NECL2/TSLC1在肺癌细胞中的表达调控研究
|
摘要
肺癌的早期诊断和治疗成为临床上一大难题。随着分子生物学技术的飞速发展,研究癌症的基因发病机制、基因诊断和基因治疗受到大家的关注。近期研究已表明肺癌细胞中常存在DNA甲基化状态的改变,DNA异常甲基化是一种表观遗传(epigenetic)改变而非基因水平改变。这种现象不仅可以激活原癌基因的表达,而且可使一些抑癌基因转录功能失活。
中国医学科学院基础研究所生化实验室新发现的基因NECL2在多种肿瘤中表达下降或失活,国外报道与启动子区甲基化相关,用去甲基化试剂处理的一些肿瘤细胞系中,NECL2的表达水平恢复到正常,这提示NECL2启动子区的甲基化可能是导致NECL2表达下调的原因。考虑到NECL2在肿瘤抑制的过程中有着重要作用,并依据启动子区甲基化与NECL2表达下调相关的线索和甲基化可逆性特征.本文在对感兴趣的肺癌细胞系中,初步研究了NECL2的表达调控机制。
首先,我们在一例正常脑组织和三种肺癌细胞系(A549、NCI-H446、Calu-3)中检测了NECL2的表达谱,运用bisulfite sequencing(亚硫酸氢盐修饰后测序)和甲基化酶抑制剂5-AzaC处理方法得知,NECL2在肺癌细胞系中的表达缺失是由启动子区的甲基化引起,去甲基化使NECL2恢复表达,DNA甲基化检测及DNA甲基化的可逆性特征为临床肿瘤诊断和治疗提供了依据。
下一步,根据生物信息学分析的结果,构建了人NECL2基因5'非编码区系列缺失报告基因质粒,应用双荧光素酶报告基因实验,推测人NECL2的核心启动子区可能位于ATG上游的—68到—329区域内。
我们克隆了Spl的编码区序列,构建了带V5标签的Spl真核表达质粒,与带有人NECL2基因核心启动子区的报告基因质粒共转染A549和NCI-H446细胞,以检测Sp1对人NECL2启动子的转录活性的影响。应用双荧光素酶报告基因实验,我们发现在肺癌细胞中,过表达外源性Sp1对人NECL2启动子的转录活性无明显影响。
最后,用化疗药顺铂处理体外培养的A549和NCI-H446细胞,用流式细胞仪检测了处理后细胞发生的变化,结果表明,顺铂可能是通过破坏细胞周期和引起细胞凋亡而杀死肺癌细胞,可以为以后的深入研究提供一点线索。
综上所述,本文通过bisulfite sequencing和5-AzaC处理实验,再次验证了NECL2在肺癌细胞中的表达缺失是由启动子区的甲基化引起。DNA甲基化程度检测可能成为癌症早期诊断的分子标志物,DNA甲基化的可逆性特征为临床抗肿瘤治疗提供了一种新途径。通过双荧光素酶报告基因实验,首次推测了人NECL2可能的核心启动子区,并对转录因子Sp1对人NECL2启动子转录活性的影响进行了初步分析,为阐明人NECL2的表达调控机制奠定了基础。
In previous study of National Laboratory of Medical Biology,we have discovered a new cDNA clone whose structure is similar with those of nectin family,so named nectin-like molecule 2,that is NECL2.NECL2 belongs to the immunoglobulin superfamily and is widely expressed in multiple tissues. The structure of NECL2 is:a signal peptide,three immunoglobulin domains, a transmembrane region and a short cytoplasmic region.Up to now NECL2 has the following functions:Ca~(2+)-independent cell-cell adhesion,synapse formation and tumor suppression.Down regulation or silent of NECL2 expression is found in many types of tumors.Others' work showed that in those tumors exhibiting loss of heterozygosity,inactivating mutation was rarely found,whereas promoter methylation was observed with high incidence and was correlated with down regulation of NECL2 expression. Furthermore,expression level of NECL2 was restored after treating cancer cell lines with 5-Aza-2-deoxycytidine.These results suggest that promoter methylation is perhaps the cause of down regulation of NECL2 expression. Considering the importance of NECL2 in neural development and tumor suppression,and the correlation between promoter methylation and down regulation of NECL2 expression,we studied the regulation of NECL2 expression in lung cancer cell lines which we are interested with.
First,we detected the expression pattern of NECL2 in one human normal brain tissue and three lung cancer cell lines(A549,NCI-H446,Calu-3).Based on the prediction result which shows that there is a CpG island in 5' non-coding region,we know promoter methylation can lead to the silent of NECL2 in lung cancer cell lines using bisulfite sequencing and 5-AzaC treatment.
Then,we used bioinformatics method to analyze the 5' non-coding region of human NECL2.The result showed that there are six conserved nucleotide sequences within the 4Kb region just upstream the human/mouse NECL2 translation start site.We also predicted the possible cis-acting elements.
Next,we extracted the genome from normal brain tissue and cloned a 1.7Kb fragment of human NECL2 5' non-coding region.Based on the result of bioinformatics analysis,we generated a series of promoter deletion constructs.With dual luciferase reporter assay,we deduced that the possible core promoter for human NECL2 is from -68 to -329,designated ATG as+1.
Bioinformatics analysis also showed that there are three putative Spl binding sites within the core promoter of human NECL2.Therefore we cloned the complete coding region of Sp1 and obtained an eukaryocyte expression plasmid of Sp1.We co-transfected this plasmid with the core promoter-reporter plasmid to test the possible effect of Sp1 on human NECL2 promoter transcription activity.With dual luciferase reporter assay,we found that over-expression of Sp1 in lung cancer cell lines does not affect the human NECL2 promoter transcription activity significantly.
Finally,we treated cultured A549 and NCI-H446 cell lines with DDP and detected the changes of cells by cell cytometry.The result showed DDP killed lung cancer cells possibly by destroying cell cycle and inducing cell apoptosis, this can provide some clues for further study.
In conclusion,we validated promoter methylation of NECL2 could lead to the silent of NECL2 in lung cancer cells by bisulfite sequencing and 5-AzaC treatment;with dual luciferase reporter assay,we deduced the possible core promoter for human NECL2 for the first time.We also analyzed the effect of Sp1 transcription factor on human NECL2 promoter transcription activity.These work provide basis for further elucidation of the regulation of human NECL2 expression.
中国医学科学院基础研究所生化实验室新发现的基因NECL2在多种肿瘤中表达下降或失活,国外报道与启动子区甲基化相关,用去甲基化试剂处理的一些肿瘤细胞系中,NECL2的表达水平恢复到正常,这提示NECL2启动子区的甲基化可能是导致NECL2表达下调的原因。考虑到NECL2在肿瘤抑制的过程中有着重要作用,并依据启动子区甲基化与NECL2表达下调相关的线索和甲基化可逆性特征.本文在对感兴趣的肺癌细胞系中,初步研究了NECL2的表达调控机制。
首先,我们在一例正常脑组织和三种肺癌细胞系(A549、NCI-H446、Calu-3)中检测了NECL2的表达谱,运用bisulfite sequencing(亚硫酸氢盐修饰后测序)和甲基化酶抑制剂5-AzaC处理方法得知,NECL2在肺癌细胞系中的表达缺失是由启动子区的甲基化引起,去甲基化使NECL2恢复表达,DNA甲基化检测及DNA甲基化的可逆性特征为临床肿瘤诊断和治疗提供了依据。
下一步,根据生物信息学分析的结果,构建了人NECL2基因5'非编码区系列缺失报告基因质粒,应用双荧光素酶报告基因实验,推测人NECL2的核心启动子区可能位于ATG上游的—68到—329区域内。
我们克隆了Spl的编码区序列,构建了带V5标签的Spl真核表达质粒,与带有人NECL2基因核心启动子区的报告基因质粒共转染A549和NCI-H446细胞,以检测Sp1对人NECL2启动子的转录活性的影响。应用双荧光素酶报告基因实验,我们发现在肺癌细胞中,过表达外源性Sp1对人NECL2启动子的转录活性无明显影响。
最后,用化疗药顺铂处理体外培养的A549和NCI-H446细胞,用流式细胞仪检测了处理后细胞发生的变化,结果表明,顺铂可能是通过破坏细胞周期和引起细胞凋亡而杀死肺癌细胞,可以为以后的深入研究提供一点线索。
综上所述,本文通过bisulfite sequencing和5-AzaC处理实验,再次验证了NECL2在肺癌细胞中的表达缺失是由启动子区的甲基化引起。DNA甲基化程度检测可能成为癌症早期诊断的分子标志物,DNA甲基化的可逆性特征为临床抗肿瘤治疗提供了一种新途径。通过双荧光素酶报告基因实验,首次推测了人NECL2可能的核心启动子区,并对转录因子Sp1对人NECL2启动子转录活性的影响进行了初步分析,为阐明人NECL2的表达调控机制奠定了基础。
In previous study of National Laboratory of Medical Biology,we have discovered a new cDNA clone whose structure is similar with those of nectin family,so named nectin-like molecule 2,that is NECL2.NECL2 belongs to the immunoglobulin superfamily and is widely expressed in multiple tissues. The structure of NECL2 is:a signal peptide,three immunoglobulin domains, a transmembrane region and a short cytoplasmic region.Up to now NECL2 has the following functions:Ca~(2+)-independent cell-cell adhesion,synapse formation and tumor suppression.Down regulation or silent of NECL2 expression is found in many types of tumors.Others' work showed that in those tumors exhibiting loss of heterozygosity,inactivating mutation was rarely found,whereas promoter methylation was observed with high incidence and was correlated with down regulation of NECL2 expression. Furthermore,expression level of NECL2 was restored after treating cancer cell lines with 5-Aza-2-deoxycytidine.These results suggest that promoter methylation is perhaps the cause of down regulation of NECL2 expression. Considering the importance of NECL2 in neural development and tumor suppression,and the correlation between promoter methylation and down regulation of NECL2 expression,we studied the regulation of NECL2 expression in lung cancer cell lines which we are interested with.
First,we detected the expression pattern of NECL2 in one human normal brain tissue and three lung cancer cell lines(A549,NCI-H446,Calu-3).Based on the prediction result which shows that there is a CpG island in 5' non-coding region,we know promoter methylation can lead to the silent of NECL2 in lung cancer cell lines using bisulfite sequencing and 5-AzaC treatment.
Then,we used bioinformatics method to analyze the 5' non-coding region of human NECL2.The result showed that there are six conserved nucleotide sequences within the 4Kb region just upstream the human/mouse NECL2 translation start site.We also predicted the possible cis-acting elements.
Next,we extracted the genome from normal brain tissue and cloned a 1.7Kb fragment of human NECL2 5' non-coding region.Based on the result of bioinformatics analysis,we generated a series of promoter deletion constructs.With dual luciferase reporter assay,we deduced that the possible core promoter for human NECL2 is from -68 to -329,designated ATG as+1.
Bioinformatics analysis also showed that there are three putative Spl binding sites within the core promoter of human NECL2.Therefore we cloned the complete coding region of Sp1 and obtained an eukaryocyte expression plasmid of Sp1.We co-transfected this plasmid with the core promoter-reporter plasmid to test the possible effect of Sp1 on human NECL2 promoter transcription activity.With dual luciferase reporter assay,we found that over-expression of Sp1 in lung cancer cell lines does not affect the human NECL2 promoter transcription activity significantly.
Finally,we treated cultured A549 and NCI-H446 cell lines with DDP and detected the changes of cells by cell cytometry.The result showed DDP killed lung cancer cells possibly by destroying cell cycle and inducing cell apoptosis, this can provide some clues for further study.
In conclusion,we validated promoter methylation of NECL2 could lead to the silent of NECL2 in lung cancer cells by bisulfite sequencing and 5-AzaC treatment;with dual luciferase reporter assay,we deduced the possible core promoter for human NECL2 for the first time.We also analyzed the effect of Sp1 transcription factor on human NECL2 promoter transcription activity.These work provide basis for further elucidation of the regulation of human NECL2 expression.
引文
1. Holliday R. DNA methylation and epigenetic mechanisms. Cell Biophys 1989;15:15-20.
2. Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science. 1997, 286:481-486.
3. Hsieh J, Gage FH. Epigenetic control of neural stem cell fate. Current Opinion in Genetics & Development. 2004, 14: 461-469.
4. Abdolmaleky HM. Methylomics in Psychiatry: Modulation of Gene-Environment Interactions may be Through DNA Methylation. American Journal of Medical Genetics 2004(127B): 51-59
5. Fan G, Huthick L. Methylo-CpG proteins in the nervous system. Cell research, 2005,15(4): 255-261.
6. Akira M., hiroyuki N,Kazushi K,et al. Nectin: an adhesion molecule involved in formation of synapses. J. Cell Biol. 2002,156:555-565.
7. Watabe K, Ito A, Koma YI and Kitamura Y. IGSF4: a new intercellular adhesion molecule that is called by three names, TSLC1, SgIGSF and SynCAM, by virture of its diverse function. Histol Histopathol. 2003, 18: 1321-1329.
8. Shingai T, Ikeda W, Kakunaga S, Morimoto K, Takekuni K, Itoh S, Satoh K, Takeuchi M, Imai T, Monden M, Takai Y. Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells. J Biol Chem. 2003 Sep 12;278(37): 35421-7.
9. Yageta M, Kuramochi M, Masuda M, Fukami T, Fukuhara H, Maruyama T, Shibuya M,Murakami Y. Direct association of TSLC1 and DAL-1, two distinct tumor suppressor proteins in lung cancer. Cancer Res. 2002 Sep 15; 62(18): 5129-33.
10. Thomas Biederer, Yildirim sara, Marina Mozhayeva, Keniz Atasoy, Xinran Liu, Ege t.Kavalali, Thomas c. Sudhof. SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 2002,297(30) 1525-31
11. Kuramochi M, Fukuhara H, Nobukuni T, Kanbe T, Maruyama T, Ghosh HP, Pletcher M,Isomura M, Onizuka M, Kitamura T, Sekiya T, Reeves RH, Murakami Y.TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer. Nat Genet. 2001 Apr; 27(4): 427-30.
12. Fukuhara H, Kuramochi M, Fukami T, Kasahara K, Furuhata M, Nobukuni T,Maruyama T, Isogai K, Sekiya T, Shuin T, Kitamura T, Reeves RH, Murakami Y.Promoter methylation of TSLCl and tumor suppression by its gene product in human prostate cancer. Jpn J Cancer Res. 2002 Jun; 93(6): 605-9.
13. Allinen M, Peri L, Kujala S, Lahti-Domenici J, Outila K, Karppinen SM, Launonen V,Winqvist R. Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLCl promoter hypermethylation. Genes Chromosomes Cancer. 2002 Aug; 34(4): 384-9.
14. Jansen M, Fukushima N, Rosty C, Walter K, Altink R, Heek TV, Hruban R, Offerhaus JG, Goggins M.Aberrant methylation of the 5' CpG island of TSLCl is common in pancreatic ductal adenocarcinoma and is first manifest in high-grade PanlNs. Cancer Biol Ther. 2002 May-Jun; 1(3): 293-6.
15. Lo KW, Huang DP.Genetic and epigenetic changes in nasopharyngeal carcinoma. Semin Cancer Biol. 2002 Dec; 12(6): 451-62.
16. Ito A, Okada M, Uchino K, Wakayama T, Koma Y, Iseki S, Tsubota N, Okita Y,Kitamura Y. Expression of the TSLCl adhesion molecule in pulmonary epithelium and its down-regulation in pulmonary adenocarcinoma other than bronchioloalveolar carcinoma. Lab Invest. 2003 Aug; 83(8): 1175-83.
17. Fukami T, Fukuhara H, Kuramochi M, Maruyama T, Isogai K, Sakamoto M, Takamoto S, Murakami Y. Promoter methylation of the TSLCl gene in advanced lung tumors and various cancer cell lines. Int J Cancer. 2003 Oct 20; 107(1): 53-9.
18. Uchino K, Ito A, Wakayama T, Koma Y, Okada T, Ohbayashi C, Iseki S, Kitamura Y,Tsubota N, Okita Y, Okada M.Clinical implication and prognostic significance of the tumor suppressor TSLCl gene detected in adenocarcinoma of the lung. Cancer. 2003 Sep1;98(5): 1002-7.
19. Ito T, Shimada Y, Hashimoto Y, Kaganoi J, Kan T, Watanabe G, Murakami Y, Imamura M. Involvement of TSLCl in progression of esophageal squamous cell carcinoma.Cancer Res. 2003 Oct 1; 63(19): 6320-6.
20. Hui AB, Lo KW, Kwong J, Lam EC, Chan SY, Chow LS, Chan AS, Teo PM, Huang DP.Epigenetic inactivation of TSLCl gene in nasopharyngeal carcinoma. Mol Carcinog.2003 Dec; 38(4): 170-8.
21. Honda T, Tamura G, Waki T, Jin Z, Sato K, Motoyama T, Kawata S, Kimura W,Nishizuka S, Murakami Y. Hypermethylation of the TSLCl gene promoter in primary gastric cancers and gastric cancer cell lines. Jpn J Cancer Res. 2002 Aug; 93(8):857-60.
22. Tamura G Promoter methylation status of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia. Histol Histopathol. 2004 Jan; 19(1):221-8.
23. Steenbergen RD, Kramer D, Braakhuis BJ, Stern PL, Verheijen RH, Meijer CJ,Snijders PJ.TSLC1 gene silencing in cervical cancer cell lines and cervical neoplasia. J Natl Cancer Inst. 2004 Feb 18; 96(4): 294-305
24. Fleming JB, Minna JD.Inactivation of tumor suppressor genes by promoter methylation is important in the pathogenesis of pancreatic cancer. Cancer Biol Ther. 2002 May-Jun;1(3):297-9.
25. Surace EI, Lusis E, Murakami Y, Scheithauer BW, Perry A, Gutmann DH. Loss of tumor suppressor in lung cancer-1 (TSLC1) expression in meningioma correlates with increased malignancy grade and reduced patient survival. J Neuropathol Exp Neurol.2004 Oct; 63(10): 1015-27.
26. Li J, Zhang Z, Bidder M, Funk MC, Nguyen L, Goodfellow PJ, Rader JS. IGSF4 promoter methylation and expression silencing in human cervical cancer. Gynecol Oncol. 2005 Jan; 96(1): 150-8.
27.金冬雁等译。分子克隆实验指南。1998；科学出版社
28.黄培堂等。细胞实验指南。2001；科学出版社
29. Sherf BA, Navarro SL, Hannah RR, Wood KV. Dual-LuciferaseTM reporter assay: an advanced co-reporter technology integrating firefly and renilla luciferase assays.Promega Notes. 1996, 56:02
30. Fukami T, Satoh H, Fujita E, Maruyama T, Fukuhara H, Kuramochi M, Takamoto S,Momoi T, Murakami Y. Identification of the Tslc1 gene, a mouse orthologue of the human tumor suppressor TSLCl gene. Gene. 2002 Jul 24; 295(1): 7-12.
31. Masuda M, Yageta M, Fukuhara H, Kuramochi M, Maruyama T, Nomoto A, Murakami Y. The tumor suppressor protein TSLC1 is involved in cell-cell adhesion. J Biol Chem.2002 Aug 23; 277(34): 31014-9.
32. Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I.VISTA:visualizing global DNA sequence alignments of arbitrary length.Bioinformatics.2000 Nov,16(11):1046-7.
33.Larsen F,Gundersen G,Lopez R,Prydz H.CpG islands as gene markers in the human genome.Genomics 1992 Aug;13(4):1095-107
34.Gardiner-Garden M,Frommer M.CpG islands in vertebrate genomes.J Mol Biol.1987Jul 20;196(2):261-82.
35.Li L,He S,Sun J,and Davie JR.Gene regulation by Sp1 and Sp3.Biochem.Cell Biol.2004,82:460-471.
36.Macleod D,Charlton J,Mullins J,and Bird AP.Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island.Genes Dev.1994:8:2282-2292.
37.Brandeis M,Frank D,Keshet I,Siegfied Z,Mendelsohn M,Nemes A.et al.Sp1elements protect a CpG island from de novo methylation.Nature(London).1994,371:435-438.
38.刘文哲,李金瀚,胡义德等。顺铂诱导A549细胞凋亡的研究。中国肺癌杂志,2002年8月第5卷第4期。
39.孙恒义,胡义德,钱频等。顺铂诱导肺腺癌A549细胞线粒体基因组及凋亡相关基因的差异表达。解放军医学杂志,2004年12月第29卷第12期。
40.Ng HH,Bird A.DNA methylation and chromatin modification.Current Opinion in Genetics and Development.1999,9(2):158-163.
41.Manel E,Montserrat SC,Rafael R,et al.Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients.Cancer Res,1999,59:67-70.
42.Sabine ZM,Fong KM,Virmani AK,et al.Aberrant promoter methylation of multiple genes in non-small cell lung cancers[J].Cancer Res,2001,61 φ249-255.
43.Christman JK.5-azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation:Mechanistic studies and their implications for cancer therapy[J].Oncogene,2002,21:5483-5495.
44.Watt F,Molloy PL.Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter.Genes and development.1998;2:1136-43.
45. Takizawa T, Nakashima K, Namihira M, et al. DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain. Dev Cell 2001;1:749-58.
46. Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998, 72:141-196
47. Faridi J, Wang L, Endemann G, Roth RA. Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo. Clin Cancer Res. 2003 Aug 1 ;9(8):2933-9.
1.Holliday R. DNA methylation and epigenetic mechanisms. Cell Biophys 1989; 15:15-20.
2. James G, Herman, MD.Epigenetics in Lung Cancer. Chest 2004; 125:119s-122s.
3. Zochbauer-Muller S, Fong KM, Virmani AK, etal. Aberrant promoter methylation of multiple genes in non-small cell lung cancer. Cancer Res 2001 61:249-255
4. Estsller M, fraga MF, Guo M, etal. DNA methyaltion patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum Mol Genet 2002; 10:3001-3007.
5. Paz MF, Fraga MF, Avilia S, etal. A systematic profile of DNA methylation in human cancer cell cell lines. Cancer Res 2003; 63:1114-1121.
6. Usadel H, Brabender J, Danenberg KD, et al. Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer Res 2002;62:371-5.
7. Eeteller M, Sanchez-Cespedes M, Rosell R, et al. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cellcancer patients. Cancer Res 1999;59:67-70.
8. Zhou XM, Shao SJ, XU GD, et al. Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Feb 25;816(1-2):145-51.
9. Ramireaz JL, Taron M, Balana C, et al. Serum DNA as a tool for cancer patient management. Rocz Akad Med Bialymst 2003 ;48:34-41.
10. Fujiwara K, Fujimoto N, Tabata M, et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. Clin Cancer Res.2005 Feb 1;11(3):1219-25.
11. Grote HJ, Schmiemann V, Geddert H, et al, Aberrant promoter methylation of p16(INK4a), RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer. Cancer. 2005 Sep 20;116(5):720-5.
12. Palmisano WA, Divine KK, Saccomanno G, et al. Prediting lung cancer by detecing abrrant promoter methylation in sputum. Cancer Res, 2000, 60:5954-5958.
13. Rosell R, Taron M, Ariza A, et al. Molecular predictors of response to chemotherapy in lung cancer. Semin Oncol. 2004 Feb;31(1 Suppl 1):20-7.
14. Plumb JA, Steele N, Finn PW. Et al. Epigenetic approaches to cancer therapy.Biochem Soc Trans. 2004 Dec;32(Pt 6): 1095-7.
15. Boivin AJ, Momparler LF, Hurtubise A, et al. Antineoplastic action of 5-aza-2'-deoxycytidine and phenylbutyrate on human lung carcinoma cells. Anticancer Drugs. 2002 Sep;13(8):869-74.
16. Soria JC, Rodriguez M, Liu DD, et al. Aberrant DNA promoter methylation in lung cancer. Biological and implications. Oncologist, 2002, 7:451-457.
17. Kim DH, Kim JS, Ji YI, et al. Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer. Cancer Res. 2003 Jul 1;63(13):3743-6.
18. Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of pl6INK4A is an early event in lung cancer and a potential biomakerfor early diagnosis. Proc Natl Acad Sci US A, 1998; 95: 11891-11896.
19. Wu MF, Cheng YW, Lai JC, et al. Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan. Int J Cancer. 2005 Jan 20;113(3):440-5.
20. Piyathilake CJ, Henao O, Frost AR, et al. Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung (United States). Cancer Causes Control. 2003 Feb;14(1):37-42.
21.Toyooka S, Maruyama R, Toyooka KO, et al. Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer.Int J Cancer. 2003 Jan 10;103(2): 153-60.
22. Tetsuo Ito,Yutaka Shimada,Yosuke Hashimoto,et al. Involvement of TSLC1 in Progression of Esophageal Squamous Cell Carcinoma. Cancer Res 2003; 63:6320-6326.
23. Mika Yageta,Masami Kuramochi,Mari Masuda,et al.Direct Association of TSLC1 and DAL-1, Two Distinct Tumor Suppressor Proteins in Lung Cancer. Cancer Res 2002; 62:5129-5133.
24. Shinji Kikuchi,Daisuke Yamada,Takeshi Fukami,et al.Promoter Methylation of DAL-1/4.IB Predicts Poor Prognosis in Non-Small Cell Lung Cancer. Clin Cancer Res 2005; 11(8): 2954-2961.
25. Takeshi Fukami, Hiroshi Fukuhara,Masami Kuramochi,et al.PROMOTER METHYLATION OF THE TSLC1 GENE IN ADVANCED LUNG TUMORS AND VARIOUS CANCER CELL LINES.Int.J.Cancer 2003;107:53-59.
26. Tatsushi Shingai, Wataru Dceda, Shigedi Kakunaga, et al. Implications of Nectin-like Molecule-2/IGSF4/RA175/SgIGSF/TSLCl/SynCAM1 in Cell-Cell Adhesion and Transmembrane Protein Localization in Epithelial Cells. The Journal Of Biological Chemistry 2003;278:35421-35427.
27. Makoto Sonobe, MD, Fumihiro Tanaka, MD, and Hiromi Wada, MD. Lung Cancer-related Genes in the Blood .Ann Thorac Cardiovasc Surg 2004; 10:213-217.
28. Ito A, Okada M, Uchino K, Wakayama T, Koma Y, et al. Expression of the TSLC1 adhesion molecule in pulmonary epithelium and its down-regulation in pulmonary adenocarcinoma other than bronchioloalveolar carcinoma. Lab Invest 2003;83(8):1175-83.
29. Uchino K, Ito A, Wakayama T, Koma Y, Okada T, Ohbayashi C, Iseki S, Kitamura Y,Tsubota N, Okita Y, Okada M. Clinical implication and prognostic significance of the tumor suppressor TSLC1 gene detected in adenocarcinoma of the lung. Cancer Rse 2003;98(5): 1002-7.
30. Fukami T, Fukuhara H, Kuramochi M, Maruyama T, Isogai K,Sakamoto S, Murakami Y. Promoter methylation of the TSLC1 gene in adcanced lung tumors and various eancer cell lines. Int J Cancer 2003;107(l):53-9.
31. Xinliang Mao, Eric Seidlitz, Kakoli Ghosh, et al.The Cytoplasmic Domain Is Critical to the Tumor Suppressor Activity of TS LC1 in Non-Small Cell Lung Cancer.Cancer Res 2003;63:7979-7985.
32. Xinliang Mao, Eric Seidlitz, Ray Truant, et al. Re-expression of TSLC1 in a non -small-cell lung cancer cell line induces apoptosis and inhibits tumor growth.Oncogene 2004;1-11.
33. Kent S.Boies, Winfried Barchet, Tom Diacovo, et al. The tumor suppressor TSLC1/NECL-2 triggers NK cell and CD8 T cell responses through the cell surface receptor CRTAM. Blood 2005; 10.
34. David GB, Eva F, Sabine ZM, et al. Epigenetic inactivation of RASSF1A in lung and breast cancer and malignant phenotype suppression. JNCI, 2001;93(9):691-699
35. Bearzatto A, Conte D, Frattini M, et al. p16(INK4A)hypermethylation detected by fluorescent methylationspecific PCR in plasmas from non-small cell lung cancer. Clin Cancer Res 2002;8:3782-7.
36. Liu JY, An Q, Xu GD, et al, Hypermethylation of p16 gene in clinical specimens of patients with lung cancer Zhonghua Zhong Liu Za Zhi. 2004 Feb;26(2):75-7.
37. Sabine Z, Kwun MF, Anirban M, et al. 5CpG island methylation of the FHIT gene is correlated with loss of dene expression in lung and breast cancer.Cancer Res.2001,61:3581-3585.
38. Kim h, Kwon YM, Kim JS,et al. Tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer. J Clin Oncol. 2004 Jun 15;22(12):2363-70.
39. Jan B,Henning U, Kathleen D, et al. Adenomatous polyposis coligene promoter hypermethylation in non-small cell lung cancer is associated with survival.Oncogene,2001,20:3528-3532
40. Grote HJ, Schmiemann V, Kiel S, et al. Aberrant methylation of the adenomatous polyposis coli promoter 1A in bronchial aspirates from patients with suspected lung cancer. J Cancer. 2004 Jul 10; 110(5):751 -5.
2. Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science. 1997, 286:481-486.
3. Hsieh J, Gage FH. Epigenetic control of neural stem cell fate. Current Opinion in Genetics & Development. 2004, 14: 461-469.
4. Abdolmaleky HM. Methylomics in Psychiatry: Modulation of Gene-Environment Interactions may be Through DNA Methylation. American Journal of Medical Genetics 2004(127B): 51-59
5. Fan G, Huthick L. Methylo-CpG proteins in the nervous system. Cell research, 2005,15(4): 255-261.
6. Akira M., hiroyuki N,Kazushi K,et al. Nectin: an adhesion molecule involved in formation of synapses. J. Cell Biol. 2002,156:555-565.
7. Watabe K, Ito A, Koma YI and Kitamura Y. IGSF4: a new intercellular adhesion molecule that is called by three names, TSLC1, SgIGSF and SynCAM, by virture of its diverse function. Histol Histopathol. 2003, 18: 1321-1329.
8. Shingai T, Ikeda W, Kakunaga S, Morimoto K, Takekuni K, Itoh S, Satoh K, Takeuchi M, Imai T, Monden M, Takai Y. Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells. J Biol Chem. 2003 Sep 12;278(37): 35421-7.
9. Yageta M, Kuramochi M, Masuda M, Fukami T, Fukuhara H, Maruyama T, Shibuya M,Murakami Y. Direct association of TSLC1 and DAL-1, two distinct tumor suppressor proteins in lung cancer. Cancer Res. 2002 Sep 15; 62(18): 5129-33.
10. Thomas Biederer, Yildirim sara, Marina Mozhayeva, Keniz Atasoy, Xinran Liu, Ege t.Kavalali, Thomas c. Sudhof. SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 2002,297(30) 1525-31
11. Kuramochi M, Fukuhara H, Nobukuni T, Kanbe T, Maruyama T, Ghosh HP, Pletcher M,Isomura M, Onizuka M, Kitamura T, Sekiya T, Reeves RH, Murakami Y.TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer. Nat Genet. 2001 Apr; 27(4): 427-30.
12. Fukuhara H, Kuramochi M, Fukami T, Kasahara K, Furuhata M, Nobukuni T,Maruyama T, Isogai K, Sekiya T, Shuin T, Kitamura T, Reeves RH, Murakami Y.Promoter methylation of TSLCl and tumor suppression by its gene product in human prostate cancer. Jpn J Cancer Res. 2002 Jun; 93(6): 605-9.
13. Allinen M, Peri L, Kujala S, Lahti-Domenici J, Outila K, Karppinen SM, Launonen V,Winqvist R. Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLCl promoter hypermethylation. Genes Chromosomes Cancer. 2002 Aug; 34(4): 384-9.
14. Jansen M, Fukushima N, Rosty C, Walter K, Altink R, Heek TV, Hruban R, Offerhaus JG, Goggins M.Aberrant methylation of the 5' CpG island of TSLCl is common in pancreatic ductal adenocarcinoma and is first manifest in high-grade PanlNs. Cancer Biol Ther. 2002 May-Jun; 1(3): 293-6.
15. Lo KW, Huang DP.Genetic and epigenetic changes in nasopharyngeal carcinoma. Semin Cancer Biol. 2002 Dec; 12(6): 451-62.
16. Ito A, Okada M, Uchino K, Wakayama T, Koma Y, Iseki S, Tsubota N, Okita Y,Kitamura Y. Expression of the TSLCl adhesion molecule in pulmonary epithelium and its down-regulation in pulmonary adenocarcinoma other than bronchioloalveolar carcinoma. Lab Invest. 2003 Aug; 83(8): 1175-83.
17. Fukami T, Fukuhara H, Kuramochi M, Maruyama T, Isogai K, Sakamoto M, Takamoto S, Murakami Y. Promoter methylation of the TSLCl gene in advanced lung tumors and various cancer cell lines. Int J Cancer. 2003 Oct 20; 107(1): 53-9.
18. Uchino K, Ito A, Wakayama T, Koma Y, Okada T, Ohbayashi C, Iseki S, Kitamura Y,Tsubota N, Okita Y, Okada M.Clinical implication and prognostic significance of the tumor suppressor TSLCl gene detected in adenocarcinoma of the lung. Cancer. 2003 Sep1;98(5): 1002-7.
19. Ito T, Shimada Y, Hashimoto Y, Kaganoi J, Kan T, Watanabe G, Murakami Y, Imamura M. Involvement of TSLCl in progression of esophageal squamous cell carcinoma.Cancer Res. 2003 Oct 1; 63(19): 6320-6.
20. Hui AB, Lo KW, Kwong J, Lam EC, Chan SY, Chow LS, Chan AS, Teo PM, Huang DP.Epigenetic inactivation of TSLCl gene in nasopharyngeal carcinoma. Mol Carcinog.2003 Dec; 38(4): 170-8.
21. Honda T, Tamura G, Waki T, Jin Z, Sato K, Motoyama T, Kawata S, Kimura W,Nishizuka S, Murakami Y. Hypermethylation of the TSLCl gene promoter in primary gastric cancers and gastric cancer cell lines. Jpn J Cancer Res. 2002 Aug; 93(8):857-60.
22. Tamura G Promoter methylation status of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia. Histol Histopathol. 2004 Jan; 19(1):221-8.
23. Steenbergen RD, Kramer D, Braakhuis BJ, Stern PL, Verheijen RH, Meijer CJ,Snijders PJ.TSLC1 gene silencing in cervical cancer cell lines and cervical neoplasia. J Natl Cancer Inst. 2004 Feb 18; 96(4): 294-305
24. Fleming JB, Minna JD.Inactivation of tumor suppressor genes by promoter methylation is important in the pathogenesis of pancreatic cancer. Cancer Biol Ther. 2002 May-Jun;1(3):297-9.
25. Surace EI, Lusis E, Murakami Y, Scheithauer BW, Perry A, Gutmann DH. Loss of tumor suppressor in lung cancer-1 (TSLC1) expression in meningioma correlates with increased malignancy grade and reduced patient survival. J Neuropathol Exp Neurol.2004 Oct; 63(10): 1015-27.
26. Li J, Zhang Z, Bidder M, Funk MC, Nguyen L, Goodfellow PJ, Rader JS. IGSF4 promoter methylation and expression silencing in human cervical cancer. Gynecol Oncol. 2005 Jan; 96(1): 150-8.
27.金冬雁等译。分子克隆实验指南。1998；科学出版社
28.黄培堂等。细胞实验指南。2001；科学出版社
29. Sherf BA, Navarro SL, Hannah RR, Wood KV. Dual-LuciferaseTM reporter assay: an advanced co-reporter technology integrating firefly and renilla luciferase assays.Promega Notes. 1996, 56:02
30. Fukami T, Satoh H, Fujita E, Maruyama T, Fukuhara H, Kuramochi M, Takamoto S,Momoi T, Murakami Y. Identification of the Tslc1 gene, a mouse orthologue of the human tumor suppressor TSLCl gene. Gene. 2002 Jul 24; 295(1): 7-12.
31. Masuda M, Yageta M, Fukuhara H, Kuramochi M, Maruyama T, Nomoto A, Murakami Y. The tumor suppressor protein TSLC1 is involved in cell-cell adhesion. J Biol Chem.2002 Aug 23; 277(34): 31014-9.
32. Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I.VISTA:visualizing global DNA sequence alignments of arbitrary length.Bioinformatics.2000 Nov,16(11):1046-7.
33.Larsen F,Gundersen G,Lopez R,Prydz H.CpG islands as gene markers in the human genome.Genomics 1992 Aug;13(4):1095-107
34.Gardiner-Garden M,Frommer M.CpG islands in vertebrate genomes.J Mol Biol.1987Jul 20;196(2):261-82.
35.Li L,He S,Sun J,and Davie JR.Gene regulation by Sp1 and Sp3.Biochem.Cell Biol.2004,82:460-471.
36.Macleod D,Charlton J,Mullins J,and Bird AP.Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island.Genes Dev.1994:8:2282-2292.
37.Brandeis M,Frank D,Keshet I,Siegfied Z,Mendelsohn M,Nemes A.et al.Sp1elements protect a CpG island from de novo methylation.Nature(London).1994,371:435-438.
38.刘文哲,李金瀚,胡义德等。顺铂诱导A549细胞凋亡的研究。中国肺癌杂志,2002年8月第5卷第4期。
39.孙恒义,胡义德,钱频等。顺铂诱导肺腺癌A549细胞线粒体基因组及凋亡相关基因的差异表达。解放军医学杂志,2004年12月第29卷第12期。
40.Ng HH,Bird A.DNA methylation and chromatin modification.Current Opinion in Genetics and Development.1999,9(2):158-163.
41.Manel E,Montserrat SC,Rafael R,et al.Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients.Cancer Res,1999,59:67-70.
42.Sabine ZM,Fong KM,Virmani AK,et al.Aberrant promoter methylation of multiple genes in non-small cell lung cancers[J].Cancer Res,2001,61 φ249-255.
43.Christman JK.5-azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation:Mechanistic studies and their implications for cancer therapy[J].Oncogene,2002,21:5483-5495.
44.Watt F,Molloy PL.Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter.Genes and development.1998;2:1136-43.
45. Takizawa T, Nakashima K, Namihira M, et al. DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain. Dev Cell 2001;1:749-58.
46. Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998, 72:141-196
47. Faridi J, Wang L, Endemann G, Roth RA. Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo. Clin Cancer Res. 2003 Aug 1 ;9(8):2933-9.
1.Holliday R. DNA methylation and epigenetic mechanisms. Cell Biophys 1989; 15:15-20.
2. James G, Herman, MD.Epigenetics in Lung Cancer. Chest 2004; 125:119s-122s.
3. Zochbauer-Muller S, Fong KM, Virmani AK, etal. Aberrant promoter methylation of multiple genes in non-small cell lung cancer. Cancer Res 2001 61:249-255
4. Estsller M, fraga MF, Guo M, etal. DNA methyaltion patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum Mol Genet 2002; 10:3001-3007.
5. Paz MF, Fraga MF, Avilia S, etal. A systematic profile of DNA methylation in human cancer cell cell lines. Cancer Res 2003; 63:1114-1121.
6. Usadel H, Brabender J, Danenberg KD, et al. Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer Res 2002;62:371-5.
7. Eeteller M, Sanchez-Cespedes M, Rosell R, et al. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cellcancer patients. Cancer Res 1999;59:67-70.
8. Zhou XM, Shao SJ, XU GD, et al. Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Feb 25;816(1-2):145-51.
9. Ramireaz JL, Taron M, Balana C, et al. Serum DNA as a tool for cancer patient management. Rocz Akad Med Bialymst 2003 ;48:34-41.
10. Fujiwara K, Fujimoto N, Tabata M, et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. Clin Cancer Res.2005 Feb 1;11(3):1219-25.
11. Grote HJ, Schmiemann V, Geddert H, et al, Aberrant promoter methylation of p16(INK4a), RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer. Cancer. 2005 Sep 20;116(5):720-5.
12. Palmisano WA, Divine KK, Saccomanno G, et al. Prediting lung cancer by detecing abrrant promoter methylation in sputum. Cancer Res, 2000, 60:5954-5958.
13. Rosell R, Taron M, Ariza A, et al. Molecular predictors of response to chemotherapy in lung cancer. Semin Oncol. 2004 Feb;31(1 Suppl 1):20-7.
14. Plumb JA, Steele N, Finn PW. Et al. Epigenetic approaches to cancer therapy.Biochem Soc Trans. 2004 Dec;32(Pt 6): 1095-7.
15. Boivin AJ, Momparler LF, Hurtubise A, et al. Antineoplastic action of 5-aza-2'-deoxycytidine and phenylbutyrate on human lung carcinoma cells. Anticancer Drugs. 2002 Sep;13(8):869-74.
16. Soria JC, Rodriguez M, Liu DD, et al. Aberrant DNA promoter methylation in lung cancer. Biological and implications. Oncologist, 2002, 7:451-457.
17. Kim DH, Kim JS, Ji YI, et al. Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer. Cancer Res. 2003 Jul 1;63(13):3743-6.
18. Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of pl6INK4A is an early event in lung cancer and a potential biomakerfor early diagnosis. Proc Natl Acad Sci US A, 1998; 95: 11891-11896.
19. Wu MF, Cheng YW, Lai JC, et al. Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan. Int J Cancer. 2005 Jan 20;113(3):440-5.
20. Piyathilake CJ, Henao O, Frost AR, et al. Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung (United States). Cancer Causes Control. 2003 Feb;14(1):37-42.
21.Toyooka S, Maruyama R, Toyooka KO, et al. Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer.Int J Cancer. 2003 Jan 10;103(2): 153-60.
22. Tetsuo Ito,Yutaka Shimada,Yosuke Hashimoto,et al. Involvement of TSLC1 in Progression of Esophageal Squamous Cell Carcinoma. Cancer Res 2003; 63:6320-6326.
23. Mika Yageta,Masami Kuramochi,Mari Masuda,et al.Direct Association of TSLC1 and DAL-1, Two Distinct Tumor Suppressor Proteins in Lung Cancer. Cancer Res 2002; 62:5129-5133.
24. Shinji Kikuchi,Daisuke Yamada,Takeshi Fukami,et al.Promoter Methylation of DAL-1/4.IB Predicts Poor Prognosis in Non-Small Cell Lung Cancer. Clin Cancer Res 2005; 11(8): 2954-2961.
25. Takeshi Fukami, Hiroshi Fukuhara,Masami Kuramochi,et al.PROMOTER METHYLATION OF THE TSLC1 GENE IN ADVANCED LUNG TUMORS AND VARIOUS CANCER CELL LINES.Int.J.Cancer 2003;107:53-59.
26. Tatsushi Shingai, Wataru Dceda, Shigedi Kakunaga, et al. Implications of Nectin-like Molecule-2/IGSF4/RA175/SgIGSF/TSLCl/SynCAM1 in Cell-Cell Adhesion and Transmembrane Protein Localization in Epithelial Cells. The Journal Of Biological Chemistry 2003;278:35421-35427.
27. Makoto Sonobe, MD, Fumihiro Tanaka, MD, and Hiromi Wada, MD. Lung Cancer-related Genes in the Blood .Ann Thorac Cardiovasc Surg 2004; 10:213-217.
28. Ito A, Okada M, Uchino K, Wakayama T, Koma Y, et al. Expression of the TSLC1 adhesion molecule in pulmonary epithelium and its down-regulation in pulmonary adenocarcinoma other than bronchioloalveolar carcinoma. Lab Invest 2003;83(8):1175-83.
29. Uchino K, Ito A, Wakayama T, Koma Y, Okada T, Ohbayashi C, Iseki S, Kitamura Y,Tsubota N, Okita Y, Okada M. Clinical implication and prognostic significance of the tumor suppressor TSLC1 gene detected in adenocarcinoma of the lung. Cancer Rse 2003;98(5): 1002-7.
30. Fukami T, Fukuhara H, Kuramochi M, Maruyama T, Isogai K,Sakamoto S, Murakami Y. Promoter methylation of the TSLC1 gene in adcanced lung tumors and various eancer cell lines. Int J Cancer 2003;107(l):53-9.
31. Xinliang Mao, Eric Seidlitz, Kakoli Ghosh, et al.The Cytoplasmic Domain Is Critical to the Tumor Suppressor Activity of TS LC1 in Non-Small Cell Lung Cancer.Cancer Res 2003;63:7979-7985.
32. Xinliang Mao, Eric Seidlitz, Ray Truant, et al. Re-expression of TSLC1 in a non -small-cell lung cancer cell line induces apoptosis and inhibits tumor growth.Oncogene 2004;1-11.
33. Kent S.Boies, Winfried Barchet, Tom Diacovo, et al. The tumor suppressor TSLC1/NECL-2 triggers NK cell and CD8 T cell responses through the cell surface receptor CRTAM. Blood 2005; 10.
34. David GB, Eva F, Sabine ZM, et al. Epigenetic inactivation of RASSF1A in lung and breast cancer and malignant phenotype suppression. JNCI, 2001;93(9):691-699
35. Bearzatto A, Conte D, Frattini M, et al. p16(INK4A)hypermethylation detected by fluorescent methylationspecific PCR in plasmas from non-small cell lung cancer. Clin Cancer Res 2002;8:3782-7.
36. Liu JY, An Q, Xu GD, et al, Hypermethylation of p16 gene in clinical specimens of patients with lung cancer Zhonghua Zhong Liu Za Zhi. 2004 Feb;26(2):75-7.
37. Sabine Z, Kwun MF, Anirban M, et al. 5CpG island methylation of the FHIT gene is correlated with loss of dene expression in lung and breast cancer.Cancer Res.2001,61:3581-3585.
38. Kim h, Kwon YM, Kim JS,et al. Tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer. J Clin Oncol. 2004 Jun 15;22(12):2363-70.
39. Jan B,Henning U, Kathleen D, et al. Adenomatous polyposis coligene promoter hypermethylation in non-small cell lung cancer is associated with survival.Oncogene,2001,20:3528-3532
40. Grote HJ, Schmiemann V, Kiel S, et al. Aberrant methylation of the adenomatous polyposis coli promoter 1A in bronchial aspirates from patients with suspected lung cancer. J Cancer. 2004 Jul 10; 110(5):751 -5.