肿瘤抑制基因的DNA甲基化在肝移植后肝癌复发预测中的价值研究
|
摘要
肝细胞癌(hepatocellular carcinoma, HCC)是最常见的肝脏恶性肿瘤,占全世界肿瘤发生率的第五位。我国作为乙肝大国,国民中乙型肝炎病毒携带率高,作为肝炎-肝硬化-肝癌这一进展链的终末端,肝癌的高发成为不争的事实,从而使我国成为全世界肝癌的最高发地区之一。不可否认的是,经过几十年的不断努力,我国在肝癌的诊治上取得了举世瞩目的成就。但是以手术切除为基础的各种综合治疗(包括放射、化学、生物、靶向、肝移植等治疗手段)依然无法解决HCC总体预后不佳这一难题。随着肝脏外科水平的进步和肝癌受者入选标准的进一步规范,肝脏移植被认为是治疗肝癌的一种理想治疗手段,但是肝脏移植术后肿瘤的转移复发这个瓶颈问题依然严重制约肝癌的治疗效果。近年来,围绕肝移植后肝癌复发转移这一核心问题,有关预测肝癌复发的生物学标记的研究不断升温,成为重要的研究方向。
随着对肿瘤转移认识的不断深入和人类基因组学的长足发展,人们已经意识到肝癌的发生发展是一个涉及到多基因改变、调控的复杂过程,基于恶性肿瘤基因改变、调控的相关研究成为热点。而作为新兴的表观遗传学最重要的研究内容,DNA甲基化所致抑癌基因转录失活,进而影响肿瘤发生、发展这一问题也成为研究者关注的焦点。然而,通过文献回顾发现,目前有关新肿瘤抑制基因PCDH10以及基于多个肿瘤抑制基因同时甲基化的分子表型——CpG岛甲基化表型(CpGislands methylator phenotype CIMP)在肝移植后肝癌复发预测中的价值国内外尚无报道,因此我们此前研究的基础上,首次尝试建立一个能反映肝癌生物学特征的CpG岛甲基化表型,探讨其在肝移植后肝癌复发预测中的价值；并以新型TSG—-PCDH10作为研究对象,系统研究作为优化的CIMP的候选TSG在肝细胞肝癌进展中的作用及其调控机制,以期建立一个与复发密切相关的CIMP模型,为进一步提高肝癌肝移植的早期复发预测能力,建立肝癌肝移植“中国标准”提供候选的新的肿瘤生物学参数。
第一部分肿瘤抑制基因的DNA甲基化及相关CpG岛甲基化表型对肝移植后肝癌复发预测的价值研究
目的：
基因启动子区甲基化是最重要的表观遗传修饰,通过结合甲基化结合域(MBD)和组蛋白乙酰化能引起基因的表达沉默,尤其是抑癌基因启动子区的甲基化在多种肿瘤的发生发展的过程中发挥了非常重要的作用。肝癌中多个基因启动子区的协同甲基化所形成的CpG岛的甲基化表型被证实与肝癌病理,如甲胎蛋白的升高有密切关系。本实验通过检测肝癌中多基因启动子区的甲基化,观察肿瘤相关基因甲基化在肝癌中的发生情况,建立与肝移植肝癌复发预后相关CpG岛甲基化表型,并分析CpG岛甲基化表型与临床病理指标之间的联系。
方法：
选取P16, CDH1, GSTP1, DAPK, MGMT, XAF1, TIMP3, SOCS1, SFRP1, TMS1, SYK, DKK1共12个肿瘤抑制基因启动子区域CpG岛上甲基化位点(上述基因已知可能与人类不同恶性肿瘤,特别是消化道肿瘤的发生、侵袭、转移乃至预后相关),应用甲基化特异性PCR (Methylation-Specific PCR)首先对小规模20例HCC肝移植病人的病肝组织DNA进行甲基化状况的初筛,将其中甲基化频率高于40%的7个肿瘤抑制基因(P16, CDH1, GSTP1, DAPK, XAF1, SOCS1, SYK)作为CpG岛甲基化表型的标志基因,并将上述位点的甲基化检测扩大到65例HCC肝移植病人的病肝组织DNA样本,然后根据甲基化状况的不同对65例病人进行CpG岛的甲基化表型(CIMP)分类；另一方面对肝癌肝移植病人进行临床病理资料的收集,对病人进行随访,比较不同CpG岛甲基化表型与病人临床病理指标及移植术后的3年无复发生存之间的关系。
结果：
所检测的临床样本中12个基因的甲基化频率各不相同：从MGMT的5%到XAF1的66%不等；所选择的7个CpG岛甲基化表型的标志基因中,存在协同甲基化现象,即P16与SYK同时甲基化(P=0.001),P16与CDH1同时甲基化(P=0.008),CDH1与DAPK同时甲基化(P=0.003)。在分析了单个肿瘤抑制基因与临床病理学指标及预后信息后发现：GSTP1的高甲基化与低组织学分级(P=0.003)和多发的肿瘤个数有关(P=0.011)；XAF1的高甲基化与病人术前AFP水平存在相关性(P=0.007),此外DAPK的甲基化状况与肿瘤的大小也存在一定的联系(P=0.016),但其他单个基因的甲基化状况与病人的临床病理学指标均无统计学差异,并且所有单个基因的甲基化状况与病人的复发预后也无明显统计学差异(P>0.05)。应用Log-rank检验,分析甲基化数目与复发生存的关系,发现在所选择的7个抑癌基因中,大于等于3个基因发生甲基化与肝移植后肝癌复发具有最显著的相关性(P=0.004),因而将CpG岛的甲基化表型(CIMP)分类为CIMP阳性(甲基化数目大于等于3个)和CIMP阴性(甲基化数目小于3个)。随后的分析发现,CpG岛的甲基化表型状态与术前AFP水平和肿瘤大小存在相关性(P值分别为0.017和0.007),但与病人的其它临床病理学指标如性别、年龄、是否合并门静脉癌栓(PVTT)、组织学分级、肿瘤大小均无关(P>0.05)。同时,结合我们中心之前提出的肝移植杭州标准,证实超越杭州标准的病人比符合杭州标准的病人具有更高的CIMP阳性发生率(84%vs.57%,P=0.017)。之后我们运用Kaplan-Meier生存曲线分析CpG岛的甲基化表型与肝癌肝移植病人生存期关系时发现CpG岛的甲基化表型阳性组(CIMP+)的病人的移植后三年无复发生存期比CpG岛的甲基化表型阴性组(CIMP-)病人要短,且具有显著性差异(15.3月vs.30.3月,P=0.004)。Cox单因素分析结果显示：除了术前AFP水平、肿瘤大小以外,CpG岛的甲基化表型状态也与复发显著相关。多因素分析结果表明：除肿瘤大小外,CpG岛的甲基化表型状态(危险系数：3.581；95%可信区间：(1.473-8.710)；P=0.005)也是影响复发的一个独立的危险因素。
结论：
CpG岛的甲基化表型是影响移植后肝癌复发的独立危险因素,对移植后肝癌复发的预测具有潜在的重要价值。
第二部分新肿瘤抑制基因PCDH10在肝细胞肝癌中的实验研究
目的：原钙粘蛋白10 (PCDH10),是新近发现的钙粘蛋白超家族的一名成员蛋白。早期关于PCDH10的研究主要集中在神经生理方面,认为PCDH10蛋白在大脑发育,特别是纹状体轴突延伸和丘脑皮质投射的形成中发挥了重要作用。最近有研究表明,在鼻咽癌,白血病,宫颈癌,乳腺癌和胃癌等多种肿瘤细胞株和组织中存在由于PCDH10基因启动子的异常甲基化而导致表达缺失现象,而通过外源性的过度表达PCDH10蛋白能够显著抑制肿瘤细胞的增殖,侵袭和转移,并诱导肿瘤细胞的凋亡。在宫颈癌的研究中则认为PCDH10的甲基化可以作为宫颈癌特异性诊断的一个潜在的标志物。而在胃癌的最新临床研究中不仅证实PCDH10是一个典型的肿瘤抑制基因,而且PCDH10的高甲基化和病人不良预后有着密切的联系。但PCDH10在肝癌中的临床和基础研究目前尚未报道,PCDH10蛋白在肝癌细胞中的生物学功能也还不得而知。
方法：
采用Western blotting和免疫组化的方法检测PCDH10在HCC患者肿瘤组织,癌旁以及正常肝脏组织中蛋白表达情况。用RT-PCR方法检测去甲基化药物(5'-Aza-dC)用药前后肝癌细胞株中PCDH10的mRNA表达水平,研究其表观遗传的调控机制,同时采用甲基化特异性PCR (MSP)和重亚硫酸盐修饰测序PCR(BSP)的方法验证目的基因的甲基化变化情况,并分析65例肝癌肝移植患者的PCDH10的甲基化状况与临床病理指标及复发预后的相关性；应用RNA干扰和质粒转染技术,分别模拟PCDH10缺失和过表达两种情况,进而从正反两方面来研究PCDH10蛋白对肝癌细胞增殖、凋亡、侵袭和克隆形成能力的影响。
结果：
在我们所选的5种不同肝癌细胞株中,80%肝癌细胞株存在PCDH10mRNA表达水平的降低或缺失,而PCDH10基因的启动子区域的高甲基化则是引起基因表达缺失的主要原因。临床组织标本中则发现DNA甲基化导致的PCDH10蛋白表达缺失具有肿瘤特异性,即肝癌组织中的表达相对于癌旁组织和正常组织普遍降低,并且发现PCDH10基因高甲基化的肝癌肝移植病人更容易发生术后的肿瘤复发,且预后不良。体外实验则发现,PCDH10的过度表达能够抑制肝癌细胞株的增殖,降低细胞的侵袭和克隆形成能力,并诱导凋亡。
结论：
在肝癌中,PCDH10基因启动子区域的DNA甲基化是导致其蛋白表达缺失的主要因素,但不是唯一因素；PCDH10基因的甲基化状态是预测肝移植后肝癌复发的潜在的生物学标记物。PCDH10蛋白过表达能够显著抑制肿瘤细胞的生长,促进肝癌细胞凋亡并降低细胞的侵袭和克隆形成能力。
Hepatocellular carcinoma (HCC) is the most common hepatic malignant tumor, ranking the fifth among tumors in the world. Our country is a big country for hepatitis B, with a high carrying rate of hepatitis B virus in the population. As the terminal end of the progression chain of hepatitis-liver cirrhosis-HCC, the high incidence of HCC is an indisputable fact, making our country the place with the highest HCC incidence in the world. Undeniably, after persistent effort in the past decades, our country has obtained remarkable achievements in the diagnosis and treatment of HCC. However, various comprehensive treatments on the basis of surgicial excision (including radiotherapy, chemotherapy, biological, targeted treatment and liver transplantation) still can not resolve the general poor prognosis of HCC. With development of hepatological surgery and standardization of inclusion criteria for HCC patients, liver transplantation is considered an ideal therapy for HCC. However, tumor metastasis and recurrence after transplantation still greatly restrict the therapeutic effect. In the recent years, arrounding recurrence and metastasis after liver transplantation, studies on biomarkers of HCC recurrence have become an important research aspect.
Meanwhile, with deeper understanding of tumor metastasis and development of human genomics, it is realized that the pathogenesis and progression of HCC is a complex process involving changes and regulation of multiple genes. Studies related to malignant tumor gene change and regulation have become a hot pot. As the most important content of the newly emerged epigenetics, inactivated transcription of tumor suppressor gene caused by DNA methylation, which further impacts tumor development has attracted many researchers'attentions. However, through literature review, we find that there is no report of predictive value of the new tumor suppressor gene PCDH10 and CIMP in tumor recurrence of patients with hepatocellular carcinoma after liver transplantation. Thus, on the basis of previous research, we first attempted to establish CIMP to reflect the biological features of HCC and discuss its predictive value in tumor recurrence after liver transplantation; we also used the novol TSG- PCDH10 to systemically investigate the role and regulation mechanism of candidate TSGs for optimized CIMP in the development of HCC, so as to establish a CIMP model closedly related to recurrence, further improve early prediction of recurrence after liver transplantation, establish HCC liver transplantation "China standard" and provide new candidate tumor biological parameters.
Part I Predictive value of DNA methylation of tumor suppressor genes and related CpG island methylator phenotype in tumor recurrence of patients with hepatocellular carcinoma after liver transplantation
Objective:
The promoter methylation of gene is an important epigenetic modification, which can bind methyl-CpG-binding domain (MBD) and histone acetylation cause gene expression silence, especially the methylation of tumor suppressor gene causing expression silence which plays an extremely important role in carcinogenesis and progression. CpG island methylator phenotype (CIMP), in which multiple genes are concurrently methylated has been recognized as one of the important mechanisms in hepatocellular carcinoma and associated with elevated serum alpha-fetoprotein level. We wanted to investigate the promoter methylation of gene, confirm CIMP associated prognositic in HCC and analyze relationship between CIMP status and clinicopathologic features.
Methods:
Methylation status of CpG islands on 12 tumor suppressor gene promoter regions were studied, including P16, CDH1, GSTP1, DAPK, MGMT, XAF1, TIMP3, SOCS1, SFRP1, TMS1, SYK and DKK1 (these genes are known to be associated with different human malignant tumors, especially carcinogenesis, invasion, metastasis and even prognosis of gastrointestinal tumor). Preliminary screening with Methylation-Specific PCR (MSP) was first performed to assess the DNA methylation status of tumor tissues from 20 patients with HCC treated with liver transplantation.7 tumor suppressor genes (P16, CDH1, GSTP1, DAPK, XAF1, SOCS1, SYK) whose methylation frequency was higher than 40% were selected as the marker genes of CIMP. Methylation detection of the above sites was expanded to tumor tissues from 65 patients with HCC treated with liver transplantation. Then according to the methylation status, CIMP classification was conducted for these 65 patients. On the other hand, clinical pathological materials of the patients with HCC liver transplantation were collected. Follow up was performed to evaluate the relationship between CIMP and clinical pathological parameters and 3-year recurrence-free survival after transplantation.
Results:
The methylation frequencies of 12 gene in our clinical samples were different from 5% for MGMT and 66% for XAF1; among the 7 selected marker genes of CIMP, there was concordant methylation phenomenon, i.e. P16 and SYK were hypermethylated concurrently (P=0.001), P16 and CDH11 were hypermethylated concurrently (P=0.008), CDH1 and DAPK were hypermethylated concurrently (p=0.003). After analyzing individual tumor suppressor gene, clinical pathological parameters and prognostic information, it was found that hypermethylation of GSTP1 was associated with low histologic grade (P=0.003) and multiple tumor numbers (P=0.011); hypermethylation of XAF1 was associated with pre-operative AFP level (P=0.007); besides, methylation status of DAPK was associated with tumor size (P=0.016). There was no significant relationship between methylation status of other individual genes and clinical pathological parameters. No statistic relationship was found between the methylation status of individual genes and tumor recurrence (P>0.05). Log-rank test was used to analyze relationship between number of hypermethylated genes and recurrence. The results indicated that among the 7 selected tumor suppressor genes, there was the most significant relationship between hypermethylation of≥3 genes and HCC recurrence after liver transplantation (P=0.004). Therefore, CIMP was classified into CIMP positive (hypermehtylation of≥3 genes) and CIMP negative (hypermethylation of< 3 genes). The subsequent analysis showed that there was correlation between CIMP status and pre-operative AFP level as well as tumor size (P=0.017 and 0.007 respectively). However, CIMP status was not related to other clinical pathological parameters, such as gender, age, PVTT, histological grade or tumor size (P>0.05). Meanwhile, in combination with Liver Transplantation Hangzhou Criteria proposed previously by our center, it was confirmed that patients beyond Hangzhou Criteria had the higher CIMP positive rate than patients meeting Hangzhou Criteria (84% vs.57%, P=0.017). Then, we used Kaplan-Meier survival curve to analyze the relationship between CIMP and survival of patients with HCC liver transplantation. It was found that 3-year currence-free survival after liver transplantation of patients in CIMP+group was shorter than that of patients in CIMP-group, with significant difference (15.3 months vs.30.3 months, P=0.004). Results of Cox univariate analysis showed that besides pre-operative AFP level and tumor size, CIMP status was also related to recurrence. Multivariate analysis indicated that besides tumor size, CIMP status (risk coefficient:3.581; 95%CI:(1.473-8.710); P=0.005) was also an independent risk factor influencing recurrence.
Conclusion:
CIMP was an independent risk factor influencing HCC recurrence after liver transplantation and thus had potential value in predicting HCC recurrence after liver transplantation.
Part II The study of a novel tumor suppressor gene, PCDH10 in hepatocellular carcinoma.
Objective:
Protocadherin 10 (PCDH10) is a newly identified protocadherin of the cadherin family. Early studies on PCDH 10 mainly focused on neurophysiology. It is considered that PCDH10 is essential for brain development, especially in elongating striatal axons and guiding thalamocortical projections. Recent studies indicated that in nasopharyngeal carcinoma, leukemia, cervical carcinoma, breast cancer and gastric carcinoma cell strains and tissues, there was expression loss caused by hypermethylation of PCDH 10 promoter region. Through exogenous over expression of PCDH 10 protein, the proliferation, invasion and metastasis of tumor cells could be significantly inhibited and apoptosis of tumor cells could be induced. In the study of cervical carcinoma, it was considered that hypermethylation of PCDH10 could serve as a potential specific diagnostic marker of cervical carcinoma. In the latest clinical study on gastric carcinoma, it was not only confirmed that PCDH10 was a typical tumor suppressor gene but also that hypermethylation of PCDH10 was closely associated with poor prognosis. However, there is no report on clinical and basic research of PCDH10 in hepatocellular carcinoma and the biological effect of PCDH10 protein in hepatocellular carcinoma cells is still unknown.
Methods:
Western blotting and immunohistochemistry were used to detect expression of PCDH10 in tumor tissues and paraneoplastic tissues from HCC patients as well as normal liver tissues. RT-PCR was used to detect PCDH10 mRNA expression level in hepatocellular carcinoma cells before and after application of demethylation agent 5'-Aza-dC. The mechanism of epigenetic regulation was studied. Meanwhile, MSP and BSP were used to verify methylation status change of the target genes. The correlation between PCDH10 methylation and clinical pathological parameters as well as recurrence was analyzed in 65 patients with HCC liver transplantation; RNA interference and plasmid transfection were used to respectively mimic PCDH10 absence and over expression, so as to study the effect of PCDH10 protein on proliferation, apoptosis, invasion and clone formation ability of hepatocellular carcinoma cells in both sides.
Results:
Among our selected 5 hepatocellular carcinoma cell lines,80% cell lines had reduced or absent PCDH10 mRNA expression. Hypermethylation of PCDH10 promoter region was the main reason resulting in gene expression loss. In clinical specimens, it was found that PCDH10 expression loss caused by DNA methylation was tumor specific, i.e. the expression in hepatocellular carcinoma tissues was relatively lower than that in paraneoplastic tissues and normal tissues. It was also revealed that patients with HCC liver transplantation with hymethylation of PCDH10 were more common to have recurrence after operation and had poor prognosis. In vitro experiment also found that over expression of PCDH10 could inhibit proliferation of hepatocellular carcinoma cell lines, reduce cellular invasion and clone formation, and induce apoptosis.
Conclusion:
In hepatocellular carcinoma, DNA methylation of PCDH10 promoter region is a main factor causing loss of protein expression but not the sole factor; methylation status of PCDH10 is a potential biological marker for HCC recurrence after liver transplantation. Over expression of PCDH10 protein can significantly inhibit growth of tumor cells, promote apoptosis of HCC cells and reduce cell invasion and clone formation.
随着对肿瘤转移认识的不断深入和人类基因组学的长足发展,人们已经意识到肝癌的发生发展是一个涉及到多基因改变、调控的复杂过程,基于恶性肿瘤基因改变、调控的相关研究成为热点。而作为新兴的表观遗传学最重要的研究内容,DNA甲基化所致抑癌基因转录失活,进而影响肿瘤发生、发展这一问题也成为研究者关注的焦点。然而,通过文献回顾发现,目前有关新肿瘤抑制基因PCDH10以及基于多个肿瘤抑制基因同时甲基化的分子表型——CpG岛甲基化表型(CpGislands methylator phenotype CIMP)在肝移植后肝癌复发预测中的价值国内外尚无报道,因此我们此前研究的基础上,首次尝试建立一个能反映肝癌生物学特征的CpG岛甲基化表型,探讨其在肝移植后肝癌复发预测中的价值；并以新型TSG—-PCDH10作为研究对象,系统研究作为优化的CIMP的候选TSG在肝细胞肝癌进展中的作用及其调控机制,以期建立一个与复发密切相关的CIMP模型,为进一步提高肝癌肝移植的早期复发预测能力,建立肝癌肝移植“中国标准”提供候选的新的肿瘤生物学参数。
第一部分肿瘤抑制基因的DNA甲基化及相关CpG岛甲基化表型对肝移植后肝癌复发预测的价值研究
目的：
基因启动子区甲基化是最重要的表观遗传修饰,通过结合甲基化结合域(MBD)和组蛋白乙酰化能引起基因的表达沉默,尤其是抑癌基因启动子区的甲基化在多种肿瘤的发生发展的过程中发挥了非常重要的作用。肝癌中多个基因启动子区的协同甲基化所形成的CpG岛的甲基化表型被证实与肝癌病理,如甲胎蛋白的升高有密切关系。本实验通过检测肝癌中多基因启动子区的甲基化,观察肿瘤相关基因甲基化在肝癌中的发生情况,建立与肝移植肝癌复发预后相关CpG岛甲基化表型,并分析CpG岛甲基化表型与临床病理指标之间的联系。
方法：
选取P16, CDH1, GSTP1, DAPK, MGMT, XAF1, TIMP3, SOCS1, SFRP1, TMS1, SYK, DKK1共12个肿瘤抑制基因启动子区域CpG岛上甲基化位点(上述基因已知可能与人类不同恶性肿瘤,特别是消化道肿瘤的发生、侵袭、转移乃至预后相关),应用甲基化特异性PCR (Methylation-Specific PCR)首先对小规模20例HCC肝移植病人的病肝组织DNA进行甲基化状况的初筛,将其中甲基化频率高于40%的7个肿瘤抑制基因(P16, CDH1, GSTP1, DAPK, XAF1, SOCS1, SYK)作为CpG岛甲基化表型的标志基因,并将上述位点的甲基化检测扩大到65例HCC肝移植病人的病肝组织DNA样本,然后根据甲基化状况的不同对65例病人进行CpG岛的甲基化表型(CIMP)分类；另一方面对肝癌肝移植病人进行临床病理资料的收集,对病人进行随访,比较不同CpG岛甲基化表型与病人临床病理指标及移植术后的3年无复发生存之间的关系。
结果：
所检测的临床样本中12个基因的甲基化频率各不相同：从MGMT的5%到XAF1的66%不等；所选择的7个CpG岛甲基化表型的标志基因中,存在协同甲基化现象,即P16与SYK同时甲基化(P=0.001),P16与CDH1同时甲基化(P=0.008),CDH1与DAPK同时甲基化(P=0.003)。在分析了单个肿瘤抑制基因与临床病理学指标及预后信息后发现：GSTP1的高甲基化与低组织学分级(P=0.003)和多发的肿瘤个数有关(P=0.011)；XAF1的高甲基化与病人术前AFP水平存在相关性(P=0.007),此外DAPK的甲基化状况与肿瘤的大小也存在一定的联系(P=0.016),但其他单个基因的甲基化状况与病人的临床病理学指标均无统计学差异,并且所有单个基因的甲基化状况与病人的复发预后也无明显统计学差异(P>0.05)。应用Log-rank检验,分析甲基化数目与复发生存的关系,发现在所选择的7个抑癌基因中,大于等于3个基因发生甲基化与肝移植后肝癌复发具有最显著的相关性(P=0.004),因而将CpG岛的甲基化表型(CIMP)分类为CIMP阳性(甲基化数目大于等于3个)和CIMP阴性(甲基化数目小于3个)。随后的分析发现,CpG岛的甲基化表型状态与术前AFP水平和肿瘤大小存在相关性(P值分别为0.017和0.007),但与病人的其它临床病理学指标如性别、年龄、是否合并门静脉癌栓(PVTT)、组织学分级、肿瘤大小均无关(P>0.05)。同时,结合我们中心之前提出的肝移植杭州标准,证实超越杭州标准的病人比符合杭州标准的病人具有更高的CIMP阳性发生率(84%vs.57%,P=0.017)。之后我们运用Kaplan-Meier生存曲线分析CpG岛的甲基化表型与肝癌肝移植病人生存期关系时发现CpG岛的甲基化表型阳性组(CIMP+)的病人的移植后三年无复发生存期比CpG岛的甲基化表型阴性组(CIMP-)病人要短,且具有显著性差异(15.3月vs.30.3月,P=0.004)。Cox单因素分析结果显示：除了术前AFP水平、肿瘤大小以外,CpG岛的甲基化表型状态也与复发显著相关。多因素分析结果表明：除肿瘤大小外,CpG岛的甲基化表型状态(危险系数：3.581；95%可信区间：(1.473-8.710)；P=0.005)也是影响复发的一个独立的危险因素。
结论：
CpG岛的甲基化表型是影响移植后肝癌复发的独立危险因素,对移植后肝癌复发的预测具有潜在的重要价值。
第二部分新肿瘤抑制基因PCDH10在肝细胞肝癌中的实验研究
目的：原钙粘蛋白10 (PCDH10),是新近发现的钙粘蛋白超家族的一名成员蛋白。早期关于PCDH10的研究主要集中在神经生理方面,认为PCDH10蛋白在大脑发育,特别是纹状体轴突延伸和丘脑皮质投射的形成中发挥了重要作用。最近有研究表明,在鼻咽癌,白血病,宫颈癌,乳腺癌和胃癌等多种肿瘤细胞株和组织中存在由于PCDH10基因启动子的异常甲基化而导致表达缺失现象,而通过外源性的过度表达PCDH10蛋白能够显著抑制肿瘤细胞的增殖,侵袭和转移,并诱导肿瘤细胞的凋亡。在宫颈癌的研究中则认为PCDH10的甲基化可以作为宫颈癌特异性诊断的一个潜在的标志物。而在胃癌的最新临床研究中不仅证实PCDH10是一个典型的肿瘤抑制基因,而且PCDH10的高甲基化和病人不良预后有着密切的联系。但PCDH10在肝癌中的临床和基础研究目前尚未报道,PCDH10蛋白在肝癌细胞中的生物学功能也还不得而知。
方法：
采用Western blotting和免疫组化的方法检测PCDH10在HCC患者肿瘤组织,癌旁以及正常肝脏组织中蛋白表达情况。用RT-PCR方法检测去甲基化药物(5'-Aza-dC)用药前后肝癌细胞株中PCDH10的mRNA表达水平,研究其表观遗传的调控机制,同时采用甲基化特异性PCR (MSP)和重亚硫酸盐修饰测序PCR(BSP)的方法验证目的基因的甲基化变化情况,并分析65例肝癌肝移植患者的PCDH10的甲基化状况与临床病理指标及复发预后的相关性；应用RNA干扰和质粒转染技术,分别模拟PCDH10缺失和过表达两种情况,进而从正反两方面来研究PCDH10蛋白对肝癌细胞增殖、凋亡、侵袭和克隆形成能力的影响。
结果：
在我们所选的5种不同肝癌细胞株中,80%肝癌细胞株存在PCDH10mRNA表达水平的降低或缺失,而PCDH10基因的启动子区域的高甲基化则是引起基因表达缺失的主要原因。临床组织标本中则发现DNA甲基化导致的PCDH10蛋白表达缺失具有肿瘤特异性,即肝癌组织中的表达相对于癌旁组织和正常组织普遍降低,并且发现PCDH10基因高甲基化的肝癌肝移植病人更容易发生术后的肿瘤复发,且预后不良。体外实验则发现,PCDH10的过度表达能够抑制肝癌细胞株的增殖,降低细胞的侵袭和克隆形成能力,并诱导凋亡。
结论：
在肝癌中,PCDH10基因启动子区域的DNA甲基化是导致其蛋白表达缺失的主要因素,但不是唯一因素；PCDH10基因的甲基化状态是预测肝移植后肝癌复发的潜在的生物学标记物。PCDH10蛋白过表达能够显著抑制肿瘤细胞的生长,促进肝癌细胞凋亡并降低细胞的侵袭和克隆形成能力。
Hepatocellular carcinoma (HCC) is the most common hepatic malignant tumor, ranking the fifth among tumors in the world. Our country is a big country for hepatitis B, with a high carrying rate of hepatitis B virus in the population. As the terminal end of the progression chain of hepatitis-liver cirrhosis-HCC, the high incidence of HCC is an indisputable fact, making our country the place with the highest HCC incidence in the world. Undeniably, after persistent effort in the past decades, our country has obtained remarkable achievements in the diagnosis and treatment of HCC. However, various comprehensive treatments on the basis of surgicial excision (including radiotherapy, chemotherapy, biological, targeted treatment and liver transplantation) still can not resolve the general poor prognosis of HCC. With development of hepatological surgery and standardization of inclusion criteria for HCC patients, liver transplantation is considered an ideal therapy for HCC. However, tumor metastasis and recurrence after transplantation still greatly restrict the therapeutic effect. In the recent years, arrounding recurrence and metastasis after liver transplantation, studies on biomarkers of HCC recurrence have become an important research aspect.
Meanwhile, with deeper understanding of tumor metastasis and development of human genomics, it is realized that the pathogenesis and progression of HCC is a complex process involving changes and regulation of multiple genes. Studies related to malignant tumor gene change and regulation have become a hot pot. As the most important content of the newly emerged epigenetics, inactivated transcription of tumor suppressor gene caused by DNA methylation, which further impacts tumor development has attracted many researchers'attentions. However, through literature review, we find that there is no report of predictive value of the new tumor suppressor gene PCDH10 and CIMP in tumor recurrence of patients with hepatocellular carcinoma after liver transplantation. Thus, on the basis of previous research, we first attempted to establish CIMP to reflect the biological features of HCC and discuss its predictive value in tumor recurrence after liver transplantation; we also used the novol TSG- PCDH10 to systemically investigate the role and regulation mechanism of candidate TSGs for optimized CIMP in the development of HCC, so as to establish a CIMP model closedly related to recurrence, further improve early prediction of recurrence after liver transplantation, establish HCC liver transplantation "China standard" and provide new candidate tumor biological parameters.
Part I Predictive value of DNA methylation of tumor suppressor genes and related CpG island methylator phenotype in tumor recurrence of patients with hepatocellular carcinoma after liver transplantation
Objective:
The promoter methylation of gene is an important epigenetic modification, which can bind methyl-CpG-binding domain (MBD) and histone acetylation cause gene expression silence, especially the methylation of tumor suppressor gene causing expression silence which plays an extremely important role in carcinogenesis and progression. CpG island methylator phenotype (CIMP), in which multiple genes are concurrently methylated has been recognized as one of the important mechanisms in hepatocellular carcinoma and associated with elevated serum alpha-fetoprotein level. We wanted to investigate the promoter methylation of gene, confirm CIMP associated prognositic in HCC and analyze relationship between CIMP status and clinicopathologic features.
Methods:
Methylation status of CpG islands on 12 tumor suppressor gene promoter regions were studied, including P16, CDH1, GSTP1, DAPK, MGMT, XAF1, TIMP3, SOCS1, SFRP1, TMS1, SYK and DKK1 (these genes are known to be associated with different human malignant tumors, especially carcinogenesis, invasion, metastasis and even prognosis of gastrointestinal tumor). Preliminary screening with Methylation-Specific PCR (MSP) was first performed to assess the DNA methylation status of tumor tissues from 20 patients with HCC treated with liver transplantation.7 tumor suppressor genes (P16, CDH1, GSTP1, DAPK, XAF1, SOCS1, SYK) whose methylation frequency was higher than 40% were selected as the marker genes of CIMP. Methylation detection of the above sites was expanded to tumor tissues from 65 patients with HCC treated with liver transplantation. Then according to the methylation status, CIMP classification was conducted for these 65 patients. On the other hand, clinical pathological materials of the patients with HCC liver transplantation were collected. Follow up was performed to evaluate the relationship between CIMP and clinical pathological parameters and 3-year recurrence-free survival after transplantation.
Results:
The methylation frequencies of 12 gene in our clinical samples were different from 5% for MGMT and 66% for XAF1; among the 7 selected marker genes of CIMP, there was concordant methylation phenomenon, i.e. P16 and SYK were hypermethylated concurrently (P=0.001), P16 and CDH11 were hypermethylated concurrently (P=0.008), CDH1 and DAPK were hypermethylated concurrently (p=0.003). After analyzing individual tumor suppressor gene, clinical pathological parameters and prognostic information, it was found that hypermethylation of GSTP1 was associated with low histologic grade (P=0.003) and multiple tumor numbers (P=0.011); hypermethylation of XAF1 was associated with pre-operative AFP level (P=0.007); besides, methylation status of DAPK was associated with tumor size (P=0.016). There was no significant relationship between methylation status of other individual genes and clinical pathological parameters. No statistic relationship was found between the methylation status of individual genes and tumor recurrence (P>0.05). Log-rank test was used to analyze relationship between number of hypermethylated genes and recurrence. The results indicated that among the 7 selected tumor suppressor genes, there was the most significant relationship between hypermethylation of≥3 genes and HCC recurrence after liver transplantation (P=0.004). Therefore, CIMP was classified into CIMP positive (hypermehtylation of≥3 genes) and CIMP negative (hypermethylation of< 3 genes). The subsequent analysis showed that there was correlation between CIMP status and pre-operative AFP level as well as tumor size (P=0.017 and 0.007 respectively). However, CIMP status was not related to other clinical pathological parameters, such as gender, age, PVTT, histological grade or tumor size (P>0.05). Meanwhile, in combination with Liver Transplantation Hangzhou Criteria proposed previously by our center, it was confirmed that patients beyond Hangzhou Criteria had the higher CIMP positive rate than patients meeting Hangzhou Criteria (84% vs.57%, P=0.017). Then, we used Kaplan-Meier survival curve to analyze the relationship between CIMP and survival of patients with HCC liver transplantation. It was found that 3-year currence-free survival after liver transplantation of patients in CIMP+group was shorter than that of patients in CIMP-group, with significant difference (15.3 months vs.30.3 months, P=0.004). Results of Cox univariate analysis showed that besides pre-operative AFP level and tumor size, CIMP status was also related to recurrence. Multivariate analysis indicated that besides tumor size, CIMP status (risk coefficient:3.581; 95%CI:(1.473-8.710); P=0.005) was also an independent risk factor influencing recurrence.
Conclusion:
CIMP was an independent risk factor influencing HCC recurrence after liver transplantation and thus had potential value in predicting HCC recurrence after liver transplantation.
Part II The study of a novel tumor suppressor gene, PCDH10 in hepatocellular carcinoma.
Objective:
Protocadherin 10 (PCDH10) is a newly identified protocadherin of the cadherin family. Early studies on PCDH 10 mainly focused on neurophysiology. It is considered that PCDH10 is essential for brain development, especially in elongating striatal axons and guiding thalamocortical projections. Recent studies indicated that in nasopharyngeal carcinoma, leukemia, cervical carcinoma, breast cancer and gastric carcinoma cell strains and tissues, there was expression loss caused by hypermethylation of PCDH 10 promoter region. Through exogenous over expression of PCDH 10 protein, the proliferation, invasion and metastasis of tumor cells could be significantly inhibited and apoptosis of tumor cells could be induced. In the study of cervical carcinoma, it was considered that hypermethylation of PCDH10 could serve as a potential specific diagnostic marker of cervical carcinoma. In the latest clinical study on gastric carcinoma, it was not only confirmed that PCDH10 was a typical tumor suppressor gene but also that hypermethylation of PCDH10 was closely associated with poor prognosis. However, there is no report on clinical and basic research of PCDH10 in hepatocellular carcinoma and the biological effect of PCDH10 protein in hepatocellular carcinoma cells is still unknown.
Methods:
Western blotting and immunohistochemistry were used to detect expression of PCDH10 in tumor tissues and paraneoplastic tissues from HCC patients as well as normal liver tissues. RT-PCR was used to detect PCDH10 mRNA expression level in hepatocellular carcinoma cells before and after application of demethylation agent 5'-Aza-dC. The mechanism of epigenetic regulation was studied. Meanwhile, MSP and BSP were used to verify methylation status change of the target genes. The correlation between PCDH10 methylation and clinical pathological parameters as well as recurrence was analyzed in 65 patients with HCC liver transplantation; RNA interference and plasmid transfection were used to respectively mimic PCDH10 absence and over expression, so as to study the effect of PCDH10 protein on proliferation, apoptosis, invasion and clone formation ability of hepatocellular carcinoma cells in both sides.
Results:
Among our selected 5 hepatocellular carcinoma cell lines,80% cell lines had reduced or absent PCDH10 mRNA expression. Hypermethylation of PCDH10 promoter region was the main reason resulting in gene expression loss. In clinical specimens, it was found that PCDH10 expression loss caused by DNA methylation was tumor specific, i.e. the expression in hepatocellular carcinoma tissues was relatively lower than that in paraneoplastic tissues and normal tissues. It was also revealed that patients with HCC liver transplantation with hymethylation of PCDH10 were more common to have recurrence after operation and had poor prognosis. In vitro experiment also found that over expression of PCDH10 could inhibit proliferation of hepatocellular carcinoma cell lines, reduce cellular invasion and clone formation, and induce apoptosis.
Conclusion:
In hepatocellular carcinoma, DNA methylation of PCDH10 promoter region is a main factor causing loss of protein expression but not the sole factor; methylation status of PCDH10 is a potential biological marker for HCC recurrence after liver transplantation. Over expression of PCDH10 protein can significantly inhibit growth of tumor cells, promote apoptosis of HCC cells and reduce cell invasion and clone formation.
引文
[1]Parkin DM:Global cancer statistics in the year 2000. Lancet Oncol 2001,2(9): 533-543.
[2]El-Serag HB, Rudolph KL:Hepatocellular carcinoma:epidemiology and molecular carcinogenesis. Gastroenterology 2007,132(7):2557-76.
[3]Wild CP, Hall AJ:Primary prevention of hepatocellular carcinoma in developing countries. Mutat Res 2000,462(2-3):381-393.
[4]Yoo HY, Patt CH, Geschwind JF, Thuluvath PJ:The outcome of liver transplantation in patients with hepatocellular carcinoma in the United States between 1988 and 2001:5-year survival has improved significantly with time. J Clin Oncol 2003,21(23):4329-4335.
[5]Zhang F, Wu LM, Zhou L, Chen QX, Xie HY, Feng XW, Zheng SS:Predictive value of expression and promoter hypermethylation of XAF1 in hepatitis B virus-associated hepatocellular carcinoma treated with transplantation. Ann Surg Oncol 2008,15(12):3494-3502.
[6]Wu L, Xu X, Shen J, Xie H, Yu S, Liang T, Wang W, Shen Y, Zhang M, Zheng S:MDR1 gene polymorphisms and risk of recurrence in patients with hepatocellular carcinoma after liver transplantation. J Surg Oncol 2007,96(1): 62-68.
[7]Wu LM, Zhang F, Xie HY, Xu X, Chen QX, Yin SY, Liu XC, Zhou L, Xu XB, Sun YL, et al.:MMP2 promoter polymorphism (C-1306T) and risk of recurrence in patients with hepatocellular carcinoma after transplantation. Clin Genet 2008,73(3):273-278.
[8]Herman JG, Baylin SB:Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003,349(21):2042-2054.
[9]de Maat MF, Umetani N, Sunami E, Turner RR, Hoon DS:Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles. Mol Cancer Res 2007,5(5): 461-471.
[10]Kusano M, Toyota M, Suzuki H, Akino K, Aoki F, Fujita M, Hosokawa M, Shinomura Y, Imai K, Tokino T:Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus. Cancer 2006,106(7):1467-1479.
[11]Ogino S, Nosho K, Irahara N, Meyerhardt JA, Baba Y, Shima K, Glickman JN, Ferrone CR, Mino-Kenudson M, Tanaka N, et al:Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and CpG island methylator phenotype. Clin Cancer Res 2009,15(20):6412-6420.
[12]Yuan Y, Wang J, Li J, Wang L, Li M, Yang Z, Zhang C, Dai JL:Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res 2006,12(22):6687-6695.
[13]Yamada S, Nomoto S, Fujii T, Takeda S, Kanazumi N, Sugimoto H, Nakao A: Frequent promoter methylation of M-cadherin in hepatocellular carcinoma is associated with poor prognosis. Anticancer Res 2007,27(4B):2269-2274.
[14]Tanemura A, Terando AM, Sim MS, van Hoesel AQ, de Maat MF, Morton DL, Hoon DS:CpG island methylator phenotype predicts progression of malignant melanoma. Clin Cancer Res 2009,15(5):1801-1807.
[15]Liu Z, Zhao J, Chen XF, Li W, Liu R, Lei Z, Liu X, Peng X, Xu K, Chen J, et al.:CpG island methylator phenotype involving tumor suppressor genes located on chromosome 3p in non-small cell lung cancer. Lung cancer 2008, 62(1):15-22.
[16]Roman-Gomez J, Jimenez-Velasco A, Agirre X, Prosper F, Heiniger A, Torres A:Lack of CpG island methylator phenotype defines a clinical subtype of T-cell acute lymphoblastic leukemia associated with good prognosis. J Clin Oncol 2005,23(28):7043-7049.
[17]Abe M, Ohira M, Kaneda A, Yagi Y, Yamamoto S, Kitano Y, Takato T, Nakagawara A, Ushijima T:CpG island methylator phenotype is a strong determinant of poor prognosis in neuroblastomas. Cancer Res 2005,65(3): 828-834.
[18]Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, Peters JH, DeMeester SR, DeMeester TR, Skinner KA, et al.:Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 2001, 61(8):3410-3418.
[19]Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, Giovannucci EL, Fuchs CS:CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 2009, 58(1):90-96.
[20]Zhang C, Li Z, Cheng Y, Jia F, Li R, Wu M, Li K, Wei L:CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma. Clin Cancer Res 2007,13(3):944-952.
[21]Cheng Y, Zhang C, Zhao J, Wang C, Xu Y, Han Z, Jiang G, Guo X, Li R, Bu X, et al.:Correlation of CpG island methylator phenotype with poor prognosis in hepatocellular carcinoma. Exp Mol Pathol, in press.
[22]Yin D, Xie D, Hofmann WK, Miller CW, Black KL, Koeffler HP:Methylation, expression, and mutation analysis of the cell cycle control genes in human brain tumors. Oncogene 2002,21(54):8372-8377.
[23]Graziano F, Arduini F, Ruzzo A, Bearzi I, Humar B, More H, Silva R, Muretto P, Guilford P, Testa E, et al.:Prognostic analysis of E-cadherin gene promoter hypermethylation in patients with surgically resected, node-positive, diffuse gastric cancer. Clin Cancer Res 2004,10(8):2784-2789.
[24]Esteller M, Corn PG, Urena JM, Gabrielson E, Baylin SB, Herman JG: Inactivation of glutathone S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 1998,58(20):4515-4518.
[25]Kato K, Iida S, Uetake H, Takagi Y, Yamashita T, Inokuchi M, Yamada H, Kojima K, Sugihara K:Methylated TMS1 and DAPK genes predict prognosis and response to chemotherapy in gastric cancer. Int J Cancer 2008,122(3): 603-608.
[26]Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG:Inactivation of the DNA repair gene O6-methylguanine DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999,59(4):793-797.
[27]Byun DS, Cho K, Ryu BK, Lee MG, Kang MJ, Kim HR, Chi SG: Hypermethylation of XIAP-associated factor1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res 2003,63(21):7068-7075.
[28]Darnton SJ, Hardie LJ, Muc RS, Wild CP, Casson AG:Tissue inhibitor of metalloproteinase-3 (TIMP-3) gene is methylated in the development of esophageal adenocarcinoma:Loss of expression correlates with poor prognosis. Int J Cancer 2005,115(3):351-358.
[29]Yang B, Guo M, Herman JG, Clark DP:Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003, 163(3):1101-1107.
[30]Takagi H, Sasaki S, Suzuki H, Toyota M, Maruyama R, Nojima M, Yamamoto H, Omata M, Tokino T, Imai K, et al.:Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma. J Gastroenterol 2008,43(5):378-389.
[31]Sato H, Suzuki H, Toyota M, Nojima M, Maruyama R, Sasaki S, Takagi H, Sogabe Y, Sasaki Y, Idogawa M, et al.:Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors. Carcinogenesis 2007,28(12):2459-66.
[32]Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB:Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996,93(18):9821-9826.
[33]Toyota M, Ahuja N, Suzuki H, Itoh F, Ohe-Toyota M, Imai K, Baylin SB, Issa JP:Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res 1999,59(21):5438-5442.
[34]Oue N, Mitani Y, Motoshita J, Matsumura S, Yoshida K, Kuniyasu H, Nakayama H, Yasui W:Accumulation of DNA methylation is associated with tumor stage in gastric cancer. Cancer 2006,106(6):1250-1259.
[35]Ottini L, Falchetti M, Lupi R, Rizzolo P, Agnese V, Colucci G, Bazan V, Russo A:Patterns of genomic instability in gastric cancer:clinical implications and perspectives. Ann Oncol 2006,17(Suppl 7):vii97-102.
[36]Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D, Buchanan D et al.:CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006,38(7): 787-793.
[37]Yang Q, Kiernan CM, Tian Y, Salwen HR, Chlenski A, Brumback BA; London WB, Cohn SL:Methylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome. Clin Cancer Res 2007,13(11):3191-3197.
[38]Zheng SS, Xu X, Wu J, Chen J, Wang WL, Zhang M, Liang TB, Wu LM:Liver transplantation for hepatocellular carcinoma:Hangzhou experiences. Transplantation 2008,85(12):1726-1732.
[39]Cillo U, Vitale A, Bassanello M, Boccagni P, Brolese A, Zanus G, Burra P, Fagiuoli S, Farinati F, Rugge M, D'Amico DF:Liver transplantation for the treatment of moderately or well-differentiated hepatocellular carcinoma. Ann Surg 2004,239(2):150-159.
[1]Wolverton T, Lalande M. Identification and characterization of three members of a novel subclass of protocadherins. Genomics.2001; 76:66-72.
[2]Uemura M, Nakao S, Suzuki ST, et al. OL-Protocadherin is essential for growth of striatal axons and thalamocortical projections. Nat Neurosci.2007; 10:1151-9
[3]Hammond C, Jeffers L, Carr BI, Simon D. Multiple genetic alterations,4q28, a new suppressor region, and potential gender differences in human hepatocellular carcinoma. Hepatology 1999; 29:1479-85
[4]Knosel T, Schluns K, Stein U. et al. Chromosomal alterations during lymphatic and liver metastasis formation of colorectal cancer. Neoplasia.2004; 6:23-8.
[5]Matsui S, LaDuca J, Rossi MR, et al. Molecular characterization of a consistent 4.5-megabase deletion at 4q28 in prostate cancer cells. Cancer Genet Cytogenet 2005; 159:18-26.
[6]Nowak NJ, Gaile D, Conroy JM. et al. Genome-wide aberrations in pancreatic adenocarcinoma. Cancer Genet Cytogenet 2005; 161:36-50.
[7]Price GR, Armes JE, Ramus SJ etal. Phenotype-directed analysis of genotype in early-onset, familial breast cancers. Pathology 2006; 38:520-7.
[8]Ying J, Li H, Seng TJ et al. Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene 2006; 25: 1070-80.
[9]Miyamoto K, Fukutomi T, Akashi-Tanaka S et al. Identification of 20 genes aberrantly methylated in human breast cancers. Int J Cancer 2005; 116: 407-14.
[10]Ying J, Gao Z, Li H et al. Frequent epigenetic silencing of protocadherin 10 by methylation in multiple haematologic malignancies. Br J Haematol 2007; 136: 829-32.
[11]Yu J, Cheng YY, Tao Q. et al. Methylation of protocadherin 10, a novel tumor suppressor, is associated with poor prognosis in patients with gastric cancer. Gastroenterology 2008; 136:640-51
[12]Wang KH, Liu HW, Lin SR, et al. Field methylation silencing of the protocadherin 10 gene in cervical carcinogenesis as a potential specific diagnostic test from cervical scrapings. Cancer Sci.2009; 100(11):2175-80.
[13]Shelby RD, Hahn KM, Sullivan KF. Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells. J Cell Biol,1996,135(3):545.
[14]ZlokarnikG, Negulescu PA, Knapp TE, et al. Quantitation of transcription and clonal selection of single living cell with B-lactamase as reporter. Science. 1998; 279(5347):84.
[15]Jongho Sun,Gabriella H,Kelemen,et al.Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor. Microbiology.1999; 145:2221.
[16]Gumbiner BM, Yamada KM. Cell-to-cell contact and extracellular matrix. Curr Opin Cell Biol.1995; 7(5):615-8.
[17]Shapiro L, Fannon AM, Kwong PD. et al. Structural basis of cell-cell adhesion by cadherins. Nature.1995; 374(6520):327-37.
[18]Angst BD, Marcozzi C, Magee AI. et al. The cadherin superfamily:diversity in form and function. J Cell Sci.2001; 114:629-641.
[19]Yagi T, Takeichi M. Cadherin superfamily genes:functions, genomic organization, and neurologic diversity. Genes Dev.2000; 14(10):1169-80.
[20]Wu Q, Maniatis T. Large exons encoding multiple ectodomains are a characteristic feature of protocadherin genes. Proc Nat Acad Sci U S A.2000; 97(7):3124-3129.
[21]Frank M, Kemler R. Protocadherins. Curr Opin Cell Biol.2002; 14(5):557-62.
[22]Suzuki ST. Recent progress in protocadherin research. Exp Cell Res.2000; 261(1):13-18.
[23]Berx G, Cleton-Jansen AM, Nollet F, et al. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J.1995; 14(24):6107-6115.
[24]Perl AK, Wilgenbus P, Dahl U, et al. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature.1998; 392(6672):190-193.
[25]Ghaffari SR, Dastan J, Rafati M. et al. Novel human pathological mutations. Gene symbol:CDH1. Disease:gastric cancer. Hum Genet.2009; 125(3):337.
[26]Takeuchi T, Liang SB, Matsuyoshi N, et al. Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma. Lab Invest. 2002 Aug;82(8):1023-9.
[27]Gupta RA, Shah N, Wang KC, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature.2010; 464(7291):1071-1076.
[28]Su PF, Lee TC, Lin PJ, et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. Int J Cancer.2007; 121: 1257-1264.
[29]Lesurtel M, Mullhaupt B, Pestalozzi BC, et al. Transarterial chemoembolization as a bridge to liver transplantation for hepatocellular carcinoma:an evidence-based analysis. Am J Transplant.2006;6:2644-2650.
[30]Bharat A, Brown DB, Crippin JS, et al. Pre-liver transplantation locoregional adjuvant therapy for hepatocellular carcinoma as a strategy to improve longterm survival. J Am Coll Surg.2006;203:411-420.
[1]Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability and tumors promoted by DNA hypomethylation. Science,2003; 300(5618):455
[2]Lee S, Lee H J, Kim J H, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol,2003; 163(4):1371-1378
[3]Lunn R M, Zhang Y J, Wang L Y, et al. p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. Cancer Res,1997; 57:3471-3477
[4]de La Costa A, Romagnolo B, Billuart P, et al. Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci USA,1998; 95:8847-8851
[5]Matsuda Y, Ichida T, Matsuzawa J, et al. p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology,1999; 116:394-400
[6]Kwon GY, Yoo BC, Koh KC, et al. Promoter methylation of E-cadherin in hepatocellular carcinomas and dysplastic nodules. J Korean Med Sci,2005; 20 (2):242-247
[7]Huang J, Zhang Y L, Teng X M, et al. Down-regulation of SFRP1 as a tumor suppressor gene can contribute to human hepatocellular carcinoma. BMC Cancer,2007; 7:126
[8]Yang B, Guo M, Herman JG, Clark DP, et al. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003; 163(3):1101-1107.
[9]Weber M, Hellmann I, Stadler MB, et al. Distribution, sileneing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet,2007; 39(4):457-466
[10]Strathdee G, Brown R. Aberrant DNA methylation in cancer:potential clinical interventions. Expert Rev Mol Med,2002; 4(4):1-17
[11]Chalitchagorn K, Shuangshoti S, Hourpai N, et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene.2004; 23(54):8841-8846
[12]Sato N, Fukushima N, Maehara N, et al. SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor-stromal interactions. Oncogene.2003; 22(32):5021-5030
[13]Kim Y I, Giuliano A, Hatch K D, et al. Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer.1994; 74:893-899
[14]Bedford MT, van Helden PD, et al. Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Res.1987; 47:5274-5276
[15]Strathdee G, MacKean M J, Illand M, et al. A role for methylation of the hMLH1 promoter in loss of hMLH1 expression and drug resistance in ovarian cancer. Oncogene.1999; 18:2335-2341
[16]Ushijima T, Morimura K, Hosoya Y, et al. Establishment of methylation sensitive representational difference analysis and isolation of hypo-and hypermethylated genomic fragments in mouse liver tumors. Proc Natl Acad Sci USA.1997; 94(6):2284-2289
[17]Calvisi DF, Ladu S, Gorden A, et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma.J Clin Invest.2007; 117(9):2713-2722
[18]Lin CH, Hsieh SY, Sheen IS, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res.2001; 61:4238-4243
[19]Chen R Z, Pettersson U, Beard C, et al. DNA hypomethylation leads to elevated mutation rates. Nature,1998; 395(6697):89-93
[20]Gaudet F, Hodgson JG, Edcn A, et al. Induction of tumors in mice by genomic hypomethylation. Science,2003,300(5618):489-492
[21]Florl AR, Lower R, Schmitz-Drager B J, et al. DNA methylation and expression of LINE-1 and HERV-k provirus sequences in urothelial and renal cell carcinomas. Br J Cancer.1999; 80:1312-1321
[22]Takai D, Yagi Y, Habib N, et al. Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis. Jpn J Clin Oncol.2000; 30(7):306-309
[23]Saito Y, Kanai Y, Sakamoto M, et al. Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis. Hepatology.2001; 33(3):561-568
[24]Nambu S, Inoue K, Saski H, et al. Site-specific hypomethylation of the c-myc oncogene in human hepatocellular carcinoma. Jpn J Cancer Res.1987; 78(7):695-704
[25]Tao L, Li Y, Kramer PM, et al. Hypomethylation of DNA and the insulin-like growth factor-Ⅱ gene in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Toxicology.2004; 196(1-2):127-36
[26]Okada H, Kimura MT, Tan D, et al. Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcnomas. Int J Oncol.2005; 26(2):369-377
[27]Sherr CJ. Cancer cell cycles. Science.1996; 274(5293):1672-1677
[28]Liew CT, LI HM, Lo KW, et al. High frequency of P16INK4A gene alterations in hepatocellular carcinoma. Oncogene.1999; 18(3):789-795
[29]Chanbert P, Gayer R, Zimmermann A, et al. Germ-line mutations of the P16INK4(MTS1) gene occur in a subset of patients with hepatocellular carcinoma. Hepatology.1997; 25(6):1376-1381
[30]Baek MJ, Piao BS, Kim NG, et al. P16 is a major inactivation target in Hepatocellular Carcinoma. Cancer.2000; 89(1):60-68.
[31]Anzola M, Cuevas N, Lopez-Martinez M, et al. P16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy. J Gastroenterol Hepatol.2004; 19(4):397-405
[32]Yu J, Ni M,Xu J, eta al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC Cancer. 2002,2:29.
[33]Narimatsu T, Tamori A, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervimlogy, 2004,47(1):26-31
[34]CohenO, Feinstein E, Kimehi A, et al. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO.1997; 16(5):998-1008.
[35]KimWS, Son HJ, Park JO, et al. Promoter methylation and down-regulation of DAPK is associated with gastric atrophy. Int J Mol Med.2003; 12(6):827-830
[36]Herman JG, Baylin SB.Gene silencing in cancer is association with promoter hypermethylation. N Engl J Med.2003; 349(21):2042-2054.
[37]Yang IT, Liu SV, Wang Y, et al. Detection of hypermethylated genes in tumor and plasma of cervical cancer patients. Gynecol Oncol.2004,93(2):435-440
[38]Toyooka S, Toyooka KO, Miyajima K, et al. Epigenetic down-regulation of death-associated protein kinase in lung cancers. Clinical Cancer Research. 2003; 9(8):3034-3041
[39]Lehmann U, Berg-Ribbe I, Wingen LU, et al. Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling. Cancer Res.2005; 11(10):3654-3660
[40]钱波,朱立新,耿小平等.肝细胞癌MGMT、DAPK、 THBS1和RIZI基因 的甲基化研究。中华普通外科杂志.2005；20(5)：291-294
[41]林勃,陈龙邦,唐永明.原发性肝癌患者血清死亡相关蛋白激酶基因启动子甲基化的检测.中西医结合肝病杂志.2006；16(3)：167-169
[42]Kato K, Inda S, Uetake H, et al. Methylated TMS 1 and DAPK genes predict prognosis and response to chemotherapy in gastric cancer. Int J Cancer.2008; 122:603-608
[43]Tchou JC, Lin X, Freije D, et al. GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. Int J Oncol.2000; 16(4):663-676
[44]Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol.2003; 163(4):1371-1378
[45]Yoshikawa H, Matsubara K, Qian GS, et al. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet.2001; 28(1): 29-35
[46]Okochi O, Hi K, Sakai M, et al.Methylation-mediated silencing of SOCS-1 gene in hepatocellular Carcinoma derived from cirrhosis. Clin Cancer Res. 2003; 9(14):5295-5298
[47]Calvisi DF, Ladu S, Gorden A, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology.2006; 130(4):1117-1128
[48]Plenchette S, Cheung HH, Fong WG, et al. The role of XAF1 in cancer. Curr Opin Investig Drugs.2007; 8(6):469-76
[49]Byun DS, Cho K, Ryu BK, et al. Hypermethylation of XIAP-associated factor 1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res 2003;63:7068-7075.
[50]Fang X, Liu Z, Fan Y, et al. Switch to full-length of XAF1 mRNA expression in prostate cancer cells by the DNA methylation inhibitor. Int J Cancer.2006; 118:2485-2489.
[51]Wang J, Peng Y, Sun YW, et al.All-trans retinoic acid induces XAF1 expression through an interferon regulatory factor-1 element in colon cancer. Gastroenterology.2006; 130:747-58.
[52]Lee MG, Huh JS, Chung SK, et al. Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses. Oncogene.2006; 25:5807-5822.
[53]Zou B, Chim CS, Zeng H, et al.Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers. Gastroenterology.2006; 131:1835-1843.
[54]Chung SK, Lee MG, Ryu BK, et al. Frequent alteration of XAF1 in human colorectal cancers:implication for tumor cell resistance to apoptotic stresses. Gastroenterology.2007; 132:2459-2477
[55]Kempkensteffen C, Hinz S, Schrader M, et al. Gene expression and promoter methylation of the XIAP-associated Factor 1 in renal cell carcinomas: correlations with pathology and outcome. Cancer Lett.2007; 254:227-235.
[56]Zhang F, Wu LM, Zhou L, et al. Predictive value of expression and promoter hypermethylation of XAF1 in hepatitis B virus-associated hepatocellular carcinoma treated with transplantation. Ann Surg Oncol.2008; 15(12): 3494-3502.
[57]Kuno Y, Abe A, Emi N, et al. Constitutive kinase activation of the TEL-Syk fusion gene in myelodysplastic syndrome with t(9;12) (q22;p12). Blood.2001; 97(4):1050-1053
[58]Hoeller C, Thallinger C, Pratscher B, et al. The non-receptor-associated tyrosine kinase Syk is a regulator of metastatic behavior in human melanoma cells. J Invest Dermatol 2005; 124:1293-1299.
[59]Coopman PJ, Do MT, Barth M, et al. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells. Nature 2000; 406:742-747.
[60]Yuan Y, Wang J, Li J, Wang L, et al. Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res. 2006; 12(22):6687-6695.
[61]Wei Y, Van Nhieu JT, Prigent S, et al. Altered expression of E-cadherin in hepatocellular carcinoma:correlations with genetic alterations, beta-catenin expression, and clinical features. Hepatology.2002; 36 (3):692-701
[62]Fukunaga Y, Liu H, Shimizu M, et al. Defining the roles of beta-catenin and plakoglobin in cell-cell adhesion:isolation of beta-catenin/plakoglobin-deficient F9 cells. Cell Struct Funct.2005; 30 (2):25-34
[63]Shen X, Kramer RH. Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor. Am J Pathol.2004; 165(4):1315-1329
[64]Fiorentino M, Altimari A, Ravaioli M, et al. Predictive value of biological markers for hepatocellular carcinoma patients treated with orthotopic livertransplantation. Clin Cancer Res.2004; 10(5):1789-1795
[65]Kanai Y, Saito Y, Ushijima S, et al. Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis. J Cancer Res Clin Oncol. 2004; 130(11):636-644
[66]Saito Y, Kanai Y, Sakamoto M, et al. Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis. Proc Natl Acad Sci USA.2002; 99(15):10060-10065
[67]Oh BK, Kim H, Park HJ, et al. DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation. Int J Mol Med.2007; 20(1):65-73.
[68]Park IY, Chung YH, Surzycki SJ, et al. Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology. 2007; 132(4):1476-1494
[69]Kihslinger JE, Godley LA. The use of hypomethylating agents in the treatment of hematologic malignancies. Leuk Lymphoma.2007; 48(9):1676-1695.
[70]Cheng JC, Weisenberger DJ, Gongzales FA, et al. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol.2004; 24(3):1270-1278.
[71]Ben-Kasus T, Ben-Zv IZ, Marquez VE, et al. Metabolic activation of zebularine, a novel DNA methylation inhibitor in human bladder carcinoma cells. Biochem Pharmacol.2005; 70(1):121-133.
[72]LaiIberte J, Marquezv E, Momparler RL. Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2'-deoxycytidine by human cytidine deaminase. Cancer Chemother Pharmacol.1992; 30(1):7-11.
[73]Villar-Garea A, Fraga MF, Espada J, et al. Procaine is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells.Cancer Res.2003; 63(16):4984-4989
[74]Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med.2001; 134 (7): 573-586
[2]El-Serag HB, Rudolph KL:Hepatocellular carcinoma:epidemiology and molecular carcinogenesis. Gastroenterology 2007,132(7):2557-76.
[3]Wild CP, Hall AJ:Primary prevention of hepatocellular carcinoma in developing countries. Mutat Res 2000,462(2-3):381-393.
[4]Yoo HY, Patt CH, Geschwind JF, Thuluvath PJ:The outcome of liver transplantation in patients with hepatocellular carcinoma in the United States between 1988 and 2001:5-year survival has improved significantly with time. J Clin Oncol 2003,21(23):4329-4335.
[5]Zhang F, Wu LM, Zhou L, Chen QX, Xie HY, Feng XW, Zheng SS:Predictive value of expression and promoter hypermethylation of XAF1 in hepatitis B virus-associated hepatocellular carcinoma treated with transplantation. Ann Surg Oncol 2008,15(12):3494-3502.
[6]Wu L, Xu X, Shen J, Xie H, Yu S, Liang T, Wang W, Shen Y, Zhang M, Zheng S:MDR1 gene polymorphisms and risk of recurrence in patients with hepatocellular carcinoma after liver transplantation. J Surg Oncol 2007,96(1): 62-68.
[7]Wu LM, Zhang F, Xie HY, Xu X, Chen QX, Yin SY, Liu XC, Zhou L, Xu XB, Sun YL, et al.:MMP2 promoter polymorphism (C-1306T) and risk of recurrence in patients with hepatocellular carcinoma after transplantation. Clin Genet 2008,73(3):273-278.
[8]Herman JG, Baylin SB:Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003,349(21):2042-2054.
[9]de Maat MF, Umetani N, Sunami E, Turner RR, Hoon DS:Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles. Mol Cancer Res 2007,5(5): 461-471.
[10]Kusano M, Toyota M, Suzuki H, Akino K, Aoki F, Fujita M, Hosokawa M, Shinomura Y, Imai K, Tokino T:Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus. Cancer 2006,106(7):1467-1479.
[11]Ogino S, Nosho K, Irahara N, Meyerhardt JA, Baba Y, Shima K, Glickman JN, Ferrone CR, Mino-Kenudson M, Tanaka N, et al:Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and CpG island methylator phenotype. Clin Cancer Res 2009,15(20):6412-6420.
[12]Yuan Y, Wang J, Li J, Wang L, Li M, Yang Z, Zhang C, Dai JL:Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res 2006,12(22):6687-6695.
[13]Yamada S, Nomoto S, Fujii T, Takeda S, Kanazumi N, Sugimoto H, Nakao A: Frequent promoter methylation of M-cadherin in hepatocellular carcinoma is associated with poor prognosis. Anticancer Res 2007,27(4B):2269-2274.
[14]Tanemura A, Terando AM, Sim MS, van Hoesel AQ, de Maat MF, Morton DL, Hoon DS:CpG island methylator phenotype predicts progression of malignant melanoma. Clin Cancer Res 2009,15(5):1801-1807.
[15]Liu Z, Zhao J, Chen XF, Li W, Liu R, Lei Z, Liu X, Peng X, Xu K, Chen J, et al.:CpG island methylator phenotype involving tumor suppressor genes located on chromosome 3p in non-small cell lung cancer. Lung cancer 2008, 62(1):15-22.
[16]Roman-Gomez J, Jimenez-Velasco A, Agirre X, Prosper F, Heiniger A, Torres A:Lack of CpG island methylator phenotype defines a clinical subtype of T-cell acute lymphoblastic leukemia associated with good prognosis. J Clin Oncol 2005,23(28):7043-7049.
[17]Abe M, Ohira M, Kaneda A, Yagi Y, Yamamoto S, Kitano Y, Takato T, Nakagawara A, Ushijima T:CpG island methylator phenotype is a strong determinant of poor prognosis in neuroblastomas. Cancer Res 2005,65(3): 828-834.
[18]Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, Peters JH, DeMeester SR, DeMeester TR, Skinner KA, et al.:Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 2001, 61(8):3410-3418.
[19]Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, Giovannucci EL, Fuchs CS:CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 2009, 58(1):90-96.
[20]Zhang C, Li Z, Cheng Y, Jia F, Li R, Wu M, Li K, Wei L:CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma. Clin Cancer Res 2007,13(3):944-952.
[21]Cheng Y, Zhang C, Zhao J, Wang C, Xu Y, Han Z, Jiang G, Guo X, Li R, Bu X, et al.:Correlation of CpG island methylator phenotype with poor prognosis in hepatocellular carcinoma. Exp Mol Pathol, in press.
[22]Yin D, Xie D, Hofmann WK, Miller CW, Black KL, Koeffler HP:Methylation, expression, and mutation analysis of the cell cycle control genes in human brain tumors. Oncogene 2002,21(54):8372-8377.
[23]Graziano F, Arduini F, Ruzzo A, Bearzi I, Humar B, More H, Silva R, Muretto P, Guilford P, Testa E, et al.:Prognostic analysis of E-cadherin gene promoter hypermethylation in patients with surgically resected, node-positive, diffuse gastric cancer. Clin Cancer Res 2004,10(8):2784-2789.
[24]Esteller M, Corn PG, Urena JM, Gabrielson E, Baylin SB, Herman JG: Inactivation of glutathone S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 1998,58(20):4515-4518.
[25]Kato K, Iida S, Uetake H, Takagi Y, Yamashita T, Inokuchi M, Yamada H, Kojima K, Sugihara K:Methylated TMS1 and DAPK genes predict prognosis and response to chemotherapy in gastric cancer. Int J Cancer 2008,122(3): 603-608.
[26]Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG:Inactivation of the DNA repair gene O6-methylguanine DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999,59(4):793-797.
[27]Byun DS, Cho K, Ryu BK, Lee MG, Kang MJ, Kim HR, Chi SG: Hypermethylation of XIAP-associated factor1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res 2003,63(21):7068-7075.
[28]Darnton SJ, Hardie LJ, Muc RS, Wild CP, Casson AG:Tissue inhibitor of metalloproteinase-3 (TIMP-3) gene is methylated in the development of esophageal adenocarcinoma:Loss of expression correlates with poor prognosis. Int J Cancer 2005,115(3):351-358.
[29]Yang B, Guo M, Herman JG, Clark DP:Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003, 163(3):1101-1107.
[30]Takagi H, Sasaki S, Suzuki H, Toyota M, Maruyama R, Nojima M, Yamamoto H, Omata M, Tokino T, Imai K, et al.:Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma. J Gastroenterol 2008,43(5):378-389.
[31]Sato H, Suzuki H, Toyota M, Nojima M, Maruyama R, Sasaki S, Takagi H, Sogabe Y, Sasaki Y, Idogawa M, et al.:Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors. Carcinogenesis 2007,28(12):2459-66.
[32]Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB:Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996,93(18):9821-9826.
[33]Toyota M, Ahuja N, Suzuki H, Itoh F, Ohe-Toyota M, Imai K, Baylin SB, Issa JP:Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res 1999,59(21):5438-5442.
[34]Oue N, Mitani Y, Motoshita J, Matsumura S, Yoshida K, Kuniyasu H, Nakayama H, Yasui W:Accumulation of DNA methylation is associated with tumor stage in gastric cancer. Cancer 2006,106(6):1250-1259.
[35]Ottini L, Falchetti M, Lupi R, Rizzolo P, Agnese V, Colucci G, Bazan V, Russo A:Patterns of genomic instability in gastric cancer:clinical implications and perspectives. Ann Oncol 2006,17(Suppl 7):vii97-102.
[36]Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D, Buchanan D et al.:CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006,38(7): 787-793.
[37]Yang Q, Kiernan CM, Tian Y, Salwen HR, Chlenski A, Brumback BA; London WB, Cohn SL:Methylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome. Clin Cancer Res 2007,13(11):3191-3197.
[38]Zheng SS, Xu X, Wu J, Chen J, Wang WL, Zhang M, Liang TB, Wu LM:Liver transplantation for hepatocellular carcinoma:Hangzhou experiences. Transplantation 2008,85(12):1726-1732.
[39]Cillo U, Vitale A, Bassanello M, Boccagni P, Brolese A, Zanus G, Burra P, Fagiuoli S, Farinati F, Rugge M, D'Amico DF:Liver transplantation for the treatment of moderately or well-differentiated hepatocellular carcinoma. Ann Surg 2004,239(2):150-159.
[1]Wolverton T, Lalande M. Identification and characterization of three members of a novel subclass of protocadherins. Genomics.2001; 76:66-72.
[2]Uemura M, Nakao S, Suzuki ST, et al. OL-Protocadherin is essential for growth of striatal axons and thalamocortical projections. Nat Neurosci.2007; 10:1151-9
[3]Hammond C, Jeffers L, Carr BI, Simon D. Multiple genetic alterations,4q28, a new suppressor region, and potential gender differences in human hepatocellular carcinoma. Hepatology 1999; 29:1479-85
[4]Knosel T, Schluns K, Stein U. et al. Chromosomal alterations during lymphatic and liver metastasis formation of colorectal cancer. Neoplasia.2004; 6:23-8.
[5]Matsui S, LaDuca J, Rossi MR, et al. Molecular characterization of a consistent 4.5-megabase deletion at 4q28 in prostate cancer cells. Cancer Genet Cytogenet 2005; 159:18-26.
[6]Nowak NJ, Gaile D, Conroy JM. et al. Genome-wide aberrations in pancreatic adenocarcinoma. Cancer Genet Cytogenet 2005; 161:36-50.
[7]Price GR, Armes JE, Ramus SJ etal. Phenotype-directed analysis of genotype in early-onset, familial breast cancers. Pathology 2006; 38:520-7.
[8]Ying J, Li H, Seng TJ et al. Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene 2006; 25: 1070-80.
[9]Miyamoto K, Fukutomi T, Akashi-Tanaka S et al. Identification of 20 genes aberrantly methylated in human breast cancers. Int J Cancer 2005; 116: 407-14.
[10]Ying J, Gao Z, Li H et al. Frequent epigenetic silencing of protocadherin 10 by methylation in multiple haematologic malignancies. Br J Haematol 2007; 136: 829-32.
[11]Yu J, Cheng YY, Tao Q. et al. Methylation of protocadherin 10, a novel tumor suppressor, is associated with poor prognosis in patients with gastric cancer. Gastroenterology 2008; 136:640-51
[12]Wang KH, Liu HW, Lin SR, et al. Field methylation silencing of the protocadherin 10 gene in cervical carcinogenesis as a potential specific diagnostic test from cervical scrapings. Cancer Sci.2009; 100(11):2175-80.
[13]Shelby RD, Hahn KM, Sullivan KF. Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells. J Cell Biol,1996,135(3):545.
[14]ZlokarnikG, Negulescu PA, Knapp TE, et al. Quantitation of transcription and clonal selection of single living cell with B-lactamase as reporter. Science. 1998; 279(5347):84.
[15]Jongho Sun,Gabriella H,Kelemen,et al.Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor. Microbiology.1999; 145:2221.
[16]Gumbiner BM, Yamada KM. Cell-to-cell contact and extracellular matrix. Curr Opin Cell Biol.1995; 7(5):615-8.
[17]Shapiro L, Fannon AM, Kwong PD. et al. Structural basis of cell-cell adhesion by cadherins. Nature.1995; 374(6520):327-37.
[18]Angst BD, Marcozzi C, Magee AI. et al. The cadherin superfamily:diversity in form and function. J Cell Sci.2001; 114:629-641.
[19]Yagi T, Takeichi M. Cadherin superfamily genes:functions, genomic organization, and neurologic diversity. Genes Dev.2000; 14(10):1169-80.
[20]Wu Q, Maniatis T. Large exons encoding multiple ectodomains are a characteristic feature of protocadherin genes. Proc Nat Acad Sci U S A.2000; 97(7):3124-3129.
[21]Frank M, Kemler R. Protocadherins. Curr Opin Cell Biol.2002; 14(5):557-62.
[22]Suzuki ST. Recent progress in protocadherin research. Exp Cell Res.2000; 261(1):13-18.
[23]Berx G, Cleton-Jansen AM, Nollet F, et al. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J.1995; 14(24):6107-6115.
[24]Perl AK, Wilgenbus P, Dahl U, et al. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature.1998; 392(6672):190-193.
[25]Ghaffari SR, Dastan J, Rafati M. et al. Novel human pathological mutations. Gene symbol:CDH1. Disease:gastric cancer. Hum Genet.2009; 125(3):337.
[26]Takeuchi T, Liang SB, Matsuyoshi N, et al. Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma. Lab Invest. 2002 Aug;82(8):1023-9.
[27]Gupta RA, Shah N, Wang KC, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature.2010; 464(7291):1071-1076.
[28]Su PF, Lee TC, Lin PJ, et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. Int J Cancer.2007; 121: 1257-1264.
[29]Lesurtel M, Mullhaupt B, Pestalozzi BC, et al. Transarterial chemoembolization as a bridge to liver transplantation for hepatocellular carcinoma:an evidence-based analysis. Am J Transplant.2006;6:2644-2650.
[30]Bharat A, Brown DB, Crippin JS, et al. Pre-liver transplantation locoregional adjuvant therapy for hepatocellular carcinoma as a strategy to improve longterm survival. J Am Coll Surg.2006;203:411-420.
[1]Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability and tumors promoted by DNA hypomethylation. Science,2003; 300(5618):455
[2]Lee S, Lee H J, Kim J H, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol,2003; 163(4):1371-1378
[3]Lunn R M, Zhang Y J, Wang L Y, et al. p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. Cancer Res,1997; 57:3471-3477
[4]de La Costa A, Romagnolo B, Billuart P, et al. Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci USA,1998; 95:8847-8851
[5]Matsuda Y, Ichida T, Matsuzawa J, et al. p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology,1999; 116:394-400
[6]Kwon GY, Yoo BC, Koh KC, et al. Promoter methylation of E-cadherin in hepatocellular carcinomas and dysplastic nodules. J Korean Med Sci,2005; 20 (2):242-247
[7]Huang J, Zhang Y L, Teng X M, et al. Down-regulation of SFRP1 as a tumor suppressor gene can contribute to human hepatocellular carcinoma. BMC Cancer,2007; 7:126
[8]Yang B, Guo M, Herman JG, Clark DP, et al. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003; 163(3):1101-1107.
[9]Weber M, Hellmann I, Stadler MB, et al. Distribution, sileneing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet,2007; 39(4):457-466
[10]Strathdee G, Brown R. Aberrant DNA methylation in cancer:potential clinical interventions. Expert Rev Mol Med,2002; 4(4):1-17
[11]Chalitchagorn K, Shuangshoti S, Hourpai N, et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene.2004; 23(54):8841-8846
[12]Sato N, Fukushima N, Maehara N, et al. SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor-stromal interactions. Oncogene.2003; 22(32):5021-5030
[13]Kim Y I, Giuliano A, Hatch K D, et al. Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer.1994; 74:893-899
[14]Bedford MT, van Helden PD, et al. Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Res.1987; 47:5274-5276
[15]Strathdee G, MacKean M J, Illand M, et al. A role for methylation of the hMLH1 promoter in loss of hMLH1 expression and drug resistance in ovarian cancer. Oncogene.1999; 18:2335-2341
[16]Ushijima T, Morimura K, Hosoya Y, et al. Establishment of methylation sensitive representational difference analysis and isolation of hypo-and hypermethylated genomic fragments in mouse liver tumors. Proc Natl Acad Sci USA.1997; 94(6):2284-2289
[17]Calvisi DF, Ladu S, Gorden A, et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma.J Clin Invest.2007; 117(9):2713-2722
[18]Lin CH, Hsieh SY, Sheen IS, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res.2001; 61:4238-4243
[19]Chen R Z, Pettersson U, Beard C, et al. DNA hypomethylation leads to elevated mutation rates. Nature,1998; 395(6697):89-93
[20]Gaudet F, Hodgson JG, Edcn A, et al. Induction of tumors in mice by genomic hypomethylation. Science,2003,300(5618):489-492
[21]Florl AR, Lower R, Schmitz-Drager B J, et al. DNA methylation and expression of LINE-1 and HERV-k provirus sequences in urothelial and renal cell carcinomas. Br J Cancer.1999; 80:1312-1321
[22]Takai D, Yagi Y, Habib N, et al. Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis. Jpn J Clin Oncol.2000; 30(7):306-309
[23]Saito Y, Kanai Y, Sakamoto M, et al. Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis. Hepatology.2001; 33(3):561-568
[24]Nambu S, Inoue K, Saski H, et al. Site-specific hypomethylation of the c-myc oncogene in human hepatocellular carcinoma. Jpn J Cancer Res.1987; 78(7):695-704
[25]Tao L, Li Y, Kramer PM, et al. Hypomethylation of DNA and the insulin-like growth factor-Ⅱ gene in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Toxicology.2004; 196(1-2):127-36
[26]Okada H, Kimura MT, Tan D, et al. Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcnomas. Int J Oncol.2005; 26(2):369-377
[27]Sherr CJ. Cancer cell cycles. Science.1996; 274(5293):1672-1677
[28]Liew CT, LI HM, Lo KW, et al. High frequency of P16INK4A gene alterations in hepatocellular carcinoma. Oncogene.1999; 18(3):789-795
[29]Chanbert P, Gayer R, Zimmermann A, et al. Germ-line mutations of the P16INK4(MTS1) gene occur in a subset of patients with hepatocellular carcinoma. Hepatology.1997; 25(6):1376-1381
[30]Baek MJ, Piao BS, Kim NG, et al. P16 is a major inactivation target in Hepatocellular Carcinoma. Cancer.2000; 89(1):60-68.
[31]Anzola M, Cuevas N, Lopez-Martinez M, et al. P16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy. J Gastroenterol Hepatol.2004; 19(4):397-405
[32]Yu J, Ni M,Xu J, eta al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC Cancer. 2002,2:29.
[33]Narimatsu T, Tamori A, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervimlogy, 2004,47(1):26-31
[34]CohenO, Feinstein E, Kimehi A, et al. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO.1997; 16(5):998-1008.
[35]KimWS, Son HJ, Park JO, et al. Promoter methylation and down-regulation of DAPK is associated with gastric atrophy. Int J Mol Med.2003; 12(6):827-830
[36]Herman JG, Baylin SB.Gene silencing in cancer is association with promoter hypermethylation. N Engl J Med.2003; 349(21):2042-2054.
[37]Yang IT, Liu SV, Wang Y, et al. Detection of hypermethylated genes in tumor and plasma of cervical cancer patients. Gynecol Oncol.2004,93(2):435-440
[38]Toyooka S, Toyooka KO, Miyajima K, et al. Epigenetic down-regulation of death-associated protein kinase in lung cancers. Clinical Cancer Research. 2003; 9(8):3034-3041
[39]Lehmann U, Berg-Ribbe I, Wingen LU, et al. Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling. Cancer Res.2005; 11(10):3654-3660
[40]钱波,朱立新,耿小平等.肝细胞癌MGMT、DAPK、 THBS1和RIZI基因 的甲基化研究。中华普通外科杂志.2005；20(5)：291-294
[41]林勃,陈龙邦,唐永明.原发性肝癌患者血清死亡相关蛋白激酶基因启动子甲基化的检测.中西医结合肝病杂志.2006；16(3)：167-169
[42]Kato K, Inda S, Uetake H, et al. Methylated TMS 1 and DAPK genes predict prognosis and response to chemotherapy in gastric cancer. Int J Cancer.2008; 122:603-608
[43]Tchou JC, Lin X, Freije D, et al. GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. Int J Oncol.2000; 16(4):663-676
[44]Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol.2003; 163(4):1371-1378
[45]Yoshikawa H, Matsubara K, Qian GS, et al. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet.2001; 28(1): 29-35
[46]Okochi O, Hi K, Sakai M, et al.Methylation-mediated silencing of SOCS-1 gene in hepatocellular Carcinoma derived from cirrhosis. Clin Cancer Res. 2003; 9(14):5295-5298
[47]Calvisi DF, Ladu S, Gorden A, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology.2006; 130(4):1117-1128
[48]Plenchette S, Cheung HH, Fong WG, et al. The role of XAF1 in cancer. Curr Opin Investig Drugs.2007; 8(6):469-76
[49]Byun DS, Cho K, Ryu BK, et al. Hypermethylation of XIAP-associated factor 1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res 2003;63:7068-7075.
[50]Fang X, Liu Z, Fan Y, et al. Switch to full-length of XAF1 mRNA expression in prostate cancer cells by the DNA methylation inhibitor. Int J Cancer.2006; 118:2485-2489.
[51]Wang J, Peng Y, Sun YW, et al.All-trans retinoic acid induces XAF1 expression through an interferon regulatory factor-1 element in colon cancer. Gastroenterology.2006; 130:747-58.
[52]Lee MG, Huh JS, Chung SK, et al. Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses. Oncogene.2006; 25:5807-5822.
[53]Zou B, Chim CS, Zeng H, et al.Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers. Gastroenterology.2006; 131:1835-1843.
[54]Chung SK, Lee MG, Ryu BK, et al. Frequent alteration of XAF1 in human colorectal cancers:implication for tumor cell resistance to apoptotic stresses. Gastroenterology.2007; 132:2459-2477
[55]Kempkensteffen C, Hinz S, Schrader M, et al. Gene expression and promoter methylation of the XIAP-associated Factor 1 in renal cell carcinomas: correlations with pathology and outcome. Cancer Lett.2007; 254:227-235.
[56]Zhang F, Wu LM, Zhou L, et al. Predictive value of expression and promoter hypermethylation of XAF1 in hepatitis B virus-associated hepatocellular carcinoma treated with transplantation. Ann Surg Oncol.2008; 15(12): 3494-3502.
[57]Kuno Y, Abe A, Emi N, et al. Constitutive kinase activation of the TEL-Syk fusion gene in myelodysplastic syndrome with t(9;12) (q22;p12). Blood.2001; 97(4):1050-1053
[58]Hoeller C, Thallinger C, Pratscher B, et al. The non-receptor-associated tyrosine kinase Syk is a regulator of metastatic behavior in human melanoma cells. J Invest Dermatol 2005; 124:1293-1299.
[59]Coopman PJ, Do MT, Barth M, et al. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells. Nature 2000; 406:742-747.
[60]Yuan Y, Wang J, Li J, Wang L, et al. Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res. 2006; 12(22):6687-6695.
[61]Wei Y, Van Nhieu JT, Prigent S, et al. Altered expression of E-cadherin in hepatocellular carcinoma:correlations with genetic alterations, beta-catenin expression, and clinical features. Hepatology.2002; 36 (3):692-701
[62]Fukunaga Y, Liu H, Shimizu M, et al. Defining the roles of beta-catenin and plakoglobin in cell-cell adhesion:isolation of beta-catenin/plakoglobin-deficient F9 cells. Cell Struct Funct.2005; 30 (2):25-34
[63]Shen X, Kramer RH. Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor. Am J Pathol.2004; 165(4):1315-1329
[64]Fiorentino M, Altimari A, Ravaioli M, et al. Predictive value of biological markers for hepatocellular carcinoma patients treated with orthotopic livertransplantation. Clin Cancer Res.2004; 10(5):1789-1795
[65]Kanai Y, Saito Y, Ushijima S, et al. Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis. J Cancer Res Clin Oncol. 2004; 130(11):636-644
[66]Saito Y, Kanai Y, Sakamoto M, et al. Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis. Proc Natl Acad Sci USA.2002; 99(15):10060-10065
[67]Oh BK, Kim H, Park HJ, et al. DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation. Int J Mol Med.2007; 20(1):65-73.
[68]Park IY, Chung YH, Surzycki SJ, et al. Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology. 2007; 132(4):1476-1494
[69]Kihslinger JE, Godley LA. The use of hypomethylating agents in the treatment of hematologic malignancies. Leuk Lymphoma.2007; 48(9):1676-1695.
[70]Cheng JC, Weisenberger DJ, Gongzales FA, et al. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol.2004; 24(3):1270-1278.
[71]Ben-Kasus T, Ben-Zv IZ, Marquez VE, et al. Metabolic activation of zebularine, a novel DNA methylation inhibitor in human bladder carcinoma cells. Biochem Pharmacol.2005; 70(1):121-133.
[72]LaiIberte J, Marquezv E, Momparler RL. Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2'-deoxycytidine by human cytidine deaminase. Cancer Chemother Pharmacol.1992; 30(1):7-11.
[73]Villar-Garea A, Fraga MF, Espada J, et al. Procaine is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells.Cancer Res.2003; 63(16):4984-4989
[74]Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med.2001; 134 (7): 573-586