65 el-Deiry, W.S. et al. (1991) High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer. Proc Natl Acad Sci U S A 88, 3470-3474, PubMed ID: 91195373

66 Issa, J.P. et al. (1993) Increased cytosine DNAmethyltransferase activity during colon cancer progression. J Natl Cancer Inst 85, 1235-1240, PubMed ID: 93323146

67 Belinsky, S.A. et al. (1996) Increased cytosine DNA-methyltransferase activity is target-cellspecific and an early event in lung cancer. Proc Natl Acad Sci U S A 93, 4045-4050, PubMed ID: 96210590

68 Melki, J.R. et al. (1998) Increased DNA methyltransferase expression in leukaemia. Leukemia 12, 311-316, PubMed ID: 98187973

69 Lee, P.J. et al. (1996) Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation. Proc Natl Acad Sci U S A 93, 10366-10370, PubMed ID: 96413652

70 Eads, C.A. et al. (1999) CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. Cancer Res 59, 2302-2306, PubMed ID: 99274530

Abstract: The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (ß-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer. 
 

71 Kanai, Y. et al. (2001) DNA methyltransferase expression and DNA methylation of CPG islands and peri-centromeric satellite regions in human colorectal and stomach cancers. Int J Cancer 91, 205-212, PubMed ID: 20581958

72 Rhee, I. et al. (2000) CpG methylation is maintained in human cancer cells lacking DNMT1. Nature 404, 1003-1007, PubMed ID:20259068

Abstract: Hypermethylation is associated with the silencing of tumour susceptibility genes in several forms of cancer; however, the mechanisms responsible for this aberrant methylation are poorly understood. The prototypic DNA methyltransferase, DNMT1, has been widely assumed to be responsible for most of the methylation of the human genome, including the abnormal methylation found in cancers. To test this hypothesis, we disrupted the DNMT1 gene through homologous recombination in human colorectal carcinoma cells. Here we show that cells lacking DNMT1 exhibited markedly decreased cellular DNA methyltransferase activity, but there was only a 20% decrease in overall genomic methylation. Although juxtacentromeric satellites became significantly demethylated, most of the loci that we analysed, including the tumour suppressor gene p16INK4a, remained fully methylated and silenced. These results indicate that DNMT1 has an unsuspected degree of regional specificity in human cells and that methylating activities other than DNMT1 can maintain the methylation of most of the genome. 
 

73 Robertson, K.D. et al. (1999) The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res 27, 2291-2298, PubMed ID: 9263031

74 Mizuno, S. et al. (2001) Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood 97, 1172-1179, PubMed ID: 21124318

75 Chuang, L.S. et al. (1997) Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 277, 1996-2000, PubMed ID: 97451025

Abstract: DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. Here, MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT, occurs in intact cells at foci of newly replicated DNA, and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21^WAF1 can disrupt the MCMT-PCNA interaction, which suggests that p21^WAF1 may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21^WAF1 may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells. 
 

76 De Marzo, A.M. et al. (1999) Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis. Cancer Res 59, 3855-3860, PubMed ID: 99391210

Abstract: Somatic changes in CpG dinucleotide methylation occur quite commonly in human cancer cell DNA. Relative to DNA from normal human colonic cells, DNA from human colorectal cancer cells typically displays regional CpG dinucleotide hypermethylation amid global CpG dinucleotide hypomethylation. The role of the maintenance DNA methyltransferase (DNMT1) in the acquisition of such abnormal CpG dinucleotide methylation changes in colorectal cancer cells remains controversial; in one study, 60–200-fold increases in DNMT1 mRNA expression were detected in colorectal polyps and cancers relative to normal colonic tissue [W. S. El-Deiry et al., Proc. Natl. Acad. Sci. USA, 88: 3470–3474, 1991], whereas in another study, only small increases in DNMT1 mRNA expression, commensurate with differences in cell proliferation accompanying colonic tumorigenesis, were observed [P. J. Lee et al., Proc. Natl. Acad. Sci. USA, 93: 10366–10370, 1996]. To definitively ascertain whether abnormal DNMT1 expression might accompany human colorectal carcinogenesis, we subjected a series of normal and neoplastic colonic tissues to immunohistochemical staining using a polyclonal antiserum raised against a DNMT1 polypeptide. A concordance of DNMT1 expression with the expression of PCNA and other cell proliferation markers, such as Ki-67 and DNA topoisomerase II, was observed in normal colonic epithelial cells and in cells comprising other normal epithelia and lymphoid tissues. The polypeptide p21, which has been reported to undermine DNMT1 binding to proliferating cell nuclear antigen at DNA replication sites, was not expressed by normal colonic cells containing DNMT1 and other cell proliferation markers. In adenomatous polyps, although DNMT1 expression coincided with the expression of other cell proliferation markers, many DNMT1-expressing cells also expressed p21. The fidelity of DNMT1 expression was further undermined in colorectal carcinomas, in which a striking heterogeneity in DNMT1 expression, with some carcinoma cells containing very high DNMT1 levels and others containing very low DNMT1 levels, was observed. These results indicate that human colorectal carcinogenesis is accompanied by a progressive dysregulation of DNMT1 expression and suggest that abnormalities in DNMT1 expression may contribute to the abnormal CpG dinucleotide methylation changes characteristic of human colorectal carcinoma cell DNA. 
 

77 Caffo, O. et al. (1996) Prognostic value of p21(WAF1) and p53 expression in breast carcinoma: an immunohistochemical study in 261 patients with long-term follow-up. Clin Cancer Res 2, 1591-1599, PubMed ID: 99035186

Abstract: p21 protein (p21) inhibitor of cyclin-dependent kinases is a critical downstream effector in the p53-specific pathway of growth control and can also be induced by p53-independent pathways in relation to terminal differentiation. We investigated p21 immunoreactivity in 261 breast carcinomas (141 node negative and 120 node positive) with long-term follow-up (median, 73 months; range, 37-119). p21 was seen in 214 (82%) infiltrating tumors, staining was nuclear and heterogeneous, and the p21 labeling index ranged from 0 to 90%. Sixty-eight (32%) patients showed p21 overexpression (>10% of reactive tumor cells). p21 overexpression was associated with large tumor size, positive nodal status, high histological grade, and high mitotic count and was related to short disease-free survival (DFS) in the whole series of patients (P = 0.04), in the node-negative subgroup (P = 0.004), and in the group of patients who did not undergo systemic adjuvant therapy (P = 0.003). In patients treated with systemic adjuvant therapy, bivariate analysis of the combined p21 and p53 phenotypes showed that p21+/p53+ tumors were associated with long DFS and overall survival (OS), whereas p21-/p53+ tumors had the worst prognosis. In treated patients, multivariate analysis showed that the p21-/53+ phenotype was independently associated with short DFS and OS. Our present data support the hypothesis that p21/p53 heterogeneous expression may be of clinical relevance for the therapeutic response to chemotherapy/hormonotherapy. The p21-/p53+ phenotype could correspond to a situation where p53 overexpression really reflects complete abrogation of p53 function. These cases with disrupted p53 function should have impaired the G1 checkpoint and may not be able to activate the apoptotic cascade in response to DNA-damaging drugs. 
 

78 Caputi, M. et al. (1998) p21waf1/cip1mda-6 expression in non-small-cell lung cancer: relationship to survival. Am J Respir Cell Mol Biol 18, 213-217, PubMed ID: 9813577579

79 Qin, L.F. et al. (1998) p21/WAF1, p53 and PCNA expression and p53 mutation status in hepatocellular carcinoma. Int J Cancer 79, 424-428, PubMed ID: 98363117

80 Barboule, N. et al. (1998) Increased level of p21 in human ovarian tumors is associated with increased expression of cdk2, cyclin A and PCNA. Int J Cancer 76, 891-896, PubMed ID: 98289660

Abstract: We have demonstrated over-expression of the cyclin-dependent kinase inhibitor p21 in various ovarian-cancer cell lines as well as in ovarian-tumor biopsies. This increase in p21 expression relative to that observed in normal ovarian epithelial cells is unrelated to proliferation index. In the present study, we found that p21 is functional, since the protein extracted from IGROVI cells is still able to inhibit cdk2-kinase activity. We then investigated how IGROVI cells overcome the growth-inhibitory function of p21. Immunofluorescence assays and subcellular fractionation showed that p21 is located in cytoplasm and nucleus both in normal and in tumoral cells. Compared with normal ovarian epithelial cells in culture, the increase in level of p21 in IGROVI cells was found to be associated with increased expression of cdk2, cyclin-A and PCNA proteins. In IGROVI cells, p21 is associated with inactive cdk2/cyclin-A complex, indicating that it acts as an inhibitory factor rather than an assembly factor. Over-expression of cdk2 and of cyclin A observed in IGROVI cells allows them to escape to p21-inhibitory activity. The fact that cells from ovarian-tumor biopsies exhibited a concomitant increase in p21 and in its partners cdk2 and PCNA suggest that ovarian-tumor cells can tolerate high levels of functional p21 via over-expression of other cell-cycle-regulatory proteins.

DNA甲基化与肿瘤

张丕显

（05级博士研究生） 
 

摘要：本文综述了DNA甲基化的研究进展。哺乳动物DNA甲基化主要发生在5’-CpG-3’的C上，生成5-甲基胞嘧啶(5mC)。DNA的甲基化可致基因突变（C®T）与基因沉默。基因的甲基化沉默机制有两种： 由于启动子区的甲基化导致启动子区结构改变，启动困难；‚甲基化引起组蛋白脱乙酰化而致染色质结构改变，关闭基因。目前，甲基化检测常用甲基化特异的PCR（MSP），检出限可达0.1%。

    肿瘤中普遍存在DNA甲基化状态的改变。表现为总体的甲基化水平降低与局部的甲基化水平升高。表现为抑癌基因与修复基因的高甲基化与反转录转座子、癌基因的去甲基化。造成肿瘤甲基化改变的原因可能与甲基转移酶、p21^WAF1及染色质结构改变有关,而甲基转移酶的调控机制尚不清楚. 
 

关键词: DNA, 甲基化, 肿瘤 
 

引子

   人类对基因本质的认识逐步深入, 目前正经历着更全面的认知过程。早期遗传学家认为, 基因是一个遗传的功能单位, 决定某种遗传性状, 它们在染色体上占有一定的位置, 并可发生突变和交换;随着DNA 作为蛋白质遗传密码载体的发现, 分子遗传学和遗传工程技术的迅速发展, 遗传学界基本上接受了下述定义: 基因是编码一条多肽链的特定DNA 片段。近几年来随着学科的进展, 一些研究者对上述看法提出了质疑^[1～3]。人类基因组计划中DNA 测序工作的基本完成, 只确定了3 万多个基因, 仅是果蝇的2 倍多, 很难想象DNA 含有充分的遗传信息以调控人类如此复杂有机体发育和生存的全过程^[4]。实际上在人类细胞中只有数千个基因有活性, 因此维持细胞的正常功能, 决定什么样的一组基因有功能, 而另一组基因无功能, 都是十分重要的。如果出错就会引起严重的后果, 据估计至少3 个基因错误表达就能诱发正常细胞癌变^[2]。这样在人类基因组含有两类遗传学信息, 传统意义上的遗传学信息提供了生命所必需的蛋白质的模板; 而表遗传学的信息提供了何时、何地和何种方式应用这些遗传学信息的指令^[4]。

   表遗传学1942 年由Waddington 首先提出, 研究基因型产生表型的过程, 此后Holliday进行了一系列的探讨^[5]。目前认为, 表遗传学是研究没有DNA 序列变化、可遗传的基因表达(活性)的改变。还有研究者从不同角度进行描述, 例如相对于DNA 序列质的改变的遗传学研究, 表遗传学被定义为研究基因表达水平(量变) 信息的遗传。也有研究者从遗传学角度, 把表遗传学定义为非孟德尔遗传, 或没有DNA 序列改变的核遗传。2003年10月，人类表观基因组协会（Human Epigenome Con-sortium,HEC）宣布开始投资实施人类表观基因组计划(Human Epigenome Project,HEP),标志着生命科学的研究已悄然进入后基因(表基因)时代.而甲基化是表遗传学作用的主要形式. 
 

DNA甲基化

　DNA 甲基化是指生物体在DNA 甲基转移酶(DNA methyltransferase ,DMT) 的催化下,以s-腺苷甲硫氨酸(SAM) 为甲基供体,将甲基转移到特定的碱基上的过程。DNA甲基化可以发生在腺嘌呤的N -6位、胞嘧啶的N -4位、鸟嘌呤的N -7位或胞嘧啶的C-5位等^[8]。但在哺乳动物,DNA甲基化主要发生在5’-CpG-3’的C上.生成5-甲基胞嘧啶(5mC) ^[9].反应如下: 
 

               图1 DNA甲基化 
 

       人类的Cp G以两种形式存在,一种是分散于DNA 中,另一种是CpG结构高度聚集的CpG岛。在正常组织里,70 %～90 %的散在的CpG是被甲基修饰的,而CpG岛则是非甲基化的^[10] 。 
 

DNA甲基化分析的方法

   分析DNA甲基化的位点与程度的实验方法有两类：一类（M SREs）利用对甲基化碱基敏感的限制性内切酶。该酶不能切割甲基化的碱基位点，从而产生片段差异，电泳后，根据片段与量的差异找到甲基化位点与甲基化程度^[6]；另一类是利用将没有甲基化的C变为其它碱基或其它物质，而甲基化的C不会发生相应变化来识别甲基化位点。甲基化特异的PCR (M ethylation-specific PCR,MSP)^[7]是较好的常用的方法。该法用 HSO₃^-处理单链DNA，使所有未甲基化的C脱氨转变为U,而甲基化的C则保持不变。然后经特异性扩增放大，测序。C位点就是甲基化位点，C的量即甲基化量。该法灵敏度高, 可检出比例为千分之一的甲基化等位片段,且对DNA 的质和量要求也低, 能用于微量的DNA 或石腊包埋组织DNA 的甲基化分析。

甲基化酶

    目前, 在真核生物中发现了3 类DNA 甲基转移酶(Dnmt1、Dnmt2、Dnmt3a、Dnmt 3b): Dnmt1 主要是维持DNA 的甲基化; Dnmt2 可与DNA上特异位点结合, 但具体作用尚不清楚; Dnmt3 主要是参与DNA 的从头甲基化。Dnmt3b 基因的RNA和蛋白在肿瘤组织中明显的高表达, 而Dnmt1 和Dnmt3a 在肿瘤组织中仅适度过表达。对10 例肿瘤组织中上述基因的表达分析发现, 5 个样本中Dnm t3a 高表达; 6 个样本中Dnmt1 高表达; 8 个样本中Dnmt3b 高表达^{[ 11 ]}。 
 

甲基化的作用 
 

1．基因C →T突变

   DNA 甲基化引起基因突变的机制主要是由于DMT催化反应形成。DMT可以加快C(胞嘧啶) 和5mC 脱氨,封闭U(尿嘧啶) 的修复,并且使U →T 改变,故DMT 促使CpG序列的C →T突变^{[12 ]} 。

       图2 C®T突变 
 
 

   抑癌基因p53就是一个典型的例证。50% 实体瘤病人出现p53基因突变。突变中24% 是CpG 甲基化后脱氨引起的C→T 突变。 
 

2　影响基因错配修复　

   DNA 错配修复系统(DNAmismatch repair system ,MMR) 是指存在人类细胞中的一种修复DNA 碱基错配的安全保障体系,它是由一系列特异修复DNA碱基错配的酶分子组成。Ahujia 等^{[13 ]} 研究发现MMR 缺陷时,CpG岛的甲基化增强,并认为MMR 与DNA 甲基化有关。在基因错配修复过程中甲基化具有导向识别作用,而在错配修复基因表达缺陷的原因中基因突变和基因启动子区的高甲基化是其主要原因^{[14 ]} 。 
 

3．基因沉默

   目前认为,甲基化影响基因表达的机制有下列几种: ①直接作用。基因的甲基化改变了基因的构型,影响DNA特异顺序与转录因子的结合,使基因不能转录; ②间接作用。基因5′端调控序列甲基化后与核内甲基化CG序列结合蛋白(methyl CG-binding p rotein)结合,阻止了转录因子与基因形成转录复合物; ③DNA去甲基化为基因的表达创造了一个良好的染色质环境。DNA去甲基化常与DNase I高敏感区同时出现,后者为基因活化的标志^[15]。

如图所示: 具有转录活性的DNA,在甲基化后与MBD(methyl-binding domain )蛋白如MBD2、MeCP2结合, 而该蛋白上连着的组蛋白脱乙酰基酶(HDAC1和2)使组蛋白脱乙酰化,导致染色质结构变化,转录抑制.( MTA2, metastasis-associated protein 2; RbAp46/48, retinoblastoma-associated protein 46/48;RNA pol II, RNA polymerase II; SAP18/30, Sin3-associated polypeptides 18/30)^[16] 
 

甲基化与肿瘤 
 

   现在的研究认为DNA甲基化与肿瘤密切相关。肿瘤的DNA甲基化改变表现为总体的甲基化水平降低与启动子区CpG岛的甲基化水平升高。抑癌基因与修复基因的甲基化导致抑癌基因沉默与修复基因失活,造成肿瘤抑制丧失与基因损伤增加; 而总体地低甲基化使反转录转座子、癌基因活化，使染色体不稳定。 
 

   高甲基化

   在肿瘤中，常见的被甲基化的抑癌基因与修复基因列于表1。它们与细胞周期调控（如p16INK4a, p15INK4a, Rb, p14ARF)、 DNA修复(BRCA1, MGMT)、细胞凋亡(DAPK, TMS1)、抗药性、分化、血管生成与转移等相关联。 
 

表1 常见的被甲基化的抑癌基因与修复基因及其作用

   P16基因: p16基因5’-CpG岛甲基化已被证实并且与多种肿瘤中p16的转录抑制有密切的关系。而且,用5-aza-dC干预甲基化的细胞系可导致启动子区域甲基化水平的严重下降而使p16 基因重新表达和随后的G1/S细胞周期循环阻滞^[44] 。Boltze等^[45]发现甲状腺肿瘤中p16 基因的突变或等位基因缺失并不多见,并认为p16 基因启动子甲基化是p16 在甲状腺肿瘤发生过程中失活的机制。采用MSP (methylation-specific PCR)检测77例甲状腺肿瘤和15例正常甲状腺组织中p16 启动子区的甲基化状态,结果显示高甲基化发生在13%的正常组织、33%的滤泡状腺瘤、44%的乳头状癌(papillary thyroid cancer, PTC)、50%的滤泡状癌( follicular thyroid cancer, FTC)、75%的差分化癌、85%的未分化癌中。同时伴随着启动子的高甲基化p16蛋白表达缺失。上述结果提示p16启动子区的高甲基化是甲状腺肿瘤发生中的早期事件并与肿瘤的进展和分化有关。

  RASSF1A 基因: RASSF1A 基因定位于3p21.3,是一种新的肿瘤抑制基因,与多种人类肿瘤有关。RASSF1A基因是继p16基因以来所发现的在肿瘤中甲基化程度最高、最广泛的基因之一,大量研究表明RASSF1A 表达缺失和启动子区高甲基化有着广泛的肿瘤谱,故该基因有望在多种肿瘤的早期诊断、预后判断中发挥重大作用。研究表明甲状腺癌中RASSF1A 启动子CpG岛区在9个甲状腺癌细胞系中完全甲基化,其基因表达缺失,经去甲基化处理后其基因表达恢复^[46] 。在38例原发性甲状腺癌中, 71%的样本存在RASSF1ACpG岛的高甲基化。Xing等^[47]用适时定量MSP研究了包括良性腺瘤在内的各种甲状腺肿瘤中RASSF1A 的甲基化状况。结果发现, RASSF1A 的异常甲基化在早期的良性腺瘤即是一个多发事件,而在癌中甲基化水平更显著。RASSF1A 启动子区CpG岛的高甲基化能致RASSF1A 基因的转录失活,这种表遗传的失活是甲状腺肿瘤发生中的一个早期步骤,在甲状腺肿瘤的恶性发展中可能发挥着重大的作用。

    有些基因，在肿瘤中，普遍被甲基化，如p16、RASSF1A。而有的基因只在特定的肿瘤中被甲基化，如GSTP1，在90%前列腺癌中高甲基化，相反，在急性髓细胞样白血病中大多没有甲基化^[30，36]。目前，研究得最详细的肿瘤是肺癌，发现其40多种基因存在一定程度的DNA甲基化改变，而其中高甲基化的是RARb、RASSF1A、CDNK2A、CHD13和 APC^.[48] 
 

   低甲基化

   低甲基化的缺陷在恶性肿瘤（malignancies）中广泛存在^[49,50]。在实体瘤如转移的肝细胞癌^[51]、在颈癌^[50], 前列腺癌^[52]以及在恶性血液病如B-细胞慢性淋巴细胞性白血病^[34]中都很普遍。总体的低甲基化在许多癌症中观察到，如乳腺癌、颈癌、脑瘤等。并且，其程度与恶性程度有正比关系^[53]。癌细胞基因组低甲基化主要发生在卫星系列、重复系列、中心粒区域以及原癌基因系列^[54,55].

   03年，美国《science》杂志同期刊登的三篇文章^{[ 56, 57, 58 ]}已经肯定了DNA 的总体低甲基化导致的基因不稳定性对肿瘤的发生起着构成原因的作用。在老鼠模型中，目前已经证明从胚胎到成体发育过程中，利用DNA甲基化转移酶( DNMT )的突变导致染色体去甲基化，可以引起老鼠基因组不稳定和导致淋巴瘤^[56,57]。

   低甲基化致癌的机制有三种：u低甲基化促进了有丝分裂的重组，导致杂合性丢失（LOH）和核型重排。中心粒系列去甲基化使染色体非整倍体化^[59]。v转座元件的再活化，如本来出于沉默状态的LINES核Alu等重复序列由于去甲基化而活跃起来，可能移动到基因组其它位置，破坏基因的功能^[60]。w 基因组印记的丢失^[61]。印记丢失可能导致某些与细胞增殖和转化相关基因过表达，而与细胞分化和凋亡相关基因的表达受阻。

值得提出是：人们绝对相信低甲基化可直接激活癌基因。实验中也的确观察到癌基因的低甲基化，如 H-ras ^[62] 和 c-myc ^[63]。但并没有找到它们的低甲基化与转录增加的相关性。是技术性问题，还是在本质上它们的低甲基化就与转录没有关系？

肿瘤甲基化改变的原因 
 

  是增加了甲基化酶的表达吗？

  一谈到甲基化的调控因素，人们首先想到的是甲基化酶。Vertino ^{[ 64]}发现在immortalized human fibroblasts中DNMT1高表达，并使 CpG 岛甲基化增加。在其它不同的肿瘤中也同样观察DNMT1的高表达^{[65，66，67，68]}。但随后的研究表明，DNMT1增加的水平与肿瘤标志物相比却是下降了^{[69，70，71]}。这提示DNMT1水平的升高是肿瘤细胞增殖的结果。DNMT1是甲基化维持酶。肿瘤细胞有较高的甲基化水平。随着肿瘤细胞数量的增加，DNMT1应该具有较高的水平才能维持肿瘤细胞的甲基化水平。更令人不解的是，当Rhee, I等^[72]灭活了DNMT1gene后，发现对HCT116结肠癌细胞的甲基化水平一点影响也没有，也不能使高甲基化的p16等抑癌基因去甲基化。这些研究结果，使人们难以相信DNMT1在肿瘤的甲基化改变中发挥着重要作用。

  至于DNMT3a与DNMT3b，报道是相矛盾的，有的说观察到了他们的升高^[73，74]，有的说没有^[70，71]。目前看来，甲基化酶对肿瘤甲基化异常的直接作用只能打上“？”了。 
 

  是p21^waf1的作用吗？

   在这个故事里，人们提出了p21^waf1占位的假说：肿瘤中出现的总体低甲基化与CpG岛高甲基化是由于DNMT1锚定目标进行甲基化的位置被其它物件所占据，使之不能进行甲基化，导致整个基因的甲基化水平降低。而自由的、没事干的DNMT1错误地将CpG岛上的C甲基化了,导致了CpG岛高甲基化. 该假设的根据是:uDNMT1与p21^waf1(周期素依赖性蛋白激酶抑制剂)在PCNA(proliferating cell nuclear antigen)上有相同的结合域^[75],而DNMT1与PCNA的结合对于DNMT1锚定复制复合体非常重要;v来自p21^waf1的小肽的确对DNMT1-PCNA复合体有很强的抑制作用^[75];w在正常细胞中, DNMT1与p21^waf1的表达是互相排斥的, 而在肿瘤细胞中两者能同时表达^[76];x p21^waf1在乳腺癌、肺癌、卵巢癌及肝细胞癌的早期高表达^{[77, 78, 79, 80]}. 
 

  其它因素

对肿瘤甲基化异常的解释假说还很多，如大复合体学说、组蛋白的乙酰化与染色质结构异常等。其中染色质结构异常比较有力。在正常细胞中，散在的C是被甲基化的，而CpG岛没有被甲基化.这本身说明了是CpG岛的整体结构抑制了对它的甲基化,是它的结构特点或是与它结合的蛋白抑制了甲基化酶对它的甲基化.当某种因素导致它的结构特点以及与它连接的那个蛋白结构改变时,它便被甲基化.可能是CpG岛被甲基化后的结构更易被甲基化导致CpG岛高甲基化.事实上, CpG岛上C含量高,自然是高甲基化. 
 

问题与展望

   DNA的甲基化涉及基因的“开”与“关”，因此意义重大。但对DNA甲基化我们还知之太少，可以说DNA甲基化研究才刚刚开始，许多急待解决：

u怎样提高甲基化检测的灵敏与准确度？

v正常组织与肿瘤组织的甲基化精细“图谱”是怎样的？

w甲基化的选择性

x甲基化的调控

高效的甲基化治疗

    解决这些问题的难度不小，但相信通过广大科研工作者的共同努力一定会迎来甲基化的完全理解与控制。为人民卫生服务。