CHFR基因DNA和组蛋白异常调控与喉癌发生机制研究
|
摘要
前言
目前研究表明,恶性肿瘤的发生与发展是一个多因素、多阶段的复杂过程,涉及多种基因的功能异常。引起基因表达异常的机制有两种：一为遗传学机制即DNA结构改变。二为表遗传学机制。表遗传学是研究非DNA序列变化引起的,可遗传的基因表达的改变。肿瘤发生中的主要表遗传学改变是肿瘤抑制基因发生DNA高甲基化和染色质中的组蛋白修饰。近几年来,表遗传学改变的研究取得了长足进展,也成为喉癌临床基础研究的前沿。
现在普遍认为DNA甲基化是除缺失与突变之外导致基因失活的第三种机制,在肿瘤的发生发展中起着不可忽视的作用。肿瘤甚至可以被认为是由DNA的突变和表观遗传学的改变共同导致的,而且表观遗传学的变化在肿瘤发生中早于DNA的突变。组蛋白的甲基化作为另一种表遗传学机制,可以改变染色体的状态,进而调节基因的转录,间接导致肿瘤的发生。H3-K9甲基化可以抑制基因表达；而H3-K4甲基化则具有激活效应。H3-K9的甲基化与DNA的甲基化在基因的沉默机制中具有协同作用,而H3-K4的甲基化拮抗DNA甲基化所产生的基因沉默,研究表明在DNA甲基化及组蛋白甲基化之间存在着相互关系,在肿瘤的发生发展中起着重要的作用。
喉癌是最常见的头颈部恶性肿瘤之一,其发病率在我国北方地区位于头颈部恶性肿瘤的第一位,在我国南方地区,仅次于鼻咽癌居于第二位；在世界范围内,居于全身恶性肿瘤的第十一位。其发病率有逐年上升的趋势,大约占新发恶性肿瘤的1%左右。近30年虽然外科技术,放化疗手段不同程度的得到提高,但是目前喉癌仍是仅有的两个五年生存率未增长的肿瘤之一。原因之一就是目前喉癌发病的分子学机制仍不清楚,早期诊断及治疗不理想。因此深入研究喉癌的发病机制,寻找早期诊断及早期治疗的新靶点非常重要。有研究表明检测特定基因甲基化状态有望为肿瘤的早期诊断提供帮助,但目前在喉癌中的研究刚刚起步,比较理想的靶基因非常少。另外一个原因可能是喉癌的治疗方面不理想,目前在我国喉癌的治疗主要是手术治疗和放射治疗,手术给患者带来很大的痛苦,远期疗效尤其是晚期患者不尽人意,从长远看,化疗联合治疗方案更能够保全器官和功能,化疗效果值得深入研究,目前去甲基化治疗已经成为全世界肿瘤研究的热点,为攻克肿瘤这一顽疾提供新的思路。
CHFR基因是一个新的有丝分裂前期检查点基因。在正常组织中广泛表达,位于纺锤体组装稽查点的上游。当存在有丝分裂应激时,CHFR通路激活,通过泛素化降解Plkl(Polo-like kinase 1),Plkl调控Cdc25和’Weel激酶的磷酸化,在G2期进入M期时控制Cdc2激酶的活性,而延迟染色体凝集,使细胞周期停滞于有丝分裂早期,纺锤体不能形成,直至错误被纠正。CHFR基因如发生改变不能阻断异常细胞通过G2期进入有丝分裂中前期,从而导致细胞的恶性增殖。已有研究发现,MAD,BUB等有丝分裂检查基因在多数肿瘤中很少失活,而CHFR在胃癌,肺癌,食道癌等恶性肿瘤中因启动子区甲基化表现为失活或低表达,与肿瘤的发生关系密切,因其具有肿瘤特异性,受到越来越多的人关注,而与喉癌发生的关系及DNA甲基化和组蛋白甲基化对其的调控目前国内外未见报道。
目的
通过探讨喉癌组织及Hep-2人喉癌细胞系中CHFR基因表达,启动子区甲基化以及组蛋白H3-K4,H3-K9甲基化,以及5-Aza-dC及TSA对基因表达和甲基化水平的影响,从而明确其与喉癌发生发展的关系,探讨喉癌的发生分子学机制及其早期诊断及治疗靶点。
材料与方法
一、实验对象
Hep-2人喉癌细胞系、50例喉癌患者肿瘤及15例正常粘膜。
二、实验方法
1、细胞培养
Hep-2人喉癌细胞按常规培养于含10%胎牛血清RPMI-1640的培养液,NaHCO2浓度2g／L,青霉素G浓度100U／L,链霉素浓度100μg／L,37℃含5%CO2的湿润空气的恒温密闭式培养箱培养。
2、实验分组
(1)Hep-2人喉癌细胞
对照组：同期培养不加药的Hep-2人喉癌细胞。
实验组：①5-Aza-dC组：加5umol/L 5-Aza-dC培养72小时；②TSA组：加TSA 300 nmol培养24小时；③5-Aza-dC及TSA组：加5umol/L 5-Aza-dC培养48小时后加TSA 300nmol继续培养24小时。
(2)喉癌组织标本
临床病例的收集：选择2008年1月-2009年1月中国医科大学附属盛京医院耳鼻喉科住院的喉鳞状细胞癌患者50例(喉癌组),男44例,女6例,年龄40-74岁,平均58±9.76岁。选取其中的15例喉全切除术标本中,距离肿瘤边缘>2.0cm的粘膜组织作为对照组。按照2002年UICC喉癌分期标准将本研究的喉癌标本进行分期：早期(Ⅰ期+Ⅱ期)22例(T1N0 5例T2N0 17例),声门上型12例,声门型10例。中晚期(Ⅲ期+Ⅳ期)28例(T2N1 1例,T2N2 1例,T3N0 9例,T3N18例,T3N2 4例,T4N0 7例),声门上型16例,声门型12例,所有喉癌组织均经病理证实为鳞癌。术前均未经放、化疗治疗。
3、引物设计
MSP、实时荧光定量PCR、CHIP引物序列和反应条件见表1-3,引物由TaKaRa公合成。
4、甲基化特异性PCR (MSP)法检测DNA甲基化水平
分别提取喉癌细胞系,喉癌组织中DNA。紫外分光光度仪定性、定量。亚硫酸氢钠修饰DNA, Wizard DNA Clean-up Systerm(Promega)纯化。离心沉淀DNA。采用甲基化特异性PCR方法。PCR反应条件：95℃12min后,95℃变性30s,56℃退火30s,72℃延伸30s,45个循环,72℃再延伸7min,琼脂糖凝胶电泳,EB染色。结果经Alpha Image 2000自动成像仪成像采集数据,分析电泳结果。实验重复三次,取其平均值进行统计分析。
5、实时荧光定量PCR (Realtime fluro-genetic quantitative PCR)法检测mRNA水平
用TRIzol试剂一步法分别提取喉癌细胞系,喉癌组织总RNA(按说明书进行)。按照反转录试剂盒说明将其反转录成cDNA第一链。无RNA酶的双蒸水将cDNA10倍稀释。进行荧光定量PCR扩增,由PCR反应曲线得到Ct值,以GAPDH为内参,采用2-ΔΔct方法进行相对定量。实验重复三次,取其平均值进行统计分析。
6、染色质免疫沉淀分析(chromatin immunoprecipitation assay,ChIP)检测组蛋白H3K9,H3K4甲基化水平
收集细胞,甲醛交联组蛋白和DNA。酶切后分别加H3-K9,H3-K4甲基化抗体,免疫血清或Tris-EDTA缓冲液。收集抗体／蛋白复合物,沉淀下来的染色质通过PCR扩增检测结果。结果经Alpha Image 2000自动成像仪成像采集数据,分析电泳结果。实验重复三次,取其平均值进行统计分析。
7、Western Blot法检测CHFR蛋白、H3总蛋白、H3K9甲基化蛋白、H3K4甲基化蛋白水平
提取蛋白,电泳,PVDF膜转膜,BSA封闭。分别加入CHFR, H3, H3K4甲基化、H3K9甲基化、actin兔单克隆抗体,然后加入单克隆二抗,成像,采集数据。采用条带密度值与相应actin密度值的比值作为指标进行比较,实验重复三次,取平均值进行分析。
三、统计学处理
应用SPSS17.0软件分析,mRNA表达水平定量测定结果用t检验,启动子甲基化结果用χ2检验(Fisher,精确概率法),mRNA的表达和临床病理参数的关系采用t检验,甲基化和临床病理参数的关系采用χ2检验。Western印迹的蛋白结果应用χ2检验统计,P<0.05,认为差异具有统计学意义。
结果
1、喉癌组织中CHFR基因启动子区甲基化情况
在喉癌组织中,正常对照组未发现CHFR基因启动子区甲基化,在喉癌组中CHFR基因启动子区甲基化率为22%(11／50),两者相比,差异显著,有统计学意义(P<0.01)。其中T1+T2期10例,T3期1例,T4期未发现甲基化,T1+T2期甲基化率(41.6%1／24)明显高于T3+T4期(3.8%1／26),甲基化程度在临床T分期方面具有统计学意义P<0.01(x2=7.50)。而在年龄、性别、分化程度、肿瘤大小、病理分化和有无淋巴结转移方面无统计学意义(P>0.05)。
2、喉癌组织中CHFR基因mRNA表达
CHFR基因在对照组mRNA全部表达。在喉癌组mRNA有2例表达缺失(4%),48例表达量明显下调,有统计学意义(P<0.01)。50例喉癌组织中,T1+T2期CHFR基因mRNA相对表达量0.29±0.18,T3+T4期相对表达量0.69±0.15,前者明显低于后者,两者差异有统计学意义(P<0.01),在不同的性别、年龄、肿瘤位置、分化程度和有无淋巴结转移方面CHFR基因mRNA水平没有统计学差异(P>0.05)。甲基化组CHFR基因mRNA表达量为0.16±0.13,非甲基化组mRNA表达量为0.65±0.17,前者明显低于后者,二者有密切关系。(P<0.05)。
3、在Hep-2人喉癌细胞系中5-Aza-dC和TSA处理前后CHFR基因CHFR启动子区甲基化情况
在Hep-2人喉癌细胞系中,CHFR启动子区表现为高甲基化,5-Aza-dC作用后DNA甲基化程度减弱,出现半甲基化,TSA作用后DNA甲基化程度无明显变化。联合使用5-Aza-dC+TSA后DNA甲基化程度明显减弱。
4、在Hep-2人喉癌细胞系中5-Aza-dC和TSA处理前后CHFR基因mRNA表达情况
同对照组比较,用5-Aza-dC作用后CHFR基因mRNA表达量上调(1.75±0.21)。用TSA作用后mRNA表达量(1.05±0.13)无明显变化。联合使用5-Aza-dC+TSA后mRNA表达量明显上调(2.15±0.18)
5、在Hep-2人喉癌细胞系中CHFR基因启动子区组蛋白H3-K4甲基化,组蛋白H3-K9甲基化情况及与DNA甲基化的关系
在Hep-2人喉癌细胞系中,CHFR基因启动子区组蛋白H3K9甲基化与DNA甲基化正相关,而组蛋白H3K4甲基化与DNA的甲基化负相关。基因启动子区组蛋白H3K9甲基化与DNA的甲基化在基因的表达下调机制中具有协同作用,而组蛋白H3K4甲基化拮抗DNA甲基化所产生的基因表达下调。
6、在Hep-2人喉癌细胞系中,5-Aza-dC和TSA处理前后的CHFR基因启动子区域组蛋白H3K9甲基化、H3K4甲基化状态分析
(1) 5-Aza-dC对启动子区域H3K9甲基化有明显作用,5-Aza-dC明显降低启动子区H3K9甲基化程度,TSA对启动子区甲基化无影响,联合给予5-Aza-dC和TSA与单独给予5-Aza-dC的作用相似。
(2)5-Aza-dC对启动子区域H3K4甲基化的作用与对启动子区域H3K9甲基化的作用恰好相反,5-Aza-dC明显提高启动子区H3K4甲基化程度,TSA对启动子区域H3K4甲基化无影响,联合给予5-Aza-dC和TSA与单独给予5-Aza-dC的作用相似。
7、Western Blot法分析喉癌组织中CHFR蛋白表达
喉癌组46.0%(23／50)的喉鳞状细胞癌未检测到CHFR蛋白表达；所有黏膜均有CHFR蛋白表达。β-actin蛋白在喉鳞癌和正常黏膜中表达水平相似。CHFR蛋白表达水平的差异有统计学意义(χ2=13.85；P<0.001)
8、Western Blot法分析5-Aza-dC和TSA处理前后Hep-2人喉癌细胞系中CHFR蛋白,H3蛋白,甲基化组蛋白H3-K9,甲基化组蛋白H3-K4表达
5-Aza-dC作用后,CHFR蛋白表达量能明显增加,TSA对CHFR蛋白表达无明显影响,联合给予5-Aza-dC和TSA与单独给予5-Aza-dC的作用相似。5-Aza-dC及TSA作用后甲基化H3K9蛋白,甲基化H3K4蛋白,总H3蛋白无明显变化。
结论
1、在喉癌组织中抑癌基因CHFR启动子区CpG岛过甲基化与mRNA和蛋白表达缺失或下调有关,可能与喉癌的发生发展有关,与临床T分期的关系可能更为密切,检测其甲基化状态可有助于喉癌早期诊断。
2、在Hep-2人喉癌细胞系中甲基转移酶抑制剂能够逆转抑癌基因CHFR启动子区甲基化状态,同时mRNA和蛋白表达上调,为临床治疗喉癌提供新思路。
3、在Hep-2人喉癌细胞系中抑癌基因CHFR基因启动子区组蛋白H3K9甲基化与DNA甲基化在其基因转录调控中呈正相关,而组蛋白H3K4甲基化与DNA的甲基化呈负相关。DNA甲基化与组蛋白H3-K9甲基化在其基因表达缺失或下调中起协调作用,而DNA甲基化与组蛋白H3-K4甲基化起拮抗作用。
4、在Hep-2人喉癌细胞系中甲基转移酶抑制剂能明显影响抑癌基因CHFR启动子区组蛋白H3K9甲基化和H3K4甲基化程度,而组蛋白脱乙酰基抑制剂对启动子区甲基化无明显影响。
Researches suggest that the tumorigenesis and development of malignant neoplasm is a multivariate, multistage and complicated process with the functional abnormality of multiple genes. The abnormal expression of genes includes genetic and epigenetic changes. The epigenetic change is heritable, which can not alter DNA sequence. DNA methylation and histone modification of tumor suppressor genes are main epigenetic changes. In recent years, the study of epigenetic changes has been developed sufficiently and become the clinical and investigative frontier of Laryngeal squamous cell carcinoma (LSCC). Recently, it has been discovored that the epigenetic alteration and DNA mutation are the same in tumorigenesis, in which the former is earlier than the latter.
It has been kowmn that aberrant methylation of DNA has been described as an alternative mechanism of gene inactivation besides mutation and deletion. As one of epigenetic mechanisms, histone methylation can alter the chromosome status and control the gene transcription, which can lead tumorigenesis. The methylation of histone H3 lysine 9 is associated with gene repression. But the methylation histone H3 lysine 4 is associated with active gene transcription. The methylation of DNA and H3 lysine 9 are both involved in genes silencing, in which the methylation of DNA and H3 lysine 9 are synergistic, but the methylation of histone H3 lysine 4 is antagonistic to them.It appears that DNA and histone methylation likely have a mutually reinforcing relationship, and play a vital role of the the tumorigenesis and development of tumors.
Laryngeal carcinoma is a common head and neck malignancy and the 11th common malignancy in the world, with a tendency towards an increasingly occurrence of new cases and deaths annually. It was shown that LSCC was one of the tumor with no increase of five-year survival rates. Therefore, it is essential to investigate the molecular mechanism of LSCC in order to identify the early diagnostic and therapeutic marker genes. Some studies have shown that the detection of the specific methylation status may be helpful to the early diagnosis of tumors. Therefore the further study of the effect on DNA and histone abnormal regulation of tumor suppressor genes plays an important role on the tumorigenesis of laryngeal carcinoma.
CHFR (checkpoint with forkhead and ring finger) is a new cell cycle mitotic stress checkpoint gene.Recently, it has been known that the abnormal CHFR involed in the primary carcinogens, and closely related to gastric cancer, lung cancer and esophagal cancer.Its encoding product is ubiquitin ligase of Plkl. Plkl regulates both the Wee 1 kinase and the Cdc25 phosphatase, which in turn control the Cdc2 kinase activity at the G2 to M transition. The CHFR gene can ubiquitinate and degenerate the Plkl, which prevents cells from entering prophase and metaphase. CHFR gene expresses in normal tissue while it is low expressed or inactivated in cancer, in which losses the ability to prevent abnormal cells proliferating from G2 to M phase, thus cells abnormal differentiation and proliferation occur.
In this study, we investigated the expression, the methylation of the promoter CpG islands and the methylation histone histone H3 lysine 9, histone H3 lysine 4 of CHFR in LSCC, and the effect on 5-Aza-dCand/orTSA to the Hep-2 cell line by MSP, Real-time PCR Western Blotting and CHIP. We explore the relationship between the level of expression and methylation of the CHFR and the tumorigenesis and development of LSCC. This investigation revealed a new pathway for the laryngeal tumorigenesis and early diagnosis and therapy.
Material and Methods
1. Subjects
50 speciments of LSCC.Hep-2 cell lines, derived from human LSCC.
2. Methods
The subjects were 50 patients with LSCC diagnosed and treated between January 2007 and January 2009 at Shengjing Hospital of China Medical University. All patients were diagnosed pathologically to be LSCC preoperatively and received no radiation and chemotherapy. We collected control mucosa from 15 patients under larngectomy and 2.0 cm away from the margin of tumor was considered as safe. The mucosa samples were histologically normal. Liquid nitrogen was used to snap-frozen all the samples and then stored in-80℃freezer. The collection of all the samples was informed patients. We extracted clinopathologic data (age, gender, primary site, T stage, clinical stage, differentiation and the nodal status) of patients were extracted from the patients' files. And the staging of the tumors was carried out according to UICC 2002 TNM classification.
(1) Cell culture
Hep-2 cell line was cultured in RPMI 1640 supplemented with 10% fetal bovine serum(Gibco), penicillin (100 IU/mL) and streptomycin (100 mg/mL), and incubated in a humidified incubator containing 50 mL/L CO2 at 37℃.
(2) Experimental teams
5-Aza-dC (5mmol/L) was used for 72h in the treatment. Culture medium containing 5-Aza-dC was exchanged every 24 h. TSA (300 nmol/L) was used for only 24 h in the treatment.5-Aza-dC was used for 48 h followed by TSA for an additional 24 h in the combined treatment.
(3) Primer design
The primer sequences and reaction conditions of MSP, Real-time PCR and CHIP were described in table 3. The primer synthesis were in TaKaRa company.
(4) mRNA extraction, expression analysis of CHFR using Real-time PCR
Total-RNA was extracted and purified from the LSCC specimens and cell line using an RNeasy mini-Kit (TAKARA) following the manufacturer's instructions. cDNA was synthesized with 1μg of total-RNA using a SuperScript II Reverse Transcriptase kit (TAKARA). The mRNA expression levels of CHFR in the laryngeal tissues were quantified by SYBR Green I real-time quantitative PCR. The relative expression was normalized to a human glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) endogenous reference standard relative to a calibrator and was evaluated by 2 -△△CT
(5) DNA extraction and methylation-specific PCR (MSP) analysis of CHFR
DNA was extracted from 65 laryngeal specimens using liquid-based cytology with a DNA Tissue Kit (TAKARA). Genomic DNA was denatured by NaOH and modified by sodium bisulfite.then purfied using Wizad DNA clean-up. An aliquot of bisulfite-treated genomic DNA (2μl) was used as the MSP template solution.10×Taq buffer, Taq enzyme and dNTP (TaKaRa) were used in the PCR analysis. PCR products were separated by electrophoresis on 2% agarose gels and quantitated with the FluorChem 2.0 system. The primers used for MSP and additional PCR conditions are described elsewhere.
(6) Chromatin immunoprecipitation assay (CHIP)
Detected methylation of histone H3 lysine 9 and histone H3 lysine 4 by chromatin immunoprecipitation assay (CHIP). Collected cell and treated it with Formaldehyde. Treated by H3-K9, H3-K4 methylation antibody, Sera or Tris-EDTA buffer. Then collected antibody-protein complex. PCR amplificated precipitated chromosome. Alpha Image 2000 collected imaging and analysed the result. All the experiments repeated three times. The average value is used to be statistically analysed.
(7) Western Blot
Detected CHFR protein,H3 general protein, histone H3 lysine 9 methylation protein and histone H3 lysine 4 methylation protein. Extracted protein, electrophoresis, transmembrane by PVDF membrane, and then closed by BSA. Treated by monoclonal antibody of CHFR, H3, H3K9 methylation, H3K4 methylation and actin. Then hybrided with second antibody, imaging and collected result. All the experiments repeated three times. The average value is used to be statistically analysed.
3. Statistical analysis
The data were processed by SPSS 17.0 statistical software. Measurement data were expressed as mean±SD. The 2-sample T test was used to examine the expression levels of mRNA and correlations between CHFR mRNA expression and clinicopathological parameters. The Fisher's exact test was used to analyze the DNA hypermethylation of CHFR as well as correlations of aberrant DNA hypermethylation of CHFR with clinicopathological parameters., respectively. P<0.05 was considered as significance.
Results
1. CHFR promoter methylation in LSCCs speciments
The frequency of the aberrant hypermethylation of CHFR promoter in LSCCs was 22%(11/50), including 10 in the stage T1+T2, and 1 in the stage T3, whereas there was no aberrant hypermethylation of CHFR promoter in the control specimens. The frequency of the aberrant hypermethylation in the early stage (41.6% 10/24) was significantly higher than that in the advanced stage (3.8% 1/26) (P<0.05). There was no significant difference in the others (including ages, sexes, the size of the tumor, the pathogenic differentiation and the lymphatic metastasis.
2. CHFR mRNA expression in LSCCs speciments
The results showed that CHFR expression was down-regulated in all LSCCs compared with the normal laryngeal tissues (P<0.05). Moreover, the CHFR gene silencing was discovered in 2 LSCCs specimens (4%). The relative ratio of CHFR mRNA between the two groups was 0.50±0.12, which was 0.29±0.18 at the early stage of LSCCs and 0.69±0.15 at advanced ones. There was significant difference in the clinical stages (P<0.05) but no significant difference in the others (including ages, sexes, the size of the tumor,, the differentiation and the lymphatic metastasis. The CHFR mRNA with the aberrant methylation in LSCC specimens (0.16±0.13) was much lower than that one with un-methylation (0.65±0.17).The CHFR gene silencing was discovered in 2 LSCCs with hypermethylation (4% 2/50).
3. CHFR promoter methylation in Hep-2 cell line
The cell line showed a characteristic DNA methylation status.5-Aza-dC and combined 5-Aza-dC and TSA resulted in demethylation of CHFR. In contrast, TSA alone did not affect the DNA methylation status of CHFR.
4. CHFR mRNA expression in Hep-2 cell line
Compared with the control team,5-Aza-dC alone reactivated expression of the CHFR in Hep-2 cell line (1.75±0.21). TSA had no effect on gene expression (1.05±0.13). The combined treatment with 5-Aza-dC and TSA increased gene expression (2.15±0.18).
5. CHFR histone H3 Lysine 9, H3 Lysine 4 in Hep-2 cell line
Histone H3 Lysine 9 methylation directly correlated with DNA methylation, but histone H3 Lysine 4 methylation inversely correlated with DNA methylation of CHFR. Gene down-expression associated with DNA methylation was accompanied by histone H3 Lysine 9 hypermethylation, but the methylation of H3 Lysine 4 is antagonistic to them.
5-Aza-dC had effects on histone H3 Lysine 9 methylation. TSA alone had no effect on histone H3 Lysine 9 methylation. The combination of 5-Aza-dC and TSA had similar effects on histone H3 Lysine 9 methylation to that of 5-Aza-dC.
TSA did not affect histone H3 Lysine 4 methylation.5-Aza-dC, or the combination of 5-Aza-dC and TSA increased histone H3 Lysine 4 methylation.
6. CHFR, H3, H3-K9methylation, H3-K4 methylation protein
CHFR protein of 50 tumor and 15 control mucosa specimens were detected. 46.0% (23/50) LSCC specimens had not CHFR protein expression; and all of the mucosa specimens showed detectable CHFR protein.β-actin expression were similar in all LSCCs and control mucosa samples. The differences were statistically significant (χ2=13.85; P<0.001).
Compared with the control team,5-Aza-dC alone reactivated protein expression of the CHFR in Hep-2 cell line. TSA had no effect on gene protein expression. The combined treatment with 5-Aza-dC and TSA increased gene protein expression. But 5-Aza-dC and TSA didn't affecd the protein level of histone H3, methylation-H3 K9, methylation-H3 K4.
Conclusion
The aberrant methylation of CHFR promoter was related to the down-expression or inactivation, which closely related to the clinical stage. The detection of aberrant methylation of CHFR may be helpful to the early diagnosis of LSCC.
5-Aza-dC and combined 5-Aza-dC and TSA can reverse the methylation of CHFR, simultaneously elevated the mRNA and protein expression level.
Histone H3 Lysine 9 methylation positively correlated with DNA methylation, while histone H3 Lysine 4 methylation negatively correlated with DNA methylation of the tumor suppressor gene CHFR.
Methyltransferase inhibitor 5-Aza-dC affected the ststus of Histone H3 Lysine 9 and Histone H3 Lysine 4.Therefore TSA had not effect on them.
目前研究表明,恶性肿瘤的发生与发展是一个多因素、多阶段的复杂过程,涉及多种基因的功能异常。引起基因表达异常的机制有两种：一为遗传学机制即DNA结构改变。二为表遗传学机制。表遗传学是研究非DNA序列变化引起的,可遗传的基因表达的改变。肿瘤发生中的主要表遗传学改变是肿瘤抑制基因发生DNA高甲基化和染色质中的组蛋白修饰。近几年来,表遗传学改变的研究取得了长足进展,也成为喉癌临床基础研究的前沿。
现在普遍认为DNA甲基化是除缺失与突变之外导致基因失活的第三种机制,在肿瘤的发生发展中起着不可忽视的作用。肿瘤甚至可以被认为是由DNA的突变和表观遗传学的改变共同导致的,而且表观遗传学的变化在肿瘤发生中早于DNA的突变。组蛋白的甲基化作为另一种表遗传学机制,可以改变染色体的状态,进而调节基因的转录,间接导致肿瘤的发生。H3-K9甲基化可以抑制基因表达；而H3-K4甲基化则具有激活效应。H3-K9的甲基化与DNA的甲基化在基因的沉默机制中具有协同作用,而H3-K4的甲基化拮抗DNA甲基化所产生的基因沉默,研究表明在DNA甲基化及组蛋白甲基化之间存在着相互关系,在肿瘤的发生发展中起着重要的作用。
喉癌是最常见的头颈部恶性肿瘤之一,其发病率在我国北方地区位于头颈部恶性肿瘤的第一位,在我国南方地区,仅次于鼻咽癌居于第二位；在世界范围内,居于全身恶性肿瘤的第十一位。其发病率有逐年上升的趋势,大约占新发恶性肿瘤的1%左右。近30年虽然外科技术,放化疗手段不同程度的得到提高,但是目前喉癌仍是仅有的两个五年生存率未增长的肿瘤之一。原因之一就是目前喉癌发病的分子学机制仍不清楚,早期诊断及治疗不理想。因此深入研究喉癌的发病机制,寻找早期诊断及早期治疗的新靶点非常重要。有研究表明检测特定基因甲基化状态有望为肿瘤的早期诊断提供帮助,但目前在喉癌中的研究刚刚起步,比较理想的靶基因非常少。另外一个原因可能是喉癌的治疗方面不理想,目前在我国喉癌的治疗主要是手术治疗和放射治疗,手术给患者带来很大的痛苦,远期疗效尤其是晚期患者不尽人意,从长远看,化疗联合治疗方案更能够保全器官和功能,化疗效果值得深入研究,目前去甲基化治疗已经成为全世界肿瘤研究的热点,为攻克肿瘤这一顽疾提供新的思路。
CHFR基因是一个新的有丝分裂前期检查点基因。在正常组织中广泛表达,位于纺锤体组装稽查点的上游。当存在有丝分裂应激时,CHFR通路激活,通过泛素化降解Plkl(Polo-like kinase 1),Plkl调控Cdc25和’Weel激酶的磷酸化,在G2期进入M期时控制Cdc2激酶的活性,而延迟染色体凝集,使细胞周期停滞于有丝分裂早期,纺锤体不能形成,直至错误被纠正。CHFR基因如发生改变不能阻断异常细胞通过G2期进入有丝分裂中前期,从而导致细胞的恶性增殖。已有研究发现,MAD,BUB等有丝分裂检查基因在多数肿瘤中很少失活,而CHFR在胃癌,肺癌,食道癌等恶性肿瘤中因启动子区甲基化表现为失活或低表达,与肿瘤的发生关系密切,因其具有肿瘤特异性,受到越来越多的人关注,而与喉癌发生的关系及DNA甲基化和组蛋白甲基化对其的调控目前国内外未见报道。
目的
通过探讨喉癌组织及Hep-2人喉癌细胞系中CHFR基因表达,启动子区甲基化以及组蛋白H3-K4,H3-K9甲基化,以及5-Aza-dC及TSA对基因表达和甲基化水平的影响,从而明确其与喉癌发生发展的关系,探讨喉癌的发生分子学机制及其早期诊断及治疗靶点。
材料与方法
一、实验对象
Hep-2人喉癌细胞系、50例喉癌患者肿瘤及15例正常粘膜。
二、实验方法
1、细胞培养
Hep-2人喉癌细胞按常规培养于含10%胎牛血清RPMI-1640的培养液,NaHCO2浓度2g／L,青霉素G浓度100U／L,链霉素浓度100μg／L,37℃含5%CO2的湿润空气的恒温密闭式培养箱培养。
2、实验分组
(1)Hep-2人喉癌细胞
对照组：同期培养不加药的Hep-2人喉癌细胞。
实验组：①5-Aza-dC组：加5umol/L 5-Aza-dC培养72小时；②TSA组：加TSA 300 nmol培养24小时；③5-Aza-dC及TSA组：加5umol/L 5-Aza-dC培养48小时后加TSA 300nmol继续培养24小时。
(2)喉癌组织标本
临床病例的收集：选择2008年1月-2009年1月中国医科大学附属盛京医院耳鼻喉科住院的喉鳞状细胞癌患者50例(喉癌组),男44例,女6例,年龄40-74岁,平均58±9.76岁。选取其中的15例喉全切除术标本中,距离肿瘤边缘>2.0cm的粘膜组织作为对照组。按照2002年UICC喉癌分期标准将本研究的喉癌标本进行分期：早期(Ⅰ期+Ⅱ期)22例(T1N0 5例T2N0 17例),声门上型12例,声门型10例。中晚期(Ⅲ期+Ⅳ期)28例(T2N1 1例,T2N2 1例,T3N0 9例,T3N18例,T3N2 4例,T4N0 7例),声门上型16例,声门型12例,所有喉癌组织均经病理证实为鳞癌。术前均未经放、化疗治疗。
3、引物设计
MSP、实时荧光定量PCR、CHIP引物序列和反应条件见表1-3,引物由TaKaRa公合成。
4、甲基化特异性PCR (MSP)法检测DNA甲基化水平
分别提取喉癌细胞系,喉癌组织中DNA。紫外分光光度仪定性、定量。亚硫酸氢钠修饰DNA, Wizard DNA Clean-up Systerm(Promega)纯化。离心沉淀DNA。采用甲基化特异性PCR方法。PCR反应条件：95℃12min后,95℃变性30s,56℃退火30s,72℃延伸30s,45个循环,72℃再延伸7min,琼脂糖凝胶电泳,EB染色。结果经Alpha Image 2000自动成像仪成像采集数据,分析电泳结果。实验重复三次,取其平均值进行统计分析。
5、实时荧光定量PCR (Realtime fluro-genetic quantitative PCR)法检测mRNA水平
用TRIzol试剂一步法分别提取喉癌细胞系,喉癌组织总RNA(按说明书进行)。按照反转录试剂盒说明将其反转录成cDNA第一链。无RNA酶的双蒸水将cDNA10倍稀释。进行荧光定量PCR扩增,由PCR反应曲线得到Ct值,以GAPDH为内参,采用2-ΔΔct方法进行相对定量。实验重复三次,取其平均值进行统计分析。
6、染色质免疫沉淀分析(chromatin immunoprecipitation assay,ChIP)检测组蛋白H3K9,H3K4甲基化水平
收集细胞,甲醛交联组蛋白和DNA。酶切后分别加H3-K9,H3-K4甲基化抗体,免疫血清或Tris-EDTA缓冲液。收集抗体／蛋白复合物,沉淀下来的染色质通过PCR扩增检测结果。结果经Alpha Image 2000自动成像仪成像采集数据,分析电泳结果。实验重复三次,取其平均值进行统计分析。
7、Western Blot法检测CHFR蛋白、H3总蛋白、H3K9甲基化蛋白、H3K4甲基化蛋白水平
提取蛋白,电泳,PVDF膜转膜,BSA封闭。分别加入CHFR, H3, H3K4甲基化、H3K9甲基化、actin兔单克隆抗体,然后加入单克隆二抗,成像,采集数据。采用条带密度值与相应actin密度值的比值作为指标进行比较,实验重复三次,取平均值进行分析。
三、统计学处理
应用SPSS17.0软件分析,mRNA表达水平定量测定结果用t检验,启动子甲基化结果用χ2检验(Fisher,精确概率法),mRNA的表达和临床病理参数的关系采用t检验,甲基化和临床病理参数的关系采用χ2检验。Western印迹的蛋白结果应用χ2检验统计,P<0.05,认为差异具有统计学意义。
结果
1、喉癌组织中CHFR基因启动子区甲基化情况
在喉癌组织中,正常对照组未发现CHFR基因启动子区甲基化,在喉癌组中CHFR基因启动子区甲基化率为22%(11／50),两者相比,差异显著,有统计学意义(P<0.01)。其中T1+T2期10例,T3期1例,T4期未发现甲基化,T1+T2期甲基化率(41.6%1／24)明显高于T3+T4期(3.8%1／26),甲基化程度在临床T分期方面具有统计学意义P<0.01(x2=7.50)。而在年龄、性别、分化程度、肿瘤大小、病理分化和有无淋巴结转移方面无统计学意义(P>0.05)。
2、喉癌组织中CHFR基因mRNA表达
CHFR基因在对照组mRNA全部表达。在喉癌组mRNA有2例表达缺失(4%),48例表达量明显下调,有统计学意义(P<0.01)。50例喉癌组织中,T1+T2期CHFR基因mRNA相对表达量0.29±0.18,T3+T4期相对表达量0.69±0.15,前者明显低于后者,两者差异有统计学意义(P<0.01),在不同的性别、年龄、肿瘤位置、分化程度和有无淋巴结转移方面CHFR基因mRNA水平没有统计学差异(P>0.05)。甲基化组CHFR基因mRNA表达量为0.16±0.13,非甲基化组mRNA表达量为0.65±0.17,前者明显低于后者,二者有密切关系。(P<0.05)。
3、在Hep-2人喉癌细胞系中5-Aza-dC和TSA处理前后CHFR基因CHFR启动子区甲基化情况
在Hep-2人喉癌细胞系中,CHFR启动子区表现为高甲基化,5-Aza-dC作用后DNA甲基化程度减弱,出现半甲基化,TSA作用后DNA甲基化程度无明显变化。联合使用5-Aza-dC+TSA后DNA甲基化程度明显减弱。
4、在Hep-2人喉癌细胞系中5-Aza-dC和TSA处理前后CHFR基因mRNA表达情况
同对照组比较,用5-Aza-dC作用后CHFR基因mRNA表达量上调(1.75±0.21)。用TSA作用后mRNA表达量(1.05±0.13)无明显变化。联合使用5-Aza-dC+TSA后mRNA表达量明显上调(2.15±0.18)
5、在Hep-2人喉癌细胞系中CHFR基因启动子区组蛋白H3-K4甲基化,组蛋白H3-K9甲基化情况及与DNA甲基化的关系
在Hep-2人喉癌细胞系中,CHFR基因启动子区组蛋白H3K9甲基化与DNA甲基化正相关,而组蛋白H3K4甲基化与DNA的甲基化负相关。基因启动子区组蛋白H3K9甲基化与DNA的甲基化在基因的表达下调机制中具有协同作用,而组蛋白H3K4甲基化拮抗DNA甲基化所产生的基因表达下调。
6、在Hep-2人喉癌细胞系中,5-Aza-dC和TSA处理前后的CHFR基因启动子区域组蛋白H3K9甲基化、H3K4甲基化状态分析
(1) 5-Aza-dC对启动子区域H3K9甲基化有明显作用,5-Aza-dC明显降低启动子区H3K9甲基化程度,TSA对启动子区甲基化无影响,联合给予5-Aza-dC和TSA与单独给予5-Aza-dC的作用相似。
(2)5-Aza-dC对启动子区域H3K4甲基化的作用与对启动子区域H3K9甲基化的作用恰好相反,5-Aza-dC明显提高启动子区H3K4甲基化程度,TSA对启动子区域H3K4甲基化无影响,联合给予5-Aza-dC和TSA与单独给予5-Aza-dC的作用相似。
7、Western Blot法分析喉癌组织中CHFR蛋白表达
喉癌组46.0%(23／50)的喉鳞状细胞癌未检测到CHFR蛋白表达；所有黏膜均有CHFR蛋白表达。β-actin蛋白在喉鳞癌和正常黏膜中表达水平相似。CHFR蛋白表达水平的差异有统计学意义(χ2=13.85；P<0.001)
8、Western Blot法分析5-Aza-dC和TSA处理前后Hep-2人喉癌细胞系中CHFR蛋白,H3蛋白,甲基化组蛋白H3-K9,甲基化组蛋白H3-K4表达
5-Aza-dC作用后,CHFR蛋白表达量能明显增加,TSA对CHFR蛋白表达无明显影响,联合给予5-Aza-dC和TSA与单独给予5-Aza-dC的作用相似。5-Aza-dC及TSA作用后甲基化H3K9蛋白,甲基化H3K4蛋白,总H3蛋白无明显变化。
结论
1、在喉癌组织中抑癌基因CHFR启动子区CpG岛过甲基化与mRNA和蛋白表达缺失或下调有关,可能与喉癌的发生发展有关,与临床T分期的关系可能更为密切,检测其甲基化状态可有助于喉癌早期诊断。
2、在Hep-2人喉癌细胞系中甲基转移酶抑制剂能够逆转抑癌基因CHFR启动子区甲基化状态,同时mRNA和蛋白表达上调,为临床治疗喉癌提供新思路。
3、在Hep-2人喉癌细胞系中抑癌基因CHFR基因启动子区组蛋白H3K9甲基化与DNA甲基化在其基因转录调控中呈正相关,而组蛋白H3K4甲基化与DNA的甲基化呈负相关。DNA甲基化与组蛋白H3-K9甲基化在其基因表达缺失或下调中起协调作用,而DNA甲基化与组蛋白H3-K4甲基化起拮抗作用。
4、在Hep-2人喉癌细胞系中甲基转移酶抑制剂能明显影响抑癌基因CHFR启动子区组蛋白H3K9甲基化和H3K4甲基化程度,而组蛋白脱乙酰基抑制剂对启动子区甲基化无明显影响。
Researches suggest that the tumorigenesis and development of malignant neoplasm is a multivariate, multistage and complicated process with the functional abnormality of multiple genes. The abnormal expression of genes includes genetic and epigenetic changes. The epigenetic change is heritable, which can not alter DNA sequence. DNA methylation and histone modification of tumor suppressor genes are main epigenetic changes. In recent years, the study of epigenetic changes has been developed sufficiently and become the clinical and investigative frontier of Laryngeal squamous cell carcinoma (LSCC). Recently, it has been discovored that the epigenetic alteration and DNA mutation are the same in tumorigenesis, in which the former is earlier than the latter.
It has been kowmn that aberrant methylation of DNA has been described as an alternative mechanism of gene inactivation besides mutation and deletion. As one of epigenetic mechanisms, histone methylation can alter the chromosome status and control the gene transcription, which can lead tumorigenesis. The methylation of histone H3 lysine 9 is associated with gene repression. But the methylation histone H3 lysine 4 is associated with active gene transcription. The methylation of DNA and H3 lysine 9 are both involved in genes silencing, in which the methylation of DNA and H3 lysine 9 are synergistic, but the methylation of histone H3 lysine 4 is antagonistic to them.It appears that DNA and histone methylation likely have a mutually reinforcing relationship, and play a vital role of the the tumorigenesis and development of tumors.
Laryngeal carcinoma is a common head and neck malignancy and the 11th common malignancy in the world, with a tendency towards an increasingly occurrence of new cases and deaths annually. It was shown that LSCC was one of the tumor with no increase of five-year survival rates. Therefore, it is essential to investigate the molecular mechanism of LSCC in order to identify the early diagnostic and therapeutic marker genes. Some studies have shown that the detection of the specific methylation status may be helpful to the early diagnosis of tumors. Therefore the further study of the effect on DNA and histone abnormal regulation of tumor suppressor genes plays an important role on the tumorigenesis of laryngeal carcinoma.
CHFR (checkpoint with forkhead and ring finger) is a new cell cycle mitotic stress checkpoint gene.Recently, it has been known that the abnormal CHFR involed in the primary carcinogens, and closely related to gastric cancer, lung cancer and esophagal cancer.Its encoding product is ubiquitin ligase of Plkl. Plkl regulates both the Wee 1 kinase and the Cdc25 phosphatase, which in turn control the Cdc2 kinase activity at the G2 to M transition. The CHFR gene can ubiquitinate and degenerate the Plkl, which prevents cells from entering prophase and metaphase. CHFR gene expresses in normal tissue while it is low expressed or inactivated in cancer, in which losses the ability to prevent abnormal cells proliferating from G2 to M phase, thus cells abnormal differentiation and proliferation occur.
In this study, we investigated the expression, the methylation of the promoter CpG islands and the methylation histone histone H3 lysine 9, histone H3 lysine 4 of CHFR in LSCC, and the effect on 5-Aza-dCand/orTSA to the Hep-2 cell line by MSP, Real-time PCR Western Blotting and CHIP. We explore the relationship between the level of expression and methylation of the CHFR and the tumorigenesis and development of LSCC. This investigation revealed a new pathway for the laryngeal tumorigenesis and early diagnosis and therapy.
Material and Methods
1. Subjects
50 speciments of LSCC.Hep-2 cell lines, derived from human LSCC.
2. Methods
The subjects were 50 patients with LSCC diagnosed and treated between January 2007 and January 2009 at Shengjing Hospital of China Medical University. All patients were diagnosed pathologically to be LSCC preoperatively and received no radiation and chemotherapy. We collected control mucosa from 15 patients under larngectomy and 2.0 cm away from the margin of tumor was considered as safe. The mucosa samples were histologically normal. Liquid nitrogen was used to snap-frozen all the samples and then stored in-80℃freezer. The collection of all the samples was informed patients. We extracted clinopathologic data (age, gender, primary site, T stage, clinical stage, differentiation and the nodal status) of patients were extracted from the patients' files. And the staging of the tumors was carried out according to UICC 2002 TNM classification.
(1) Cell culture
Hep-2 cell line was cultured in RPMI 1640 supplemented with 10% fetal bovine serum(Gibco), penicillin (100 IU/mL) and streptomycin (100 mg/mL), and incubated in a humidified incubator containing 50 mL/L CO2 at 37℃.
(2) Experimental teams
5-Aza-dC (5mmol/L) was used for 72h in the treatment. Culture medium containing 5-Aza-dC was exchanged every 24 h. TSA (300 nmol/L) was used for only 24 h in the treatment.5-Aza-dC was used for 48 h followed by TSA for an additional 24 h in the combined treatment.
(3) Primer design
The primer sequences and reaction conditions of MSP, Real-time PCR and CHIP were described in table 3. The primer synthesis were in TaKaRa company.
(4) mRNA extraction, expression analysis of CHFR using Real-time PCR
Total-RNA was extracted and purified from the LSCC specimens and cell line using an RNeasy mini-Kit (TAKARA) following the manufacturer's instructions. cDNA was synthesized with 1μg of total-RNA using a SuperScript II Reverse Transcriptase kit (TAKARA). The mRNA expression levels of CHFR in the laryngeal tissues were quantified by SYBR Green I real-time quantitative PCR. The relative expression was normalized to a human glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) endogenous reference standard relative to a calibrator and was evaluated by 2 -△△CT
(5) DNA extraction and methylation-specific PCR (MSP) analysis of CHFR
DNA was extracted from 65 laryngeal specimens using liquid-based cytology with a DNA Tissue Kit (TAKARA). Genomic DNA was denatured by NaOH and modified by sodium bisulfite.then purfied using Wizad DNA clean-up. An aliquot of bisulfite-treated genomic DNA (2μl) was used as the MSP template solution.10×Taq buffer, Taq enzyme and dNTP (TaKaRa) were used in the PCR analysis. PCR products were separated by electrophoresis on 2% agarose gels and quantitated with the FluorChem 2.0 system. The primers used for MSP and additional PCR conditions are described elsewhere.
(6) Chromatin immunoprecipitation assay (CHIP)
Detected methylation of histone H3 lysine 9 and histone H3 lysine 4 by chromatin immunoprecipitation assay (CHIP). Collected cell and treated it with Formaldehyde. Treated by H3-K9, H3-K4 methylation antibody, Sera or Tris-EDTA buffer. Then collected antibody-protein complex. PCR amplificated precipitated chromosome. Alpha Image 2000 collected imaging and analysed the result. All the experiments repeated three times. The average value is used to be statistically analysed.
(7) Western Blot
Detected CHFR protein,H3 general protein, histone H3 lysine 9 methylation protein and histone H3 lysine 4 methylation protein. Extracted protein, electrophoresis, transmembrane by PVDF membrane, and then closed by BSA. Treated by monoclonal antibody of CHFR, H3, H3K9 methylation, H3K4 methylation and actin. Then hybrided with second antibody, imaging and collected result. All the experiments repeated three times. The average value is used to be statistically analysed.
3. Statistical analysis
The data were processed by SPSS 17.0 statistical software. Measurement data were expressed as mean±SD. The 2-sample T test was used to examine the expression levels of mRNA and correlations between CHFR mRNA expression and clinicopathological parameters. The Fisher's exact test was used to analyze the DNA hypermethylation of CHFR as well as correlations of aberrant DNA hypermethylation of CHFR with clinicopathological parameters., respectively. P<0.05 was considered as significance.
Results
1. CHFR promoter methylation in LSCCs speciments
The frequency of the aberrant hypermethylation of CHFR promoter in LSCCs was 22%(11/50), including 10 in the stage T1+T2, and 1 in the stage T3, whereas there was no aberrant hypermethylation of CHFR promoter in the control specimens. The frequency of the aberrant hypermethylation in the early stage (41.6% 10/24) was significantly higher than that in the advanced stage (3.8% 1/26) (P<0.05). There was no significant difference in the others (including ages, sexes, the size of the tumor, the pathogenic differentiation and the lymphatic metastasis.
2. CHFR mRNA expression in LSCCs speciments
The results showed that CHFR expression was down-regulated in all LSCCs compared with the normal laryngeal tissues (P<0.05). Moreover, the CHFR gene silencing was discovered in 2 LSCCs specimens (4%). The relative ratio of CHFR mRNA between the two groups was 0.50±0.12, which was 0.29±0.18 at the early stage of LSCCs and 0.69±0.15 at advanced ones. There was significant difference in the clinical stages (P<0.05) but no significant difference in the others (including ages, sexes, the size of the tumor,, the differentiation and the lymphatic metastasis. The CHFR mRNA with the aberrant methylation in LSCC specimens (0.16±0.13) was much lower than that one with un-methylation (0.65±0.17).The CHFR gene silencing was discovered in 2 LSCCs with hypermethylation (4% 2/50).
3. CHFR promoter methylation in Hep-2 cell line
The cell line showed a characteristic DNA methylation status.5-Aza-dC and combined 5-Aza-dC and TSA resulted in demethylation of CHFR. In contrast, TSA alone did not affect the DNA methylation status of CHFR.
4. CHFR mRNA expression in Hep-2 cell line
Compared with the control team,5-Aza-dC alone reactivated expression of the CHFR in Hep-2 cell line (1.75±0.21). TSA had no effect on gene expression (1.05±0.13). The combined treatment with 5-Aza-dC and TSA increased gene expression (2.15±0.18).
5. CHFR histone H3 Lysine 9, H3 Lysine 4 in Hep-2 cell line
Histone H3 Lysine 9 methylation directly correlated with DNA methylation, but histone H3 Lysine 4 methylation inversely correlated with DNA methylation of CHFR. Gene down-expression associated with DNA methylation was accompanied by histone H3 Lysine 9 hypermethylation, but the methylation of H3 Lysine 4 is antagonistic to them.
5-Aza-dC had effects on histone H3 Lysine 9 methylation. TSA alone had no effect on histone H3 Lysine 9 methylation. The combination of 5-Aza-dC and TSA had similar effects on histone H3 Lysine 9 methylation to that of 5-Aza-dC.
TSA did not affect histone H3 Lysine 4 methylation.5-Aza-dC, or the combination of 5-Aza-dC and TSA increased histone H3 Lysine 4 methylation.
6. CHFR, H3, H3-K9methylation, H3-K4 methylation protein
CHFR protein of 50 tumor and 15 control mucosa specimens were detected. 46.0% (23/50) LSCC specimens had not CHFR protein expression; and all of the mucosa specimens showed detectable CHFR protein.β-actin expression were similar in all LSCCs and control mucosa samples. The differences were statistically significant (χ2=13.85; P<0.001).
Compared with the control team,5-Aza-dC alone reactivated protein expression of the CHFR in Hep-2 cell line. TSA had no effect on gene protein expression. The combined treatment with 5-Aza-dC and TSA increased gene protein expression. But 5-Aza-dC and TSA didn't affecd the protein level of histone H3, methylation-H3 K9, methylation-H3 K4.
Conclusion
The aberrant methylation of CHFR promoter was related to the down-expression or inactivation, which closely related to the clinical stage. The detection of aberrant methylation of CHFR may be helpful to the early diagnosis of LSCC.
5-Aza-dC and combined 5-Aza-dC and TSA can reverse the methylation of CHFR, simultaneously elevated the mRNA and protein expression level.
Histone H3 Lysine 9 methylation positively correlated with DNA methylation, while histone H3 Lysine 4 methylation negatively correlated with DNA methylation of the tumor suppressor gene CHFR.
Methyltransferase inhibitor 5-Aza-dC affected the ststus of Histone H3 Lysine 9 and Histone H3 Lysine 4.Therefore TSA had not effect on them.
引文
1 Genden EM, Ferlito A, Silver CE, et al. Evolution of the management of laryngeal cancer. Oral Oncol.2007; 43:431-439.
2 Marioniq G, Marchese-Ragona R, CarteiG et al. Current opinion in diagnosis and treatment of laryngeal carcinoma. Cancer Treat Rev.2006; 32:504-515.
3 Almadori G, Bussu F, Cadoni G, et al. Molecular markers in laryngeal squamous cell carcinoma:towards an integrated clinicobiological approach. Eur JCancer.2005; 41:683-693.
4 Wu C, Morris JR. Genesgenetics, andepigenetics:acorrespondence. Science.2001; 10:1103-1105.
5 Qiu G, Fang J, He Y.5'CpG island methylation analysis identifies the MAGE-A1 and MAGE-A3 genes as potential markers of HCC. Clin Biochem,2006; 39:259-266.
6 Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet,2006; 7:21-33.
7 Scolnick DM, Halazonetis TD.Chfr defines a mitotic stress checkpoint that delays entry into metaphase.Nature.2000; 406:430-435.
8 Mizuno K, Osada H, Konishi H, et al. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene.2002; 21:2328-2333.
9 Milne AN, Sitarz R, Carvalho R, et al. Molecular analysis of primary gastric can cer, corresponding xenografts, and 2 novel gastriccarcinom a cell lines reveals novel alterations in gastric carcinogenesis. Hum Pathof.2007; 38:903-913.
10 Shibata Y, H aruki N, Kuwabara Y, et al. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Crcf Psfs.2002; 23:1695-1699.
11 Guo MZ, Alumkal, J, Drachova T, et al. CHFR methylation strongly correlates with methylation of DNA damage repair and apoptotic pathway genes in non small cell lung cancer. AACR Meeting Abstracts.2007; 4:383-383.
12 Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nature reviews.2006; 7:21-33.
13 Rodenhiser D, Mann M. Epigenetics and human disease:translating basic biology into clinical applications. Cmaj.2006; 174:341-348.
14 Mizuno K, Osada H, Konishi H, et al. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene.2002; 21:2328-2333.
15 Corn PG, Heath El, Heitmiller R, et al. Frequent hypermethylation of the 5'CpG island of E-cadherin in esophageal adenocarcicinoma. Clin Cancer Rea.2001; 7:2765-2769.
16 Toyota M, Sasaki Y, Satoh A, et al.Epigenetic inactivation of CHFR in human tumors.Proc Natl Acad Sci U S A.2003; 100:7818-7823.
17 Summers M K, Bothos T, Halazonetis TD. The CHFR mitotic checkpoint protein delays cell cycleprogression by excluding Cyclin B1 from thenucleus. Oncogene.2005; 24:2589-2598.
18 Yu X, Minter-Dykhouse K, Malureanu L, et al. Chfr is required for tumor suppression and Aurora A regulation. Nat Genet 2005; 37:401-406Tsai MD. FHA:a signal transduction domain with diverse specificity and function. Structure.2002; 10:887-888.
19 Matsusaka L Pines T. Cracts with the p38 stress kinases to block entry to mitosis in mamm alian cells. CP Biol.2004; 166:507-516.
20 Oh YM, Yoo SJ, Seol JH. Deubiquitination of Chfr acheckpoint protein, by USP7/HAUSP regulates its stability an d activity. Biochem Biophgs Res Commun.2007; 357:615-619.
21 Syed N, Smith P, Sullivan A, et al. Transcriptional silencing of Chfr predicts clinical outcome to chemoradiotherapy in head and neck cancer, AACR Meeting Abstracts.2005; 4:506-507.
22 Chen K, Sawhney R, Khan M, et al. Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell. Archives of Ocolaryngology. 2007; 133:1131-1134.
23 Maddika S, Shirley M H, Chen J. DNA:replication, repair, recombination, and chromosome dynamics:Functional Interaction between Chfr and Kif22 Controls Genomic Stability. Biol Chem.2009; 284:12998-13003.
24 Morioka Y, Hibi K, Sakai M, et al. Aberrant methylation of the CHFR gene in digestive tract cancer. Anticancer Res.2006; 26:1791-1795.
25 Jones PA, Laird PW. Cancer epigenetics comes of age. Net Genet.1999; 21:163-167.
26 Tomita M, Toyota M, Ishikawa C, et al. Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-kappaB ctivity.Int J Cancer.2009; 124:2607-2615.
27 Hu W, Kavanagh JJ, Deaver M, et al. Frequent overexpression of STK15/Aurora-A/BTAK and chromosomal instability in tumorigenic cell cultures derived from human ovarian cancer. Oncol Res.2005; 15:49-57.
28 McCabe M T, Brandes J C, Vertino P M.Cancer DNA Methylation:Molecular Mechanisms and Clinical Implications Clin. Cancer Res.2009; 15:3927-3937.
29 Banno K, Yanokura M, Kawaguchi M, et al. Epigenetic inactivation of the CHFR gene in cervical cancer contributes to sensitivity to taxanes. Int J Oncol.2007; 31:713-720.
30 Koga Y, Kitajima Y, Miyoshi A, et al. The significance of aberrant CHFR methylation for clinical response to microtubule inhibitors in gastric cancer. J Gastroenterol.2006; 41:133-139.
31 Satoh A, Toyota M, Itoh F, et al. Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer. Cancer Res.2003; 63:8606-8613.
32 Li JJ, Weroha SJ, Lingle WL, et al. Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats. Proc Natl Acad Sci USA.2004; 101:18123-18128.
33 Schubeler D, MacAlpine D M, Scalzo D, et al. The histonemodification pattern of active genes revealed through ge-nome-wide chromatinanalysis of a higher eukaryote.Genes Dev.2004; 18: 1263-1271.
34 Liang GN, Lin JC, Wei V, et al. Distinct localization of histone H3 acetylation and H3-K4 methylation to the tran-scription start sites in the human genome.Proc Natl Acad Sci USA. 2004; 101:7357-7362.
35 Wolffe AP, Matzke MA. Epigenetics:regulation through repression. Science.1999; 286: 481-486.
36 Pal S, Sif S. Interplay between chromatin remodelers and protein arginine methyltransferases. Cell Physiol.2007; 213:3-4.
37 Koga Y, Kitajima Y, Miyoshi A, et al. The significance of aberrant CHFR methylation for
clinical response to microtubule inhibitors in gastric cancer. J Gastroenterol.2006; 41:133-139.
38 Cameron EE, Bachman KE, Myohanen S, et al. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet.1999; 21: 103-107.
39 Johnson LM, Cao XF, Jacobsen SE. DNA Methylation and Histone H3 Lysine 9 Methylation. Curr Biol.2002; 12:1360-1367.
40 Fahrner JA, Eguchi S, Herman JG.Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res.2002; 62:7213-7218.
41 Nguyen CT, Weisenberger DJ, Velicescu M, et al. Histone H3-Lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-Aza-2'-deoxycytidine.Cancer Research.2002; 62:6456-6461.
42 Wolffe AP, Matzke MA. Epigenetics:regulation through repression. Science.1999; 286: 481-486.
43 Fahrner JA, Eguchi S, Herman JG Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res.2002; 62:7213-7218.
44 Bachman KE, Park I, Rhee, H, et al. Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene.Cancer Cell.2003; 3:89-95.
45 Zinner R, Albiez H, Walter J et al. Histone lysine methylation patterns in human cell types are arranged in distinct three-dimensional nuclear zones.Histochem Cell Biol.2006; 125:3-19.
46 Zegerman P, Canas B. Histone H3 lysine 4 methylation disrupts binding of nucleosome remodelingand deacetylase (NuRD) repressor complex. J Biol Chem.2002; 277:11621-11624.
47 Lachner M, Carroll D, Rea S. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.Nature.2001; 410:116-120.
48 Stwart MD, Li J, Wong J. Relationship between histone H3 Lysine 9methylation, transcription repression, and heterochromatin protein lrecruitment. Mol Cell Biol.2005; 25:2525-2538.
49 Kim KC, Geng L, Huang S.Inactivation of histone methyltransferase by mutations in human cancers.Cancer Res.2003; 63:7619-7623.
50 Egger Q, Liang A, Paricio A, et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature.2004; 429:457-463.
51 Zelent A, Waxman S, Cardueei M, et al. State of the translation science:summary of Baltimore workshop on gene re-expression as a therapeutic target in cancer. Clin Cancer Res.2004; 10: 4622-4629.
52 Dobosy JR, Roberts JL, Fu VX, et al. The expanding role of epigenetics in the development, diagnosis and treatment of prostate cancer and benign prostatic hyperplasia. J Urol.2007; 177: 822-831.
53 Momparler RL, Bovenzi V. DNA methylation and cancer. J Cell Physiol.2000; 183:145-154.
54 Strathdee G, Brown R. Epigenetic cancer therapies:DNA methyltransferase inhibitors. Expert Opin Investig Drugs.2002; 11:747-754.
55 Kaminskas E, Farrell A, Abraham S, et al. Approval Summary:Azacitidine for Treatment of Myelodysplastic Syndrome Subtypes. Clinical Cancer Research.2005; 11:3604-3608.
1 Bradbury J. Human epigenome project-up and running Plos BioL.2003; 1:316-319.
2 Wolffe AP, Matzke MA. Epigenetics:regulation througrepression Science.1999; 286:481-486.
3 Hollidy R. Abstract of the 8 thInternational Conference on Environmental Mutagens. Mutat Res.2001; 20:1-8.
4 Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001; 293:1089-1093.
5 Jones PA, Laird PW. Cancer epigenetics comes of age. Net Genet.1999; 21:163-167.
6 Robertson KD. DNA methylation, methytransferases and cancer.Oncogene.2001; 20: 4139-4155.
7 Momparler RL.Cancer epigenetics. Oncogene.2003; 22:6479-6483.
8 Farrell WE, Clayton RN. Epigenetic change in pituitary tumorigenesis. Endocr Relat Cancer, 2003; 10:323-330.
9 Jing F, Zhang J, Tao Y, et al. Hypermethylation of tumor suppressor genes BRCA1, p16 and 14-3-3sigma in serum of sporadic breast cancer patients. Onkologie.2007; 30:14-19.
10 Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Lett.2003; 190:125-133.
11 Robertson KD, Jones PA, Balin SB, et al. DNA methylation:pastpresent and future directions [J]. Carcinogenesis.2000; 21:461-467.
12 Scolnick DM, Halazonetis TD. Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature.2000; 406:430-435.
13 Chaturvedi P, Sudakin V, BobiakML, et al. Chfr regulates a mito-tic stress pathway through its RNG-finger domain with ubiquitin lig-ase activity. Cancer Res.2002; 62:1797-1801
14 Kang D, Chen J, Wong J, et al. The checkpoint protein Chfr is a li-gase that ubiquitinates Plkl and inhibits Cdc2 at the G2 to M transi-tion. J Cell Biol.2002; 156:249-259
15 Mizuno K, Osada H, Konishi H, et al. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers.Oncogene.2002; 21:2328-2333.
16 Corn PG, Summers MK, Fogt F, et al. Frequent hypermethylation of the 5'CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer. Carcinogenesis.2003; 24:47-51.
17 Shibata Y, Haruki N, Kuwabara Y, et al. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Carcinogenesis.2002; 23:1695-1699.
18 Corn PG, Heath EI, Heitmiller R, et al. Frequent hypermethylation of the 5'CpG island of E-cadherin in esophageal adenocarcicinoma. Clin Cancer Rea.2001; 7:2765-2769.
19 Morioka Y, Hibi K, Sakai M, et al. Aberrant methylation of the CHFR gene in digestive tract cancer. Anticancer Res.2006; 26:1791-1795.
20 Summers M K, Bothos T, Halazonetis TD. The CHFR mitotic checkpoint protein delays cell cycleprogression by excluding Cyclin B1 from the nucleus. Oncogene.2005; 24:2589-2598.
21 Yu X, Minter-Dykhouse K, Malureanu L, et al. Chfr is required for tumor suppression and Aurora A regulation. Nat Genet.2005; 37:401-406.
22 Matsusaka L Pines T. Cracts with the p38 stress kinases to block entry to mitosis in mamm alian cells. CP Bio1.2004; 166:507-516.
23 Oh YM, Yoo SJ, Seol JH. Deubiquitination of Chf acheckpoint protein, by USP7/HAUSP regulates its stability an d activity. Biochem Biophgs Res Commun.2007; 357:615-619.
24 Shibata Y, Haruki N, Kuwabara Y, et al. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Carcinogenesis.2002; 23:1695-1699.
25 Chen K, Sawhney R, Khan M, et al. Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell.Archives of Ocolaryngology. 2007; 133:1131-1138.
26 Esteller M, Toyota M, Sanchez-CM Hamilton, et al. Inactivation of the DNA Repair Gene 06-Methylguanine-DNA Methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis. Cancer Res.2000; 60:2368-2371.
27 Peterson LA, Thomson NM, Crankshaw DL, et al.Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs:Implicationsfor4-(methylnitrosamino)-1-(3-pyridl)-1-butanone-induced lung tumorigenesis. Cancer Res.2001; 61:5757-5763.
28 Zhong Song, Kong Wei-jia, Liu Zheng. Promoter hypermethylation of DNA repair gene 06-me-ylguanine DNA methyltransferase in laryngeal squamous cell carcinoma.Chin J Otorhinolaryngol.2004; 39:152-156.
29 Maruya S, Issa J P, Weber R S, et al. Dfferential methylation status of tumor-associated genes in head and neck squamous carcinoma:i ncidence and potential implication. Clin Cancer Res.2004; 10:3825-2830.
30 Temam S, Benard J, Dugas C, et al. Molecular detection of early-stage laryngopharyngeal squa-mous cell carcinomas. Clin Cancer Res.2005; 11:2547-2551.
31 Krecicki T, Smigiel R, Fraczek M, et al. Studies of the cell cycle regulatory proteins P16, cyc-lin D1 and retinoblastoma protein in laryngeal carcino-ma tissue. J Laryngol Otol.2004; 118:676-680.
32 Dammann R, Li C, Yoon JH, et al. Epigenetic inactivation of a ras association domain family protein from the lung tumor suppressor locus 3p21.3. Nat Genet.2000; 25:315-319.
33 Reinhard D, Glen Y, GeM P. Hypermethylation of the CpGisland of RASSFIA, a putative TSG from the 3p21.3 locus, occurs in a large percentage of human breast cancer. Cancer Res.2002; 61:3105-3109.
34 Xing M, Cohen Y, Mambo E, et al. Early occurrence of RASSFIA hypermeththylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis. Cancer Res.2004; 64: 1664-1668.
35 Kwong J, Lo KW, To, KF. et al. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res.2002; 8:131-131.
36 Xu Cheng-bi, Teng Bo, Li Chang-qing. Jin Chun-shun.Promoter methylation of RASSF1 A gene in laryngeal squamous cell carcinom and protein expression. Journal of Jilin Universit. 2006; 32:326-329.
37 Carvalho AL, Jeronimo C, Henrique R, et al. Evaluation of promoter hypermethylation in head and neck squamous carcinoma and normal tissue using quantitative-MSP. AACR Meeting Abstracts.2004; 2004:207-207.
38 Youssef E M, Lotan D, Issa J P, et al. Hypermethylation of the Retinoic acid receptor-β2 gene
in head and neck carcinogenesis. Clin Cancer Res.2004; 10:1733-1742.
39 Ohta M, Inoue H, CotticelliM G, et al. The FHIT gene, span ningthe chromosome 3p14.2 fragile site and renal carcinoma-associated (3.8) breakpoint is abnormal in digestive tract cancers. Cell.1996; 84:587-597.
40 Yuge T, Nibu K, Kondok K, et al. Loss of FHIT expression in squamous cell carcinoma and premalignant lesions of the larynx. Ann Otol Rhinol Laryngol.2005; 114:127-131.
41 Wu H, Luan X, Zhou W, et al. The relationshipbetween exp ressionof FHIT and biological behaviors of hypopharyngeal carcinoma. Lin Chuang Er Bi Hou Ke Za Zhi.2006; 20:249-250.
42 Yin D, Dong M. Methylation of promoter and expression of FH ITgene in laryngeal squamous cell carcinoma. Lin Chuang Er Bi Yan Hou Ke Za Zhi.2005; 6:16-21.
43 Worsham MJ, Chen KM, Meduri, et al. Epigenetic events of disease progression in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Sury.2006; 132:668-677.
44 Rosas SL, Koch W, Carvalho MG, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res.2001; 61:939-942.
45 Ogi K, Toyota M, Ohe-Toyota, M, et al Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma. Clin Cancer Res.2002; 8: 3164-3164.
46 Viswanathan M, Tsuchida N, Shanmugam G.Promoterhypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cellcarcinoma. Int J Cancer.2003; 20:41-46.
47 Demokan S, Suoglu Y, Demir D, et al. Microsatellite instability and methylation of the DNA mismatch repair genes in head and neck cancer.Ann Onc.2006; 17:995-999.
48 Shaw RJ, Hall GL, Lowe D, The role of pyrosequencing in head and neck cancer epigenetics: correlation of quantitative methylation data with gene expression, Arch Otolaryngol Head Neck Surg.2008; 134:251-256.
49 Linggi B E, Brandt S J, Sun Zw, et al. Translating the histone-code into Leukemia. J Cell Biochem.2005; 96:938-950.
50 Liang G N, Lin J C, Wei V, et al. Distinct localization ofhistone H3 acetylation and H3-K4 methylation to the trans-cription start sites in the human genome Proc Natl Acad Sci USA. 2004; 101:7357-7362.
51 Xin Z H, Tachibana M, Guggiari M, et al. Role of histonemethyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center. J Biol Chem.2003; 278: 14996-15000.
52 Lehnertz B, Ueda Y, Derijck A A, et al. Suv39h-mediatedhistone H3 lysine 9 methylation directs DNA methylation Tomajor satelliter epeatsatperi centrichetero chromatin. CurrBiol. 2003; 13:1192-1200.
53 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PC novel PCR assay for methylation status of CpG islands.Proc NAcad Sci USA.1996;93:9821-9826.
54 Rosas SL, Koch W,Carvalho MG, et al. Prom hypermethylation patterns of pO6-methylguanine-DNA-methyltransferase, and death-associ protein kinase in tumors and saliva of head and neck cancer patie Cancer Res.2001;61:939-942.
55 Feltus FA, Lee EK, Costello JF, et al. Predicting aberrant CpG island methylation. PNA online. 2003; 100:12253-12258.
56 Herman JG, Baylin SB. Gene Silencing in Cancer in Association with Promot Hypermethylation.The New England of journal of medicine.2003; 349:2042-2054.
57 Palmisano WA, Divine KK, Saccomanno G, et al. Predicting lung cancer by detecti aberrant promoter methylation in sputum. Cancer Research.2000; 60:5954-5958.
58 Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of p16INK 4a is an earevent in lung cancer and a potential biomarker for early diagnosis. PNAS online.2003; 95: 11891-11896.
59 Seike M, Gemma A, Yoko Hosoya, et al. Increase in the Frequency of p16INK4 Ge Inactivation by Hypermethylation in Lung Cancer during the Process of Metastasis and Relation to the Status of p53. Clinical Cancer Research.2004; 6:4307-4313.
60 Bailey, V J. Easwaran H, Zhang, Y et al. MS-qFRET:A quantum dot-based method for analysis of DNA methylation. Genome Res.2009; 19:1455-1461.
61 Rosas S, Koch W, Carvalho M, et al. Promoter hypermethylation patterns of p16, O-methylgua2nine2DNA 2methylt ransferase, and death2associated protein k inase in tumors and saliva of head and neck cancer pat ients. Cancer Res.2004; 61:939-942.
62 Righini CA, Fraipint FD, Timsit JF, et al. Tumor-specific methylation in saliva-a promising biomarker for early detection of head and neckcancer recurrence.Clin Cancer Res.2007; 13: 1179-1185.
63 Fergnson AT, Vertino PM, Spitzneri JR.Role of Estrogen Receptor Gene Demethylation and DNA Methyltransferase. DNA Adduct Formation in 5-Aza-2-deoxycytidine-induced Gytotoxidry In Human Breast Cancer Biochem.1997; 272:32260-32266.
2 Marioniq G, Marchese-Ragona R, CarteiG et al. Current opinion in diagnosis and treatment of laryngeal carcinoma. Cancer Treat Rev.2006; 32:504-515.
3 Almadori G, Bussu F, Cadoni G, et al. Molecular markers in laryngeal squamous cell carcinoma:towards an integrated clinicobiological approach. Eur JCancer.2005; 41:683-693.
4 Wu C, Morris JR. Genesgenetics, andepigenetics:acorrespondence. Science.2001; 10:1103-1105.
5 Qiu G, Fang J, He Y.5'CpG island methylation analysis identifies the MAGE-A1 and MAGE-A3 genes as potential markers of HCC. Clin Biochem,2006; 39:259-266.
6 Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet,2006; 7:21-33.
7 Scolnick DM, Halazonetis TD.Chfr defines a mitotic stress checkpoint that delays entry into metaphase.Nature.2000; 406:430-435.
8 Mizuno K, Osada H, Konishi H, et al. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene.2002; 21:2328-2333.
9 Milne AN, Sitarz R, Carvalho R, et al. Molecular analysis of primary gastric can cer, corresponding xenografts, and 2 novel gastriccarcinom a cell lines reveals novel alterations in gastric carcinogenesis. Hum Pathof.2007; 38:903-913.
10 Shibata Y, H aruki N, Kuwabara Y, et al. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Crcf Psfs.2002; 23:1695-1699.
11 Guo MZ, Alumkal, J, Drachova T, et al. CHFR methylation strongly correlates with methylation of DNA damage repair and apoptotic pathway genes in non small cell lung cancer. AACR Meeting Abstracts.2007; 4:383-383.
12 Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nature reviews.2006; 7:21-33.
13 Rodenhiser D, Mann M. Epigenetics and human disease:translating basic biology into clinical applications. Cmaj.2006; 174:341-348.
14 Mizuno K, Osada H, Konishi H, et al. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene.2002; 21:2328-2333.
15 Corn PG, Heath El, Heitmiller R, et al. Frequent hypermethylation of the 5'CpG island of E-cadherin in esophageal adenocarcicinoma. Clin Cancer Rea.2001; 7:2765-2769.
16 Toyota M, Sasaki Y, Satoh A, et al.Epigenetic inactivation of CHFR in human tumors.Proc Natl Acad Sci U S A.2003; 100:7818-7823.
17 Summers M K, Bothos T, Halazonetis TD. The CHFR mitotic checkpoint protein delays cell cycleprogression by excluding Cyclin B1 from thenucleus. Oncogene.2005; 24:2589-2598.
18 Yu X, Minter-Dykhouse K, Malureanu L, et al. Chfr is required for tumor suppression and Aurora A regulation. Nat Genet 2005; 37:401-406Tsai MD. FHA:a signal transduction domain with diverse specificity and function. Structure.2002; 10:887-888.
19 Matsusaka L Pines T. Cracts with the p38 stress kinases to block entry to mitosis in mamm alian cells. CP Biol.2004; 166:507-516.
20 Oh YM, Yoo SJ, Seol JH. Deubiquitination of Chfr acheckpoint protein, by USP7/HAUSP regulates its stability an d activity. Biochem Biophgs Res Commun.2007; 357:615-619.
21 Syed N, Smith P, Sullivan A, et al. Transcriptional silencing of Chfr predicts clinical outcome to chemoradiotherapy in head and neck cancer, AACR Meeting Abstracts.2005; 4:506-507.
22 Chen K, Sawhney R, Khan M, et al. Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell. Archives of Ocolaryngology. 2007; 133:1131-1134.
23 Maddika S, Shirley M H, Chen J. DNA:replication, repair, recombination, and chromosome dynamics:Functional Interaction between Chfr and Kif22 Controls Genomic Stability. Biol Chem.2009; 284:12998-13003.
24 Morioka Y, Hibi K, Sakai M, et al. Aberrant methylation of the CHFR gene in digestive tract cancer. Anticancer Res.2006; 26:1791-1795.
25 Jones PA, Laird PW. Cancer epigenetics comes of age. Net Genet.1999; 21:163-167.
26 Tomita M, Toyota M, Ishikawa C, et al. Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-kappaB ctivity.Int J Cancer.2009; 124:2607-2615.
27 Hu W, Kavanagh JJ, Deaver M, et al. Frequent overexpression of STK15/Aurora-A/BTAK and chromosomal instability in tumorigenic cell cultures derived from human ovarian cancer. Oncol Res.2005; 15:49-57.
28 McCabe M T, Brandes J C, Vertino P M.Cancer DNA Methylation:Molecular Mechanisms and Clinical Implications Clin. Cancer Res.2009; 15:3927-3937.
29 Banno K, Yanokura M, Kawaguchi M, et al. Epigenetic inactivation of the CHFR gene in cervical cancer contributes to sensitivity to taxanes. Int J Oncol.2007; 31:713-720.
30 Koga Y, Kitajima Y, Miyoshi A, et al. The significance of aberrant CHFR methylation for clinical response to microtubule inhibitors in gastric cancer. J Gastroenterol.2006; 41:133-139.
31 Satoh A, Toyota M, Itoh F, et al. Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer. Cancer Res.2003; 63:8606-8613.
32 Li JJ, Weroha SJ, Lingle WL, et al. Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats. Proc Natl Acad Sci USA.2004; 101:18123-18128.
33 Schubeler D, MacAlpine D M, Scalzo D, et al. The histonemodification pattern of active genes revealed through ge-nome-wide chromatinanalysis of a higher eukaryote.Genes Dev.2004; 18: 1263-1271.
34 Liang GN, Lin JC, Wei V, et al. Distinct localization of histone H3 acetylation and H3-K4 methylation to the tran-scription start sites in the human genome.Proc Natl Acad Sci USA. 2004; 101:7357-7362.
35 Wolffe AP, Matzke MA. Epigenetics:regulation through repression. Science.1999; 286: 481-486.
36 Pal S, Sif S. Interplay between chromatin remodelers and protein arginine methyltransferases. Cell Physiol.2007; 213:3-4.
37 Koga Y, Kitajima Y, Miyoshi A, et al. The significance of aberrant CHFR methylation for
clinical response to microtubule inhibitors in gastric cancer. J Gastroenterol.2006; 41:133-139.
38 Cameron EE, Bachman KE, Myohanen S, et al. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet.1999; 21: 103-107.
39 Johnson LM, Cao XF, Jacobsen SE. DNA Methylation and Histone H3 Lysine 9 Methylation. Curr Biol.2002; 12:1360-1367.
40 Fahrner JA, Eguchi S, Herman JG.Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res.2002; 62:7213-7218.
41 Nguyen CT, Weisenberger DJ, Velicescu M, et al. Histone H3-Lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-Aza-2'-deoxycytidine.Cancer Research.2002; 62:6456-6461.
42 Wolffe AP, Matzke MA. Epigenetics:regulation through repression. Science.1999; 286: 481-486.
43 Fahrner JA, Eguchi S, Herman JG Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res.2002; 62:7213-7218.
44 Bachman KE, Park I, Rhee, H, et al. Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene.Cancer Cell.2003; 3:89-95.
45 Zinner R, Albiez H, Walter J et al. Histone lysine methylation patterns in human cell types are arranged in distinct three-dimensional nuclear zones.Histochem Cell Biol.2006; 125:3-19.
46 Zegerman P, Canas B. Histone H3 lysine 4 methylation disrupts binding of nucleosome remodelingand deacetylase (NuRD) repressor complex. J Biol Chem.2002; 277:11621-11624.
47 Lachner M, Carroll D, Rea S. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.Nature.2001; 410:116-120.
48 Stwart MD, Li J, Wong J. Relationship between histone H3 Lysine 9methylation, transcription repression, and heterochromatin protein lrecruitment. Mol Cell Biol.2005; 25:2525-2538.
49 Kim KC, Geng L, Huang S.Inactivation of histone methyltransferase by mutations in human cancers.Cancer Res.2003; 63:7619-7623.
50 Egger Q, Liang A, Paricio A, et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature.2004; 429:457-463.
51 Zelent A, Waxman S, Cardueei M, et al. State of the translation science:summary of Baltimore workshop on gene re-expression as a therapeutic target in cancer. Clin Cancer Res.2004; 10: 4622-4629.
52 Dobosy JR, Roberts JL, Fu VX, et al. The expanding role of epigenetics in the development, diagnosis and treatment of prostate cancer and benign prostatic hyperplasia. J Urol.2007; 177: 822-831.
53 Momparler RL, Bovenzi V. DNA methylation and cancer. J Cell Physiol.2000; 183:145-154.
54 Strathdee G, Brown R. Epigenetic cancer therapies:DNA methyltransferase inhibitors. Expert Opin Investig Drugs.2002; 11:747-754.
55 Kaminskas E, Farrell A, Abraham S, et al. Approval Summary:Azacitidine for Treatment of Myelodysplastic Syndrome Subtypes. Clinical Cancer Research.2005; 11:3604-3608.
1 Bradbury J. Human epigenome project-up and running Plos BioL.2003; 1:316-319.
2 Wolffe AP, Matzke MA. Epigenetics:regulation througrepression Science.1999; 286:481-486.
3 Hollidy R. Abstract of the 8 thInternational Conference on Environmental Mutagens. Mutat Res.2001; 20:1-8.
4 Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001; 293:1089-1093.
5 Jones PA, Laird PW. Cancer epigenetics comes of age. Net Genet.1999; 21:163-167.
6 Robertson KD. DNA methylation, methytransferases and cancer.Oncogene.2001; 20: 4139-4155.
7 Momparler RL.Cancer epigenetics. Oncogene.2003; 22:6479-6483.
8 Farrell WE, Clayton RN. Epigenetic change in pituitary tumorigenesis. Endocr Relat Cancer, 2003; 10:323-330.
9 Jing F, Zhang J, Tao Y, et al. Hypermethylation of tumor suppressor genes BRCA1, p16 and 14-3-3sigma in serum of sporadic breast cancer patients. Onkologie.2007; 30:14-19.
10 Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Lett.2003; 190:125-133.
11 Robertson KD, Jones PA, Balin SB, et al. DNA methylation:pastpresent and future directions [J]. Carcinogenesis.2000; 21:461-467.
12 Scolnick DM, Halazonetis TD. Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature.2000; 406:430-435.
13 Chaturvedi P, Sudakin V, BobiakML, et al. Chfr regulates a mito-tic stress pathway through its RNG-finger domain with ubiquitin lig-ase activity. Cancer Res.2002; 62:1797-1801
14 Kang D, Chen J, Wong J, et al. The checkpoint protein Chfr is a li-gase that ubiquitinates Plkl and inhibits Cdc2 at the G2 to M transi-tion. J Cell Biol.2002; 156:249-259
15 Mizuno K, Osada H, Konishi H, et al. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers.Oncogene.2002; 21:2328-2333.
16 Corn PG, Summers MK, Fogt F, et al. Frequent hypermethylation of the 5'CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer. Carcinogenesis.2003; 24:47-51.
17 Shibata Y, Haruki N, Kuwabara Y, et al. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Carcinogenesis.2002; 23:1695-1699.
18 Corn PG, Heath EI, Heitmiller R, et al. Frequent hypermethylation of the 5'CpG island of E-cadherin in esophageal adenocarcicinoma. Clin Cancer Rea.2001; 7:2765-2769.
19 Morioka Y, Hibi K, Sakai M, et al. Aberrant methylation of the CHFR gene in digestive tract cancer. Anticancer Res.2006; 26:1791-1795.
20 Summers M K, Bothos T, Halazonetis TD. The CHFR mitotic checkpoint protein delays cell cycleprogression by excluding Cyclin B1 from the nucleus. Oncogene.2005; 24:2589-2598.
21 Yu X, Minter-Dykhouse K, Malureanu L, et al. Chfr is required for tumor suppression and Aurora A regulation. Nat Genet.2005; 37:401-406.
22 Matsusaka L Pines T. Cracts with the p38 stress kinases to block entry to mitosis in mamm alian cells. CP Bio1.2004; 166:507-516.
23 Oh YM, Yoo SJ, Seol JH. Deubiquitination of Chf acheckpoint protein, by USP7/HAUSP regulates its stability an d activity. Biochem Biophgs Res Commun.2007; 357:615-619.
24 Shibata Y, Haruki N, Kuwabara Y, et al. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Carcinogenesis.2002; 23:1695-1699.
25 Chen K, Sawhney R, Khan M, et al. Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell.Archives of Ocolaryngology. 2007; 133:1131-1138.
26 Esteller M, Toyota M, Sanchez-CM Hamilton, et al. Inactivation of the DNA Repair Gene 06-Methylguanine-DNA Methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis. Cancer Res.2000; 60:2368-2371.
27 Peterson LA, Thomson NM, Crankshaw DL, et al.Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs:Implicationsfor4-(methylnitrosamino)-1-(3-pyridl)-1-butanone-induced lung tumorigenesis. Cancer Res.2001; 61:5757-5763.
28 Zhong Song, Kong Wei-jia, Liu Zheng. Promoter hypermethylation of DNA repair gene 06-me-ylguanine DNA methyltransferase in laryngeal squamous cell carcinoma.Chin J Otorhinolaryngol.2004; 39:152-156.
29 Maruya S, Issa J P, Weber R S, et al. Dfferential methylation status of tumor-associated genes in head and neck squamous carcinoma:i ncidence and potential implication. Clin Cancer Res.2004; 10:3825-2830.
30 Temam S, Benard J, Dugas C, et al. Molecular detection of early-stage laryngopharyngeal squa-mous cell carcinomas. Clin Cancer Res.2005; 11:2547-2551.
31 Krecicki T, Smigiel R, Fraczek M, et al. Studies of the cell cycle regulatory proteins P16, cyc-lin D1 and retinoblastoma protein in laryngeal carcino-ma tissue. J Laryngol Otol.2004; 118:676-680.
32 Dammann R, Li C, Yoon JH, et al. Epigenetic inactivation of a ras association domain family protein from the lung tumor suppressor locus 3p21.3. Nat Genet.2000; 25:315-319.
33 Reinhard D, Glen Y, GeM P. Hypermethylation of the CpGisland of RASSFIA, a putative TSG from the 3p21.3 locus, occurs in a large percentage of human breast cancer. Cancer Res.2002; 61:3105-3109.
34 Xing M, Cohen Y, Mambo E, et al. Early occurrence of RASSFIA hypermeththylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis. Cancer Res.2004; 64: 1664-1668.
35 Kwong J, Lo KW, To, KF. et al. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res.2002; 8:131-131.
36 Xu Cheng-bi, Teng Bo, Li Chang-qing. Jin Chun-shun.Promoter methylation of RASSF1 A gene in laryngeal squamous cell carcinom and protein expression. Journal of Jilin Universit. 2006; 32:326-329.
37 Carvalho AL, Jeronimo C, Henrique R, et al. Evaluation of promoter hypermethylation in head and neck squamous carcinoma and normal tissue using quantitative-MSP. AACR Meeting Abstracts.2004; 2004:207-207.
38 Youssef E M, Lotan D, Issa J P, et al. Hypermethylation of the Retinoic acid receptor-β2 gene
in head and neck carcinogenesis. Clin Cancer Res.2004; 10:1733-1742.
39 Ohta M, Inoue H, CotticelliM G, et al. The FHIT gene, span ningthe chromosome 3p14.2 fragile site and renal carcinoma-associated (3.8) breakpoint is abnormal in digestive tract cancers. Cell.1996; 84:587-597.
40 Yuge T, Nibu K, Kondok K, et al. Loss of FHIT expression in squamous cell carcinoma and premalignant lesions of the larynx. Ann Otol Rhinol Laryngol.2005; 114:127-131.
41 Wu H, Luan X, Zhou W, et al. The relationshipbetween exp ressionof FHIT and biological behaviors of hypopharyngeal carcinoma. Lin Chuang Er Bi Hou Ke Za Zhi.2006; 20:249-250.
42 Yin D, Dong M. Methylation of promoter and expression of FH ITgene in laryngeal squamous cell carcinoma. Lin Chuang Er Bi Yan Hou Ke Za Zhi.2005; 6:16-21.
43 Worsham MJ, Chen KM, Meduri, et al. Epigenetic events of disease progression in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Sury.2006; 132:668-677.
44 Rosas SL, Koch W, Carvalho MG, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res.2001; 61:939-942.
45 Ogi K, Toyota M, Ohe-Toyota, M, et al Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma. Clin Cancer Res.2002; 8: 3164-3164.
46 Viswanathan M, Tsuchida N, Shanmugam G.Promoterhypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cellcarcinoma. Int J Cancer.2003; 20:41-46.
47 Demokan S, Suoglu Y, Demir D, et al. Microsatellite instability and methylation of the DNA mismatch repair genes in head and neck cancer.Ann Onc.2006; 17:995-999.
48 Shaw RJ, Hall GL, Lowe D, The role of pyrosequencing in head and neck cancer epigenetics: correlation of quantitative methylation data with gene expression, Arch Otolaryngol Head Neck Surg.2008; 134:251-256.
49 Linggi B E, Brandt S J, Sun Zw, et al. Translating the histone-code into Leukemia. J Cell Biochem.2005; 96:938-950.
50 Liang G N, Lin J C, Wei V, et al. Distinct localization ofhistone H3 acetylation and H3-K4 methylation to the trans-cription start sites in the human genome Proc Natl Acad Sci USA. 2004; 101:7357-7362.
51 Xin Z H, Tachibana M, Guggiari M, et al. Role of histonemethyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center. J Biol Chem.2003; 278: 14996-15000.
52 Lehnertz B, Ueda Y, Derijck A A, et al. Suv39h-mediatedhistone H3 lysine 9 methylation directs DNA methylation Tomajor satelliter epeatsatperi centrichetero chromatin. CurrBiol. 2003; 13:1192-1200.
53 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PC novel PCR assay for methylation status of CpG islands.Proc NAcad Sci USA.1996;93:9821-9826.
54 Rosas SL, Koch W,Carvalho MG, et al. Prom hypermethylation patterns of pO6-methylguanine-DNA-methyltransferase, and death-associ protein kinase in tumors and saliva of head and neck cancer patie Cancer Res.2001;61:939-942.
55 Feltus FA, Lee EK, Costello JF, et al. Predicting aberrant CpG island methylation. PNA online. 2003; 100:12253-12258.
56 Herman JG, Baylin SB. Gene Silencing in Cancer in Association with Promot Hypermethylation.The New England of journal of medicine.2003; 349:2042-2054.
57 Palmisano WA, Divine KK, Saccomanno G, et al. Predicting lung cancer by detecti aberrant promoter methylation in sputum. Cancer Research.2000; 60:5954-5958.
58 Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of p16INK 4a is an earevent in lung cancer and a potential biomarker for early diagnosis. PNAS online.2003; 95: 11891-11896.
59 Seike M, Gemma A, Yoko Hosoya, et al. Increase in the Frequency of p16INK4 Ge Inactivation by Hypermethylation in Lung Cancer during the Process of Metastasis and Relation to the Status of p53. Clinical Cancer Research.2004; 6:4307-4313.
60 Bailey, V J. Easwaran H, Zhang, Y et al. MS-qFRET:A quantum dot-based method for analysis of DNA methylation. Genome Res.2009; 19:1455-1461.
61 Rosas S, Koch W, Carvalho M, et al. Promoter hypermethylation patterns of p16, O-methylgua2nine2DNA 2methylt ransferase, and death2associated protein k inase in tumors and saliva of head and neck cancer pat ients. Cancer Res.2004; 61:939-942.
62 Righini CA, Fraipint FD, Timsit JF, et al. Tumor-specific methylation in saliva-a promising biomarker for early detection of head and neckcancer recurrence.Clin Cancer Res.2007; 13: 1179-1185.
63 Fergnson AT, Vertino PM, Spitzneri JR.Role of Estrogen Receptor Gene Demethylation and DNA Methyltransferase. DNA Adduct Formation in 5-Aza-2-deoxycytidine-induced Gytotoxidry In Human Breast Cancer Biochem.1997; 272:32260-32266.