DNA甲基化與腫瘤

1、DNA甲基化與腫瘤張丕顯（05級(jí)博士研究生）摘要：本文綜述了DNA甲基化的研究進(jìn)展。哺乳動(dòng)物DNA甲基化主要發(fā)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變（C®T）與基因沉默?；虻募谆聊瑱C(jī)制有兩種：由于啟動(dòng)子區(qū)的甲基化導(dǎo)致啟動(dòng)子區(qū)結(jié)構(gòu)改變，啟動(dòng)困難；甲基化引起組蛋白脫乙?；氯旧|(zhì)結(jié)構(gòu)改變，關(guān)閉基因。目前，甲基化檢測(cè)常用甲基化特異的PCR（MSP），檢出限可達(dá)0.1%。 腫瘤中普遍存在DNA甲基化狀態(tài)的改變。表現(xiàn)為總體的甲基化水平降低與局部的甲基化水平升高。表現(xiàn)為抑癌基因與修復(fù)基因的高甲基化與反轉(zhuǎn)錄轉(zhuǎn)座子、癌基因的去甲基化。造成腫瘤甲基化

2、改變的原因可能與甲基轉(zhuǎn)移酶、p21WAF1及染色質(zhì)結(jié)構(gòu)改變有關(guān),而甲基轉(zhuǎn)移酶的調(diào)控機(jī)制尚不清楚.關(guān)鍵詞: DNA, 甲基化, 腫瘤引子人類對(duì)基因本質(zhì)的認(rèn)識(shí)逐步深入, 目前正經(jīng)歷著更全面的認(rèn)知過程。早期遺傳學(xué)家認(rèn)為, 基因是一個(gè)遺傳的功能單位, 決定某種遺傳性狀, 它們?cè)谌旧w上占有一定的位置, 并可發(fā)生突變和交換;隨著DNA 作為蛋白質(zhì)遺傳密碼載體的發(fā)現(xiàn), 分子遺傳學(xué)和遺傳工程技術(shù)的迅速發(fā)展, 遺傳學(xué)界基本上接受了下述定義: 基因是編碼一條多肽鏈的特定DNA 片段。近幾年來隨著學(xué)科的進(jìn)展, 一些研究者對(duì)上述看法提出了質(zhì)疑13。人類基因組計(jì)劃中DNA 測(cè)序工作的基本完成, 只確定了3 萬多個(gè)基因

3、, 僅是果蠅的2 倍多, 很難想象DNA 含有充分的遺傳信息以調(diào)控人類如此復(fù)雜有機(jī)體發(fā)育和生存的全過程4。實(shí)際上在人類細(xì)胞中只有數(shù)千個(gè)基因有活性, 因此維持細(xì)胞的正常功能, 決定什么樣的一組基因有功能, 而另一組基因無功能, 都是十分重要的。如果出錯(cuò)就會(huì)引起嚴(yán)重的后果, 據(jù)估計(jì)至少3 個(gè)基因錯(cuò)誤表達(dá)就能誘發(fā)正常細(xì)胞癌變2。這樣在人類基因組含有兩類遺傳學(xué)信息, 傳統(tǒng)意義上的遺傳學(xué)信息提供了生命所必需的蛋白質(zhì)的模板; 而表遺傳學(xué)的信息提供了何時(shí)、何地和何種方式應(yīng)用這些遺傳學(xué)信息的指令4。表遺傳學(xué)1942 年由Waddington 首先提出, 研究基因型產(chǎn)生表型的過程, 此后Holliday進(jìn)行了一

4、系列的探討5。目前認(rèn)為, 表遺傳學(xué)是研究沒有DNA 序列變化、可遺傳的基因表達(dá)(活性)的改變。還有研究者從不同角度進(jìn)行描述, 例如相對(duì)于DNA 序列質(zhì)的改變的遺傳學(xué)研究, 表遺傳學(xué)被定義為研究基因表達(dá)水平(量變) 信息的遺傳。也有研究者從遺傳學(xué)角度, 把表遺傳學(xué)定義為非孟德爾遺傳, 或沒有DNA 序列改變的核遺傳。2003年10月，人類表觀基因組協(xié)會(huì)（Human Epigenome Con-sortium,HEC）宣布開始投資實(shí)施人類表觀基因組計(jì)劃(Human Epigenome Project,HEP),標(biāo)志著生命科學(xué)的研究已悄然進(jìn)入后基因(表基因)時(shí)代.而甲基化是表遺傳學(xué)作用的主要形式.D

5、NA甲基化DNA 甲基化是指生物體在DNA 甲基轉(zhuǎn)移酶(DNA methyltransferase ,DMT) 的催化下,以s-腺苷甲硫氨酸(SAM) 為甲基供體,將甲基轉(zhuǎn)移到特定的堿基上的過程。DNA甲基化可以發(fā)生在腺嘌呤的N -6位、胞嘧啶的N -4位、鳥嘌呤的N -7位或胞嘧啶的C-5位等8。但在哺乳動(dòng)物,DNA甲基化主要發(fā)生在5-CpG-3的C上.生成5-甲基胞嘧啶(5mC) 9.反應(yīng)如下:圖1 DNA甲基化人類的Cp G以兩種形式存在,一種是分散于DNA 中,另一種是CpG結(jié)構(gòu)高度聚集的CpG島。在正常組織里,70 %90 %的散在的CpG是被甲基修飾的,而CpG島則是非甲基化的10

6、 。DNA甲基化分析的方法分析DNA甲基化的位點(diǎn)與程度的實(shí)驗(yàn)方法有兩類：一類（M SREs）利用對(duì)甲基化堿基敏感的限制性內(nèi)切酶。該酶不能切割甲基化的堿基位點(diǎn)，從而產(chǎn)生片段差異，電泳后，根據(jù)片段與量的差異找到甲基化位點(diǎn)與甲基化程度6；另一類是利用將沒有甲基化的C變?yōu)槠渌鼔A基或其它物質(zhì)，而甲基化的C不會(huì)發(fā)生相應(yīng)變化來識(shí)別甲基化位點(diǎn)。甲基化特異的PCR (M ethylation-specific PCR,MSP)7是較好的常用的方法。該法用 HSO3-處理單鏈DNA，使所有未甲基化的C脫氨轉(zhuǎn)變?yōu)閁,而甲基化的C則保持不變。然后經(jīng)特異性擴(kuò)增放大，測(cè)序。C位點(diǎn)就是甲基化位點(diǎn)，C的量即甲基化量。該法靈敏

7、度高, 可檢出比例為千分之一的甲基化等位片段,且對(duì)DNA 的質(zhì)和量要求也低, 能用于微量的DNA 或石臘包埋組織DNA 的甲基化分析。 甲基化酶 目前, 在真核生物中發(fā)現(xiàn)了3 類DNA 甲基轉(zhuǎn)移酶(Dnmt1、Dnmt2、Dnmt3a、Dnmt 3b): Dnmt1 主要是維持DNA 的甲基化; Dnmt2 可與DNA上特異位點(diǎn)結(jié)合, 但具體作用尚不清楚; Dnmt3 主要是參與DNA 的從頭甲基化。Dnmt3b 基因的RNA和蛋白在腫瘤組織中明顯的高表達(dá), 而Dnmt1 和Dnmt3a 在腫瘤組織中僅適度過表達(dá)。對(duì)10 例腫瘤組織中上述基因的表達(dá)分析發(fā)現(xiàn), 5 個(gè)樣本中Dnm t3a 高表達(dá)

8、; 6 個(gè)樣本中Dnmt1 高表達(dá); 8 個(gè)樣本中Dnmt3b 高表達(dá) 11 。甲基化的作用1基因C T突變DNA 甲基化引起基因突變的機(jī)制主要是由于DMT催化反應(yīng)形成。DMT可以加快C(胞嘧啶) 和5mC 脫氨,封閉U(尿嘧啶) 的修復(fù),并且使U T 改變,故DMT 促使CpG序列的C T突變12 。圖2 C®T突變抑癌基因p53就是一個(gè)典型的例證。50% 實(shí)體瘤病人出現(xiàn)p53基因突變。突變中24% 是CpG 甲基化后脫氨引起的CT 突變。2影響基因錯(cuò)配修復(fù)DNA 錯(cuò)配修復(fù)系統(tǒng)(DNAmismatch repair system ,MMR) 是指存在人類細(xì)胞中的一種修復(fù)DNA 堿基

9、錯(cuò)配的安全保障體系,它是由一系列特異修復(fù)DNA堿基錯(cuò)配的酶分子組成。Ahujia 等13 研究發(fā)現(xiàn)MMR 缺陷時(shí),CpG島的甲基化增強(qiáng),并認(rèn)為MMR 與DNA 甲基化有關(guān)。在基因錯(cuò)配修復(fù)過程中甲基化具有導(dǎo)向識(shí)別作用,而在錯(cuò)配修復(fù)基因表達(dá)缺陷的原因中基因突變和基因啟動(dòng)子區(qū)的高甲基化是其主要原因14 。3基因沉默目前認(rèn)為,甲基化影響基因表達(dá)的機(jī)制有下列幾種: 直接作用?；虻募谆淖兞嘶虻臉?gòu)型,影響DNA特異順序與轉(zhuǎn)錄因子的結(jié)合,使基因不能轉(zhuǎn)錄; 間接作用?；?端調(diào)控序列甲基化后與核內(nèi)甲基化CG序列結(jié)合蛋白(methyl CG-binding p rotein)結(jié)合,阻止了轉(zhuǎn)錄因子與基因形成

10、轉(zhuǎn)錄復(fù)合物; DNA去甲基化為基因的表達(dá)創(chuàng)造了一個(gè)良好的染色質(zhì)環(huán)境。DNA去甲基化常與DNase I高敏感區(qū)同時(shí)出現(xiàn),后者為基因活化的標(biāo)志15。如圖所示: 具有轉(zhuǎn)錄活性的DNA,在甲基化后與MBD(methyl-binding domain )蛋白如MBD2、MeCP2結(jié)合, 而該蛋白上連著的組蛋白脫乙?；?HDAC1和2)使組蛋白脫乙?；?導(dǎo)致染色質(zhì)結(jié)構(gòu)變化,轉(zhuǎn)錄抑制.( MTA2, metastasis-associated protein 2; RbAp46/48, retinoblastoma-associated protein 46/48;RNA pol II, RNA poly

11、merase II; SAP18/30, Sin3-associated polypeptides 18/30)16甲基化與腫瘤現(xiàn)在的研究認(rèn)為DNA甲基化與腫瘤密切相關(guān)。腫瘤的DNA甲基化改變表現(xiàn)為總體的甲基化水平降低與啟動(dòng)子區(qū)CpG島的甲基化水平升高。抑癌基因與修復(fù)基因的甲基化導(dǎo)致抑癌基因沉默與修復(fù)基因失活,造成腫瘤抑制喪失與基因損傷增加; 而總體地低甲基化使反轉(zhuǎn)錄轉(zhuǎn)座子、癌基因活化，使染色體不穩(wěn)定。高甲基化在腫瘤中，常見的被甲基化的抑癌基因與修復(fù)基因列于表1。它們與細(xì)胞周期調(diào)控（如p16INK4a, p15INK4a, Rb, p14ARF)、 DNA修復(fù)(BRCA1, MGMT)、細(xì)胞凋

12、亡(DAPK, TMS1)、抗藥性、分化、血管生成與轉(zhuǎn)移等相關(guān)聯(lián)。表1 常見的被甲基化的抑癌基因與修復(fù)基因及其作用基因基因沉默對(duì)腫瘤的意義腫瘤類型APC對(duì)細(xì)胞增殖、遷移、粘附、骨架重組及染色質(zhì)穩(wěn)定性失去調(diào)節(jié)作用乳腺癌17、肺癌18、食管癌、結(jié)腸癌、胃癌、胰、 肝癌BRCA1與DNA 修復(fù)與轉(zhuǎn)錄激活有關(guān)乳腺癌19、卵巢癌20 CDKN2A/p16周期素依賴性蛋白激酶抑制劑GIT 21、頭與頸部瘤22、NHL23、肺癌 21DAPK1鈣/鈣調(diào)素-依賴的絲氨酸/蘇氨酸磷酸化酶; 凋亡抑制肺癌24E-cadherin增強(qiáng)增殖、侵襲與轉(zhuǎn)移乳腺癌 25、甲狀腺癌26、胃癌27ER激素抵抗乳腺癌28、前列腺

13、癌29GSTP1失去對(duì)致癌物活性代謝產(chǎn)物的解毒作用前列腺癌30、乳腺癌31、腎癌31hMLH1缺損DNA錯(cuò)配修復(fù),基因點(diǎn)突變結(jié)腸癌32、胃癌27、子宮內(nèi)膜瘤33、卵巢癌34MGMTp53-相關(guān)基因，與DNA 修復(fù)及耐藥性有關(guān)肺癌24、腦瘤35P15細(xì)胞的過度激活與增殖非白血性白血病36、淋巴瘤37, 38、鱗狀細(xì)胞癌、肺癌 RASSF1A失去了對(duì)G1/S負(fù)調(diào)控抑制作用肺癌39、乳腺癌39、卵巢癌39、腎癌40、鼻咽癌41Rb不能抑制DNA復(fù)制和細(xì)胞分裂必需的基因轉(zhuǎn)錄成視網(wǎng)膜細(xì)胞瘤42、少突神經(jīng)膠質(zhì)(細(xì)胞)瘤43VHL錯(cuò)誤的降解RNA結(jié)合蛋白質(zhì)，改變RNA穩(wěn)定性腎細(xì)胞癌40縮寫: APC, ad

14、enomatous polyposis coli; BRCA1, breast cancer 1; CDKN2A/p16, cyclin-dependent kinase 2A; DAPK1, death-associated protein kinase 1; ER, estrogen receptor; GSTP1, glutathione S-transferase Pi 1; hMLH1, Mut L homologue 1; MGMT, O-6 methylguanine-DNA methyltransferase; RASSF1A, Ras association domain f

15、amily member 1; Rb, retinoblastoma; VHL, von Hippel-Lindau; GIT, gastrointestinal tract; NHL, non-Hodgkins lymphoma.P16基因: p16基因5-CpG島甲基化已被證實(shí)并且與多種腫瘤中p16的轉(zhuǎn)錄抑制有密切的關(guān)系。而且,用5-aza-dC干預(yù)甲基化的細(xì)胞系可導(dǎo)致啟動(dòng)子區(qū)域甲基化水平的嚴(yán)重下降而使p16 基因重新表達(dá)和隨后的G1/S細(xì)胞周期循環(huán)阻滯44 。Boltze等45發(fā)現(xiàn)甲狀腺腫瘤中p16 基因的突變或等位基因缺失并不多見,并認(rèn)為p16 基因啟動(dòng)子甲基化是p16 在甲狀腺腫瘤發(fā)

16、生過程中失活的機(jī)制。采用MSP (methylation-specific PCR)檢測(cè)77例甲狀腺腫瘤和15例正常甲狀腺組織中p16 啟動(dòng)子區(qū)的甲基化狀態(tài),結(jié)果顯示高甲基化發(fā)生在13%的正常組織、33%的濾泡狀腺瘤、44%的乳頭狀癌(papillary thyroid cancer, PTC)、50%的濾泡狀癌( follicular thyroid cancer, FTC)、75%的差分化癌、85%的未分化癌中。同時(shí)伴隨著啟動(dòng)子的高甲基化p16蛋白表達(dá)缺失。上述結(jié)果提示p16啟動(dòng)子區(qū)的高甲基化是甲狀腺腫瘤發(fā)生中的早期事件并與腫瘤的進(jìn)展和分化有關(guān)。RASSF1A 基因: RASSF1A 基因

17、定位于3p21.3,是一種新的腫瘤抑制基因,與多種人類腫瘤有關(guān)。RASSF1A基因是繼p16基因以來所發(fā)現(xiàn)的在腫瘤中甲基化程度最高、最廣泛的基因之一,大量研究表明RASSF1A 表達(dá)缺失和啟動(dòng)子區(qū)高甲基化有著廣泛的腫瘤譜,故該基因有望在多種腫瘤的早期診斷、預(yù)后判斷中發(fā)揮重大作用。研究表明甲狀腺癌中RASSF1A 啟動(dòng)子CpG島區(qū)在9個(gè)甲狀腺癌細(xì)胞系中完全甲基化,其基因表達(dá)缺失,經(jīng)去甲基化處理后其基因表達(dá)恢復(fù)46 。在38例原發(fā)性甲狀腺癌中, 71%的樣本存在RASSF1ACpG島的高甲基化。Xing等47用適時(shí)定量MSP研究了包括良性腺瘤在內(nèi)的各種甲狀腺腫瘤中RASSF1A 的甲基化狀況。結(jié)果

18、發(fā)現(xiàn), RASSF1A 的異常甲基化在早期的良性腺瘤即是一個(gè)多發(fā)事件,而在癌中甲基化水平更顯著。RASSF1A 啟動(dòng)子區(qū)CpG島的高甲基化能致RASSF1A 基因的轉(zhuǎn)錄失活,這種表遺傳的失活是甲狀腺腫瘤發(fā)生中的一個(gè)早期步驟,在甲狀腺腫瘤的惡性發(fā)展中可能發(fā)揮著重大的作用。 有些基因，在腫瘤中，普遍被甲基化，如p16、RASSF1A。而有的基因只在特定的腫瘤中被甲基化，如GSTP1，在90%前列腺癌中高甲基化，相反，在急性髓細(xì)胞樣白血病中大多沒有甲基化30，36。目前，研究得最詳細(xì)的腫瘤是肺癌，發(fā)現(xiàn)其40多種基因存在一定程度的DNA甲基化改變，而其中高甲基化的是RARb、RASSF1A、CDNK2

19、A、CHD13和 APC.48低甲基化低甲基化的缺陷在惡性腫瘤（malignancies）中廣泛存在49,50。在實(shí)體瘤如轉(zhuǎn)移的肝細(xì)胞癌51、在頸癌50, 前列腺癌52以及在惡性血液病如B-細(xì)胞慢性淋巴細(xì)胞性白血病34中都很普遍?？傮w的低甲基化在許多癌癥中觀察到，如乳腺癌、頸癌、腦瘤等。并且，其程度與惡性程度有正比關(guān)系53。癌細(xì)胞基因組低甲基化主要發(fā)生在衛(wèi)星系列、重復(fù)系列、中心粒區(qū)域以及原癌基因系列54,55.03年，美國(guó)science雜志同期刊登的三篇文章 56, 57, 58 已經(jīng)肯定了DNA 的總體低甲基化導(dǎo)致的基因不穩(wěn)定性對(duì)腫瘤的發(fā)生起著構(gòu)成原因的作用。在老鼠模型中，目前已經(jīng)證明從胚胎

20、到成體發(fā)育過程中，利用DNA甲基化轉(zhuǎn)移酶( DNMT )的突變導(dǎo)致染色體去甲基化，可以引起老鼠基因組不穩(wěn)定和導(dǎo)致淋巴瘤56,57。低甲基化致癌的機(jī)制有三種：u低甲基化促進(jìn)了有絲分裂的重組，導(dǎo)致雜合性丟失（LOH）和核型重排。中心粒系列去甲基化使染色體非整倍體化59。v轉(zhuǎn)座元件的再活化，如本來出于沉默狀態(tài)的LINES核Alu等重復(fù)序列由于去甲基化而活躍起來，可能移動(dòng)到基因組其它位置，破壞基因的功能60。w 基因組印記的丟失61。印記丟失可能導(dǎo)致某些與細(xì)胞增殖和轉(zhuǎn)化相關(guān)基因過表達(dá)，而與細(xì)胞分化和凋亡相關(guān)基因的表達(dá)受阻。值得提出是：人們絕對(duì)相信低甲基化可直接激活癌基因。實(shí)驗(yàn)中也的確觀察到癌基因的低甲

21、基化，如 H-ras 62 和 c-myc 63。但并沒有找到它們的低甲基化與轉(zhuǎn)錄增加的相關(guān)性。是技術(shù)性問題，還是在本質(zhì)上它們的低甲基化就與轉(zhuǎn)錄沒有關(guān)系？ 腫瘤甲基化改變的原因是增加了甲基化酶的表達(dá)嗎？一談到甲基化的調(diào)控因素，人們首先想到的是甲基化酶。Vertino 64發(fā)現(xiàn)在immortalized human fibroblasts中DNMT1高表達(dá)，并使 CpG 島甲基化增加。在其它不同的腫瘤中也同樣觀察DNMT1的高表達(dá)65，66，67，68。但隨后的研究表明，DNMT1增加的水平與腫瘤標(biāo)志物相比卻是下降了69，70，71。這提示DNMT1水平的升高是腫瘤細(xì)胞增殖的結(jié)果。DNMT1是甲

22、基化維持酶。腫瘤細(xì)胞有較高的甲基化水平。隨著腫瘤細(xì)胞數(shù)量的增加，DNMT1應(yīng)該具有較高的水平才能維持腫瘤細(xì)胞的甲基化水平。更令人不解的是，當(dāng)Rhee, I等72滅活了DNMT1gene后，發(fā)現(xiàn)對(duì)HCT116結(jié)腸癌細(xì)胞的甲基化水平一點(diǎn)影響也沒有，也不能使高甲基化的p16等抑癌基因去甲基化。這些研究結(jié)果，使人們難以相信DNMT1在腫瘤的甲基化改變中發(fā)揮著重要作用。至于DNMT3a與DNMT3b，報(bào)道是相矛盾的，有的說觀察到了他們的升高73，74，有的說沒有70，71。目前看來，甲基化酶對(duì)腫瘤甲基化異常的直接作用只能打上“？”了。是p21waf1的作用嗎？在這個(gè)故事里，人們提出了p21waf1占位的

23、假說：腫瘤中出現(xiàn)的總體低甲基化與CpG島高甲基化是由于DNMT1錨定目標(biāo)進(jìn)行甲基化的位置被其它物件所占據(jù)，使之不能進(jìn)行甲基化，導(dǎo)致整個(gè)基因的甲基化水平降低。而自由的、沒事干的DNMT1錯(cuò)誤地將CpG島上的C甲基化了,導(dǎo)致了CpG島高甲基化. 該假設(shè)的根據(jù)是:uDNMT1與p21waf1(周期素依賴性蛋白激酶抑制劑)在PCNA(proliferating cell nuclear antigen)上有相同的結(jié)合域75,而DNMT1與PCNA的結(jié)合對(duì)于DNMT1錨定復(fù)制復(fù)合體非常重要;v來自p21waf1的小肽的確對(duì)DNMT1-PCNA復(fù)合體有很強(qiáng)的抑制作用75;w在正常細(xì)胞中, DNMT1與p2

24、1waf1的表達(dá)是互相排斥的, 而在腫瘤細(xì)胞中兩者能同時(shí)表達(dá)76;x p21waf1在乳腺癌、肺癌、卵巢癌及肝細(xì)胞癌的早期高表達(dá)77, 78, 79, 80. 其它因素對(duì)腫瘤甲基化異常的解釋假說還很多，如大復(fù)合體學(xué)說、組蛋白的乙?；c染色質(zhì)結(jié)構(gòu)異常等。其中染色質(zhì)結(jié)構(gòu)異常比較有力。在正常細(xì)胞中，散在的C是被甲基化的，而CpG島沒有被甲基化.這本身說明了是CpG島的整體結(jié)構(gòu)抑制了對(duì)它的甲基化,是它的結(jié)構(gòu)特點(diǎn)或是與它結(jié)合的蛋白抑制了甲基化酶對(duì)它的甲基化.當(dāng)某種因素導(dǎo)致它的結(jié)構(gòu)特點(diǎn)以及與它連接的那個(gè)蛋白結(jié)構(gòu)改變時(shí),它便被甲基化.可能是CpG島被甲基化后的結(jié)構(gòu)更易被甲基化導(dǎo)致CpG島高甲基化.事實(shí)上,

25、CpG島上C含量高,自然是高甲基化.問題與展望 DNA的甲基化涉及基因的“開”與“關(guān)”，因此意義重大。但對(duì)DNA甲基化我們還知之太少，可以說DNA甲基化研究才剛剛開始，許多急待解決：u怎樣提高甲基化檢測(cè)的靈敏與準(zhǔn)確度？v正常組織與腫瘤組織的甲基化精細(xì)“圖譜”是怎樣的？w甲基化的選擇性x甲基化的調(diào)控高效的甲基化治療解決這些問題的難度不小，但相信通過廣大科研工作者的共同努力一定會(huì)迎來甲基化的完全理解與控制。為人民衛(wèi)生服務(wù)。參考文獻(xiàn)1W u C et al . Genes, Genetics, and Epigenetics: A Correspondence.Science, 2001, 293

26、(5532) : 1103-1105.2Wolff A P. Chromatin remodeling: why it is important in cancer.Oncogene, 2001, 20 (24) : 2988-2990.3Pennisi E. Behind the Scenes of Gene Expression.Science, 2001 , 293 (24) : 1064-1067.4Robertson KD. DNA methylation, methyltransferases, and cancer. Oncogene, 2001, 20 (24) : 3135-

27、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 mo

28、lecular 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

29、 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 b

30、e 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.

31、5Holliday R. Mutation Res, 2001, 483 (Supp ll) : s36 周玉球.DNA甲基化分析技術(shù)的研究進(jìn)展.國(guó)外醫(yī)學(xué)遺傳學(xué)分冊(cè)2001,24(6):303-3087 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 pa

32、tterns 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

33、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 am

34、plification 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 in

35、herent 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 imp

36、ortant tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.8Ahmad 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 fibrob

37、lasts overexpressing DNA(cytosine- 5) - methyltransferase1Mol Cell Biol ,1996 ,16 (8) :4555Abstract: 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.

38、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

39、. 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 transfecte

40、d 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

41、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 cultu

42、re. 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 c

43、ontribute to tumor progression through CpG island methylation-mediated gene inactivation. 10Ehrlich M , Wang RY.H1 5 - methylcytosine in eukaryotic DNA1.Science,1981 ,212 :135011Keith D et al. Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G0/G1 to S

44、 phase transition in normal and tumor cells.Nucleic Acids Research, 2000; 10: 2108Abstract: 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 propose

45、d 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

46、 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 transforme

47、d 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 decr

48、ease 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 l

49、oss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.12Bender CM,Zingg JM,Jones PA. DNA methylation in bladder cancerJ . Pharm Aceutical Res,1998 ,15(2) :175.Abstract: DNA methylation is essential for normal embryonic development

50、. 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

51、 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 DN

52、A methylation.13Ahujia N. Aging and DNA methylation in colorectal mucosa and cancerJ . Cancer Res ,1997 ,58 (23) :3370-3374.14Wheeler JM,Beck NE , Kim HC , et al . Mechanisms of inactivation ofmismatch repair genes in human colorectal cancer cell lines : the predominant role of Hmlh1J . Proc Natl Ac

53、ad Sci USA ,1999 ,96 (18) :10296-10301.15 彭正良.甲狀腺腫瘤相關(guān)基因甲基化研究進(jìn)展.國(guó)外醫(yī)學(xué)·生理、病理科學(xué)與臨床分冊(cè)，2005，25（2）：126-12916 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 methyl

54、ation of the adenomatous polyposis coli (APC) gene promoter 1A in breastand lung carcinomas. Clin Cancer Res 7:1998-2004, 2001Abstract: 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 a

55、nd 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

56、 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

57、-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 SCL

58、C 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 a