靶向EBV-LMP1的脱氧核酶放射增敏的分子机制
摘要
鼻咽癌(nasopharyngeal carcinoma,NPC)是中国人群特有的高发肿瘤,鼻咽癌病理类型主要为低分化鳞癌,放射治疗是其首选治疗方式。临床上发现放疗抵抗、复发、转移是导致治疗失败的主要原因,寻找放疗增敏的策略是降低鼻咽癌复发、转移、提高病人生存率,降低死亡率的关键。
放射增敏研究是肿瘤学研究的一个热点,其不仅涉及到许多重要分子机制的新进展,同时对于改善临床放射治疗的疗效具有重要意义。EB病毒(Epstein-Barr Virus,EBV)是鼻咽癌的主要病因。其中EB病毒编码的潜伏膜蛋白LMP1是重要的致瘤蛋白,在鼻咽癌病人中阳性率超过65%。研究发现EBV编码的LMP1在放疗抵抗中具有重要作用。我们的前期研究证明靶向LMP1脱氧核酶能够通过抑制NF-κB、AP-1、Stat三条信号通路抑制细胞增殖,诱导细胞凋亡,并且能够显著抑制鼻咽癌裸鼠移植瘤的生长。在细胞和动物水平上将脱氧核酶与放疗联合应用,证实靶向LMP1的脱氧核酶能够增强放疗敏感性。
肿瘤细胞的周期调控能力及DNA损伤修复能力,是细胞辐射敏感的主要决定因素。在实验室前期工作的基础上本研究以鼻咽癌细胞为模型,以靶向脱氧核酶为主要手段,以LMP1对细胞周期与DNA损伤修复异常调控为切入点,深入研究脱氧核酶放射增敏的分子机制。1.靶向LMP1的脱氧核酶DZ1对LMP1表达的抑制及对细胞周期的影响
通过Western blotting检测靶向LMP1脱氧核酶DZ1能够抑制LMP1表达,流式细胞术发现脱氧核酶能引起LMP1阳性细胞发生S期阻滞,后者是放疗增敏的重要特征。我们以细胞周期网路通路为切入点,围绕G1/S以及G2/M两个细胞周期监测点,研究靶向EBV-LMP1脱氧核酶所致重要细胞周期分子的改变,从细胞周期素Cyclins,细胞周期依赖性蛋白激酶CDKs,以及相关CDKIs三个层次研究S期阻滞的分子机制。我们发现脱氧核酶通过抑制LMP1的表达,不同程度地下调CycD1、CDK4、CycE、CDK2的表达,通过CO-IP及免疫沉淀激酶活性实验发现脱氧核酶能够CycD1/CDK4的相互作用及激酶活性,抑制下游RB/E2F通路;另一方面,脱氧核酶抑制LMP1的表达,引起cdc2活性磷酸化水平的降低,通过ELISA实验发现脱氧核酶能够使CycB/cdc2复合物活性降低,使细胞停留在G2期不再继续行进,两方面造成了S期阻滞这一现象。Western blotting检测细胞周期负性调节因子CDKI和P53发现,脱氧核酶抑制LMP1表达后,并没有引起这些分子的表达上升,反而有不同程度的下降。因为我们认为靶向LMP1脱氧核酶能引起LMP1阳性细胞发生S期阻滞现象,这种S期阻滞的分子机制涉及到G1/S及G2/M两个细胞监测点通路多种蛋白分子及激酶活性的改变,不依赖于P53。2.靶向LMP1的脱氧核酶通过NF-κB通路抑制DNA损伤修复系统中核心分子ATM的表达
ATM基因与肿瘤放射敏感性有关,其基因表达产物能够识别电离辐射等细胞毒作用造成的DNA损伤,磷酸化相应的底物,进行DNA损伤的信号传递,参与DNA损伤的修复过程。因此,ATM在放射引起的DNA损伤修复过程中起着十分重要的作用。这一部分研究我们将检测靶向LMP1的脱氧核酶能否抑制DNA损伤修复系统中核心分子ATM的表达。
首先,在转录水平检测LMP1对ATM表达的调控以及靶向LMP1脱氧核酶处理后能否抑制这种上调。构建含有完整的ATM启动子DNA序列报告基因质粒,进行报道基因实验,在LMP1阴性细胞CNE1中瞬时转染ATM报道基因质粒并剂量梯度转染野生型的LMP1质粒,在转录水平利用报道基因检测ATM启动子的活性随着LMP1的表达呈剂量性增强。在稳定表达野生型LMP1的鼻咽癌细胞CNE1-LMP1和LMP1阴性细胞CNE1中检测,发现LMP1能上调ATM的转录活性。同时转染脱氧核酶,检测LMP1表达被阻断后,ATM的转录活性回复到正常水平。
进一步在蛋白水平检测LMP1调控ATM表达的情况。在LMP1阴性细胞CNE1中剂量性瞬时转染野生型pSG5-LMP1质粒,LMP1的表达随着转入质粒呈剂量依赖性增加,同时在蛋白水平上检测ATM的表达也呈剂量性增加。在EBV阴性的鼻咽低分化鳞癌细胞系HNE2和稳定表达EB病毒LMP1(B95.8来源)的鼻咽癌细胞系HNE2-LMP1以及CNE1和CNE1-LMP1细胞中进行实验,通过Western Blotting检测ATM的表达,发现LMP1能够上调ATM的表达。脱氧核酶处理后随着LMP1表达下降后ATM的表达也随之降低。通过转录水平和蛋白水平的检测证实ATM是一个受EBV-LMP1调控的重要的损伤修复蛋白。LMP1能够上调ATM的表达,靶向LMP1脱氧核酶能够通过阻断LMP1的表达抑制这种上调作用。
在此基础上,我们进一步研究参与这种调控的信号转导机制。生物信息学预测发现ATM启动子区GXP-480587包含了三个NF-κB转录因子结合位点,其中两个位于同一段DNA序列的正义链和反义链上。推测转录因子NF-κB可能作用于启动子,从而调节ATM的基因表达。因此,LMP1可能通过活化NF-κB信号转导通路上调ATM表达。
采用特异性阻断策略,阻断NF-κB的活性,发现随着NF-κB特异性抑制剂Bayl1-7082浓度梯度的增加,ATM的表达呈剂量依赖性减少,确定LMP1通过激活NF-κB信号转导通路上调ATM表达。利用稳定表达IκBα显性负性突变体的鼻咽癌细胞系下调NF-κB活性,Western Blotting检测其ATM表达量与亲本细胞的差异。结果证实稳定表达IκBα显性负性突变体能部分阻断LMP1上调的ATM表达。
基因的表达调控需要顺式作用元件与反式作用因子共同参与。在明确转录因子NF-κB通过NF-κB信号传导通路参与LMP1上调鼻咽癌细胞ATM表达基础上,我们进一步明确与转录因子NF-κB相互作用的顺式作用元件。在已构建含NF-κB结合位点ATM启动子荧光素酶报道基因质粒基础上,利用基于重叠延伸PCR(overlap extensionPCR)体外定点突变技术将这个质粒中两个κB位点分别突变或同时突变,构建三种突变质粒,将质粒分别转染CNE1和CNE1-LMP1细胞中,发现分别突变的κB位点能不同程度地抑制LMP1上调的ATM启动子活性,两个位点同时突变后能显著抑制ATM的启动子活性。说明位于ATM启动子区的κB位点具有功能活性,而且两个κB位点可能具有协同效应。
在明确转录因子NF-κB参与激活顺式调控元件κB基础上,根据人ATM基因启动子区的两个NF-κB位点序列构建突变质粒的突变序列分别合成生物素标记的野生型和突变型NF-κB寡核苷酸探针。利用EMSA和Supershift-EMSA方法进一步分析其与ATM基因启动子区相应DNA的结合能力及这两个二聚体转录因子的亚单位组成。结果表明,CNE1-LMP1细胞核蛋白与ATM基因中κB DNA结合能力明显高于CNE1细胞。脱氧核酶处理CNE1-LMP1细胞后其核蛋白结合ATM基因κB DNA能力明显降低。Supershift结果表明直接结合到ATM基因启动子增强子上的NF-κB亚单位至少包括p50。这从信号转导调控角度提出了靶向EBV-LMP1的脱氧核酶放射增敏的分子机制。3.抑制AIM的表达能够增强鼻咽癌细胞对放射的敏感性
我们证实了靶向LMP1的脱氧核酶能够抑制细胞中ATM的表达,并且是通过调控NF-κB信号通路,抑制转录因子NF-κB与ATM启动子区的结合。进一步深入研究在LMP1阳性细胞中关闭ATM的表达对LMP1阳性的鼻咽癌细胞的放疗敏感性的影响。
应用流式细胞仪分析放射处理后细胞凋亡,发现在siRNA沉默ATM表达的LMP1阳性鼻咽癌细胞中,相同剂量放射处理后在相应时间点检测细胞凋亡,发现细胞凋亡百分率明显增高,证实了抑制ATM的表达能协同放射线促进鼻咽癌细胞凋亡。
通过平板集落形成实验证实,在用ATMsiRNA降低ATM蛋白表达的鼻咽癌细胞中,放射后集落形成率受到明显抑制。采用线性-二次模型和多靶单击模型对细胞存活曲线进行拟合,检测放射参数,进一步证实ATM蛋白表达降低后细胞的放射敏感性增强,首次提出了在鼻咽癌中关闭ATM的表达能引起放射增敏。
本课题以鼻咽癌细胞为实验模型,首次从细胞周期及信号传导角度对脱氧核酶的放射增敏分子机制进行较为系统地探索性研究,发现脱氧核酶能够通过调控细胞周期进程及DNA损伤修复中核心分子ATM的表达参与放射增敏,为脱氧核酶在临床上的应用提供了重要的理论及实验证据,也为放疗抵抗的EBV相关肿瘤的治疗提供了新的思路。
Nasopharyngeal carcinoma(NPC) has a remarkably distinctive ethnic and geographic distribution,more than 80%of which were reported from China,Southeast Asia,and some other Asian countries.A unique feature of NPC is its strong association with Epstein-Barr Virus (EBV).Latent membrane protein 1(LMP1) is a major one with oncogenic properties among EBV encoded proteins.Since NPC is highly radiosensitive,radiotherapy(RT) has always been the main treatment of choice for this cancer.Although overall survival after RT for the patients at early stages is encouraging,there are significant rates of local failure and distant metastases subsequent to RT in the advanced stage disease. Thus,it has been a great challenge to identify biological agents as radiosensitizers that could enhance radiosensitivity for treatment of the EBV-associated NPC.In previous studies,we experimentally demonstrated that the phosphorothioate-modified“10-23”DNAzymes specifically against the LMP1 mRNA could down-regulate the expression of LMP1 in a nasopharyngeal carcinoma cell line CNE1-LMP1 and affect the down-stream pathways activated by LMP1,such as NF-κB, JNK/AP-1 and STAT signaling pathways.When tested in a mouse xenograft model,the DNAzyme was found to inhibit tumor growth and enhance radiosensitivity in vivo.We also demonstrated that when combined with the radiotherapy the DNAzyme could enhance the radiosensitivity both in vivo and in vitro.
It is known that the celluar radiosensitivity is closed related to the cell cycle regulation and DNA repairing.To explore the molecular mechanisms underlying the radiosensitizing effect of the LMP1-targeted DNAzyme in nasopharyngeal carcinoma(NPC),we used the NPC cell lines as model to investigate how EBV-LMP1 is involved in the radioresistance via cell cycle control and DNA repair mechanisms.
The LMP1-targeted DNAzyme inhibited the expression of LMP1 and affected the cell cycle
We first demonstrated when the expression of LMP1 was inhibited by DNAzyme,the NPC cells was shown to be arrested at the S phase. This cell cycle arrest was accompanied with a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DNAzyme treatment.Moreover, we observed an inhibition of CDK4 activity and a decreased cyclinD1 expression in the complexes immunoprecipitated with CDK4 antibody and the suppression of RB/E2F pathway in DNAzyme treated cells.We also found that,a reduction in cdc2 phosphorylation at Thr161,which partially stands for the cdc2 kinase activity in DNAzyme treated CNE1-LMP1 cells,although the active DNAzyme did not affect cyclinB1 and cdc2 expression.Further,we analyzed that changes in cdc2 kinase activity induced by DNAzyme and found that the down-regulation of the LMP1 expression resulted in a 5-fold reduction of cdc2 kinase activity in CNE1-LMP1,suggesting that G1/S and G2/M checkpoint pathways could contribute to the S phase arrest in LMP1-positive cells induced by the LMP 1-targeted DNAzyme.
The LMP1-targeted DNAzyme inhibited the expression of ATM,a key factor of DNA repairing systems through NF-κB pathway.
The central role of the ATM protein in DNA damage repair is now well established.Ionizing radiation(IR),not UV radiation,enhances ATM kinase activity and phosphorylates a series of target proteins(e.g. p53,BRCA1,c-ab1,etc.),which are involved in cell cycle control and repair of DNA damage.ATM-deficient cells have impaired ability to efficiently halt proliferation and repair DNA damage.We further examined if the LMP1-targeted DNAzyme could regulate the expression of ATM.
Using the induction strategy by LMP1 expression plasmid and the blockage strategy by LMP1-targeted DNAzyme,we confirmed that LMP1 up-regulated the ATM transcriptional activity and the protein level in the reporter gene assay and western blotting assay.
Based on the finding that LMP1 could up-regulate the ATM expression,we investigated the signaling pathway involved in the procession.Bioinformatic analysis revealed that there are three putative NF-κB binding sites in ATM promoter region(GenBank Accession GXP_480587).The first and third binding sites were located in the same location,but in different strands.It implied that NF-κB binding to corresponding sites might be responsible for the modulation of ATM gene expression.
By using a specific inhibitor of the NF-κB signaling pathway,the suppression of the ATM up-regulated in CNE1-LMP1 cells by the inhibitor could be achieved in a dose-dependent manner.A stable NPC cell line expressing dominant-negative mutant of IκBα(DNMIκBα) was used to further confirm the role of NF-κB pathway in regulating ATM expression.As verified by Western blotting,the stable expression DNMIκB resulted in a decrease ATM expression in HNE2-LMP1-DNMIκBαcell.
On the basis of the findings that NF-κB pathway was involved in LMP1-augmented ATM expression in human NPC cells,we attempt to confirm the precise element which NF-κB binds to.The site-directed mutagenesis by Overlap Extension PCR was used to introduce mutations into two locations of NF-κB in promoter region(the first/third and second sites).From the reporter gene assay we found that the different mutations could suppress the ATM promoter activity in different degrees.Mutations at two locations at the same time downregulate the ATM promoter activity.This suggested that a synergistic effect of the NF-κB binding to two sites may be responsible for the up-regulation of ATM expression mediated by LMP1.
To demonstrate the direct binding of NF-κB to the ATM promoter, an electrophoretic mobility supershift assay(EMSA) was further conducted.We showed that the NF-κB DNA-binding activity was much more stronger in CNE1-LMP1 cells than in CNE1 cells,indicating the role of LMP1 in mediation of NF-κB-ATM pathway.The induction of NF-κB DNA binding activity by LMP1 was clearly inhibited by LMP1-targeted DNAzyme.Supershift analysis with antibodies specific for NF-κB family members showed that NF-κB DNA/protein complex composed of p50 subunit in nuclear extracts of CNE1-LMP1 cells,which suggested that at least the NF-κB p50 directly bind to the ATM promoter. Inhibition of the ATM expression could enhance the radiosensitivity in NPC cells
We have confirmed that the ATM expression could be downregulated by LMP1-targeted DNAzyme through suppressing the transcription factor NF-κB binding to the ATM promoter.It has been demonstrated that silence of the ATM could enhance radiosensitivity in breast cancer and glioma cells.We then investigate if the radiosenstivity caused by LMP1-targeted DNAzyme is through the downregulation of the ATM expression in NPC cells.
Using the ATM-targeted siRNA and FACS assay,we found when exposed to 5 Gy,nearly 60%of the ATM-siRNA-treated cells underwent apoptosis.The colony-formation assay showed when the expression of ATM was deceased by the siRNA,the colony-formation ability was reduced.The result suggested that the down-regulation of ATM may suppress the DNA repairing signaling pathway and promote the cellular death,then enhanced the radiosensitivity of NPC cells.Comprehensive analysis of the radiobiological parameters in the single-hit multitarget model and linear quadratic equation further confirmed that the silence of the ATM expression led to sensitizing of the NPC cells to radiation.
In conclusion,our finding demonstrated that the LMP1-targeted DNAzyme inhibited the expression of LMP1 and affected the cell cycle; the LMP1-targeted DNAzyme inhibited the expression of ATM;and inhibition of the ATM expression could enhance the radiosensitivity in NPC cells.The data provided solid experimental evidence to support our hypothesis that the radiosensitization of NPC cells by LMP1-targeted DNAzyme is through the impact on cell cycle control and DNA repair systems.The results can provide a basis for the use of the LMP1-targeted DNAzyme as potential radiosensitizer for clinical treatment and supply a new way to treat the EBV-associated radioresistance carcinomas.
放射增敏研究是肿瘤学研究的一个热点,其不仅涉及到许多重要分子机制的新进展,同时对于改善临床放射治疗的疗效具有重要意义。EB病毒(Epstein-Barr Virus,EBV)是鼻咽癌的主要病因。其中EB病毒编码的潜伏膜蛋白LMP1是重要的致瘤蛋白,在鼻咽癌病人中阳性率超过65%。研究发现EBV编码的LMP1在放疗抵抗中具有重要作用。我们的前期研究证明靶向LMP1脱氧核酶能够通过抑制NF-κB、AP-1、Stat三条信号通路抑制细胞增殖,诱导细胞凋亡,并且能够显著抑制鼻咽癌裸鼠移植瘤的生长。在细胞和动物水平上将脱氧核酶与放疗联合应用,证实靶向LMP1的脱氧核酶能够增强放疗敏感性。
肿瘤细胞的周期调控能力及DNA损伤修复能力,是细胞辐射敏感的主要决定因素。在实验室前期工作的基础上本研究以鼻咽癌细胞为模型,以靶向脱氧核酶为主要手段,以LMP1对细胞周期与DNA损伤修复异常调控为切入点,深入研究脱氧核酶放射增敏的分子机制。1.靶向LMP1的脱氧核酶DZ1对LMP1表达的抑制及对细胞周期的影响
通过Western blotting检测靶向LMP1脱氧核酶DZ1能够抑制LMP1表达,流式细胞术发现脱氧核酶能引起LMP1阳性细胞发生S期阻滞,后者是放疗增敏的重要特征。我们以细胞周期网路通路为切入点,围绕G1/S以及G2/M两个细胞周期监测点,研究靶向EBV-LMP1脱氧核酶所致重要细胞周期分子的改变,从细胞周期素Cyclins,细胞周期依赖性蛋白激酶CDKs,以及相关CDKIs三个层次研究S期阻滞的分子机制。我们发现脱氧核酶通过抑制LMP1的表达,不同程度地下调CycD1、CDK4、CycE、CDK2的表达,通过CO-IP及免疫沉淀激酶活性实验发现脱氧核酶能够CycD1/CDK4的相互作用及激酶活性,抑制下游RB/E2F通路;另一方面,脱氧核酶抑制LMP1的表达,引起cdc2活性磷酸化水平的降低,通过ELISA实验发现脱氧核酶能够使CycB/cdc2复合物活性降低,使细胞停留在G2期不再继续行进,两方面造成了S期阻滞这一现象。Western blotting检测细胞周期负性调节因子CDKI和P53发现,脱氧核酶抑制LMP1表达后,并没有引起这些分子的表达上升,反而有不同程度的下降。因为我们认为靶向LMP1脱氧核酶能引起LMP1阳性细胞发生S期阻滞现象,这种S期阻滞的分子机制涉及到G1/S及G2/M两个细胞监测点通路多种蛋白分子及激酶活性的改变,不依赖于P53。2.靶向LMP1的脱氧核酶通过NF-κB通路抑制DNA损伤修复系统中核心分子ATM的表达
ATM基因与肿瘤放射敏感性有关,其基因表达产物能够识别电离辐射等细胞毒作用造成的DNA损伤,磷酸化相应的底物,进行DNA损伤的信号传递,参与DNA损伤的修复过程。因此,ATM在放射引起的DNA损伤修复过程中起着十分重要的作用。这一部分研究我们将检测靶向LMP1的脱氧核酶能否抑制DNA损伤修复系统中核心分子ATM的表达。
首先,在转录水平检测LMP1对ATM表达的调控以及靶向LMP1脱氧核酶处理后能否抑制这种上调。构建含有完整的ATM启动子DNA序列报告基因质粒,进行报道基因实验,在LMP1阴性细胞CNE1中瞬时转染ATM报道基因质粒并剂量梯度转染野生型的LMP1质粒,在转录水平利用报道基因检测ATM启动子的活性随着LMP1的表达呈剂量性增强。在稳定表达野生型LMP1的鼻咽癌细胞CNE1-LMP1和LMP1阴性细胞CNE1中检测,发现LMP1能上调ATM的转录活性。同时转染脱氧核酶,检测LMP1表达被阻断后,ATM的转录活性回复到正常水平。
进一步在蛋白水平检测LMP1调控ATM表达的情况。在LMP1阴性细胞CNE1中剂量性瞬时转染野生型pSG5-LMP1质粒,LMP1的表达随着转入质粒呈剂量依赖性增加,同时在蛋白水平上检测ATM的表达也呈剂量性增加。在EBV阴性的鼻咽低分化鳞癌细胞系HNE2和稳定表达EB病毒LMP1(B95.8来源)的鼻咽癌细胞系HNE2-LMP1以及CNE1和CNE1-LMP1细胞中进行实验,通过Western Blotting检测ATM的表达,发现LMP1能够上调ATM的表达。脱氧核酶处理后随着LMP1表达下降后ATM的表达也随之降低。通过转录水平和蛋白水平的检测证实ATM是一个受EBV-LMP1调控的重要的损伤修复蛋白。LMP1能够上调ATM的表达,靶向LMP1脱氧核酶能够通过阻断LMP1的表达抑制这种上调作用。
在此基础上,我们进一步研究参与这种调控的信号转导机制。生物信息学预测发现ATM启动子区GXP-480587包含了三个NF-κB转录因子结合位点,其中两个位于同一段DNA序列的正义链和反义链上。推测转录因子NF-κB可能作用于启动子,从而调节ATM的基因表达。因此,LMP1可能通过活化NF-κB信号转导通路上调ATM表达。
采用特异性阻断策略,阻断NF-κB的活性,发现随着NF-κB特异性抑制剂Bayl1-7082浓度梯度的增加,ATM的表达呈剂量依赖性减少,确定LMP1通过激活NF-κB信号转导通路上调ATM表达。利用稳定表达IκBα显性负性突变体的鼻咽癌细胞系下调NF-κB活性,Western Blotting检测其ATM表达量与亲本细胞的差异。结果证实稳定表达IκBα显性负性突变体能部分阻断LMP1上调的ATM表达。
基因的表达调控需要顺式作用元件与反式作用因子共同参与。在明确转录因子NF-κB通过NF-κB信号传导通路参与LMP1上调鼻咽癌细胞ATM表达基础上,我们进一步明确与转录因子NF-κB相互作用的顺式作用元件。在已构建含NF-κB结合位点ATM启动子荧光素酶报道基因质粒基础上,利用基于重叠延伸PCR(overlap extensionPCR)体外定点突变技术将这个质粒中两个κB位点分别突变或同时突变,构建三种突变质粒,将质粒分别转染CNE1和CNE1-LMP1细胞中,发现分别突变的κB位点能不同程度地抑制LMP1上调的ATM启动子活性,两个位点同时突变后能显著抑制ATM的启动子活性。说明位于ATM启动子区的κB位点具有功能活性,而且两个κB位点可能具有协同效应。
在明确转录因子NF-κB参与激活顺式调控元件κB基础上,根据人ATM基因启动子区的两个NF-κB位点序列构建突变质粒的突变序列分别合成生物素标记的野生型和突变型NF-κB寡核苷酸探针。利用EMSA和Supershift-EMSA方法进一步分析其与ATM基因启动子区相应DNA的结合能力及这两个二聚体转录因子的亚单位组成。结果表明,CNE1-LMP1细胞核蛋白与ATM基因中κB DNA结合能力明显高于CNE1细胞。脱氧核酶处理CNE1-LMP1细胞后其核蛋白结合ATM基因κB DNA能力明显降低。Supershift结果表明直接结合到ATM基因启动子增强子上的NF-κB亚单位至少包括p50。这从信号转导调控角度提出了靶向EBV-LMP1的脱氧核酶放射增敏的分子机制。3.抑制AIM的表达能够增强鼻咽癌细胞对放射的敏感性
我们证实了靶向LMP1的脱氧核酶能够抑制细胞中ATM的表达,并且是通过调控NF-κB信号通路,抑制转录因子NF-κB与ATM启动子区的结合。进一步深入研究在LMP1阳性细胞中关闭ATM的表达对LMP1阳性的鼻咽癌细胞的放疗敏感性的影响。
应用流式细胞仪分析放射处理后细胞凋亡,发现在siRNA沉默ATM表达的LMP1阳性鼻咽癌细胞中,相同剂量放射处理后在相应时间点检测细胞凋亡,发现细胞凋亡百分率明显增高,证实了抑制ATM的表达能协同放射线促进鼻咽癌细胞凋亡。
通过平板集落形成实验证实,在用ATMsiRNA降低ATM蛋白表达的鼻咽癌细胞中,放射后集落形成率受到明显抑制。采用线性-二次模型和多靶单击模型对细胞存活曲线进行拟合,检测放射参数,进一步证实ATM蛋白表达降低后细胞的放射敏感性增强,首次提出了在鼻咽癌中关闭ATM的表达能引起放射增敏。
本课题以鼻咽癌细胞为实验模型,首次从细胞周期及信号传导角度对脱氧核酶的放射增敏分子机制进行较为系统地探索性研究,发现脱氧核酶能够通过调控细胞周期进程及DNA损伤修复中核心分子ATM的表达参与放射增敏,为脱氧核酶在临床上的应用提供了重要的理论及实验证据,也为放疗抵抗的EBV相关肿瘤的治疗提供了新的思路。
Nasopharyngeal carcinoma(NPC) has a remarkably distinctive ethnic and geographic distribution,more than 80%of which were reported from China,Southeast Asia,and some other Asian countries.A unique feature of NPC is its strong association with Epstein-Barr Virus (EBV).Latent membrane protein 1(LMP1) is a major one with oncogenic properties among EBV encoded proteins.Since NPC is highly radiosensitive,radiotherapy(RT) has always been the main treatment of choice for this cancer.Although overall survival after RT for the patients at early stages is encouraging,there are significant rates of local failure and distant metastases subsequent to RT in the advanced stage disease. Thus,it has been a great challenge to identify biological agents as radiosensitizers that could enhance radiosensitivity for treatment of the EBV-associated NPC.In previous studies,we experimentally demonstrated that the phosphorothioate-modified“10-23”DNAzymes specifically against the LMP1 mRNA could down-regulate the expression of LMP1 in a nasopharyngeal carcinoma cell line CNE1-LMP1 and affect the down-stream pathways activated by LMP1,such as NF-κB, JNK/AP-1 and STAT signaling pathways.When tested in a mouse xenograft model,the DNAzyme was found to inhibit tumor growth and enhance radiosensitivity in vivo.We also demonstrated that when combined with the radiotherapy the DNAzyme could enhance the radiosensitivity both in vivo and in vitro.
It is known that the celluar radiosensitivity is closed related to the cell cycle regulation and DNA repairing.To explore the molecular mechanisms underlying the radiosensitizing effect of the LMP1-targeted DNAzyme in nasopharyngeal carcinoma(NPC),we used the NPC cell lines as model to investigate how EBV-LMP1 is involved in the radioresistance via cell cycle control and DNA repair mechanisms.
The LMP1-targeted DNAzyme inhibited the expression of LMP1 and affected the cell cycle
We first demonstrated when the expression of LMP1 was inhibited by DNAzyme,the NPC cells was shown to be arrested at the S phase. This cell cycle arrest was accompanied with a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DNAzyme treatment.Moreover, we observed an inhibition of CDK4 activity and a decreased cyclinD1 expression in the complexes immunoprecipitated with CDK4 antibody and the suppression of RB/E2F pathway in DNAzyme treated cells.We also found that,a reduction in cdc2 phosphorylation at Thr161,which partially stands for the cdc2 kinase activity in DNAzyme treated CNE1-LMP1 cells,although the active DNAzyme did not affect cyclinB1 and cdc2 expression.Further,we analyzed that changes in cdc2 kinase activity induced by DNAzyme and found that the down-regulation of the LMP1 expression resulted in a 5-fold reduction of cdc2 kinase activity in CNE1-LMP1,suggesting that G1/S and G2/M checkpoint pathways could contribute to the S phase arrest in LMP1-positive cells induced by the LMP 1-targeted DNAzyme.
The LMP1-targeted DNAzyme inhibited the expression of ATM,a key factor of DNA repairing systems through NF-κB pathway.
The central role of the ATM protein in DNA damage repair is now well established.Ionizing radiation(IR),not UV radiation,enhances ATM kinase activity and phosphorylates a series of target proteins(e.g. p53,BRCA1,c-ab1,etc.),which are involved in cell cycle control and repair of DNA damage.ATM-deficient cells have impaired ability to efficiently halt proliferation and repair DNA damage.We further examined if the LMP1-targeted DNAzyme could regulate the expression of ATM.
Using the induction strategy by LMP1 expression plasmid and the blockage strategy by LMP1-targeted DNAzyme,we confirmed that LMP1 up-regulated the ATM transcriptional activity and the protein level in the reporter gene assay and western blotting assay.
Based on the finding that LMP1 could up-regulate the ATM expression,we investigated the signaling pathway involved in the procession.Bioinformatic analysis revealed that there are three putative NF-κB binding sites in ATM promoter region(GenBank Accession GXP_480587).The first and third binding sites were located in the same location,but in different strands.It implied that NF-κB binding to corresponding sites might be responsible for the modulation of ATM gene expression.
By using a specific inhibitor of the NF-κB signaling pathway,the suppression of the ATM up-regulated in CNE1-LMP1 cells by the inhibitor could be achieved in a dose-dependent manner.A stable NPC cell line expressing dominant-negative mutant of IκBα(DNMIκBα) was used to further confirm the role of NF-κB pathway in regulating ATM expression.As verified by Western blotting,the stable expression DNMIκB resulted in a decrease ATM expression in HNE2-LMP1-DNMIκBαcell.
On the basis of the findings that NF-κB pathway was involved in LMP1-augmented ATM expression in human NPC cells,we attempt to confirm the precise element which NF-κB binds to.The site-directed mutagenesis by Overlap Extension PCR was used to introduce mutations into two locations of NF-κB in promoter region(the first/third and second sites).From the reporter gene assay we found that the different mutations could suppress the ATM promoter activity in different degrees.Mutations at two locations at the same time downregulate the ATM promoter activity.This suggested that a synergistic effect of the NF-κB binding to two sites may be responsible for the up-regulation of ATM expression mediated by LMP1.
To demonstrate the direct binding of NF-κB to the ATM promoter, an electrophoretic mobility supershift assay(EMSA) was further conducted.We showed that the NF-κB DNA-binding activity was much more stronger in CNE1-LMP1 cells than in CNE1 cells,indicating the role of LMP1 in mediation of NF-κB-ATM pathway.The induction of NF-κB DNA binding activity by LMP1 was clearly inhibited by LMP1-targeted DNAzyme.Supershift analysis with antibodies specific for NF-κB family members showed that NF-κB DNA/protein complex composed of p50 subunit in nuclear extracts of CNE1-LMP1 cells,which suggested that at least the NF-κB p50 directly bind to the ATM promoter. Inhibition of the ATM expression could enhance the radiosensitivity in NPC cells
We have confirmed that the ATM expression could be downregulated by LMP1-targeted DNAzyme through suppressing the transcription factor NF-κB binding to the ATM promoter.It has been demonstrated that silence of the ATM could enhance radiosensitivity in breast cancer and glioma cells.We then investigate if the radiosenstivity caused by LMP1-targeted DNAzyme is through the downregulation of the ATM expression in NPC cells.
Using the ATM-targeted siRNA and FACS assay,we found when exposed to 5 Gy,nearly 60%of the ATM-siRNA-treated cells underwent apoptosis.The colony-formation assay showed when the expression of ATM was deceased by the siRNA,the colony-formation ability was reduced.The result suggested that the down-regulation of ATM may suppress the DNA repairing signaling pathway and promote the cellular death,then enhanced the radiosensitivity of NPC cells.Comprehensive analysis of the radiobiological parameters in the single-hit multitarget model and linear quadratic equation further confirmed that the silence of the ATM expression led to sensitizing of the NPC cells to radiation.
In conclusion,our finding demonstrated that the LMP1-targeted DNAzyme inhibited the expression of LMP1 and affected the cell cycle; the LMP1-targeted DNAzyme inhibited the expression of ATM;and inhibition of the ATM expression could enhance the radiosensitivity in NPC cells.The data provided solid experimental evidence to support our hypothesis that the radiosensitization of NPC cells by LMP1-targeted DNAzyme is through the impact on cell cycle control and DNA repair systems.The results can provide a basis for the use of the LMP1-targeted DNAzyme as potential radiosensitizer for clinical treatment and supply a new way to treat the EBV-associated radioresistance carcinomas.
引文
[1]Bichsel VE,Liotta LA and Petricoin EF,3rd.Cancer proteomics:from biomarker discovery to signal pathway profiling.Cancer J,2001,7(1):69-78.
[2]Harris RA,Yang A,Stein RC et al.Cluster analysis of an extensive human breast cancer cell line protein expression map database.Proteomics,2002,2(2):212-23.
[3]Niedobitek G,Meru N and Delecluse HJ.Epstein-Ban virus infection and human malignancies.Int J Exp Pathol,2001,82(3):149-70.
[4]Pagano JS.Epstein-Barr virus:the first human tumor virus and its role in cancer.Proc Assoc Am Physicians,1999,111(6):573-80.
[5]Fahraeus R,Fu HL,Ernberg I et al.Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma.Int J Cancer,1988,42(3):329-38.
[6]Young LS,Dawson CW,Clark D et al.Epstein-Barr virus gene expression in nasopharyngeal carcinoma.J Gen Virol,1988,69(Pt 5):1051-65.
[7]Gires O,Kohlhuber F,Kilger E et al.Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.Embo J,1999,18(11):3064-73.
[8]Kawa K.Epstein-Barr virus--associated diseases in humans.Int J Hematol,2000,71(2):108-17.
[9]Sylla BS,Hung SC,Davidson DM et al.Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta.Proc Natl Acad Sci U S A,1998,95(17):10106-11.
[10]Song X,Tao YG,Zeng L et al.Latent membrane protein 1 encoded by Epstein-Barr virus modulates directly and synchronously cyclin D1 and p16 by newly forming a c-Jun/Jun B heterodimer in nasopharyngeal carcinoma cell line. Virus Res, 2005,113(2):89-99.
[11] Izzo JG, Papadimitrakopoulou VA, Li XQ et al. Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification. Oncogene, 1998, 17(18):2313-22.
[12] Deng L, Yang J, Zhao XR et al. Cells in G2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-kappaB and AP-1. Cell Res, 2003,13(3):187-94.
[13] Baum DA and Silverman SK. Deoxyribozymes: useful DNA catalysts in vitro and in vivo. Cell Mol Life Sci, 2008, 65(14):2156-74.
[14] Silverman SK. Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects. Chem Commun (Camb), 2008(30):3467-85.
[15] Sun LQ, Cairns MJ, Saravolac EG et al. Catalytic nucleic acids: from lab to applications. Pharmacol Rev, 2000, 52(3):325-47.
[16] Lim T, Yuan J, Zhang HM et al Antisense DNA and RNA agents against picornaviruses. Front Biosci, 2008, 13:4707-25.
[17] Fahmy RG, Dass CR, Sun LQ et al. Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med, 2003, 9(8): 1026-32.
[18] Fahmy RG, Waldman A, Zhang G et al. Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun. Nat Biotechnol, 2006, 24(7):856-63.
[19] Khachigian LM, Fahmy RG, Zhang G et al. c-Jun regulates vascular smooth muscle cell growth and neointima formation after arterial injury. Inhibition by a novel DNA enzyme targeting c-Jun. J Biol Chem, 2002,277(25):22985-91.
[20] Mitchell A, Dass CR, Sun LQ et al. Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1. Nucleic Acids Res, 2004, 32(10):3065-9.
[21] Zhang G, Dass CR, Sumithran E et al. Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents. J Natl Cancer Inst, 2004, 96(9):683-96.
[22] Lu ZX, Ye M, Yan GR et al. Effect of EBV LMP1 targeted DNAzymes on cell proliferation and apoptosis. Cancer Gene Ther, 2005, 12(7):647-54.
[23] Lu ZX, Ma XQ, Yang LF et al. DNAzymes targeted to EBV-encoded latent membrane protein-1 induce apoptosis and enhance radiosensitivity in nasopharyngeal carcinoma. Cancer Lett, 2008, 265(2):226-38.
[24] Owa T, Yoshino H, Yoshimatsu K et al. Cell cycle regulation in the G1 phase: a promising target for the development of new chemotherapeutic anticancer agents. CurrMed Chem, 2001, 8(12):1487-503.
[25] Pardee AB. G1 events and regulation of cell proliferation. Science, 1989, 246(4930):603-8.
[26] Lauper N, Beck AR, Cariou S et al. Cyclin E2: a novel CDK2 partner in the late G1 and S phases of the mammalian cell cycle. Oncogene, 1998, 17(20):2637-43.
[27] Classon M and Harlow E. The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer, 2002, 2(12):910-7.
[28] Glassford J, Holman M, Banerji L et al. Vav is required for cyclin D2 induction and proliferation of mouse B lymphocytes activated via the antigen Receptor. J Biol Chem, 2001, 276(44):41040-8.
[29] Riches LC, Lynch AM and Gooderham NJ. Early events in the mammalian response to DNA double-strand breaks. Mutagenesis, 2008, 23(5):331-9.
[30] Schmitt E, Paquet C, Beauchemin M et al. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis. J Zhejiang Univ Sci B, 2007, 8(6):377-97.
[31] Houtgraaf JH, Versmissen J and van der Giessen WJ. A concise review of DNA damage checkpoints and repair in mammalian cells. Cardiovasc Revasc Med, 2006,7(3):165-72.
[32] Nojima H. G1 and S-phase checkpoints, chromosome instability, and cancer. Methods Mol Biol, 2004, 280:3-49.
[33] Fedorov SN, Shubina LK, Bode AM et al. Dactylone inhibits epidermal growth factor-induced transformation and phenotype expression of human cancer cells and induces G1-S arrest and apoptosis. Cancer Res, 2007,67(12):5914-20.
[34] Weinberg RA. The retinoblastoma protein and cell cycle control. Cell, 1995, 81(3):323-30.
[35] Gray NS, Wodicka L, Thunnissen AM et al. Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science, 1998, 281(5376):533-8.
[36] Krek W and Nigg EA. Differential phosphorylation of vertebrate p34cdc2 kinase at the Gl/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. Embo J, 1991,10(2):305-16.
[37] Mueller PR, Coleman TR, Kumagai A et al. Mytl: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15. Science, 1995, 270(5233):86-90.
[38] Solomon MJ, Lee T and Kirschner MW. Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Mol Biol Cell, 1992, 3(1):13-27.
[39] Fuster JJ, Sanz-Gonzalez SM, Moll UM et al. Classic and novel roles of p53: prospects for anticancer therapy. Trends Mol Med, 2007,13(5): 192-9.
[40] Ljungman M. Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress. Neoplasia, 2000, 2(3):208-25.
[41] Soussi T. p53 alterations in human cancer: more questions than answers. Oncogene, 2007, 26(15):2145-56.
[42] Zhao F, Hou NB, Yang XL et al. Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection. World J Gastroenterol, 2008, 14(40):6163-70.
[43] [43] Alt JR, Gladden AB and Diehl JA. p21(Cipl) Promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export. J Biol Chem, 2002, 277(10):8517-23.
[44] Cheng M, Olivier P, Diehl JA et al. The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. Embo J, 1999, 18(6):1571-83.
[45] LaBaer J, Garrett MD, Stevenson LF et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev, 1997, 11(7):847-62.
[46] Mayhew CN, Perkin LM, Zhang X et al. Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents. Oncogene, 2004, 23(23):4107-20.
[47] Saha P, Banerjee S, Ganguly C et al. Black tea extract can modulate protein expression of H-ras, c-Myc, p53, and Bcl-2 genes during pulmonary hyperplasia, dysplasia, and carcinoma in situ. J Environ Pathol Toxicol Oncol, 2005, 24(3):211-24.
[48] Dimberg A, Bahram F, Karlberg I et al. Retinoic acid-induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27(Kipl). Blood, 2002, 99(6):2199-206.
[49] Toyoshima M. Analysis of p53 dependent damage response in sperm-irradiated mouse embryos. J Radiat Res (Tokyo), 2009, 50(1):11-7.
[50] Hermeking H and Benzinger A. 14-3-3 proteins in cell cycle regulation. Semin Cancer Biol, 2006, 16(3): 183-92.
[51 ] Stark GR and Taylor WR. Control of the G2/M transition. Mol Biotechnol, 2006, 32(3):227-48.
[52] Eastman A. Cell cycle checkpoints and their impact on anticancer therapeutic strategies. J Cell Biochem, 2004, 91(2):223-31.
[53] Facchinetti MM, De Siervi A, Toskos D et al. UCN-01-induced cell cycle arrest requires the transcriptional induction of p21(wafl1/cip1) by activation of mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Cancer Res, 2004, 64(10):3629-37.
[54] Schwartz GK. Development of cell cycle active drugs for the treatment of gastrointestinal cancers: a new approach to cancer therapy. J Clin Oncol, 2005, 23(20):4499-508.
[55] Wadler S. Perspectives for cancer therapies with cdk2 inhibitors. Drug Resist Updat,2001,4(6):347-67.
[56] Lawrence TS, Blackstock AW and McGinn C. The mechanism of action of radiosensitization of conventional chemotherapeutic agents. Semin Radiat Oncol, 2003,13(1):13-21.
[57] Pauwels B, Korst AE, Pattyn GG et al. Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro. Int J Radiat Oncol Biol Phys, 2003, 57(4): 1075-83.
[58] Lundberg AS and Weinberg RA. Control of the cell cycle and apoptosis. Eur J Cancer, 1999, 35(14): 1886-94.
[59] Schwartz GK. CDK inhibitors: cell cycle arrest versus apoptosis. Cell Cycle, 2002, 1(2):122-3.
[60] Tao D, Wu J, Feng Y et al. New method for the analysis of cell cycle-specific apoptosis. Cytometry A, 2004, 57(2):70-4.
[61] Liu DX and Greene LA. Neuronal apoptosis at the G1/S cell cycle checkpoint. Cell Tissue Res, 2001, 305(2):217-28.
[62] Liao HF, Kuo CD, Yang YC et al. Resveratrol enhances radiosensitivity of human non-small cell lung cancer NCI-H838 cells accompanied by inhibition of nuclear factor-kappa B activation. J Radiat Res (Tokyo), 2005,46(4):387-93.
[1] Hoshikawa Y, Satoh Y, Murakami M et al. Evidence of lytic infection of Epstein-Barr virus (EBV) in EBV-positive gastric carcinoma. J Med Virol, 2002, 66(3):351-9.
[2] Lo KW and Huang DP. Genetic and epigenetic changes in nasopharyngeal carcinoma. Semin Cancer Biol, 2002, 12(6):451-62.
[3] Touitou R, Bonnet-Duquenoy M and Joab I. Association of Epstein-Barr virus with human mammary carcinoma. Pros and cons. Dis Markers, 2001, 17(3):163-5.
[4] Dudziak D, Kieser A, Dirmeier U et al. Latent membrane protein 1 of Epstein-Barr virus induces CD83 by the NF-kappaB signaling pathway. J Virol, 2003, 77(15):8290-8.
[5] Eliopoulos AG, Gallagher NJ, Blake SM et al. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem, 1999, 274(23): 16085-96.
[6] Gires O, Kohlhuber F, Kilger E et al. Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. Embo J, 1999,18(ll):3064-73.
[7] Guo G, Yan-Sanders Y, Lyn-Cook BD et al. Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses. Mol Cell Biol, 2003, 23(7):2362-78.
[8] Hammarskjold ML and Simurda MC. Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-kappa B activity. J Virol, 1992, 66(11):6496-501.
[9] Laherty CD, Hu HM, Opipari AW et al. The Epstein-Barr virus LMPl gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B. J Biol Chem, 1992, 267(34):24157-60.
[10] Mainou BA, Everly DN, Jr. and Raab-Traub N. Epstein-Barr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K. Oncogene, 2005, 24(46):6917-24.
[11] Goormachtigh G, Ouk TS, Mougel A et al. Autoactivation of the Epstein-Barr virus oncogenic protein LMPl during type Ⅱ latency through opposite roles of the NF-kappaB and JNK signaling pathways. J Virol, 2006, 80(15):7382-93.
[12] Mehl AM, Floettmann JE, Jones M et al. Characterization of intercellular adhesion molecule-1 regulation by Epstein-Barr virus-encoded latent membrane protein-1 identifies pathways that cooperate with nuclear factor kappa B to activate transcription.J Biol Chem,2001,276(2):984-92.
[13]Morrison JA,Gulley ML,Pathmanathan R et al.Differential signaling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma.Cancer Res,2004,64(15):5251-60.
[14]Hatzivassiliou E and Mosialos G.Cellular signaling pathways engaged by the Epstein-Barr virus transforming protein LMP 1.Front Biosci,2002,7:d319-29.
[15]Lim DS,Kim ST,Xu B et al.ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway.Nature,2000,404(6778):613-7.
[16]Bartek J and Lukas J.Chk1 and Chk2 kinases in checkpoint control and cancer.Cancer Cell,2003,3(5):421-9.
[17]Iannuzzi CM,Atencio DP,Green S et al.ATM mutations in female breast cancer patients predict for an increase in radiation-induced late effects.Int J Radiat Oncol Biol Phys,2002,52(3):606-13.
[18]Wang HM,Wu XY,Xia YF et al.[Study on mutation of ATM/PI3K region in NPC cell lines with different radiosensitivity].Yi Chuan,2003,25(3):276-8.
[19]Wang HM,Wu XY,Xia YF et al.[Expression of ATM protein in nasopharyngeal carcinoma cell lines with different radiosensitivity].Ai Zheng,2003,22(6):579-81.
[20]Gronbaek K,Worm J,Ralfkiaer E et al.ATM mutations are associated with inactivation of the ARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma.Blood,2002,100(4):1430-7.
[21]Iliakis G,Wang Y,Guan J et al.DNA damage checkpoint control in cells exposed to ionizing radiation.Oncogene,2003,22(37):5834-47.
[22]Nieuwenhuis B,Van Assen-Bolt AJ,Van Waarde-Verhagen MA et al.BRCA1and BRCA2 heterozygosity and repair of X-ray-induced DNA damage.Int J Radiat Biol,2002,78(4):285-95.
[23]Leskov KS,Criswell T,Antonio S et al.When X-ray-inducible proteins meet DNA double strand break repair.Semin Radiat Oncol,2001,11(4):352-72.
[24]胡建林,杨和平and李前伟.经皮穿刺瘤体内植入~(125)I粒子治疗肺癌的初步评价.重庆医学,2002,31(9):774.
[25]Cahir McFarland ED,Izumi KM and Mosialos G.Epstein-barr virus transformation:involvement of latent membrane protein 1-mediated activation of NF-kappaB.Oncogene,1999,18(49):6959-64.
[26]Brach MA,Hass R,Sherman ML et al.Ionizing radiation induces expression and binding activity of the nuclear factor kappa B.J Clin Invest,1991,88(2):691-5.
[27]Chen X,Shen B,Xia L et al.Activation of nuclear factor kappaB in radioresistance of TP53-inactive human keratinocytes.Cancer Res,2002,62(4):1213-21.
[28]Tang G,Minemoto Y,Dibling B et al.Inhibition of JNK activation through NF-kappaB target genes.Nature,2001,414(6861):313-7.
[29]Deng L,Yang J,Zhao XR et al.Cells in G2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-kappaB and AP-1.Cell Res,2003,13(3):187-94.
[30]Wang F,Liang K and Yin W.[Radiation-induced apoptosis of two nasopharyngeal carcinoma cell lines].Zhonghua Zhong Liu Za Zhi,1998,20(2):119-21.
[31]Wu ZH,Shi Y,Tibbetts RS et al.Molecular linkage between the kinase ATM and NF-kappaB signaling in response to genotoxic stimuli.Science,2006,311(5764):1141-6.
[32]Gueven N,Keating K,Fukao T et al.Site-directed mutagenesis of the ATM promoter:consequences for response to proliferation and ionizing radiation.Genes Chromosomes Cancer,2003,38(2):157-67.
[33]Tyagi A,Singh RP,Agarwal C et al.Resveratrol causes Cdc2-tyr15phosphorylation via ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells.Carcinogenesis,2005,26(11):1978-87.
[34]Sylla BS,Hung SC,Davidson DM et al.Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta.Proc Natl Acad Sci U S A,1998,95(17):10106-11.
[35]罗非君,曹亚.EB病毒潜伏膜蛋白1激活AP-1与NFκB信号转导通路在鼻咽癌变中的意义.博士论文,2001:131-43.
[36]Thyss R,Virolle V,Imbert V et al.NF-kappaB/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death.Embo J,2005,24(1):128-37.
[37]Tribius S,Pidel A and Casper D.ATM protein expression correlates with radioresistance in primary glioblastoma cells in culture.Int J Radiat Oncol Biol Phys,2001,50(2):511-23.
[38]Collis SJ,Swartz MJ,Nelson WG et al.Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors.Cancer Res,2003,63(7):1550-4.
[39]Guha C,Guha U,Tribius S et al.Antisense ATM gene therapy:a strategy to increase the radiosensitivity of human tumors.Gene Ther,2000,7(10):852-8.
[40]Song X,Tao YG,Deng XY et al.Heterodimer formation between c-Jun and Jun B proteins mediated by Epstein-Barr virus encoded latent membrane protein 1.Cell Signal,2004,16(10):1153-62.
[41]Yin L,Liao W,Deng X et al.LMP1 activates NF-kappa B via degradation of I kappa B alpha in nasopharyngeal carcinoma cells.Chin Med J(Engl),2001,114(7):718-22.
[42]Z.Hu YGT,F.Q.Tang,L.F.Yang,Y.Zhao,L.Zeng,F.J.Luo,Y.Cao.Effect of JIP on The Proliferation and Apoptosis of Nasopharyngeal Carcinoma Cells Through Interaction With JNK Mediated Pathway.Progress in Biochemistry and Biophysics,2003,30:579-585.
[43]Vockerodt M,Haier B,Buttgereit P et al.The Epstein-Barr virus latent membrane protein 1 induces interleukin-10 in Burkitt's lymphoma cells but not in Hodgkin's cells involving the p38/SAPK2 pathway.Virology,2001, 280(2): 183-98.
[44] Nabel GJ and Verma IM. Proposed NF-kappa B/I kappa B family nomenclature. Genes Dev, 1993, 7(11):2063.
[45] Ghosh G, van Duyne G, Ghosh S et al. Structure of NF-kappa B p50 homodimer bound to a kappa B site. Nature, 1995, 373(6512):303-10.
[46] Gossen M, Freundlieb S, Bender G et al. Transcriptional activation by tetracyclines in mammalian cells. Science, 1995, 268(5218): 1766-9.
[47] Kuriyan J and Thanos D. Structure of the NF-kappa B transcription factor: a holistic interaction with DNA. Structure, 1995, 3(2): 135-41.
[48] Verma IM, Stevenson JK, Schwarz EM et al. Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev, 1995, 9(22):2723-35.
[49] Baeuerle PA and Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol, 1994, 12:141-79.
[50] Paine E, Scheinman RI, Baldwin AS, Jr. et al. Expression of LMP1 in epithelial cells leads to the activation of a select subset of NF-kappa B/Rel family proteins. J Virol, 1995, 69(7):4572-6.
[51] Thornburg NJ, Pathmanathan R and Raab-Traub N. Activation of nuclear factor-kappaB p50 homodimer/Bcl-3 complexes in nasopharyngeal carcinoma. Cancer Res, 2003, 63(23):8293-301.
[52] Bours V, Franzoso G, Azarenko V et al. The oncoprotein Bcl-3 directly transactivates through kappa B motifs via association with DNA-binding p50B homodimers. Cell, 1993, 72(5):729-39.
[53] Nolan GP, Fujita T, Bhatia K et al. The bcl-3 proto-oncogene encodes a nuclear I kappa B-like molecule that preferentially interacts with NF-kappa B p50 and p52 in a phosphorylation-dependent manner. Mol Cell Biol, 1993, 13(6):3557-66.
[54] Miller WE, Earp HS and Raab-Traub N. The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J Virol, 1995, 69(7):4390-8.
[55] Orlowski RZ and Baldwin AS, Jr. NF-kappaB as a therapeutic target in cancer. Trends Mol Med,2002,8(8):385-9.
[56]Herscher LL,Cook JA,Pacelli R et al.Principles of chemoradiation:theoretical and practical considerations.Oncology(Williston Park),1999,13(10 Suppl 5):11-22.
[57]Pajonk F,Pajonk K and McBride WH.Inhibition of NF-kappaB,clonogenicity,and radiosensitivity of human cancer cells.J Natl Cancer Inst,1999,91(22):1956-60.
[58]Flynn V,Jr.,Ramanitharan A,Moparty K et al.Adenovirus-mediated inhibition of NF-kappaB confers chemo-sensitization and apoptosis in prostate cancer cells.Int J Oncol,2003,23(2):317-23.
[59]Fan M,Ahmed KM,Coleman MC et al.Nuclear factor-kappaB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells.Cancer Res,2007,67(7):3220-8.
[60]Li Z,Xia L,Lee LM et al.Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation.Radiat Res,2001,155(4):543-53.
[61]Jung M,Zhang Y,Lee S et al.Correction of radiation sensitivity in ataxia telangiectasia cells by a truncated I kappa B-alpha.Science,1995,268(5217):1619-21.
[1]Nowak R.Discovery of AT gene sparks biomedical research bonanza.Science,1995,268(5218):1700-1.
[2]Tribius S,Pidel A and Casper D.ATM protein expression correlates with radioresistance in primary glioblastoma cells in culture.Int J Radiat Oncol Biol Phys,2001,50(2):511-23.
[3]Wang F,Liang K and Yin W.[Radiation-induced apoptosis of two nasopharyngeal carcinoma cell lines].Zhonghua Zhong Liu Za Zhi,1998,20(2):119-21.
[4] Wang HM, Wu XY, Xia YF et al. [Study on mutation of ATM/PI3K region in NPC cell lines with different radiosensitivity]. Yi Chuan, 2003, 25(3):276-8.
[5] Tham IW, Hee SW, Yeo RM et al. Treatment of Nasopharyngeal Carcinoma Using Intensity-Modulated Radiotherapy-The National Cancer Centre Singapore Experience. Int J Radiat Oncol Biol Phys, 2009.
[6] Malaise EP, Lambin P and Joiner MC. Radiosensitivity of human cell lines to small doses. Are there some clinical implications? Radiat Res, 1994, 138(1 Suppl):S25-7.
[7] Lee AW, Poon YF, Foo W et al. Retrospective analysis of 5037 patients with nasopharyngeal carcinoma treated during 1976-1985: overall survival and patterns of failure. Int J Radiat Oncol Biol Phys, 1992, 23(2):261-70.
[8] Huang H, Pan X and Zhou J. BHRF1 antisense oligonucleotide inhibits anti-apoptosis of nasopharyngeal carcinoma cells. Int J Mol Med, 1999, 4(6):649-53.
[9] Pernin D, Bay JO, Uhrhammer N et al. ATM heterozygote cells exhibit intermediate levels of apoptosis in response to streptonigrin and etoposide. Eur J Cancer, 1999, 35(7):1130-5.
[10] Lavin MF and Khanna KK. ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia. Int J Radiat Biol, 1999, 75(10):1201-14.
[11] Kapoor M, Hamm R, Yan W et al. Cooperative phosphorylation at multiple sites is required to activate p53 in response to UV radiation. Oncogene, 2000, 19(3):358-64.
[12] Khosravi R, Maya R, Gottlieb T et al. Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. Proc Natl Acad Sci U S A, 1999, 96(26): 14973-7.
[13] Shafman T, Khanna KK, Kedar P et al. Interaction between ATM protein and c-Abl in response to DNA damage. Nature, 1997, 387(6632):520-3.
[14] Sarkaria JN and Eshleman JS. ATM as a target for novel radiosensitizers. Semin Radiat Oncol, 2001, 11(4):316-27.
[15]Playle LC,Hicks DJ,Qualtrough D et al.Abrogation of the radiation-induced G2 checkpoint by the staurosporine derivative UCN-01 is associated with radiosensitisation in a subset of colorectal tumour cell lines.Br J Cancer,2002,87(3):352-8.
[16]Guha C,Guha U,Tribius S et al.Antisense ATM gene therapy:a strategy to increase the radiosensitivity of human tumors.Gene Ther,2000,7(10):852-8.
[17]Enns L,Murray D and Mirzayans R.Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts.Br J Cancer,1999,81(6):959-65.
[18]Fan Z,Chakravarty P,Alfieri A et al.Adenovirus-mediated antisense ATM gene transfer sensitizes prostate cancer cells to radiation.Cancer Gene Ther,2000,7(10):1307-14.
[19]古铣之,殷蔚伯and刘泰福.肿瘤放射治疗学.北京医科大学中国协和医科大学联合出版社,1993,第二版:p232-237.
[1]O'Connell MJ et al.Trends Cell Biol,2000,10(7):296-303.
[2]Brown EJ et al.Genes Dev,2003,17(5):615-628.
[3]Lee CH et al.J Biol Chem,2001,276(32):30537-30541.
[4]DeSimone JN et al.Radiat Res,2003,159(1):72-85.
[5]Vairapandi M et al.J Cell Physiol,2002,192(3):327-338.
[6]Papa S et al.Nat Cell Biol,2004,6(2):146-153.
[7]Wuerzberger-Davis SM et al.Mol Cancer Res,2005,3(6):345-353.
[8]Dimova DK et al.Oncogene,2005,24(17):2810-2826.
[9]Crosby ME et al.Oncogene,2007,26(13):1897-1909.
[10]Mayhew CN et al.Methods Mol Biol,2004,281:3-16.
[11]Jackson MW et al.J Cell Sci,2005,118(Pt 9):1821-1832.
[12]Cowell IG et al.Biochem Pharmacol,2005,71(1-2):13-20.
[13]Deng L et al.Cell Res,2003,13(3):187-194.
[14]Lu ZX et al.Cancer Gene Ther,2005,12(7):647-654.
[15]Rao ZG et al.Ai Zheng,2002,21(2):149-152.
[16]Wang T et al.J Biol Chem,2005,280(13):12593-12601.
[17]Guo G et al.Oncogene,2004,23(2):535-545.
[2]Harris RA,Yang A,Stein RC et al.Cluster analysis of an extensive human breast cancer cell line protein expression map database.Proteomics,2002,2(2):212-23.
[3]Niedobitek G,Meru N and Delecluse HJ.Epstein-Ban virus infection and human malignancies.Int J Exp Pathol,2001,82(3):149-70.
[4]Pagano JS.Epstein-Barr virus:the first human tumor virus and its role in cancer.Proc Assoc Am Physicians,1999,111(6):573-80.
[5]Fahraeus R,Fu HL,Ernberg I et al.Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma.Int J Cancer,1988,42(3):329-38.
[6]Young LS,Dawson CW,Clark D et al.Epstein-Barr virus gene expression in nasopharyngeal carcinoma.J Gen Virol,1988,69(Pt 5):1051-65.
[7]Gires O,Kohlhuber F,Kilger E et al.Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.Embo J,1999,18(11):3064-73.
[8]Kawa K.Epstein-Barr virus--associated diseases in humans.Int J Hematol,2000,71(2):108-17.
[9]Sylla BS,Hung SC,Davidson DM et al.Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta.Proc Natl Acad Sci U S A,1998,95(17):10106-11.
[10]Song X,Tao YG,Zeng L et al.Latent membrane protein 1 encoded by Epstein-Barr virus modulates directly and synchronously cyclin D1 and p16 by newly forming a c-Jun/Jun B heterodimer in nasopharyngeal carcinoma cell line. Virus Res, 2005,113(2):89-99.
[11] Izzo JG, Papadimitrakopoulou VA, Li XQ et al. Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification. Oncogene, 1998, 17(18):2313-22.
[12] Deng L, Yang J, Zhao XR et al. Cells in G2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-kappaB and AP-1. Cell Res, 2003,13(3):187-94.
[13] Baum DA and Silverman SK. Deoxyribozymes: useful DNA catalysts in vitro and in vivo. Cell Mol Life Sci, 2008, 65(14):2156-74.
[14] Silverman SK. Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects. Chem Commun (Camb), 2008(30):3467-85.
[15] Sun LQ, Cairns MJ, Saravolac EG et al. Catalytic nucleic acids: from lab to applications. Pharmacol Rev, 2000, 52(3):325-47.
[16] Lim T, Yuan J, Zhang HM et al Antisense DNA and RNA agents against picornaviruses. Front Biosci, 2008, 13:4707-25.
[17] Fahmy RG, Dass CR, Sun LQ et al. Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med, 2003, 9(8): 1026-32.
[18] Fahmy RG, Waldman A, Zhang G et al. Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun. Nat Biotechnol, 2006, 24(7):856-63.
[19] Khachigian LM, Fahmy RG, Zhang G et al. c-Jun regulates vascular smooth muscle cell growth and neointima formation after arterial injury. Inhibition by a novel DNA enzyme targeting c-Jun. J Biol Chem, 2002,277(25):22985-91.
[20] Mitchell A, Dass CR, Sun LQ et al. Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1. Nucleic Acids Res, 2004, 32(10):3065-9.
[21] Zhang G, Dass CR, Sumithran E et al. Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents. J Natl Cancer Inst, 2004, 96(9):683-96.
[22] Lu ZX, Ye M, Yan GR et al. Effect of EBV LMP1 targeted DNAzymes on cell proliferation and apoptosis. Cancer Gene Ther, 2005, 12(7):647-54.
[23] Lu ZX, Ma XQ, Yang LF et al. DNAzymes targeted to EBV-encoded latent membrane protein-1 induce apoptosis and enhance radiosensitivity in nasopharyngeal carcinoma. Cancer Lett, 2008, 265(2):226-38.
[24] Owa T, Yoshino H, Yoshimatsu K et al. Cell cycle regulation in the G1 phase: a promising target for the development of new chemotherapeutic anticancer agents. CurrMed Chem, 2001, 8(12):1487-503.
[25] Pardee AB. G1 events and regulation of cell proliferation. Science, 1989, 246(4930):603-8.
[26] Lauper N, Beck AR, Cariou S et al. Cyclin E2: a novel CDK2 partner in the late G1 and S phases of the mammalian cell cycle. Oncogene, 1998, 17(20):2637-43.
[27] Classon M and Harlow E. The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer, 2002, 2(12):910-7.
[28] Glassford J, Holman M, Banerji L et al. Vav is required for cyclin D2 induction and proliferation of mouse B lymphocytes activated via the antigen Receptor. J Biol Chem, 2001, 276(44):41040-8.
[29] Riches LC, Lynch AM and Gooderham NJ. Early events in the mammalian response to DNA double-strand breaks. Mutagenesis, 2008, 23(5):331-9.
[30] Schmitt E, Paquet C, Beauchemin M et al. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis. J Zhejiang Univ Sci B, 2007, 8(6):377-97.
[31] Houtgraaf JH, Versmissen J and van der Giessen WJ. A concise review of DNA damage checkpoints and repair in mammalian cells. Cardiovasc Revasc Med, 2006,7(3):165-72.
[32] Nojima H. G1 and S-phase checkpoints, chromosome instability, and cancer. Methods Mol Biol, 2004, 280:3-49.
[33] Fedorov SN, Shubina LK, Bode AM et al. Dactylone inhibits epidermal growth factor-induced transformation and phenotype expression of human cancer cells and induces G1-S arrest and apoptosis. Cancer Res, 2007,67(12):5914-20.
[34] Weinberg RA. The retinoblastoma protein and cell cycle control. Cell, 1995, 81(3):323-30.
[35] Gray NS, Wodicka L, Thunnissen AM et al. Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science, 1998, 281(5376):533-8.
[36] Krek W and Nigg EA. Differential phosphorylation of vertebrate p34cdc2 kinase at the Gl/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. Embo J, 1991,10(2):305-16.
[37] Mueller PR, Coleman TR, Kumagai A et al. Mytl: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15. Science, 1995, 270(5233):86-90.
[38] Solomon MJ, Lee T and Kirschner MW. Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Mol Biol Cell, 1992, 3(1):13-27.
[39] Fuster JJ, Sanz-Gonzalez SM, Moll UM et al. Classic and novel roles of p53: prospects for anticancer therapy. Trends Mol Med, 2007,13(5): 192-9.
[40] Ljungman M. Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress. Neoplasia, 2000, 2(3):208-25.
[41] Soussi T. p53 alterations in human cancer: more questions than answers. Oncogene, 2007, 26(15):2145-56.
[42] Zhao F, Hou NB, Yang XL et al. Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection. World J Gastroenterol, 2008, 14(40):6163-70.
[43] [43] Alt JR, Gladden AB and Diehl JA. p21(Cipl) Promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export. J Biol Chem, 2002, 277(10):8517-23.
[44] Cheng M, Olivier P, Diehl JA et al. The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. Embo J, 1999, 18(6):1571-83.
[45] LaBaer J, Garrett MD, Stevenson LF et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev, 1997, 11(7):847-62.
[46] Mayhew CN, Perkin LM, Zhang X et al. Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents. Oncogene, 2004, 23(23):4107-20.
[47] Saha P, Banerjee S, Ganguly C et al. Black tea extract can modulate protein expression of H-ras, c-Myc, p53, and Bcl-2 genes during pulmonary hyperplasia, dysplasia, and carcinoma in situ. J Environ Pathol Toxicol Oncol, 2005, 24(3):211-24.
[48] Dimberg A, Bahram F, Karlberg I et al. Retinoic acid-induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27(Kipl). Blood, 2002, 99(6):2199-206.
[49] Toyoshima M. Analysis of p53 dependent damage response in sperm-irradiated mouse embryos. J Radiat Res (Tokyo), 2009, 50(1):11-7.
[50] Hermeking H and Benzinger A. 14-3-3 proteins in cell cycle regulation. Semin Cancer Biol, 2006, 16(3): 183-92.
[51 ] Stark GR and Taylor WR. Control of the G2/M transition. Mol Biotechnol, 2006, 32(3):227-48.
[52] Eastman A. Cell cycle checkpoints and their impact on anticancer therapeutic strategies. J Cell Biochem, 2004, 91(2):223-31.
[53] Facchinetti MM, De Siervi A, Toskos D et al. UCN-01-induced cell cycle arrest requires the transcriptional induction of p21(wafl1/cip1) by activation of mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Cancer Res, 2004, 64(10):3629-37.
[54] Schwartz GK. Development of cell cycle active drugs for the treatment of gastrointestinal cancers: a new approach to cancer therapy. J Clin Oncol, 2005, 23(20):4499-508.
[55] Wadler S. Perspectives for cancer therapies with cdk2 inhibitors. Drug Resist Updat,2001,4(6):347-67.
[56] Lawrence TS, Blackstock AW and McGinn C. The mechanism of action of radiosensitization of conventional chemotherapeutic agents. Semin Radiat Oncol, 2003,13(1):13-21.
[57] Pauwels B, Korst AE, Pattyn GG et al. Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro. Int J Radiat Oncol Biol Phys, 2003, 57(4): 1075-83.
[58] Lundberg AS and Weinberg RA. Control of the cell cycle and apoptosis. Eur J Cancer, 1999, 35(14): 1886-94.
[59] Schwartz GK. CDK inhibitors: cell cycle arrest versus apoptosis. Cell Cycle, 2002, 1(2):122-3.
[60] Tao D, Wu J, Feng Y et al. New method for the analysis of cell cycle-specific apoptosis. Cytometry A, 2004, 57(2):70-4.
[61] Liu DX and Greene LA. Neuronal apoptosis at the G1/S cell cycle checkpoint. Cell Tissue Res, 2001, 305(2):217-28.
[62] Liao HF, Kuo CD, Yang YC et al. Resveratrol enhances radiosensitivity of human non-small cell lung cancer NCI-H838 cells accompanied by inhibition of nuclear factor-kappa B activation. J Radiat Res (Tokyo), 2005,46(4):387-93.
[1] Hoshikawa Y, Satoh Y, Murakami M et al. Evidence of lytic infection of Epstein-Barr virus (EBV) in EBV-positive gastric carcinoma. J Med Virol, 2002, 66(3):351-9.
[2] Lo KW and Huang DP. Genetic and epigenetic changes in nasopharyngeal carcinoma. Semin Cancer Biol, 2002, 12(6):451-62.
[3] Touitou R, Bonnet-Duquenoy M and Joab I. Association of Epstein-Barr virus with human mammary carcinoma. Pros and cons. Dis Markers, 2001, 17(3):163-5.
[4] Dudziak D, Kieser A, Dirmeier U et al. Latent membrane protein 1 of Epstein-Barr virus induces CD83 by the NF-kappaB signaling pathway. J Virol, 2003, 77(15):8290-8.
[5] Eliopoulos AG, Gallagher NJ, Blake SM et al. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem, 1999, 274(23): 16085-96.
[6] Gires O, Kohlhuber F, Kilger E et al. Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. Embo J, 1999,18(ll):3064-73.
[7] Guo G, Yan-Sanders Y, Lyn-Cook BD et al. Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses. Mol Cell Biol, 2003, 23(7):2362-78.
[8] Hammarskjold ML and Simurda MC. Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-kappa B activity. J Virol, 1992, 66(11):6496-501.
[9] Laherty CD, Hu HM, Opipari AW et al. The Epstein-Barr virus LMPl gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B. J Biol Chem, 1992, 267(34):24157-60.
[10] Mainou BA, Everly DN, Jr. and Raab-Traub N. Epstein-Barr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K. Oncogene, 2005, 24(46):6917-24.
[11] Goormachtigh G, Ouk TS, Mougel A et al. Autoactivation of the Epstein-Barr virus oncogenic protein LMPl during type Ⅱ latency through opposite roles of the NF-kappaB and JNK signaling pathways. J Virol, 2006, 80(15):7382-93.
[12] Mehl AM, Floettmann JE, Jones M et al. Characterization of intercellular adhesion molecule-1 regulation by Epstein-Barr virus-encoded latent membrane protein-1 identifies pathways that cooperate with nuclear factor kappa B to activate transcription.J Biol Chem,2001,276(2):984-92.
[13]Morrison JA,Gulley ML,Pathmanathan R et al.Differential signaling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma.Cancer Res,2004,64(15):5251-60.
[14]Hatzivassiliou E and Mosialos G.Cellular signaling pathways engaged by the Epstein-Barr virus transforming protein LMP 1.Front Biosci,2002,7:d319-29.
[15]Lim DS,Kim ST,Xu B et al.ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway.Nature,2000,404(6778):613-7.
[16]Bartek J and Lukas J.Chk1 and Chk2 kinases in checkpoint control and cancer.Cancer Cell,2003,3(5):421-9.
[17]Iannuzzi CM,Atencio DP,Green S et al.ATM mutations in female breast cancer patients predict for an increase in radiation-induced late effects.Int J Radiat Oncol Biol Phys,2002,52(3):606-13.
[18]Wang HM,Wu XY,Xia YF et al.[Study on mutation of ATM/PI3K region in NPC cell lines with different radiosensitivity].Yi Chuan,2003,25(3):276-8.
[19]Wang HM,Wu XY,Xia YF et al.[Expression of ATM protein in nasopharyngeal carcinoma cell lines with different radiosensitivity].Ai Zheng,2003,22(6):579-81.
[20]Gronbaek K,Worm J,Ralfkiaer E et al.ATM mutations are associated with inactivation of the ARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma.Blood,2002,100(4):1430-7.
[21]Iliakis G,Wang Y,Guan J et al.DNA damage checkpoint control in cells exposed to ionizing radiation.Oncogene,2003,22(37):5834-47.
[22]Nieuwenhuis B,Van Assen-Bolt AJ,Van Waarde-Verhagen MA et al.BRCA1and BRCA2 heterozygosity and repair of X-ray-induced DNA damage.Int J Radiat Biol,2002,78(4):285-95.
[23]Leskov KS,Criswell T,Antonio S et al.When X-ray-inducible proteins meet DNA double strand break repair.Semin Radiat Oncol,2001,11(4):352-72.
[24]胡建林,杨和平and李前伟.经皮穿刺瘤体内植入~(125)I粒子治疗肺癌的初步评价.重庆医学,2002,31(9):774.
[25]Cahir McFarland ED,Izumi KM and Mosialos G.Epstein-barr virus transformation:involvement of latent membrane protein 1-mediated activation of NF-kappaB.Oncogene,1999,18(49):6959-64.
[26]Brach MA,Hass R,Sherman ML et al.Ionizing radiation induces expression and binding activity of the nuclear factor kappa B.J Clin Invest,1991,88(2):691-5.
[27]Chen X,Shen B,Xia L et al.Activation of nuclear factor kappaB in radioresistance of TP53-inactive human keratinocytes.Cancer Res,2002,62(4):1213-21.
[28]Tang G,Minemoto Y,Dibling B et al.Inhibition of JNK activation through NF-kappaB target genes.Nature,2001,414(6861):313-7.
[29]Deng L,Yang J,Zhao XR et al.Cells in G2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-kappaB and AP-1.Cell Res,2003,13(3):187-94.
[30]Wang F,Liang K and Yin W.[Radiation-induced apoptosis of two nasopharyngeal carcinoma cell lines].Zhonghua Zhong Liu Za Zhi,1998,20(2):119-21.
[31]Wu ZH,Shi Y,Tibbetts RS et al.Molecular linkage between the kinase ATM and NF-kappaB signaling in response to genotoxic stimuli.Science,2006,311(5764):1141-6.
[32]Gueven N,Keating K,Fukao T et al.Site-directed mutagenesis of the ATM promoter:consequences for response to proliferation and ionizing radiation.Genes Chromosomes Cancer,2003,38(2):157-67.
[33]Tyagi A,Singh RP,Agarwal C et al.Resveratrol causes Cdc2-tyr15phosphorylation via ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells.Carcinogenesis,2005,26(11):1978-87.
[34]Sylla BS,Hung SC,Davidson DM et al.Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta.Proc Natl Acad Sci U S A,1998,95(17):10106-11.
[35]罗非君,曹亚.EB病毒潜伏膜蛋白1激活AP-1与NFκB信号转导通路在鼻咽癌变中的意义.博士论文,2001:131-43.
[36]Thyss R,Virolle V,Imbert V et al.NF-kappaB/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death.Embo J,2005,24(1):128-37.
[37]Tribius S,Pidel A and Casper D.ATM protein expression correlates with radioresistance in primary glioblastoma cells in culture.Int J Radiat Oncol Biol Phys,2001,50(2):511-23.
[38]Collis SJ,Swartz MJ,Nelson WG et al.Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors.Cancer Res,2003,63(7):1550-4.
[39]Guha C,Guha U,Tribius S et al.Antisense ATM gene therapy:a strategy to increase the radiosensitivity of human tumors.Gene Ther,2000,7(10):852-8.
[40]Song X,Tao YG,Deng XY et al.Heterodimer formation between c-Jun and Jun B proteins mediated by Epstein-Barr virus encoded latent membrane protein 1.Cell Signal,2004,16(10):1153-62.
[41]Yin L,Liao W,Deng X et al.LMP1 activates NF-kappa B via degradation of I kappa B alpha in nasopharyngeal carcinoma cells.Chin Med J(Engl),2001,114(7):718-22.
[42]Z.Hu YGT,F.Q.Tang,L.F.Yang,Y.Zhao,L.Zeng,F.J.Luo,Y.Cao.Effect of JIP on The Proliferation and Apoptosis of Nasopharyngeal Carcinoma Cells Through Interaction With JNK Mediated Pathway.Progress in Biochemistry and Biophysics,2003,30:579-585.
[43]Vockerodt M,Haier B,Buttgereit P et al.The Epstein-Barr virus latent membrane protein 1 induces interleukin-10 in Burkitt's lymphoma cells but not in Hodgkin's cells involving the p38/SAPK2 pathway.Virology,2001, 280(2): 183-98.
[44] Nabel GJ and Verma IM. Proposed NF-kappa B/I kappa B family nomenclature. Genes Dev, 1993, 7(11):2063.
[45] Ghosh G, van Duyne G, Ghosh S et al. Structure of NF-kappa B p50 homodimer bound to a kappa B site. Nature, 1995, 373(6512):303-10.
[46] Gossen M, Freundlieb S, Bender G et al. Transcriptional activation by tetracyclines in mammalian cells. Science, 1995, 268(5218): 1766-9.
[47] Kuriyan J and Thanos D. Structure of the NF-kappa B transcription factor: a holistic interaction with DNA. Structure, 1995, 3(2): 135-41.
[48] Verma IM, Stevenson JK, Schwarz EM et al. Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev, 1995, 9(22):2723-35.
[49] Baeuerle PA and Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol, 1994, 12:141-79.
[50] Paine E, Scheinman RI, Baldwin AS, Jr. et al. Expression of LMP1 in epithelial cells leads to the activation of a select subset of NF-kappa B/Rel family proteins. J Virol, 1995, 69(7):4572-6.
[51] Thornburg NJ, Pathmanathan R and Raab-Traub N. Activation of nuclear factor-kappaB p50 homodimer/Bcl-3 complexes in nasopharyngeal carcinoma. Cancer Res, 2003, 63(23):8293-301.
[52] Bours V, Franzoso G, Azarenko V et al. The oncoprotein Bcl-3 directly transactivates through kappa B motifs via association with DNA-binding p50B homodimers. Cell, 1993, 72(5):729-39.
[53] Nolan GP, Fujita T, Bhatia K et al. The bcl-3 proto-oncogene encodes a nuclear I kappa B-like molecule that preferentially interacts with NF-kappa B p50 and p52 in a phosphorylation-dependent manner. Mol Cell Biol, 1993, 13(6):3557-66.
[54] Miller WE, Earp HS and Raab-Traub N. The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J Virol, 1995, 69(7):4390-8.
[55] Orlowski RZ and Baldwin AS, Jr. NF-kappaB as a therapeutic target in cancer. Trends Mol Med,2002,8(8):385-9.
[56]Herscher LL,Cook JA,Pacelli R et al.Principles of chemoradiation:theoretical and practical considerations.Oncology(Williston Park),1999,13(10 Suppl 5):11-22.
[57]Pajonk F,Pajonk K and McBride WH.Inhibition of NF-kappaB,clonogenicity,and radiosensitivity of human cancer cells.J Natl Cancer Inst,1999,91(22):1956-60.
[58]Flynn V,Jr.,Ramanitharan A,Moparty K et al.Adenovirus-mediated inhibition of NF-kappaB confers chemo-sensitization and apoptosis in prostate cancer cells.Int J Oncol,2003,23(2):317-23.
[59]Fan M,Ahmed KM,Coleman MC et al.Nuclear factor-kappaB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells.Cancer Res,2007,67(7):3220-8.
[60]Li Z,Xia L,Lee LM et al.Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation.Radiat Res,2001,155(4):543-53.
[61]Jung M,Zhang Y,Lee S et al.Correction of radiation sensitivity in ataxia telangiectasia cells by a truncated I kappa B-alpha.Science,1995,268(5217):1619-21.
[1]Nowak R.Discovery of AT gene sparks biomedical research bonanza.Science,1995,268(5218):1700-1.
[2]Tribius S,Pidel A and Casper D.ATM protein expression correlates with radioresistance in primary glioblastoma cells in culture.Int J Radiat Oncol Biol Phys,2001,50(2):511-23.
[3]Wang F,Liang K and Yin W.[Radiation-induced apoptosis of two nasopharyngeal carcinoma cell lines].Zhonghua Zhong Liu Za Zhi,1998,20(2):119-21.
[4] Wang HM, Wu XY, Xia YF et al. [Study on mutation of ATM/PI3K region in NPC cell lines with different radiosensitivity]. Yi Chuan, 2003, 25(3):276-8.
[5] Tham IW, Hee SW, Yeo RM et al. Treatment of Nasopharyngeal Carcinoma Using Intensity-Modulated Radiotherapy-The National Cancer Centre Singapore Experience. Int J Radiat Oncol Biol Phys, 2009.
[6] Malaise EP, Lambin P and Joiner MC. Radiosensitivity of human cell lines to small doses. Are there some clinical implications? Radiat Res, 1994, 138(1 Suppl):S25-7.
[7] Lee AW, Poon YF, Foo W et al. Retrospective analysis of 5037 patients with nasopharyngeal carcinoma treated during 1976-1985: overall survival and patterns of failure. Int J Radiat Oncol Biol Phys, 1992, 23(2):261-70.
[8] Huang H, Pan X and Zhou J. BHRF1 antisense oligonucleotide inhibits anti-apoptosis of nasopharyngeal carcinoma cells. Int J Mol Med, 1999, 4(6):649-53.
[9] Pernin D, Bay JO, Uhrhammer N et al. ATM heterozygote cells exhibit intermediate levels of apoptosis in response to streptonigrin and etoposide. Eur J Cancer, 1999, 35(7):1130-5.
[10] Lavin MF and Khanna KK. ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia. Int J Radiat Biol, 1999, 75(10):1201-14.
[11] Kapoor M, Hamm R, Yan W et al. Cooperative phosphorylation at multiple sites is required to activate p53 in response to UV radiation. Oncogene, 2000, 19(3):358-64.
[12] Khosravi R, Maya R, Gottlieb T et al. Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. Proc Natl Acad Sci U S A, 1999, 96(26): 14973-7.
[13] Shafman T, Khanna KK, Kedar P et al. Interaction between ATM protein and c-Abl in response to DNA damage. Nature, 1997, 387(6632):520-3.
[14] Sarkaria JN and Eshleman JS. ATM as a target for novel radiosensitizers. Semin Radiat Oncol, 2001, 11(4):316-27.
[15]Playle LC,Hicks DJ,Qualtrough D et al.Abrogation of the radiation-induced G2 checkpoint by the staurosporine derivative UCN-01 is associated with radiosensitisation in a subset of colorectal tumour cell lines.Br J Cancer,2002,87(3):352-8.
[16]Guha C,Guha U,Tribius S et al.Antisense ATM gene therapy:a strategy to increase the radiosensitivity of human tumors.Gene Ther,2000,7(10):852-8.
[17]Enns L,Murray D and Mirzayans R.Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts.Br J Cancer,1999,81(6):959-65.
[18]Fan Z,Chakravarty P,Alfieri A et al.Adenovirus-mediated antisense ATM gene transfer sensitizes prostate cancer cells to radiation.Cancer Gene Ther,2000,7(10):1307-14.
[19]古铣之,殷蔚伯and刘泰福.肿瘤放射治疗学.北京医科大学中国协和医科大学联合出版社,1993,第二版:p232-237.
[1]O'Connell MJ et al.Trends Cell Biol,2000,10(7):296-303.
[2]Brown EJ et al.Genes Dev,2003,17(5):615-628.
[3]Lee CH et al.J Biol Chem,2001,276(32):30537-30541.
[4]DeSimone JN et al.Radiat Res,2003,159(1):72-85.
[5]Vairapandi M et al.J Cell Physiol,2002,192(3):327-338.
[6]Papa S et al.Nat Cell Biol,2004,6(2):146-153.
[7]Wuerzberger-Davis SM et al.Mol Cancer Res,2005,3(6):345-353.
[8]Dimova DK et al.Oncogene,2005,24(17):2810-2826.
[9]Crosby ME et al.Oncogene,2007,26(13):1897-1909.
[10]Mayhew CN et al.Methods Mol Biol,2004,281:3-16.
[11]Jackson MW et al.J Cell Sci,2005,118(Pt 9):1821-1832.
[12]Cowell IG et al.Biochem Pharmacol,2005,71(1-2):13-20.
[13]Deng L et al.Cell Res,2003,13(3):187-194.
[14]Lu ZX et al.Cancer Gene Ther,2005,12(7):647-654.
[15]Rao ZG et al.Ai Zheng,2002,21(2):149-152.
[16]Wang T et al.J Biol Chem,2005,280(13):12593-12601.
[17]Guo G et al.Oncogene,2004,23(2):535-545.