测胰腺癌Syk基因甲基化改变的研究[J].河北医学,2003,9(7)：577-579.
[43]Ulanova M, Puttagunta L, Marcet-Palacios M, et al.Syk tyrosine kinaseparticipates in betal-integrin signaling and inflammatory responses in airwayepithelial cells[J].Am J PhysiolLung Cell Mol Physiol,2005,288(3):497-507.
[44]Yamada T, Fujieda S,Yanagi S, etal.Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production[J].J Immunol,2001,166(1):538-543.
[45]Inatome R, Yanagi S,Takano T, etal. A critical role for Syk in endothelial cell proliferation and migration[J].Biochem Biophys Res Commun,2001,286(1):195-199.
[46]Tsujimura T, Yanagi S,Inatome R,et al.Syk protein-tyrosine kinase isinvolved in neuron-like differentiation of embryonal carcinoma P19 cells[J].FEBS Lett,2001,489(2-3):129-133.
[47]Tsuchida S, Yanagi S, Inatome R,et al.Purification of a 72-kDaprotein-tyrosine kinase from rat liver and its identification as Syk:involvement of Syk in signaling events of hepatocytes[J].J Biochem(Tokyo),2000,127(2):321-327.
[48]Hoeller C, Thallinger C, PratscherB, et al. The non-receptor-associatedtyrosine kinase Syk is a regulator of metastatic behavior in humanmelanoma cells[J].J Invest Dermatol,2005,124(6): 1293-1299.
[49]Yuan Y, Mendez R, Sahin A, et al.Hypermethylation leads to silencing ofthe SYK gene in human breast cancer[J].Cancer Res,2001,61(14):5558-5561.
[50]丁永斌,武正炎,王水,等.候选抑癌基因Syk启动子甲基化与乳腺癌发生转移的关[J].中华医学杂志,2004,84(4)：290-294.
[51]杨祖立,汪建平,元云飞,等.结直肠癌侵袭性与脾酪氨酸激酶基因Syk启动子甲基化的关系[J].中华实验外科杂志,2005,22(1)：68-70.
[52]陈明霞,丁永斌,蒋奎荣,等.酪氨酸激酶syk在胰腺癌中的表达检测及其临床意义[J].南京医科大学学报(自然科学版),2003,23(5)：492-493.
[53]元云飞,汪建平,李锦清,等.DNA甲基化导致肝细胞癌SYK基因失表达[J].中国肿瘤临床,2006,33(6)：307-309.
[54]Dhillon V S, Young A R, Husain SA, et al.Promoter hypermethylation of MGMT, CDH1, RAR-beta andSYK tumour suppressor genes ingranulosa cell tumours(GCTs) ofovarian origin[J]. Br J Cancer,2004,90(4):874-881.
[55]Skomedal H, Kristensen GH, Lie AK et aL Aberrant expression of the cell cycle associated proteins TP53,MDM7,P21,P27,CDK4,cuclinDl,Rb and EGFR in cervical carcinoma. Gyneonl Oncol, 1999,73(2):223-228
[56]Jones PA, Takai D. The role of DNA methylation in mamma lion epigenetics[J].Science,2001, 293 (5532):106821070.
[57]Graff JR, Gabrielson, E, Fujii H, et al. Methylation patterns of the E-cadherin 5'-CpG island are unstable and reflect the dynam2ic, heterogeneous loss of E-cadherin expression during metastatic progression[J].J Biol chem,2000,275 (4):272722732.
[58]Bender CM, PaoMM, Jones PA. Inhibition of DNA by 5-Aza-2'deoxycytidine suppresses the growth of human tumor cell lines[J].Cancer Res,1998,58 (1):952101.
[59]Wijermans P, LubbertM, Verhoef G, et al. Low2dose 5-Aza-2'-deoxycytidine, a DNA hypomethylation agent, for the treatment ofhighrisk myelodyspastic syndrome:a multicenter phase Ⅱ study in elderly patients[J].J Clin Oncol,2000,18 (5):9562960.