参考文献

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2　Wolff A P. Chromatin remodeling: why it is important in cancer.Oncogene, 2001, 20 (24) : 2988-2990.

3　Pennisi E. Behind the Scenes of Gene Expression.Science, 2001 , 293 (24) : 1064-1067.

4　Robertson KD. DNA methylation, methyltransferases, and cancer. Oncogene, 2001, 20 (24) : 3135-3155.

Abstract: The field of epigenetics has recently moved to the forefront of studies relating to diverse processes such as transcriptional regulation, chromatin structure, genome integrity, and tumorigenesis. Recent work has revealed how DNA methylation and chromatin structure are linked at the molecular level and how methylation anomalies play a direct causal role in tumorigenesis and genetic disease. Much new information has also come to light regarding the cellular methylation machinery, known as the DNA methyltransferases, in terms of their roles in mammalian development and the types of proteins they are known to interact with. This information has forced a new view for the role of DNA methyltransferases. Rather than enzymes that act in isolation to copy methylation patterns after replication, the types of interactions discovered thus far indicate that DNA methyltransferases may be components of larger complexes actively involved in transcriptional control and chromatin structure modulation. These new findings will likely enhance our understanding of the myriad roles of DNA methylation in disease as well as point the way to novel therapies to prevent or repair these defects. 
 

5　Holliday R. Mutation Res, 2001, 483 (Supp ll) : s3

6 周玉球.DNA甲基化分析技术的研究进展.国外医学遗传学分册　2001,24(6):303-308

7  Herman JG et al . Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands。Proc Natl A cad SciU SA , 1996, 93: 9821-9826.

Abstract: Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer. 
 

8　Ahmad I, Rao DN. Chemistry and biology of DNA methyltransferases. Crit Rev Biochem MolBiol, 1996, 31: 361-380.

9. Vertino PM , Yen RW, Gao J , et al. De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA(cytosine- 5) - methyltransferase1Mol Cell Biol ,1996 ,16 (8) :4555

Abstract: Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.  

10　Ehrlich M , Wang RY.H1 5 - methylcytosine in eukaryotic DNA1.Science,1981 ,212 :1350

11　Keith D et al. Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G₀/G₁ to S phase transition in normal and tumor cells.Nucleic Acids Research, 2000; 10: 2108

Abstract: DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G₀/G₁ while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G₀/G₁ arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells. 
 

12　Bender CM,Zingg JM,Jones PA. DNA methylation in bladder cancer[J ] . Pharm Aceutical Res,1998 ,15(2) :175.

Abstract: DNA methylation is essential for normal embryonic development. Distinctive genomic methylation patterns must be formed and maintained with high fidelity to ensure the inactivities of specific promoters during development. The mutagenic and epigenetic aspects of DNA methylation are especially interesting because they may lead to the inactivation of genes which are involved in human carcinogenesis. The mutagenicity of 5-Methylcytosine (5mC) and the role of promoter hypermethylation in gene silencing, particularly in cancer, suggest a clinical significance for the design of novel DNA methylation inhibitors which may be utilized to reverse the effects of DNA methylation. 
 

13　Ahujia N. Aging and DNA methylation in colorectal mucosa and cancer[J ] . Cancer Res ,1997 ,58 (23) :3370-3374.

14　Wheeler JM,Beck NE , Kim HC , et al . Mechanisms of inactivation ofmismatch repair genes in human colorectal cancer cell lines : the predominant role of Hmlh1[J ] . Proc Natl Acad Sci USA ,1999 ,96 (18) :10296-10301.

15 彭正良.甲状腺肿瘤相关基因甲基化研究进展.国外医学·生理、病理科学与临床分册，2005，25（2）：126-129

16 G. Strathdee and R. Brown. Aberrant DNA methylation in cancer:potential clinical interventions. Expert Rev Mol Med, 2002,3: 1-17.

17 Virmani AK, Rathi A, Sathyanarayana UG,et al: Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breastand lung carcinomas. Clin Cancer Res 7:1998-2004, 2001

Abstract: The adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic cancer. Despite high rates of allelic loss in lung and breast cancers, point mutations of the APC gene are infrequent in these cancer types. Aberrant methylation of the APC promoter 1A occurs in some colorectal and gastric malignancies, and we investigated whether the same mechanism occurs in lung and breast cancers. The methylation status of the APC gene promoter 1A was analyzed in 77 breast, 50 small cell (SCLC), and 106 non-small cell (NSCLC) lung cancer tumors and cell lines and in 68 nonmalignant tissues by methylation-specific PCR. Expression of the APC promoter 1A transcript was examined in a subset of cell lines by reverse transcription-PCR, and loss of heterozygosity at the gene locus was analyzed by the use of 12 microsatellite and polymorphic markers. Statistical tests were two-sided. Promoter 1A was methylated in 34 of 77 breast cancer tumors and cell lines (44%), in 56 of 106 NSCLC tumors and cell lines (53%), in 13 of 50 SCLC cell lines (26%), and in 3 of 68 nonmalignant samples (4%). Most cell lines tested contained the unmethylated or methylated form exclusively. In 27 cell lines tested, there was complete concordance between promoter methylation and silencing of its transcript. Demethylation with 5-aza-2'-deoxycytidine treatment restored transcript 1A expression in all eight methylated cell lines tested. Loss of heterozygosity at the APC locus was observed in 85% of SCLCs, 83% of NSCLCs, and 63% of breast cancer cell lines. The frequency of methylation in breast cancers increased with tumor stage and size. In summary, aberrant methylation of the 1A promoter of the APC gene and loss of its specific transcript is frequently present in breast and NSCLC cancers and cell lines and, to a lesser extent, in SCLC cell lines. Our findings may be of biological and clinical importance.  
 

18 Kawakami K, Brabender J, Lord RV, et al:Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma.J Natl Cancer Inst 92:1805-1811, 2000

19. Dobrovic A, Simpfendorfer D: Methylation of the BRCA1 gene in sporadic breast cancer. Cancer Res 57:3347-3350, 1997

20 Chan KY, Ozcelik H, Cheung AN, et al:Epigenetic factors controlling the BRCA1 and BRCA2 genes in sporadic ovarian cancer. Cancer Res 62:4151-4156, 2002

21 Herman JG, Merlo A, Mao L, et al: Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 55:4525-4530, 1995

Abstract: The tumor suppressor gene CDKN2/p16/MTS1, located on chromosome 9p21, is frequently inactivated in many human cancers through homozygous deletion. Recently, we have reported another pathway of inactivation that involves loss of transcription associated with de novo methylation of a 5' CpG island of CDKN2/p16 in lung cancers, gliomas, and head and neck squamous cell carcinomas. We now show that this aberrant CpG island methylation also occurs frequently in cell lines of breast cancer (33%), prostate cancer (60%), renal cancer (23%), and colon cancer (92%) and is associated with loss of transcription. Primary tumors of the breast (31%) and colon (40%) also displayed de novo methylation of this CpG island. This alteration of p16 in colon cancer was particularly striking, since inactivation does not occur through homozygous deletion in this tumor type. Our data show that in tumors, de novo methylation of the 5' CpG island is a frequent mode of inactivation of CDKN2/p16 and also firmly demonstrate that CDKN2/p16 is one of the most frequently altered genes in human neoplasia.  

22 Sanchez-Cespedes M, Esteller M, Wu L et al: Gene promoter hypermethylation in tumorsand serum of head and neck cancer patients.Cancer Res 60:892-895, 2000

23 Villuendas R, Sanchez-Beato M, Martinez JC, et al: Loss of p16/INK4A protein expressionin non-Hodgkin’s lymphomas is a frequent finding associated with tumor progression. Am J Pathol 153:887-997, 1998

24 Harden SV, Tokumaru Y, Westra WH, et al: Gene promoter hypermethylation in tumors and lymph nodes of stage I lung cancer patients.Clin Cancer Res 9:1370-1375, 2003

25 Graff JR, Herman JG, Lapidus RG, et al:E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas.Cancer Res 55:5195-5199, 1995

26 Graff JR, Greenberg VE, Herman JG, et al: Distinct patterns of E-cadherin CpG island methylation in papillary, follicular, Hurthle’s cell,and poorly differentiated human thyroid carcinoma.Cancer Res 58:2063-2066, 1998

27 Waki T, Tamura G, Tsuchiya T, et al: Promoter methylation status of E-cadherin,hMLH1, and p16 genes in nonneoplastic gastric epithelia. Am J Pathol 161:399-403, 2002

28 Yang X, Yan L, Davidson NE: DNA methylation in breast cancer. Endocr Relat Cancer， 8:115-127, 2001

29 Li LC, Chui R, Nakajima K, et al: Frequent methylation of estrogen receptor in prostate cancer: Correlation with tumor progression. Cancer Res 60:702-706, 2000

30 Lee WH, Morton RA, Epstein JI, 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 91:11733-11737, 1994

31 Esteller M, Corn PG, Urena JM, et al:Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 58:4515-4518, 1998

32 Veigl ML, Kasturi L, Olechnowicz J, et al. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A 95:8698-8702, 1998

33 Kondo E, Furukawa T, Yoshinaga K, et al: Not hMSH2 but hMLH1 is frequently silenced by hypermethylation in endometrial cancer but rarely silenced in pancreatic cancer with microsatellite instability. Int J Oncol 17:535-541, 2000

34 Strathdee G, MacKean MJ, Illand M, et al: A role for methylation of the hMLH1 promoter in loss of hMLH1 expression and drug resistance in ovarian cancer. Oncogene 18:2335-2341, 1999

35 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 343:1350-1354, 2000

36 Melki JR, Vincent PC, Clark SJ: Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res 59:3730-3740, 1999

37 Garcia MJ, Martinez-Delgado B, Cebrian A, et al: Different incidence and pattern of p15INK4b and p16INK4a promoter region hypermethylation in Hodgkin’s and CD30-Positive non-Hodgkin’s lymphomas. Am J Pathol 161:1007-1013, 2002

38 Herman JG, Jen J, Merlo A, et al: Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B. Cancer Res 56:722-727, 1996

39 Agathanggelou A, Honorio S, Macartney DP, et al: Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene 20:1509-1518, 2001

40 Morrissey C, Martinez A, Zatyka M, et al: Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma. Cancer Res 61:7277-7281, 2001

41 Kwong J, Lo KW, To KF, et al: Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res 8:131-137,2002

42 Stirzaker C, Millar DS, Paul CL, et al: Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res 57:2229-2237, 1997

43 Gonzalez-Gomez P, Bello MJ, Alonso ME, et al: CpG island methylation status and mutation analysis of the RB1 gene essential promoter region and protein-binding pocket domain in nervous system tumours. Br J Cancer 88:109-114, 2003

44　Merlo A, Herman JG,Mao L, et al. 5′CpG island methylation is associated with transcription silencing of the tumor suppressor p16 /CDKN2 /MTS1 in human cancers[ J ]. N atM ed, 1995, 1 (7) : 686-692.

45　Boltze C, Zack S, Quednow C, et al. Hypermethylation of the CDKN2 /p16 INK4 A promotor in thyroid carcinogenesis[ J ]. Pathol ResPract, 2003, 199 (6) : 399-404.

Abstract: Functional inactivation of the p16INK4A gene has been reported to be involved in the development of a variety of human malignancies. In thyroid carcinomas, mutations of the p16INK4A gene or homozygous deletions of the gene locus 9p21 are rare. This study investigated whether p16INK4A promotor methylation is an alternative mechanism for p16INK4A gene inactivation during thyroid carcinogenesis. A methylation-specific polymerase chain reaction protocol was applied. A total of 77 thyroid tumor specimens, including 18 follicular adenomas, 18 follicular carcinomas, 16 papillary carcinomas, 12 poorly differentiated carcinomas, and 13 undifferentiated carcinomas were analyzed longitudinally. In addition, 15 tumor-free thyroid tissues were investigated. The p16INK4A promotor status was compared with p16INK4A protein expression and patient-specific data. p16INK4A promotor hypermethylation was detected in 13% of non-tumorous tissue; in 33% of follicular adenomas; in 44% of papillary carcinomas; in 50% of follicular carcinomas; in 75% of poorly differentiated carcinomas; and in 85% of undifferentiated carcinomas. With the exception of two cases, the p16INK4A protein was lost as a result of promotor hypermethylation. Comparing the methylation status with tumor stage, no correlation was found. However, lymph node and distant metastasis status showed a statistically significant prevalence for the p16INK4A promotor methylation (p = 0.035). There was no association between p16INK4A promotor methylation and age and sex. These results suggest that hypermethylation of the p16INK4A promotor region is a frequent and an early event during thyroid carcinogenesis and is associated with tumor progression and dedifferentiation. 
 

46　Schagdarsuregin U, Gimm O, Cuong Hoang-Vu C, et al. Frequent epigenetic silencing of the CpG island promoter of RASSF1A in thyroid carcinoma[ J ]. Cancer Res, 2002, 62 (12) : 3698-3701.

47　Xing M, Cohen Y,Mambo E, et al. Early ocurrence of RASSF1A hypermethylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis[ J ]. Cancer Res, 2004, 64 (6) : 166421668.

48 Tsou JA, Hagen JA, Carpenter CL, et al: DNA methylation analysis: A powerful new tool for lung cancer diagnosis. Oncogene 21:5450-5461, 2002

49 Feinberg AP, Vogelstein B: Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301:89-92, 1983

Abstract: It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations, rearrangements and changes in methylation have been proposed to have such a role. Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9-11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture. The results of these studies have varied; depending on the techniques and systems used, an increase, decrease, or no change in the degree of methylation has been reported. To our knowledge, however, primary human tumour tissues have not been used in such studies. We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy. In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts. This hypomethylation was progressive in a metastasis from one of the patients. 
 

50 Kim YI, Giuliano A, Hatch KD, et al: Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer 74:893-899, 1994

51 Lin CH, Hsieh SY, Sheen IS, et al: Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res 61:4238-4243, 2001

52 Bedford MT, van Helden PD: Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Res 47:5274-5276,1987

53 Ehrlich M: DNA methylation in cancer:Too much, but also too little. Oncogene 21:5400-5413, 2002

54 Ji W, Hernandez R, Zhang XY, et al. DNA demethylation and pericentromeric rearrangements of chromosome 1. Mutat Res, 379: 33, 1997

Abstract: Rearrangements in the vicinity of the centromere of chromosome 1 are over-represented in many types of human cancer and are a characteristic feature of a rare genetic disease called ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies). Evidence is presented that implicates DNA hypomethylation in the formation of these pericentromeric chromosomal anomalies. The DNA methylation inhibitors 5-azadeoxycytidine and 5-azacytidine, but not other tested genotoxins, induced the preferential formation of pericentromeric rearrangements of chromosome 1 at a very high frequency in a pro-B-cell line (FLEB14) and at a lower frequency in a mature B-cell line (AHH-1). These abnormal chromosomes appear identical to the diagnostic chromosomal aberrations in the ICF syndrome. A major component of the pericentromeric DNA in chromosome 1, satellite 2, was shown to be hypomethylated in an ICF B-cell line, although DNA from this cell line did not display detectable overall hypomethylation. It is hypothesized that demethylation in certain DNA regions, including in pericentromeric satellite DNA, helps lead to pericentromeric chromosomal rearrangements in lymphocytes from ICF patients and in normal lymphoblastoid cells incubated in vitro with DNA demethylating agents. 
 

55 Crossen PE & Morrison MJ. Methylation status of the 3^rd exon of the C-myc oncogene in B-cell malignancies. Leu Res, 1999,23: 251

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58 Christoph Lengauer. CANCER:An Unstable Liaison.Science, Apr 2003; 300: 442 - 443.

59  Chen RZ, Pettersson U, Beard C, et al. DNA hypomethylation leads to elevated mutation rates.Nature,1998,395:89

60 Florl AR, Lower R, Schmitz-Drager BJ,et al. DNA methylation and expression of LINE-1 and HERV-k provirus sequences in urothelial and renal cell carcinomas. Br J Cancer,1999,80:1312

61 Feinerg AP. Imprinting of a genomic domain of 11p15 and loss of imprinting in cancer: An introduction. Cancer Res, 1999,59:1743

62 Vachtenheim, J., Horakova, I. and Novotna, H.(1994) Hypomethylation of CCGG sites in the 3'region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer. Cancer Res 54, 1145-1148, PubMed ID: 94163597

63 Cheah, M.S., Wallace, C.D. and Hoffman, R.M.(1984) Hypomethylation of DNA in human cancer cells: a site-specific change in the c-myc oncogene. J Natl Cancer Inst 73, 1057-1065,

64 Vertino, P.M. et al. (1996) De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase. Mol Cell Biol 16, 4555-4565, PubMed ID: 96315682