【作者】 马小倩；

【导师】 曹亚； 孙仑泉；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2009， 博士

【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。更多还原

【Abstract】 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 cycleWe 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 GXP₄80587）.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 cellsWe 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.更多还原