胃癌中若干肿瘤抑制基因异常甲基化的研究
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摘要
胃癌中若干肿瘤抑制基因异常甲基化的研究
胃癌的发生与其他恶性肿瘤一样是多基因多阶段变异累积形成的病理过程，而抑癌基因的甲基化是一个研究的热点，目前已鉴定的易高甲基化的基因包括：参与细胞周期、DNA修复、耐药性形成、分化、凋亡的基因及参与肿瘤转移和血管生成的基因。此研究中选择了主要与细胞周期及细胞凋亡有关的基因Apaf-1，P27kip1，DAPK1，对胃癌组织中的DAPK1，Apaf-1，P27kip1基因甲基化状态、表达进行研究，以研究胃癌组织中此三种基因表达异常的原因，探讨基因甲基化在胃癌发病中的作用。在对胃癌细胞系的研究中除对此三个基因的甲基化状态进行进一步研究，在去甲基化药物5-aza-CdR作用细胞后，亦检测目的基因的甲基化状态、表达的改变及与细胞生长状态的关系，进一步探讨抑癌基因的表达与其甲基化状态的相关性。顺铂(CDDP)作为治疗多种实体瘤临床一线用药，是胃癌的常用化疗药物，5-aza-CdR是明确的去甲基化的药物，本实验从这两株胃腺癌细胞的抑癌基因的高甲基化状态入手，通过对低剂量的顺铂与5-aza-CdR的联合用药以观察两者对胃癌细胞生长的影响，探讨5-aza-CdR是否能够作为一线化疗药物顺铂的辅助用药，使顺铂在较低剂量时发挥较大的作用。
材料与方法
1、实验材料
中国医科大学附属第一医院及中国医科大学附属第二医院胃癌患者术后胃癌组织、胃癌旁组织；标本细胞株：胃癌BGC823细胞系，胃癌SGC7901细胞系(均购自细胞生物教研室)；总RNA提取试剂：TRIZOL Reagent(GIBCO BRL公司)，RNAout(TaKaRa公司)，去甲基化试剂5-aza-2’-deoxycitydine(5-Aza-CdR)为Sigma产品，碘化丙啶(PI)、丫啶橙(AO)为Sigma产品，RT-PCR试剂盒购自TaKaRa公司，Wizard DNA Clean-up(Promega公司)，Annexin-v-FITC凋亡检测试剂盒为北京宝赛生生物技术有限公司生产，RPMI 1640培养基为Hyclone产品，Apaf-1绵羊抗人单克隆抗体(Santa cruz产品)，Taq酶及dNTP均为TaKaRa公司产品，Apaf-1绵羊抗人单克隆抗体(Santa cruz产品)，p27kip1鼠抗人单克隆抗体(博士德公司产品)，驴抗羊抗体(Santa cruz产品)，羊抗鼠抗体(北京中杉金桥生物公司产品)。
2、实验方法
MTT法检测细胞生长活性，AO染色、Annexin-v-FITC凋亡流式细胞仪检测细胞凋亡；PI染色流式细胞仪检测细胞周期及凋亡的变化；用半定量RT-PCR方法，Western-Blot方法分析胃癌细胞及组织中各目的基因的表达，用MSP对胃癌细胞及组织中各基因的启动子区甲基化情况进行研究。用杂合性丢失(Loss of heterozygosity，LOH)分析胃癌患者Apaf-1基因所在的12q22-23区域出现缺失的情况。
3、统计分析
所有资料采用SPSS11.5分析。计量资料比较采用方差分析；两两比较采用LSD法；计数资料采用χ~2检验；凋亡率资料分析采用秩和检验，相关分析采用Spearman方法。
结果
一、胃癌组织中各基因的表达及甲基化状况
1、各目的基因在胃癌组织的表达
(1)RT-PCR检测基因mRNA表达：在胃癌组织中Apaf-1基因表达下调17／35例，p27kip1基因表达下调17／35例，DAPK1基因表达下调16／35例，此三组基因的癌旁组织的mRNA表达明显高于均癌组织，具有统计学意义(均P＜0.05)。
(2)Western Blot检测基因蛋白表达：在胃癌组织中Apaf-1基因在胃癌组织中5例(5／35)有蛋白表达，癌旁组织则有29例蛋白表达(29／35)，P＜0.05，P27kip1基因在胃癌组织中3例有蛋白表达(3／35)，癌旁组织有30例蛋白表达(30／35)，此两组基因癌旁组织的蛋白表达显著高于癌组织，均具有统计学意义(P＜0.05)。
(3)各基因启动子甲基化的情况DAPK1胃癌组织及癌旁组织中甲基化率均为100％，无统计学意义；P27kip1胃癌组织中甲基化率为51.42％明显高于癌旁组织(25.71％)，具有统计学意义(P＜0.05)，Apaf-1胃癌组织及癌旁组织中甲基化率分别为48.57％，22.86％，胃癌组织的甲基化率明显高于癌旁组织，具有统计学意义(P＜0.05)。
(4)胃癌中P27kip1、Apaf-1基因的甲基化状态与其表达的关系胃癌组织中17例具有Apaf-1基因甲基化的组织16例有mRNA表达下调，而在无该基因甲基化的胃癌组织中仅有1例表达下调(相关性分析r_s=0.886，P=10~(-6))；而P27kip1基因在胃癌组织中则18例甲基化的组织中则有15例mRNA表达下调，而在无该基因甲基化的组织中有2例mRNA表达下调(相关性分析r_s=0.716，P=10~(-6))。分析结果显示此两种基因的在胃癌组织的低表达均与基因的高甲基化有关。
(5)胃癌中Apaf-1基因的5个多态位点的LOH与其表达下降的关系检出率分别是：D12S346为34.3％，D12S1706为8.3％，D12S327为57.1％，D12S1657为11.4％，D12S393为42.8％，而在两个位点以上均出现LOH的则占51.4％，其中有三个位点出现LOH的则占16.7％，出现两个位点LOH的18例组织中13例出现Apaf-1的mRNA表达下降，经相关性分析，r_s=0.487，P=0.003，有统计学意义，说明Apaf-1基因的多位点的LOH的出现与胃癌中该基因表达下降有关。
二、5-Aza-CdR对胃癌细胞系中多种肿瘤抑制基因甲基化的影响
1、5-aza-CdR对胃癌BGC823，SGC7901细胞生长的影响
MTT检测发现胃癌BGC823，SGC7901细胞在5-aza-CdR作用后，细胞生长活力明显减少，AO染色显示细胞形态亦明显改变，而流式细胞仪检测显示SGC7901细胞的细胞在用药后，10~(-6)与5×10~(-6)组中得G_0期细胞明显上升，而S期细胞数量下降(P＜0.05)，此细胞株的凋亡率亦明显上升(P＜0.05)，而BGC823细胞的细胞周期未受5-aza-CdR太多影响，但是在1×10~(-6)，5×10~(-6)组细胞凋亡率均明显上升(P＜0.05)。
2、5-aza-CdR对胃癌BGC823，SGC7901细胞的Apaf-1，P27kip1，DAPK1基因的表达影响
(1)mRNA的表达：在用药前胃癌BGC823，SGC7901细胞中Apaf-1基因及DAPK1基因是低表达状态，而经5-aza-CdR 1×10~(-6)mmol／L，5×10~(-6)mmol／L，1×10~(-5)mmol／L浓度作用下，48h、72h时各基因的表达较对照组均明显上调(P＜0.05)，但在此三个浓度组无明显的时间与剂量的依赖性，而p27kip1则在用药前为均失表达，而用药后则出现了表达的恢复(P＜0.05)，亦无明显的时间与剂量的依赖性。
(1) Western-blot检测胃癌BGC823，SGC7901细胞的Apaf-1，P27kip1蛋白表达：SGC7901中Apaf-1、P27kip1基因的表达在对照组中为失表达，而在药物作用后，基因的表达恢复，并且在5×10~(-7)mmol／L，1×10~(-6)mmol／L，5×10~(-6)mmol／L有一定的药物的剂量但无明显的时间依赖性；在BGC823中，Apaf-1基因的表达比较低，P27kip1基因的蛋白表达在对照组中为失表达，在用5-aza-CdR作用后基因表达明显上升(P＜0.05)。
3、MSP检测胃癌BGC823，SGC7901细胞中目的基因的启动子甲基化状态
在经过5-aza-CdR药物作用后，二种细胞的各基因的甲基化状态均发生了改变。各基因在二种细胞中均为高甲基化状态并且通过去甲基化的药物作用后，高甲基化的状态均出现减少的改变，同时非甲基化的条带出现，甚至在药物浓度较大或作用时间过长时，高甲基化状态完全消失。
三、5-aza-CdR与低浓度CDDP联合用药对胃癌细胞的影响
1、MTT检测细胞活性
从生长曲线可以看出SGC7901细胞与BGC823细胞在5-aza-CdR(5×10~(-6)mmol／L)的作用下生长受到一定的抑制，而在低浓度的CDDP(5×10~(-7)mmol／L)的作用下生长为接近直线，无明显的对数生长期，而在两种药物的联合作用下生长曲线出现了下降的趋势，这说明此两种药物的联合作用有协同的作用。
2、Annexin-v-FITC凋亡检测各组凋亡情况
SGC7901细胞与BGC823细胞在未用药物作用的对照组中凋亡及坏死细胞很少，而在用药物5-aza-CdR(5×10~(-6)mmol／L)作用后，凋亡及坏死细胞均有所增加，在低浓度的CDDP(5×10~(-7)mmol／L)作用后，细胞的凋亡及坏死均明显增加，而在联合用药组则细胞的凋亡及坏死则较单独用药组有明显的上升。
3、各目的基因的表达的情况
(1)RT-PCR：目的基因的mRNA的表达情况
在SGC7901细胞中Apaf-1基因在与CDDP联合用药中虽然各基因的表达均较空白对照组明显上升(P＜0.05)，但相对于单独应用CDDP的对照组细胞的各基因表达并无明显上升，而BGC823细胞中则在联合用药组该基因的表达明显上升；P27kip1基因在此二株细胞中对照组表达均阴性，在5-aza-CdR处理后，基因均表达恢复，在单独应用CDDP后，亦出现基因表达的恢复，同样的在BGC823细胞中联合用药组亦见明显的该基因表达上升(与CDDP组比较)。而DAPK1基因的表达则在用药后表达均明显上升，尤其在联合用药组，二株细胞中该基因表达均明显上升(与CDDP组比较)。
(2) Western-Blot：目的基因蛋白的表达
二株细胞的目的基因的Apaf-1，p27kip1蛋白表达与mRNA的表达基本一致，用药后均可见蛋白的恢复表达与高表达(P＜0.05vs对照组)，但在联合用药组两株细胞的Apaf-1基因的蛋白表达均较对照组，5-aza-CdR组及CDDP组明显升高(均P＜0.05)；而P27kip1的蛋白表达则无此明显的变化。
结论
1、P27kip1基因在胃癌组织中表达下降与该基因启动子甲基化相关。
2、Apaf-1基因启动子甲基化以及多态位点的LOH均是该基因在胃癌组织中表达下降的主要原因。
3、5-Aza-CdR对SGC7901细胞和BGC823细胞具有增生抑制作用，其机制与其阻滞细胞周期、诱导凋亡有关。
4、5-Aza-CdR能改变细胞中Apaf-1，DAPK1，P27kip1基因的甲基化状态，Apaf-1、DAPK1、p27kip1基因的表达情况与其甲基化状态的改变有关。
5、5-aza-CdR能够增加胃癌细胞对低浓度CDDP的敏感性。
6、在胃癌细胞中，DAPK基因在5-aza-CdR增加CDDP药敏性有关。
Research on abnormal methylation of several tumor suppressors in human gastric cancer
Effects of 5-Aza-CdR on proliferation and abnormal methylation of tumor supressor genes of human gastric cancer cell lines Gastric cancer is a kind of malignant tumor which threatens human beings' health, and its occurance is a pathological process which is many different genes involved. The gene promoter CpG island methylation is a important reason in tumor growth. In the study, we detected the effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines SGC7901 and BGC823, methylation and epression of Apaf-1, p27kipl, DAPK1 gene and discussed the relationship between the methylation and expression of Apaf-1, p27kip1, DAPK1. we detect the expression and methylation state of DAPK1, p27kip1, Apaf-1, and explore the role of these genes concurrent methylation in gastric cancer tissues. CDDP is one of the important clinical anticancer drugs, and 5-aza-CdR is a specific demethylating agents. Many studies have indicated that promoter hypermethylation is in relation to chemotherapy resistance of cisplatin in many tumors. The experiment is to study whether the 5-aza-CdR can increase the sensitivity of gastric cancer cells to low dose CDDP.
Materials and methods
Materials: 35 surgical specimens of gastric carcinoma and their adjacent nonmal gastic tissues resected at the First Affiliated Hospital of China Medical University and the second Affiliated Hospital of China Medical University. Gasric cancer lines: BGC823 cell line, SGC7901 cell line. TRIzol Reagent(GIBCOL BRL company), RNAout(TakaRa公司), demethylate agent 5-aza-2'-deoxycitydine(5-Aza-CdR) is the product of Sigma; PI is the product of Sigma; RT-PCR kit is the product of TaKaRa Company; Wizard DNA Clean-up(Promega company), Wizard DNA Clean-up(Promega company); RPMI 1640 culture medium is the product of Hyclone company. Apaf-1, p27kip1 monoclone antibody is the prduct of Santa cruz.
Methods: SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR. The proliferation of the cells was detected by MTT assay, OA stain, flow cytometry. The methylation of Apaf-1, DAPK1, P27kip1 in the two cell lines was detected by MSP, and the expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR and low dose CDDP. The proliferation of the cells was detected by MTT assay and flow cytometry. The expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. Using the semi-quantitative RT-PCR and western-blot analyses the expression of the Apaf-1, p27kip1, DAPKlin gastric cancer. Using the Loss of heterozygosity(LOH) analyses whether there is the loss of Apaf-1 gene in the domain of 12q22-23. Using the methylation specific PCR(MSP) analysis the promoter methylation of Apaf-1, p27kip1, DAPK1 gene in gastric cancer. Statistics analysis: Adopting t test of Excel.
RESULTS
Ⅰ. Supressor genes methylation detection in gastric cancer
1. The expression of Apaf-1, DAPK1, p27kip1's mRNA: Apaf-1, p27kip1, DAPK1 gene low expression is 17/35, 17/35 and 16/35(P＜0.05).
2. The expression of Apaf-1, DAPK1, p27kipl's protein: There was 5 pous expression of Apaf-1 in gastric cancer and 29 pous expression of Apaf-1 in cancer-adjacent tissues; There was 3 pou expression of Apaf-1 in gastric cancer and 30 pous expression of Apaf-1 in cancer-adjacent tissues.
3. The LOH frequencies of D12S346, D12S1706, D12S327, D12S1657 and D12S393is 34.3%, 8.3%, 57.1%, 11.4%, 42.8%. There was 50% LOH in two sites and 17% LOH in three sites.
4. Methylation of DAPK1 was present in both cancer-adjacent tissues and gastric cancer tissue. P27kip1 and Apaf-1 rate of methylation were 25.71% and 22.86% in cancer-adjacent tissues 51.42% and 48.57% in gastric cancer respectively(p＜0.05).
Ⅱ. Effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines and abnormal methylation ofApaf-1 gene
1. After SGC7901 and BGC823 cells were exposed to 5-Aza-CdR (1×10~(-7)mol/L, 5×10~(-7)mol/L, 1×10~(-6)mol/L, 5×10~(-6)mol/L), 5-Aza-CdR displayed a growth inhibitory effect on SGC7901 and BGC823 cells in a dose-and time-dependent manner, FCM analysis showed that G0/G1 phrase rate increased with exposing to 5-Aza-CdR (1×10~(-6)mol/L, 5×10~(-6)mol/L) for 72h(P＜0.01), the apoptosis rate increased too(P＜0.01).
2. The methylation and the loss of Apaf-1, P27kip1, DAPK1 mRNA and protein of Apaf-1,, P27kip1, DAPK1 in SGC7901 and BGC823 cells were changed with 5-Aza-CdR.
Ⅲ. The influence of 5-aza-CdR and low dose CDDP on gastric cell line
1. MTT assay: The proliferation of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more inactive than that only added low dose CDDP.
2. Annexin-v-FITC detecting the apoptosis: The apoptosis of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more than that only added low dose CDDP.
3. the expression of gene: Analysis of mRNA and protein indicated the DAPK is both increased more greatly in SGC7901 cell and BGC823 cell when they were added 5-aza-CdR and low dose CDDP than that only added low dose CDDP.
CONCLUSION
1. The mechanism of 5-Aza-CdR's growth inhibitory effect on SGC7901 and BGC823 cells is related with blocking cell cycles, inducing apoptosis, and changing the methylation ofApaf-1 gene.
2. The loss expression in SGC7901 and BGC823 cells is because of methylation states.
3. 5-aza-CdR can increase the gastric cells' sensitivity to low dose CDDP.
4. DAPK gene acts a important role in 5-aza-CdR increasing the gastric cells' sensitivity to low dose CDDP.
5. multiple genes concurrent methylation plays an important role in some gastric cancers, They happened at early stage.
6. The expression of Apaf-1, DAPK1, p27kip1 is low in gastric cancer.
7. gene's promoter Methylation of Apaf-1 and LOH of the gene in the domain of 12q22-23 both are the main reasons gene's promoter Methylation of p27kip1 is related with the in gastric cancer.
胃癌的发生与其他恶性肿瘤一样是多基因多阶段变异累积形成的病理过程，而抑癌基因的甲基化是一个研究的热点，目前已鉴定的易高甲基化的基因包括：参与细胞周期、DNA修复、耐药性形成、分化、凋亡的基因及参与肿瘤转移和血管生成的基因。此研究中选择了主要与细胞周期及细胞凋亡有关的基因Apaf-1，P27kip1，DAPK1，对胃癌组织中的DAPK1，Apaf-1，P27kip1基因甲基化状态、表达进行研究，以研究胃癌组织中此三种基因表达异常的原因，探讨基因甲基化在胃癌发病中的作用。在对胃癌细胞系的研究中除对此三个基因的甲基化状态进行进一步研究，在去甲基化药物5-aza-CdR作用细胞后，亦检测目的基因的甲基化状态、表达的改变及与细胞生长状态的关系，进一步探讨抑癌基因的表达与其甲基化状态的相关性。顺铂(CDDP)作为治疗多种实体瘤临床一线用药，是胃癌的常用化疗药物，5-aza-CdR是明确的去甲基化的药物，本实验从这两株胃腺癌细胞的抑癌基因的高甲基化状态入手，通过对低剂量的顺铂与5-aza-CdR的联合用药以观察两者对胃癌细胞生长的影响，探讨5-aza-CdR是否能够作为一线化疗药物顺铂的辅助用药，使顺铂在较低剂量时发挥较大的作用。
材料与方法
1、实验材料
中国医科大学附属第一医院及中国医科大学附属第二医院胃癌患者术后胃癌组织、胃癌旁组织；标本细胞株：胃癌BGC823细胞系，胃癌SGC7901细胞系(均购自细胞生物教研室)；总RNA提取试剂：TRIZOL Reagent(GIBCO BRL公司)，RNAout(TaKaRa公司)，去甲基化试剂5-aza-2’-deoxycitydine(5-Aza-CdR)为Sigma产品，碘化丙啶(PI)、丫啶橙(AO)为Sigma产品，RT-PCR试剂盒购自TaKaRa公司，Wizard DNA Clean-up(Promega公司)，Annexin-v-FITC凋亡检测试剂盒为北京宝赛生生物技术有限公司生产，RPMI 1640培养基为Hyclone产品，Apaf-1绵羊抗人单克隆抗体(Santa cruz产品)，Taq酶及dNTP均为TaKaRa公司产品，Apaf-1绵羊抗人单克隆抗体(Santa cruz产品)，p27kip1鼠抗人单克隆抗体(博士德公司产品)，驴抗羊抗体(Santa cruz产品)，羊抗鼠抗体(北京中杉金桥生物公司产品)。
2、实验方法
MTT法检测细胞生长活性，AO染色、Annexin-v-FITC凋亡流式细胞仪检测细胞凋亡；PI染色流式细胞仪检测细胞周期及凋亡的变化；用半定量RT-PCR方法，Western-Blot方法分析胃癌细胞及组织中各目的基因的表达，用MSP对胃癌细胞及组织中各基因的启动子区甲基化情况进行研究。用杂合性丢失(Loss of heterozygosity，LOH)分析胃癌患者Apaf-1基因所在的12q22-23区域出现缺失的情况。
3、统计分析
所有资料采用SPSS11.5分析。计量资料比较采用方差分析；两两比较采用LSD法；计数资料采用χ~2检验；凋亡率资料分析采用秩和检验，相关分析采用Spearman方法。
结果
一、胃癌组织中各基因的表达及甲基化状况
1、各目的基因在胃癌组织的表达
(1)RT-PCR检测基因mRNA表达：在胃癌组织中Apaf-1基因表达下调17／35例，p27kip1基因表达下调17／35例，DAPK1基因表达下调16／35例，此三组基因的癌旁组织的mRNA表达明显高于均癌组织，具有统计学意义(均P＜0.05)。
(2)Western Blot检测基因蛋白表达：在胃癌组织中Apaf-1基因在胃癌组织中5例(5／35)有蛋白表达，癌旁组织则有29例蛋白表达(29／35)，P＜0.05，P27kip1基因在胃癌组织中3例有蛋白表达(3／35)，癌旁组织有30例蛋白表达(30／35)，此两组基因癌旁组织的蛋白表达显著高于癌组织，均具有统计学意义(P＜0.05)。
(3)各基因启动子甲基化的情况DAPK1胃癌组织及癌旁组织中甲基化率均为100％，无统计学意义；P27kip1胃癌组织中甲基化率为51.42％明显高于癌旁组织(25.71％)，具有统计学意义(P＜0.05)，Apaf-1胃癌组织及癌旁组织中甲基化率分别为48.57％，22.86％，胃癌组织的甲基化率明显高于癌旁组织，具有统计学意义(P＜0.05)。
(4)胃癌中P27kip1、Apaf-1基因的甲基化状态与其表达的关系胃癌组织中17例具有Apaf-1基因甲基化的组织16例有mRNA表达下调，而在无该基因甲基化的胃癌组织中仅有1例表达下调(相关性分析r_s=0.886，P=10~(-6))；而P27kip1基因在胃癌组织中则18例甲基化的组织中则有15例mRNA表达下调，而在无该基因甲基化的组织中有2例mRNA表达下调(相关性分析r_s=0.716，P=10~(-6))。分析结果显示此两种基因的在胃癌组织的低表达均与基因的高甲基化有关。
(5)胃癌中Apaf-1基因的5个多态位点的LOH与其表达下降的关系检出率分别是：D12S346为34.3％，D12S1706为8.3％，D12S327为57.1％，D12S1657为11.4％，D12S393为42.8％，而在两个位点以上均出现LOH的则占51.4％，其中有三个位点出现LOH的则占16.7％，出现两个位点LOH的18例组织中13例出现Apaf-1的mRNA表达下降，经相关性分析，r_s=0.487，P=0.003，有统计学意义，说明Apaf-1基因的多位点的LOH的出现与胃癌中该基因表达下降有关。
二、5-Aza-CdR对胃癌细胞系中多种肿瘤抑制基因甲基化的影响
1、5-aza-CdR对胃癌BGC823，SGC7901细胞生长的影响
MTT检测发现胃癌BGC823，SGC7901细胞在5-aza-CdR作用后，细胞生长活力明显减少，AO染色显示细胞形态亦明显改变，而流式细胞仪检测显示SGC7901细胞的细胞在用药后，10~(-6)与5×10~(-6)组中得G_0期细胞明显上升，而S期细胞数量下降(P＜0.05)，此细胞株的凋亡率亦明显上升(P＜0.05)，而BGC823细胞的细胞周期未受5-aza-CdR太多影响，但是在1×10~(-6)，5×10~(-6)组细胞凋亡率均明显上升(P＜0.05)。
2、5-aza-CdR对胃癌BGC823，SGC7901细胞的Apaf-1，P27kip1，DAPK1基因的表达影响
(1)mRNA的表达：在用药前胃癌BGC823，SGC7901细胞中Apaf-1基因及DAPK1基因是低表达状态，而经5-aza-CdR 1×10~(-6)mmol／L，5×10~(-6)mmol／L，1×10~(-5)mmol／L浓度作用下，48h、72h时各基因的表达较对照组均明显上调(P＜0.05)，但在此三个浓度组无明显的时间与剂量的依赖性，而p27kip1则在用药前为均失表达，而用药后则出现了表达的恢复(P＜0.05)，亦无明显的时间与剂量的依赖性。
(1) Western-blot检测胃癌BGC823，SGC7901细胞的Apaf-1，P27kip1蛋白表达：SGC7901中Apaf-1、P27kip1基因的表达在对照组中为失表达，而在药物作用后，基因的表达恢复，并且在5×10~(-7)mmol／L，1×10~(-6)mmol／L，5×10~(-6)mmol／L有一定的药物的剂量但无明显的时间依赖性；在BGC823中，Apaf-1基因的表达比较低，P27kip1基因的蛋白表达在对照组中为失表达，在用5-aza-CdR作用后基因表达明显上升(P＜0.05)。
3、MSP检测胃癌BGC823，SGC7901细胞中目的基因的启动子甲基化状态
在经过5-aza-CdR药物作用后，二种细胞的各基因的甲基化状态均发生了改变。各基因在二种细胞中均为高甲基化状态并且通过去甲基化的药物作用后，高甲基化的状态均出现减少的改变，同时非甲基化的条带出现，甚至在药物浓度较大或作用时间过长时，高甲基化状态完全消失。
三、5-aza-CdR与低浓度CDDP联合用药对胃癌细胞的影响
1、MTT检测细胞活性
从生长曲线可以看出SGC7901细胞与BGC823细胞在5-aza-CdR(5×10~(-6)mmol／L)的作用下生长受到一定的抑制，而在低浓度的CDDP(5×10~(-7)mmol／L)的作用下生长为接近直线，无明显的对数生长期，而在两种药物的联合作用下生长曲线出现了下降的趋势，这说明此两种药物的联合作用有协同的作用。
2、Annexin-v-FITC凋亡检测各组凋亡情况
SGC7901细胞与BGC823细胞在未用药物作用的对照组中凋亡及坏死细胞很少，而在用药物5-aza-CdR(5×10~(-6)mmol／L)作用后，凋亡及坏死细胞均有所增加，在低浓度的CDDP(5×10~(-7)mmol／L)作用后，细胞的凋亡及坏死均明显增加，而在联合用药组则细胞的凋亡及坏死则较单独用药组有明显的上升。
3、各目的基因的表达的情况
(1)RT-PCR：目的基因的mRNA的表达情况
在SGC7901细胞中Apaf-1基因在与CDDP联合用药中虽然各基因的表达均较空白对照组明显上升(P＜0.05)，但相对于单独应用CDDP的对照组细胞的各基因表达并无明显上升，而BGC823细胞中则在联合用药组该基因的表达明显上升；P27kip1基因在此二株细胞中对照组表达均阴性，在5-aza-CdR处理后，基因均表达恢复，在单独应用CDDP后，亦出现基因表达的恢复，同样的在BGC823细胞中联合用药组亦见明显的该基因表达上升(与CDDP组比较)。而DAPK1基因的表达则在用药后表达均明显上升，尤其在联合用药组，二株细胞中该基因表达均明显上升(与CDDP组比较)。
(2) Western-Blot：目的基因蛋白的表达
二株细胞的目的基因的Apaf-1，p27kip1蛋白表达与mRNA的表达基本一致，用药后均可见蛋白的恢复表达与高表达(P＜0.05vs对照组)，但在联合用药组两株细胞的Apaf-1基因的蛋白表达均较对照组，5-aza-CdR组及CDDP组明显升高(均P＜0.05)；而P27kip1的蛋白表达则无此明显的变化。
结论
1、P27kip1基因在胃癌组织中表达下降与该基因启动子甲基化相关。
2、Apaf-1基因启动子甲基化以及多态位点的LOH均是该基因在胃癌组织中表达下降的主要原因。
3、5-Aza-CdR对SGC7901细胞和BGC823细胞具有增生抑制作用，其机制与其阻滞细胞周期、诱导凋亡有关。
4、5-Aza-CdR能改变细胞中Apaf-1，DAPK1，P27kip1基因的甲基化状态，Apaf-1、DAPK1、p27kip1基因的表达情况与其甲基化状态的改变有关。
5、5-aza-CdR能够增加胃癌细胞对低浓度CDDP的敏感性。
6、在胃癌细胞中，DAPK基因在5-aza-CdR增加CDDP药敏性有关。
Research on abnormal methylation of several tumor suppressors in human gastric cancer
Effects of 5-Aza-CdR on proliferation and abnormal methylation of tumor supressor genes of human gastric cancer cell lines Gastric cancer is a kind of malignant tumor which threatens human beings' health, and its occurance is a pathological process which is many different genes involved. The gene promoter CpG island methylation is a important reason in tumor growth. In the study, we detected the effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines SGC7901 and BGC823, methylation and epression of Apaf-1, p27kipl, DAPK1 gene and discussed the relationship between the methylation and expression of Apaf-1, p27kip1, DAPK1. we detect the expression and methylation state of DAPK1, p27kip1, Apaf-1, and explore the role of these genes concurrent methylation in gastric cancer tissues. CDDP is one of the important clinical anticancer drugs, and 5-aza-CdR is a specific demethylating agents. Many studies have indicated that promoter hypermethylation is in relation to chemotherapy resistance of cisplatin in many tumors. The experiment is to study whether the 5-aza-CdR can increase the sensitivity of gastric cancer cells to low dose CDDP.
Materials and methods
Materials: 35 surgical specimens of gastric carcinoma and their adjacent nonmal gastic tissues resected at the First Affiliated Hospital of China Medical University and the second Affiliated Hospital of China Medical University. Gasric cancer lines: BGC823 cell line, SGC7901 cell line. TRIzol Reagent(GIBCOL BRL company), RNAout(TakaRa公司), demethylate agent 5-aza-2'-deoxycitydine(5-Aza-CdR) is the product of Sigma; PI is the product of Sigma; RT-PCR kit is the product of TaKaRa Company; Wizard DNA Clean-up(Promega company), Wizard DNA Clean-up(Promega company); RPMI 1640 culture medium is the product of Hyclone company. Apaf-1, p27kip1 monoclone antibody is the prduct of Santa cruz.
Methods: SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR. The proliferation of the cells was detected by MTT assay, OA stain, flow cytometry. The methylation of Apaf-1, DAPK1, P27kip1 in the two cell lines was detected by MSP, and the expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR and low dose CDDP. The proliferation of the cells was detected by MTT assay and flow cytometry. The expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. Using the semi-quantitative RT-PCR and western-blot analyses the expression of the Apaf-1, p27kip1, DAPKlin gastric cancer. Using the Loss of heterozygosity(LOH) analyses whether there is the loss of Apaf-1 gene in the domain of 12q22-23. Using the methylation specific PCR(MSP) analysis the promoter methylation of Apaf-1, p27kip1, DAPK1 gene in gastric cancer. Statistics analysis: Adopting t test of Excel.
RESULTS
Ⅰ. Supressor genes methylation detection in gastric cancer
1. The expression of Apaf-1, DAPK1, p27kip1's mRNA: Apaf-1, p27kip1, DAPK1 gene low expression is 17/35, 17/35 and 16/35(P＜0.05).
2. The expression of Apaf-1, DAPK1, p27kipl's protein: There was 5 pous expression of Apaf-1 in gastric cancer and 29 pous expression of Apaf-1 in cancer-adjacent tissues; There was 3 pou expression of Apaf-1 in gastric cancer and 30 pous expression of Apaf-1 in cancer-adjacent tissues.
3. The LOH frequencies of D12S346, D12S1706, D12S327, D12S1657 and D12S393is 34.3%, 8.3%, 57.1%, 11.4%, 42.8%. There was 50% LOH in two sites and 17% LOH in three sites.
4. Methylation of DAPK1 was present in both cancer-adjacent tissues and gastric cancer tissue. P27kip1 and Apaf-1 rate of methylation were 25.71% and 22.86% in cancer-adjacent tissues 51.42% and 48.57% in gastric cancer respectively(p＜0.05).
Ⅱ. Effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines and abnormal methylation ofApaf-1 gene
1. After SGC7901 and BGC823 cells were exposed to 5-Aza-CdR (1×10~(-7)mol/L, 5×10~(-7)mol/L, 1×10~(-6)mol/L, 5×10~(-6)mol/L), 5-Aza-CdR displayed a growth inhibitory effect on SGC7901 and BGC823 cells in a dose-and time-dependent manner, FCM analysis showed that G0/G1 phrase rate increased with exposing to 5-Aza-CdR (1×10~(-6)mol/L, 5×10~(-6)mol/L) for 72h(P＜0.01), the apoptosis rate increased too(P＜0.01).
2. The methylation and the loss of Apaf-1, P27kip1, DAPK1 mRNA and protein of Apaf-1,, P27kip1, DAPK1 in SGC7901 and BGC823 cells were changed with 5-Aza-CdR.
Ⅲ. The influence of 5-aza-CdR and low dose CDDP on gastric cell line
1. MTT assay: The proliferation of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more inactive than that only added low dose CDDP.
2. Annexin-v-FITC detecting the apoptosis: The apoptosis of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more than that only added low dose CDDP.
3. the expression of gene: Analysis of mRNA and protein indicated the DAPK is both increased more greatly in SGC7901 cell and BGC823 cell when they were added 5-aza-CdR and low dose CDDP than that only added low dose CDDP.
CONCLUSION
1. The mechanism of 5-Aza-CdR's growth inhibitory effect on SGC7901 and BGC823 cells is related with blocking cell cycles, inducing apoptosis, and changing the methylation ofApaf-1 gene.
2. The loss expression in SGC7901 and BGC823 cells is because of methylation states.
3. 5-aza-CdR can increase the gastric cells' sensitivity to low dose CDDP.
4. DAPK gene acts a important role in 5-aza-CdR increasing the gastric cells' sensitivity to low dose CDDP.
5. multiple genes concurrent methylation plays an important role in some gastric cancers, They happened at early stage.
6. The expression of Apaf-1, DAPK1, p27kip1 is low in gastric cancer.
7. gene's promoter Methylation of Apaf-1 and LOH of the gene in the domain of 12q22-23 both are the main reasons gene's promoter Methylation of p27kip1 is related with the in gastric cancer.
引文
1. M Esteller. Epigenetics provides a new generation of oncogenes and tumour-suppressor genes. British Journal of Cancer (2006) 94, 179-183.
2. Yamada Y, Jackson-Grusby L, Linhart H, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci USA. 2005;102:13580-13585
3. Beier V, Mund C, Hoheisel JD. Monitoring methylation changes in cancer.Adv Biochem Eng Biotechnol. 2007;104:1-11.
4. Chim CS, Liang R, Leung MH, et al. Aberrant gene methylation implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma. J Clin Pathol. 2007 Jan;60(1):104-6.
5．陆嵘，房静远．表观遗传修饰与肿瘤．生命科学．2006；18(1)：10-14
6．张永彪，褚嘉佑．表观遗传学与人类疾病的研究进展遗传．2005；27(3)：466-472
7. Sobin LH, Fleming ID. TNM classification of malignant tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997; 80:1803-1804
8. Zhang WF, Chert JQ, Shah JX, et al. Indication of radical surgery (R2, R3) based upon the pattern of lymph node metastasis from gastric cancer.Zhonghua Zhongliu Zazhi 1987; 9: 286-289
9. Herman JG, Graft JR., Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc NatlAcad Sci USA 1996; 93:9821-9826
10. Tanaka T, Bai T, Yukawa K, et al. Radiation-induced cell death is independent of the apoptotic signals mediated by death-associated protein kinase in human cervical squamous cell carcinoma cells. Oncol Rep. 2005;14(4):949-55.
11. Shang T, Joseph J, Hillard CJ,et al. Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death. J Biol Chem. 2005 14;280(41):34644-53..
12. Bowman P, Galea CA, Lacy E, et al. Thermodynamic characterization of interactions between p27(Kipl) and activated and non-activated Cdk2: intrinsically unstructured proteins as thermodynamic tethers. Biochim Biophys Acta. 2006; 1764(2): 182-9.
13. Mikami T, Yoshida T, Shiraishi H, et al. Bottom-up cell proliferation with cyclin A and p27Kip 1 expression in ulcerative colitis-associated dysplasia. Pathol Int. 2006; 56(1): 10-6.
14. Le XF, Pruefer F, Bast RC Jr HER2-targeting antibodies modulate the cyclin-dependent kinase inhibitor p27Kipl via multiple signaling pathwaysCell Cycle. 2005 Jan;4(1):87-95. Epub 2005 Jan 10.
15.Ozkara SK, Corakci A. Significantly decreased P27 expression in endometrial carcinoma compared to complex hyperplasia with atypia (correlation with p53 expressionPathol Oncol Res. 2004; 10(2):89-97.
16. Leo C, Richter C, Horn LC,et al. Expression of Apaf-1 in cervical cancer correlates with lymphnode metasis but not with introtumor of hypoxia. Cynecol Oncol. 2005;97(2):602-6
17. Mustika r, budiganto A, Nishigori C,et al. Decreased expression of Apaf-1 with progression of melanoma. Pigment cell Des. 2005; 18(l):59-62
18. Lei PP, Zhang ZJ, Shen LJ, et al. Expression and hypermethylation of p27 kipl in hepatocarcinogenesis. World J Gastroenterol. 2005; 11 (29):4587-91.
19. Brakensiek K, Langer F, Kreipe H, et al. Absence of p21(CIP 1), p27(KIP 1) and p 57(KIP 2) methylation in MDS and AML. Leuk Res. 2005; 29(11): 1357-60.
20. Furukawa Y, Sutheesophon K, Wada T, et al. Methylation silencing of the APAF1 gene in acute leukemia. Mol Cancer Res. 2005; 3(6):325-34.
21．黄带发，富伟能，尚超，等．喉鳞癌APAF1基因表达及启动子区甲基化研究遗传学报．2004，31(12)1327．1333．
22. Jian YU, Hong Yu ZHANG, Zhen Zhong MA, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Research. 2003; 13(5):319-333
23. Murty VV, Montgomery K, Dutta S, et al. A 3-Mb high-resolution BAC/PAC contig of 12q22 encompassing the 830-kb consensus minimal deletion in male germ cell tumors. Genome Res.1999;9: 662-71.
24. Umetani N, Fujimoto A, Takeuchi H,et al. Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma.2004. Oncogene Oct 28;23(50):8292-300
25. Baldi A, Santini D, Russo P, et al. Analysis of APAF-1 expression in human cutaneous melanoma progression. Exp Dermatol. 2004;13(2):93-7.
26. Jones PA. DNA methylation errors and cancer.Cancer Res, 1996,56(11):2463-2467
27. Wijermans PW, Krulder JWM,Huijigens PC,et al. Continuous infusion of low-dose 5-aza-2'deoxycytiene in elderly patients with high risk myelodysplastic syndrome. Leukemia, 1997,11(1):1-5
28．杨丽，朱红音，程中华，等．人胃癌细胞肿瘤相关基因的表Ichihashi M，Veda M达与甲基化调控．世界华人消化杂志 2005；13(13)：1493-1498
29．杜红玲，任立敏，陈华，等．丁酸钠与5-氮杂-2’-脱氧胞苷协同诱导U266细胞p16基因重新表达．第一军医大学学报 2002；22：981-984
30．吕国丽，文剑明，张萌，等5-Aza-CdR对癌细胞TIMP-3启动子去甲基化的作用．中国病理生理杂志 2005，21(9)：1763-1768
31. Liu X. S., Kim C. N., Yang J, et al. Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c. Cell 1996 86, 147-157.
32. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell. 1997, 479-489.
33. Leo C, Richter C, Horn LC, Schutz A,et al. Expression of Apaf-1 in cervical cancer correlates with lymphnode metasis but not with introtumor of hypoxia. Cynecol Oncol, 2005 may;97(2):602-6
34. Mustika r, budiganto A, Nishigori C,. Decreased expression of Apaf-1 with progression of melanoma. Pigment cell Des. 2005 Feb; 18(1):59-62
35．高红，张志波，尚海．p57kip2和p27kip1基因mRNA在组织中的表达及意义．中国医科大学学报 2004，33(6)525-529
36. Widschwendter A, Muller HM, Fiegl H, et al. DNA methylation in serum and tumors of cervical cancer patients[J]. Clin Cancer Res, 2004, 10(2): 565-571
37. Yamaguchi S, A sao T, Nakamura J, et al. High frequency of DAP-kinase gene Promoter methylation in colorectal cancer specimens and its identification in serum[J]. Cancer Lett, 2003, 194(1):99-105
38. Toyooka S, Toyooka KO, Miyajima K, et al. Epigenetic down-regulation of death-associated protein kinase in lung cancer[J]. Clin Cancer Res,2003,9(8):3034-3041.
39. Bialik S, Kimchi A. DAP-kinase as a target for drug design in cancer and disease associated with accelerated cell death. Sem in Cancer Biol, 2004, 14 (4): 283-294
40. Kochi M, Fujii M, Kanamori N, et al. Neoadjuvant chemotherapy with S-1 and CDDP in advanced gastric cancer. J Cancer Res Clin Oncol. 2006; 132(12):781-5.
41. Matsuhashi N, Saio M, Matsuo A,et al.The evaluation of gastric cancer sensitivity to 5-FU/CDDP in terms of induction of apoptosis: time- and p53 expression-dependency of anti-cancer drugs. Oncol Rep. 2005; 14(3):609-15.
42. Iioka Y, Mishima K, Azuma N, et al. Overexpression of protein kinase Cdelta enhances cisplatin-induced cytotoxicity correlated with p53 in gastric cancer cell line. Pathobiology. 2005; 72(3): 152-9
43. Egger G, Aparicio AM, Escobar SG, et al. Inhibition of histone deacetylation does not block resilencing of p 16 after 5-aza-2'-deoxycytidine treatment. Cancer Res. 2007;67(1):346-53.
44. Christoph F, Kempkensteffen C, Weikert S, et al. Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer. Br J Cancer. 2006 18;95(12):1701-7
45．马韬，朱正刚．胃癌化疗与细胞凋亡研究进展．国外医学消化系疾病分册．2003；23：15-18
46. Francia G, Green SK, Bocci G, Man S, et al. Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents. Mol Cancer Ther. 2005; 4(10): 1484-94
47. Staub J, Chien J, Pan Y,et al. Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance. Oncogene. 2007; 19.
48. Bai T, Tanaka T, Yukawa K, et al. Int J Oncol. A novel mechanism for acquired cisplatin-resistance: suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells. 2006; 28(2):497-508.
49. Fang X, Zheng C, Liu Z, Ekman P, Xu D. Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine. Oncol Rep. 2004 Sep;12(3):523-6.
50. Qiu YY, Mirkin BL, Dwivedi RS. Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells. Cancer Detect Prev. 2005; 29(5):456-63.
51. Missiaglia E, Donadelli M, Palmieri M, et al. Growth delay of human pancreatic cancer cells by methylase inhibitor 5-aza-2'-deoxycytidine treatment is associated with activation of the interferon signalling pathway. Oncogene. 2005 ; 24(1): 199-211.
52. Koul S, McKiernan JM, Narayan G, et al. Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors. Mol Cancer. 2004 ;3:16-26.
53. Iioka Y, Mishima K, Azuma N, et al. Overexpression of protein kinase Cdelta enhances cisplatin-induced cytotoxicity correlated with p53 in gastric cancer cell line. Pathobiology.2005;72(3):152-9
54. Del Bello B, Valentini MA, Comporti M, et al. Cisplatin-induced apoptosis in melanoma cells: role of caspase-3 and caspase-7 in Apaf-1 proteolytic cleavage and in execution of the degradative phases. Ann N Y Acad Sci. 2003 ; 1010:200-4.
55. Kim WS, 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):827-30.
56. House MG, Guo M, Efron DT, et al. Tumor suppressor gene hypermethylation as a predictor of gastric stromal tumor behavior.J Gastrointest Surg. 2003 ;7(8):1004-14.
57. Schildhaus HU, Krockel I, Lippert H, et al. Promoter hypermethylation of pl6INK4a,E-cadherin, 06-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus,esophagogastric junction and proximal stomach.Int J Oncol. 2005 ;26(6):1493-500.
58. Sabbioni S, Miotto E, Veronese A, et al. Multigene methylation analysis of gastrointestinal tumors: TPEF emerges as a frequent tumor-specific aberrantly methylated marker that can be detected in peripheral blood.Mol Diagn. 2003;7(3-4):201-207.
59. Chang MS, Uozaki H, Chong JM, et al. CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus.Clin Cancer Res. 2006 ;12(10):2995-3002.
60. Zhang X, Yashiro M, Ohira M, et al. Synergic antiproliferative effect of DNA methyltransferase inhibitor in combination with anticancer drugs in gastric carcinoma.Cancer Sci.2006 ;97(9):938-44.
1 Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation (1980) Nature 284, 555-556.
2 Yuan, J., Shaham, S., Ledoux, S., et al. Elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme .Cell.1993; 75: 641-652.
3 Chew S K., Akdemir F, Chen P, et al. The apical caspase dronc governs programmed and unprogrammed cell death in drosophila. Cell. 2004; 7: 897-907.
4 Daish T J, Mills K., Kumar S. Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis Cell. 2004; 7:909-915.
5 Kuida K, Zheng T S, Na S, et al.Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature. 1996; 384:368-372.
6. Kuida K, Haydar T F, Kuan C Y, et al. Reduced Apoptosis and Cytochrome c-Mediated Caspase activation in Mice Lacking Caspase 9. Cell .1998; 94: 325-337.
7. Hakem R, Hakem A., Duncan G S, et al. Differential Requirement for Caspase 9 in Apoptotic Pathways In Vivo. Cell. 1998; 94: 339-352.
8. Zou H, Henzel W J, Liu X, et al. Apaf-1, a human protein homologous to C. elegans CED-4,Participates in Cytochrome c-Dependent Activation of Caspase-3. Cell .1997;90: 405-413.
9. Srinivasula S M, Ahmad M, Fernandes-Alnemri,et al. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell.1998; 1: 949-957.
10 Rodriguez A, Oliver H, Zou H, et al. Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway. Nat Cell Biol.1999; 1: 272-279.
11. Kanuka H, Sawamoto K, Inohara N,et al. Control of the Cell Death Pathway by Dapaf-1, a Drosophila Apaf-1/CED-4-Related Caspase Activator. Mol Cell. 1999; 4: 757-769.
12. Zhou L, Song Z, Tittel J, et al. HAC-1, a Drosophila Homolog of APAF-1 and CED-4,Functions in Developmental and Radiation-Induced Apoptosis. Mol Cell. 1999 ;4: 745-755.
13. Yuan J Y, Horvitz R H. The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death.Development .1992; 116: 309-320.
14 Martinon F, Burns K, Tschopp J. The InflammasomeA Molecular Platform Triggering Activation of Inflammatory Caspases and Processing of proIL-beta. Mol Cell. 2002; 10:417-426.
15 Inohara N, Ogura Y, Nunez G,et al. Nods: a family of cytosolic proteins that regulate the host response to pathogens. Curr. Opin. Microbiol.,2002; 5: 76-80.
16. Liu X S, Kim C N, Yang J,et al. Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome C. Cell. 1996; 86: 147-157.
17 Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell. 1997; 91: 479-489.
18 Rodriguez J,Lazebnik Y. Dark is a Drosophila homologue of Apaf-l/CED-4 and functions in an evolutionarily conserved death pathway. Genes Dev. 1999; 13:3179-3184.
19. Acehan D, Jiang X., Morgan D G, et al. Three-Dimensional Structure of the Apoptosomelmplications for Assembly, Procaspase-9 Binding, and Activation. Mol. Cell. 2002;9: 423-43
20. Hao Z, Duncan GS, Chang CC, et al. Specific ablation of the apoptotic functions of cytochrome C reveals a differential requirement for cytochrome C and Apaf-1 in apoptosis. Cell.2005;121(4):579-91.
21. Robles AI, Bemmels NA, Foraker AB, et al. APAF-1 is a transcriptional target of p53 in DNA damage-induced apoptosis. Cancer Res.2001; 61: 6660-4.
22. Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. Mol Cell. 2003; 11:577-90.
23. Fortin A, Cregan SP, MacLaurin JG, et al. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death. J Cell Biol. 2001; 155: 207-16.
24. Moroni MC, Hickman ES, Denchi EL, et al Apaf-1 is a transcriptional target for E2F and p53.Nat Cell Biol.2001; 3: 552-8.
25. Ho CK, Bush JA, Li G. Tissue-specific regulation of Apaf-1 expression by p53. Oncol Rep.2003 ;10(5):l 139-43
26. Sturm I, Bosanquet AG, Radetzki S, et al.Silencing of APAF-1 in B-CLL result in poor prognosis in the case of concomitant p53 mutation. Int J cancer. 2006; 118(9):2329-36.
27. Marsden VS, O'Connor L, O'Reilly LA, et al. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature.2002 ;419(6907):634-7.
28. Leo C, Richter C, Horn LC, et al. Expression of Apaf-1 in cervical cancer correlates with lymphnode metasis but not with introtumor of hypoxia. Cynecol Oncol: 2005 ;97(2):602-6
29. Mustika r,budiganto A, Nishigori C, et al. Decreased expression of Apaf-1 with progression of melanoma. Pigment cell Des. 2005 ; 18(1):59-62
30. Ekert PG, Read SH, Silke J, et al. Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J Cell Biol. 2004;165(6):835-42.
31. Anichini A, Mortarini R, Sensi M, et al. Apaf-1 signaling in human melanoma. Cancer Lett.2006;238(2): 168-79.
32. Kimura M, Furukawa T, Abe T, et al. Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer. Cancer Res. 1998;58: 2456-60.
33. Murty VV, Montgomery K, Dutta S, et al A 3-Mb high-resolution BAC/PAC contig of 12q22 encompassing the 830-kb consensus minimal deletion in male germ cell tumors. Genome Res.1999;9:662-71.
34. Hatta Y, Takeuchi S, Yokota J, et al. Ovarian cancer has frequent loss of heterozygosity at chromosome 12pl2.3-13.1 (region of TEL and Kip1 loci) and chromosome 12q23-ter: evidence for two new tumour-suppressor genes. Br J Cancer. 1997; 75: 1256-62.
35. Watanabe T, Hirota Y, Arakawa Y, et al. Frequent LOH at chromosome 12q22-23 and Apaf-1 inactivation in glioblastoma. Brain Pathol. 2003;13(4):431-9.
36. Umetani N, Fujimoto A, Takeuchi H,et al. Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma .Oncogene. 2004; 23(50):8292-300
37. Baldi A, Santini D, Russo P, et al. Analysis of APAF-1 expression in human cutaneous melanoma progression. Exp Dermatol. 2004; 13(2):93-7.
38. Fujimoto A, O'Day SJ, Taback B, et al. Allelic imbalance on 12q22-23 in serum circulating DNA of melanoma patients predicts disease outcome.Cancer Res. 2004 ;64(12):4085-8.
39. Fujimoto A, Takeuchi H, Taback B, et al. Allelic imbalance of 12q22-23 associated with APAF-1 locus correlates with poor disease outcome in cutaneous melanoma. Cancer Res. 2004; 64(6):2245-50.
40. Scott CL, Schuler M, Marsden VS, et al. Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation. J Cell Biol. 2004; 164(1):89-96.
41. Ekert PG, Read SH, Silke J, et al. Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J Ceil Biol. 2004;165(6):835-42.
42. Besse B, Cande C, Spano JP, et al. Nuclear localization of apoptosis protease activating factor-1 predicts survival after tumor resection in early-stage non-small cell lung cancer. Clin Cancer Res. 2004; 10(17):5665-9.
43. Fu WN, Bertoni F, Kelsey SM, et al. Role of DNA methylation in the suppression of Apaf-1 protein in human leukaemia. Oncogene.2003; 22(3):451-5
44. Furukawa Y, Sutheesophon K, Wada T, et al. Methylation silencing of the Apaf-1 gene in acute leukemia. Mol Cancer Res.2005; 3(6):325-34.
45．黄带发，富伟能，尚超，等．喉鳞癌Apaf-1基因表达及启动子区甲基化研究遗传学报．2004；31 (12)：1327-1333．
46. McLeod, Gordon Strathdee, Robert Brown. CpG Island Methylation of DNA Damage Response Genes in Advanced Ovarian Cancer. Cancer Res. 2005; 65: 8961-8967.
47．程留芳 杨云生 李红梅．Apaf-1基因转染及其对5-氟尿嘧啶诱导AGS细胞凋亡的影响．中华消化杂志．2002；22(9)：550-553．
48. Schneider BG, Rha SY, Chung HC, et al. Regions of allelic imbalance in the distal portion of chromosome 12q in gastric cancer. Mol Pathol.2003;56: 141-9.
2. Yamada Y, Jackson-Grusby L, Linhart H, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci USA. 2005;102:13580-13585
3. Beier V, Mund C, Hoheisel JD. Monitoring methylation changes in cancer.Adv Biochem Eng Biotechnol. 2007;104:1-11.
4. Chim CS, Liang R, Leung MH, et al. Aberrant gene methylation implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma. J Clin Pathol. 2007 Jan;60(1):104-6.
5．陆嵘，房静远．表观遗传修饰与肿瘤．生命科学．2006；18(1)：10-14
6．张永彪，褚嘉佑．表观遗传学与人类疾病的研究进展遗传．2005；27(3)：466-472
7. Sobin LH, Fleming ID. TNM classification of malignant tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997; 80:1803-1804
8. Zhang WF, Chert JQ, Shah JX, et al. Indication of radical surgery (R2, R3) based upon the pattern of lymph node metastasis from gastric cancer.Zhonghua Zhongliu Zazhi 1987; 9: 286-289
9. Herman JG, Graft JR., Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc NatlAcad Sci USA 1996; 93:9821-9826
10. Tanaka T, Bai T, Yukawa K, et al. Radiation-induced cell death is independent of the apoptotic signals mediated by death-associated protein kinase in human cervical squamous cell carcinoma cells. Oncol Rep. 2005;14(4):949-55.
11. Shang T, Joseph J, Hillard CJ,et al. Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death. J Biol Chem. 2005 14;280(41):34644-53..
12. Bowman P, Galea CA, Lacy E, et al. Thermodynamic characterization of interactions between p27(Kipl) and activated and non-activated Cdk2: intrinsically unstructured proteins as thermodynamic tethers. Biochim Biophys Acta. 2006; 1764(2): 182-9.
13. Mikami T, Yoshida T, Shiraishi H, et al. Bottom-up cell proliferation with cyclin A and p27Kip 1 expression in ulcerative colitis-associated dysplasia. Pathol Int. 2006; 56(1): 10-6.
14. Le XF, Pruefer F, Bast RC Jr HER2-targeting antibodies modulate the cyclin-dependent kinase inhibitor p27Kipl via multiple signaling pathwaysCell Cycle. 2005 Jan;4(1):87-95. Epub 2005 Jan 10.
15.Ozkara SK, Corakci A. Significantly decreased P27 expression in endometrial carcinoma compared to complex hyperplasia with atypia (correlation with p53 expressionPathol Oncol Res. 2004; 10(2):89-97.
16. Leo C, Richter C, Horn LC,et al. Expression of Apaf-1 in cervical cancer correlates with lymphnode metasis but not with introtumor of hypoxia. Cynecol Oncol. 2005;97(2):602-6
17. Mustika r, budiganto A, Nishigori C,et al. Decreased expression of Apaf-1 with progression of melanoma. Pigment cell Des. 2005; 18(l):59-62
18. Lei PP, Zhang ZJ, Shen LJ, et al. Expression and hypermethylation of p27 kipl in hepatocarcinogenesis. World J Gastroenterol. 2005; 11 (29):4587-91.
19. Brakensiek K, Langer F, Kreipe H, et al. Absence of p21(CIP 1), p27(KIP 1) and p 57(KIP 2) methylation in MDS and AML. Leuk Res. 2005; 29(11): 1357-60.
20. Furukawa Y, Sutheesophon K, Wada T, et al. Methylation silencing of the APAF1 gene in acute leukemia. Mol Cancer Res. 2005; 3(6):325-34.
21．黄带发，富伟能，尚超，等．喉鳞癌APAF1基因表达及启动子区甲基化研究遗传学报．2004，31(12)1327．1333．
22. Jian YU, Hong Yu ZHANG, Zhen Zhong MA, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Research. 2003; 13(5):319-333
23. Murty VV, Montgomery K, Dutta S, et al. A 3-Mb high-resolution BAC/PAC contig of 12q22 encompassing the 830-kb consensus minimal deletion in male germ cell tumors. Genome Res.1999;9: 662-71.
24. Umetani N, Fujimoto A, Takeuchi H,et al. Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma.2004. Oncogene Oct 28;23(50):8292-300
25. Baldi A, Santini D, Russo P, et al. Analysis of APAF-1 expression in human cutaneous melanoma progression. Exp Dermatol. 2004;13(2):93-7.
26. Jones PA. DNA methylation errors and cancer.Cancer Res, 1996,56(11):2463-2467
27. Wijermans PW, Krulder JWM,Huijigens PC,et al. Continuous infusion of low-dose 5-aza-2'deoxycytiene in elderly patients with high risk myelodysplastic syndrome. Leukemia, 1997,11(1):1-5
28．杨丽，朱红音，程中华，等．人胃癌细胞肿瘤相关基因的表Ichihashi M，Veda M达与甲基化调控．世界华人消化杂志 2005；13(13)：1493-1498
29．杜红玲，任立敏，陈华，等．丁酸钠与5-氮杂-2’-脱氧胞苷协同诱导U266细胞p16基因重新表达．第一军医大学学报 2002；22：981-984
30．吕国丽，文剑明，张萌，等5-Aza-CdR对癌细胞TIMP-3启动子去甲基化的作用．中国病理生理杂志 2005，21(9)：1763-1768
31. Liu X. S., Kim C. N., Yang J, et al. Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c. Cell 1996 86, 147-157.
32. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell. 1997, 479-489.
33. Leo C, Richter C, Horn LC, Schutz A,et al. Expression of Apaf-1 in cervical cancer correlates with lymphnode metasis but not with introtumor of hypoxia. Cynecol Oncol, 2005 may;97(2):602-6
34. Mustika r, budiganto A, Nishigori C,. Decreased expression of Apaf-1 with progression of melanoma. Pigment cell Des. 2005 Feb; 18(1):59-62
35．高红，张志波，尚海．p57kip2和p27kip1基因mRNA在组织中的表达及意义．中国医科大学学报 2004，33(6)525-529
36. Widschwendter A, Muller HM, Fiegl H, et al. DNA methylation in serum and tumors of cervical cancer patients[J]. Clin Cancer Res, 2004, 10(2): 565-571
37. Yamaguchi S, A sao T, Nakamura J, et al. High frequency of DAP-kinase gene Promoter methylation in colorectal cancer specimens and its identification in serum[J]. Cancer Lett, 2003, 194(1):99-105
38. Toyooka S, Toyooka KO, Miyajima K, et al. Epigenetic down-regulation of death-associated protein kinase in lung cancer[J]. Clin Cancer Res,2003,9(8):3034-3041.
39. Bialik S, Kimchi A. DAP-kinase as a target for drug design in cancer and disease associated with accelerated cell death. Sem in Cancer Biol, 2004, 14 (4): 283-294
40. Kochi M, Fujii M, Kanamori N, et al. Neoadjuvant chemotherapy with S-1 and CDDP in advanced gastric cancer. J Cancer Res Clin Oncol. 2006; 132(12):781-5.
41. Matsuhashi N, Saio M, Matsuo A,et al.The evaluation of gastric cancer sensitivity to 5-FU/CDDP in terms of induction of apoptosis: time- and p53 expression-dependency of anti-cancer drugs. Oncol Rep. 2005; 14(3):609-15.
42. Iioka Y, Mishima K, Azuma N, et al. Overexpression of protein kinase Cdelta enhances cisplatin-induced cytotoxicity correlated with p53 in gastric cancer cell line. Pathobiology. 2005; 72(3): 152-9
43. Egger G, Aparicio AM, Escobar SG, et al. Inhibition of histone deacetylation does not block resilencing of p 16 after 5-aza-2'-deoxycytidine treatment. Cancer Res. 2007;67(1):346-53.
44. Christoph F, Kempkensteffen C, Weikert S, et al. Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer. Br J Cancer. 2006 18;95(12):1701-7
45．马韬，朱正刚．胃癌化疗与细胞凋亡研究进展．国外医学消化系疾病分册．2003；23：15-18
46. Francia G, Green SK, Bocci G, Man S, et al. Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents. Mol Cancer Ther. 2005; 4(10): 1484-94
47. Staub J, Chien J, Pan Y,et al. Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance. Oncogene. 2007; 19.
48. Bai T, Tanaka T, Yukawa K, et al. Int J Oncol. A novel mechanism for acquired cisplatin-resistance: suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells. 2006; 28(2):497-508.
49. Fang X, Zheng C, Liu Z, Ekman P, Xu D. Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine. Oncol Rep. 2004 Sep;12(3):523-6.
50. Qiu YY, Mirkin BL, Dwivedi RS. Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells. Cancer Detect Prev. 2005; 29(5):456-63.
51. Missiaglia E, Donadelli M, Palmieri M, et al. Growth delay of human pancreatic cancer cells by methylase inhibitor 5-aza-2'-deoxycytidine treatment is associated with activation of the interferon signalling pathway. Oncogene. 2005 ; 24(1): 199-211.
52. Koul S, McKiernan JM, Narayan G, et al. Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors. Mol Cancer. 2004 ;3:16-26.
53. Iioka Y, Mishima K, Azuma N, et al. Overexpression of protein kinase Cdelta enhances cisplatin-induced cytotoxicity correlated with p53 in gastric cancer cell line. Pathobiology.2005;72(3):152-9
54. Del Bello B, Valentini MA, Comporti M, et al. Cisplatin-induced apoptosis in melanoma cells: role of caspase-3 and caspase-7 in Apaf-1 proteolytic cleavage and in execution of the degradative phases. Ann N Y Acad Sci. 2003 ; 1010:200-4.
55. Kim WS, 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):827-30.
56. House MG, Guo M, Efron DT, et al. Tumor suppressor gene hypermethylation as a predictor of gastric stromal tumor behavior.J Gastrointest Surg. 2003 ;7(8):1004-14.
57. Schildhaus HU, Krockel I, Lippert H, et al. Promoter hypermethylation of pl6INK4a,E-cadherin, 06-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus,esophagogastric junction and proximal stomach.Int J Oncol. 2005 ;26(6):1493-500.
58. Sabbioni S, Miotto E, Veronese A, et al. Multigene methylation analysis of gastrointestinal tumors: TPEF emerges as a frequent tumor-specific aberrantly methylated marker that can be detected in peripheral blood.Mol Diagn. 2003;7(3-4):201-207.
59. Chang MS, Uozaki H, Chong JM, et al. CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus.Clin Cancer Res. 2006 ;12(10):2995-3002.
60. Zhang X, Yashiro M, Ohira M, et al. Synergic antiproliferative effect of DNA methyltransferase inhibitor in combination with anticancer drugs in gastric carcinoma.Cancer Sci.2006 ;97(9):938-44.
1 Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation (1980) Nature 284, 555-556.
2 Yuan, J., Shaham, S., Ledoux, S., et al. Elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme .Cell.1993; 75: 641-652.
3 Chew S K., Akdemir F, Chen P, et al. The apical caspase dronc governs programmed and unprogrammed cell death in drosophila. Cell. 2004; 7: 897-907.
4 Daish T J, Mills K., Kumar S. Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis Cell. 2004; 7:909-915.
5 Kuida K, Zheng T S, Na S, et al.Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature. 1996; 384:368-372.
6. Kuida K, Haydar T F, Kuan C Y, et al. Reduced Apoptosis and Cytochrome c-Mediated Caspase activation in Mice Lacking Caspase 9. Cell .1998; 94: 325-337.
7. Hakem R, Hakem A., Duncan G S, et al. Differential Requirement for Caspase 9 in Apoptotic Pathways In Vivo. Cell. 1998; 94: 339-352.
8. Zou H, Henzel W J, Liu X, et al. Apaf-1, a human protein homologous to C. elegans CED-4,Participates in Cytochrome c-Dependent Activation of Caspase-3. Cell .1997;90: 405-413.
9. Srinivasula S M, Ahmad M, Fernandes-Alnemri,et al. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell.1998; 1: 949-957.
10 Rodriguez A, Oliver H, Zou H, et al. Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway. Nat Cell Biol.1999; 1: 272-279.
11. Kanuka H, Sawamoto K, Inohara N,et al. Control of the Cell Death Pathway by Dapaf-1, a Drosophila Apaf-1/CED-4-Related Caspase Activator. Mol Cell. 1999; 4: 757-769.
12. Zhou L, Song Z, Tittel J, et al. HAC-1, a Drosophila Homolog of APAF-1 and CED-4,Functions in Developmental and Radiation-Induced Apoptosis. Mol Cell. 1999 ;4: 745-755.
13. Yuan J Y, Horvitz R H. The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death.Development .1992; 116: 309-320.
14 Martinon F, Burns K, Tschopp J. The InflammasomeA Molecular Platform Triggering Activation of Inflammatory Caspases and Processing of proIL-beta. Mol Cell. 2002; 10:417-426.
15 Inohara N, Ogura Y, Nunez G,et al. Nods: a family of cytosolic proteins that regulate the host response to pathogens. Curr. Opin. Microbiol.,2002; 5: 76-80.
16. Liu X S, Kim C N, Yang J,et al. Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome C. Cell. 1996; 86: 147-157.
17 Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell. 1997; 91: 479-489.
18 Rodriguez J,Lazebnik Y. Dark is a Drosophila homologue of Apaf-l/CED-4 and functions in an evolutionarily conserved death pathway. Genes Dev. 1999; 13:3179-3184.
19. Acehan D, Jiang X., Morgan D G, et al. Three-Dimensional Structure of the Apoptosomelmplications for Assembly, Procaspase-9 Binding, and Activation. Mol. Cell. 2002;9: 423-43
20. Hao Z, Duncan GS, Chang CC, et al. Specific ablation of the apoptotic functions of cytochrome C reveals a differential requirement for cytochrome C and Apaf-1 in apoptosis. Cell.2005;121(4):579-91.
21. Robles AI, Bemmels NA, Foraker AB, et al. APAF-1 is a transcriptional target of p53 in DNA damage-induced apoptosis. Cancer Res.2001; 61: 6660-4.
22. Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. Mol Cell. 2003; 11:577-90.
23. Fortin A, Cregan SP, MacLaurin JG, et al. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death. J Cell Biol. 2001; 155: 207-16.
24. Moroni MC, Hickman ES, Denchi EL, et al Apaf-1 is a transcriptional target for E2F and p53.Nat Cell Biol.2001; 3: 552-8.
25. Ho CK, Bush JA, Li G. Tissue-specific regulation of Apaf-1 expression by p53. Oncol Rep.2003 ;10(5):l 139-43
26. Sturm I, Bosanquet AG, Radetzki S, et al.Silencing of APAF-1 in B-CLL result in poor prognosis in the case of concomitant p53 mutation. Int J cancer. 2006; 118(9):2329-36.
27. Marsden VS, O'Connor L, O'Reilly LA, et al. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature.2002 ;419(6907):634-7.
28. Leo C, Richter C, Horn LC, et al. Expression of Apaf-1 in cervical cancer correlates with lymphnode metasis but not with introtumor of hypoxia. Cynecol Oncol: 2005 ;97(2):602-6
29. Mustika r,budiganto A, Nishigori C, et al. Decreased expression of Apaf-1 with progression of melanoma. Pigment cell Des. 2005 ; 18(1):59-62
30. Ekert PG, Read SH, Silke J, et al. Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J Cell Biol. 2004;165(6):835-42.
31. Anichini A, Mortarini R, Sensi M, et al. Apaf-1 signaling in human melanoma. Cancer Lett.2006;238(2): 168-79.
32. Kimura M, Furukawa T, Abe T, et al. Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer. Cancer Res. 1998;58: 2456-60.
33. Murty VV, Montgomery K, Dutta S, et al A 3-Mb high-resolution BAC/PAC contig of 12q22 encompassing the 830-kb consensus minimal deletion in male germ cell tumors. Genome Res.1999;9:662-71.
34. Hatta Y, Takeuchi S, Yokota J, et al. Ovarian cancer has frequent loss of heterozygosity at chromosome 12pl2.3-13.1 (region of TEL and Kip1 loci) and chromosome 12q23-ter: evidence for two new tumour-suppressor genes. Br J Cancer. 1997; 75: 1256-62.
35. Watanabe T, Hirota Y, Arakawa Y, et al. Frequent LOH at chromosome 12q22-23 and Apaf-1 inactivation in glioblastoma. Brain Pathol. 2003;13(4):431-9.
36. Umetani N, Fujimoto A, Takeuchi H,et al. Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma .Oncogene. 2004; 23(50):8292-300
37. Baldi A, Santini D, Russo P, et al. Analysis of APAF-1 expression in human cutaneous melanoma progression. Exp Dermatol. 2004; 13(2):93-7.
38. Fujimoto A, O'Day SJ, Taback B, et al. Allelic imbalance on 12q22-23 in serum circulating DNA of melanoma patients predicts disease outcome.Cancer Res. 2004 ;64(12):4085-8.
39. Fujimoto A, Takeuchi H, Taback B, et al. Allelic imbalance of 12q22-23 associated with APAF-1 locus correlates with poor disease outcome in cutaneous melanoma. Cancer Res. 2004; 64(6):2245-50.
40. Scott CL, Schuler M, Marsden VS, et al. Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation. J Cell Biol. 2004; 164(1):89-96.
41. Ekert PG, Read SH, Silke J, et al. Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J Ceil Biol. 2004;165(6):835-42.
42. Besse B, Cande C, Spano JP, et al. Nuclear localization of apoptosis protease activating factor-1 predicts survival after tumor resection in early-stage non-small cell lung cancer. Clin Cancer Res. 2004; 10(17):5665-9.
43. Fu WN, Bertoni F, Kelsey SM, et al. Role of DNA methylation in the suppression of Apaf-1 protein in human leukaemia. Oncogene.2003; 22(3):451-5
44. Furukawa Y, Sutheesophon K, Wada T, et al. Methylation silencing of the Apaf-1 gene in acute leukemia. Mol Cancer Res.2005; 3(6):325-34.
45．黄带发，富伟能，尚超，等．喉鳞癌Apaf-1基因表达及启动子区甲基化研究遗传学报．2004；31 (12)：1327-1333．
46. McLeod, Gordon Strathdee, Robert Brown. CpG Island Methylation of DNA Damage Response Genes in Advanced Ovarian Cancer. Cancer Res. 2005; 65: 8961-8967.
47．程留芳 杨云生 李红梅．Apaf-1基因转染及其对5-氟尿嘧啶诱导AGS细胞凋亡的影响．中华消化杂志．2002；22(9)：550-553．
48. Schneider BG, Rha SY, Chung HC, et al. Regions of allelic imbalance in the distal portion of chromosome 12q in gastric cancer. Mol Pathol.2003;56: 141-9.