【作者】 孙晓南；

【导师】 郑树；

【作者基本信息】 浙江大学 ， 肿瘤学， 2011， 博士

【摘要】 由辐射引起的DNA损伤的主要类型包括碱基损伤、单链断裂(single strand breaks, SSBs)和双链断裂(DSBs).真核细胞有非常复杂的酶修复系统来消除这些暴露于辐射引起的损伤,碱基损伤和SSBs不被认为是致命的损伤,因为即使它们产生的频率比DNA双链断裂高,它们会完全和迅速地被修复。因为DNA双链断裂修复相当缓慢,并可能在不正确的错误连接时导致染色体畸变,所以DSBs属于电离辐射的致死性效应。碱基损害和SSBs通过碱基切除修复(base excision repair, BER)的途径完成修复。DSBs的修复是由两个不同的修复机制完成：被称为非同源末端连接(NHEJ)和同源重组修复(HRR)。NHEJ通路通常被认为是控制辐射对肿瘤细胞诱发致死性损伤得主要途径。虽然NHEJ涉及多个蛋白质的参与,但DNA蛋白激酶催化亚基(DNA-PKcs)是一个关键酶。当DNA双链断裂时,DNA-PKcs通过磷酸化被激活,并和它的调节亚基KU70和KU80一起稳定地附着在DNA的断裂末端。这个蛋白质复合物和其他蛋白质,如Mre11、Rad50、Nbs1、XRCC4.和DNA连接酶Ⅳ在一起完成DNA的断裂两端的再连接。共济失调毛细血管扩张症突变(ATM)是另一个重要的修复蛋白,DNA损伤时,参与对细胞周期检测点的调控。DNA损伤修复机制决定细胞对放射线的内在的放射敏感性。大多数恶性肿瘤对射线不敏感,因而导致照射后复发。如果能够抑制DNA损伤后的修复，则有可能使肿瘤细胞对放射敏感。近年来众多的研究都针对这一方向Non-homologous DNA end joining (NHEJ)是一种主要DNA损伤修复机制NHEJ需要至少六种蛋白Ku70/Ku80异聚体、DNA-PKcs、XRCC4、DNA连接酶Ⅳ和Artemis。DNA-PKcs作为主要的酶影响DNA修复的过程,因而其可以作为一种理想的分子靶。DNA-PKcs在ABCDE (T2609、S2612、T2620、S2624、T2638及T2647), PQR (S2023、S2029、S2041、S2051、S2053、S2056)等区域进行自主磷酸化。研究证明了突变以上位点造成DNAPKcs自我磷酸化障碍可以影响DNAPKcs的活性而增加放射敏感性。本课题研究中,我们设计用大分子化合物融合多肽来干扰DNA-PKcs的磷酸化,从而影响DNAPKcs的活性。融合多肽由两个功能区组成：一部分为肿瘤靶向区即入细胞区(引导肽)HIV 1-TAT部分的氨基酸序列,即TAT蛋白转导域(氨基酸序列为YGRKKRRQRRR),具有很强的穿过细胞膜和核膜的功能。另一部分为分子靶向区,选择DNA-PKcs的ABCDE结构域部分,分3个肽序列FVET (2609) QAS (2612) QGT, QTRT (2620) QEGS (2624) LS, T (2638) QQQHDFTLT(2647)。分别与HIV 1-TAT融合。我们设想：融合多肽能够穿过细胞膜和核膜；融合多肽能够增加肿瘤细胞的放射敏感性；其中融合多肽的放射增敏作用机制很可能是：通过抑制相应位点丝(S)、苏(T)氨酸的自主磷酸化(Autophosphorylation)并进一步抑制其下游的Artemis磷酸化而产生作用。1融合多肽的设计、合成及序列和纯度的验证我们将蛋白转导功能域称为肿瘤细胞靶向区,序列选择HIV 1-TAT的一部分,氨基酸序列为YGRKKRRQRRR,分子量为1560。分子靶向区选择DNA-PKcs的ABCDE结构域部分,分3个肽序列FVET(2609)QAS(2612)QGT QTRT(2620)QEGS(2624)LS, T(2638)QQQHDFTLT(2647)。分别与HIV1-TAT融合,这样便于分别研究三个多肽的放射增敏作用及相应的作用机制。同时设计随机肽序列(scrambled sequence)作为阴性对照。Biotin标记在多肽的N-端(N-terminusof the peptides)便于观察多肽的入胞情况(多肽具体序列及相对应英文缩写见图1.1)。Biotin-TAT (BT)用于观察细胞穿膜肽TAT自身的穿胞作用,Biotin-wtDIP1 (Bwl)仅为Biotin标记的分子靶向区多肽(FVET (2609) QAS (2612) QGT),未与HIV 1-TAT连接,用于观察在没有细胞穿膜肽TAT引导的情况下分子靶向区多肽是否有穿膜作用。Biotin-TAT-scDIP1 (BTs1)和Biotin-TAT-scDIP2(BTs2)为与细胞穿膜肽TAT连接的两个随机肽。Biotin-TAT-wtDIP1(BTw1)、Biotin-TAT-wtDIP2(BTw2)和Biotin-TAT-wtDIP3(BTw3)分别是HIV 1-TAT与FVET (2609) QAS (2612) QGT、QTRT (2620) QEGS (2624) LS和T (2638) QQQHDFTLT (2647)3个分子靶向区多肽融合后的多肽。多肽合成是一个重复添加氨基酸的过程,固相合成顺序一般从C端向N端合成。为了防止副反应的发生,参加反应的氨基酸的侧链都是被保护的；而羧基是游离的,并且在反应之前必须活化。化学合成方法有两种,即Fmoc和tBoc。所有合成的多肽得到产品后,经ESI-MS检测所有合成多肽的实际分子量均与各自对应的目标分子量接近,说明该多肽实际分子量与理论值相符,分子量正确(具体见图1.3A-图1.9A),序列正确。HPLC检测分析得各样品纯度均大于95%(具体见图1.3B-图1.9B)。2融合多肽入胞、细胞动力学和细胞毒作用的实验研究我们首先观察和检测融合多肽在多个细胞系中的入胞效果,所用的细胞系包括：人肠癌细胞系RKO、SW480和人宫颈癌细胞系Hela。10μM多肽加入培养的细胞中60分钟后,用免疫荧光显微镜评估肽的定位。应用共聚焦显微镜观察多肽在细胞中的滞留时间。观察融合多肽的半衰期(half-life)。用MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)检测和评价评价多肽的细胞毒作用。用DDP作为一个阳性对照药物。结果发现：合成的各种多肽(10μM)加入RKO细胞后1小时后,共聚焦显微镜观察多肽的入胞情况：干扰功能域(分子靶向区多肽)未能进入细胞(胞浆或细胞核),而TAT、与TAT融合的随机肽(BTs1和BTs1)以及与TAT融合的分子靶向区多肽(BTw1、BTw2和BTw3)明显地被肿瘤细胞摄取。入胞后,主要分布于胞浆和细胞核。细胞动力学研究结果显示：融合多肽作用4小时后,荧光强度开始明显减弱(图2.2a)。各个多肽的细胞毒验证(MTT assay)结果显示：按final concentraton设置的浓度加入多肽(0,5,10,20,40,60,80μM)和及阳性对照药物顺铂(0,5,10,20,40,60,80μM),继续培养48h,多肽对肿瘤细胞均未表现出细胞毒作用(图2.3)。而顺铂随着加药浓度的增加对肿瘤细胞的抑制作用明显增加。基于MTT有关细胞毒试验的结果,我们可以选择10μM、20μM、30μM和40μM等低于80μM浓度的多肽作为下一步一系列试验的工作浓度。3融合多肽对放射敏感性影响的实验研究这一部分试验在体外(in vitro)应用克隆形成的方法(clony-forming assays)研究融合多肽对肿瘤细胞系(主要为RKO细胞)放射敏感性的影响。通过体外实验测量γ-H2AX焦点形成(y-H2AX focus formation)研究融合多肽对肿瘤细胞DNA损伤形成损伤修复的影响。我们发现BTw1和BTw2对放射敏感性均无影响,而BTw3能够明显降低放射诱导的细胞生存。D0值代表37%肿瘤细胞存活的致死平均照射剂量。D0值是肿瘤细胞内在放射敏感性(intrinsic cellular radiosensi-tivity)的指标。RKO细胞在BTw3处理后的Do值为1.95Gy,随机肽(BTs2)处理后的Do值为2.46Gy。放射增敏比(SER)为1.25。结果提示BTw3对RKO细胞具有放射增敏作用。在0小时间点,未检测到γ-H2AX焦点,在照射0.5小时后,各组的γ-H2AX焦点数明显增加,且各组在0.5时间点的γ-H2AX焦点数无明显差异(P>0.05)。在1小时时间点BTw3组的γ-H2AX焦点数明显多于BTs1组(P=0.032),但与和BTs2组相比无明显差异(P=0.132)。在这一时间点,BTw3组的γ-H2AX焦点数均明显多于BTw1和BTw2组(P分别为0.031和0.009)。在2、4、8、24和48小时的时间点,BTw3组的γ-H2AX焦点数明显多于其他各组(单纯照射、BTs1、BTs2、BTw1和BTw2组)(P均小于0.00001)。根据结果我们可以推测：融合多肽加放射没有诱导或增加γ-H2AX焦点,但BTw3可能推迟了DNA双链断裂修复的时间,因而延长了γ-H2AX焦点存在的时间。结果提示BTw3对RKO细胞DNA双链断裂修复有抑制作用。总之,克隆形成试验和γ-H2AX foci试验结果均支持BTw3 (T (2638) QQQHDFTLT (2647))对RKO细胞具有潜在的放射增敏作用。4融合多肽(TAT-QQQHDFTLT)放射增敏的分子生物学机制研究基于以上结果,我们进一步研究了BTw3对其对应的苏氨酸位点磷酸化的影响(如对T2647位点磷酸化的影响),以及BTw3对DNA-PKcs下游分子Artemis磷酸化的影响,以及BTw3对介导细胞凋亡Caspase家族关键的执行分子caspase-3的影响。结果提示：在照射20分钟时,DNA-PKcs T2647磷酸化程度变化不明显,然而在照射60分钟后,我们观察到DNA-PKcs分子T2647磷酸化程度明显降低(见图4.1)。我们研究Artemis蛋白对于电离辐射的效应,具体观察了融合多肽BTw3(TAT-T (2638) QQQHDFTLT (2647))联合放射对Artemis S516磷酸化的的影响,用Artemis S516磷酸化特异性抗体来检测和分析Artemis蛋白磷酸化的变化。结果显示：BTw3处理RKO细胞后照射10Gy,在照射后30分钟和60分钟,融合多肽BTw3明显抑制了Artemis S516磷酸化。细胞凋亡分析caspase-3的变化结果显示：BTw3联合放射与单独放射相比,可以明显增加cleaved caspase-3蛋白表达,说明BTw3可以进一步促进放射诱导的细胞凋亡结论：1)MS分析各多肽的相对分子质量测定值与理论值相符。证明序列正确。确保了所合成的肽为高纯度目的肽。2)HPLC对合成多肽进行的纯度分析显示,所有合成多肽的纯度均在95%以上符合高纯度多肽的合成要求。3)TAT或融合其他短肽时可穿膜进入细胞,并可定位于细胞质和细胞核,同时具有较高的入胞效率。而非穿膜肽则不能进入细胞。4)BTwl和BTw2对放射敏感性均无影响,而BTw3能够明显降低RKO细胞放射后的细胞生存。即BTw3对RKO细胞有潜在的放射增敏作用。5)BTw3明显延长了放射诱导γ-H2AX焦点(γ-H2AX foci)存在的时间6)克隆形成试验和γ-H2AX foci试验结果均支持BTw3 (T (2638) QQQHDFTLT (2647))对RKO细胞具有潜在的放射增敏作用。7) BTw3 (TAT-T (2638) QQQHDFTLT (2647))可以抑制放射诱导的RKO细胞DNA-PKcs分子T2647的磷酸化。8) BTw3 (TAT-T (2638) QQQHDFTLT (2647))对放射诱导的RKO细胞ArtemisS516磷酸化具有抑制作用。9)BTw3联合放射与单独放射相比,可以明显增加cleaved caspase-3蛋白表达,说明BTw3可以进一步促进放射诱导的细胞凋亡。更多还原

【Abstract】 DNA damage caused by radiation mainly include base damage, single strand breaks (single strand breaks, SSBs), double-strand breaks (DSBs). Eukaryotic cells have a very complex enzyme repair system to eliminate these radiation induced damage. Base damage and SSBs are not considered fatal injury, because even if they produce higher breaks frequency than that of DNA double-strand breaks, they will be fully and quickly be repaired. Because repair of DNA double-strand break is quite slow, and an error in the connection leads to chromosomal aberrations, so the DSBs are lethal effects of ionizing radiation. Base damage and SSBs complete the repair by base excision repair (BER). The repair of DSBs are completed by two different mechanisms: non-homologous end joining (NHEJ) and homologous recombination repair (HRR). NHEJ pathway is generally considered the main pathway for the control of tumor cells fatal damage induced by radiation. Although many proteins involved in NHEJ, but the DNA protein kinase catalytic subunit (DNA-PKcs) is a key enzyme. When the DNA double-strand breaks, DNA-PKcs is activated by phosphorylation and attach the end of breaks stablely with its regulation subunits KU70 and KU80. The protein complexes and other proteins, such as Mre11, Rad50, Nbs1, XRCC4, and DNA ligase IV complete the breaks ends re-connection of DNA together. Ataxia telangiectasia mutated (ATM) is another important repair protein, participate in the regulation of cell cycle checkpoints when DNA damage.DNA damage repair mechanism determine intrinsic cell radiosensitivity to radiation. Most malignant tumors are not sensitive to radiation, lead to recurrence after irradiation. If we can inhibit DNA repair after radiation injury, it is possible to make tumor cells sensitive to radiation. In recent years, numerous studies have aimed in this direction. Non-homologous DNA endjoining (NHEJ) is a major DNA damage repair mechanisms, NHEJ requires at least six proteins:Ku70/Ku80 different polymers, DNA-PKcs, XRCC4, DNA ligase IV and Artemis. DNA-PKcs as a major enzyme of DNA repair process, so it can be used as an ideal molecular target. DNA-PKcs auto-phosphorylation in the regions of ABCDE (T2609, S2612, T2620, S2624, T2638 and T2647), PQR (S2023, S2029, S2041, S2051, S2053, S2056) had been observed. Studies have shown that mutations in these sites can increase the radiation sensitivity, Therefore, we design fusion peptides compounds to influence the activity of DNA-PKcs. Fusion peptide composed of two functional domains:one domain is a protein transduction domain (internalization peptide):TAT sequence for intercellular peptide delivery, the amino acid sequence part of HIV 1-TAT (Amino acid sequence: YGRKKRRQRRR). the protein transduction domains can carry our proposed peptides into cells across the cell membrane and nuclear membrane. An interference domain, select part of the ABCDE domain in DNA-PKcs. Include Peptides sequence:FVET (2609) QAS (2612) QGT, QTRT (2620) QEGS (2624) LS and T (2638) QQQHDFTLT (2647). Fused with HIV 1-TAT, respectively.We expect the fusion peptide can cross the cell membrane and nuclear membrane. fusion peptides can increase the radiosensitivity of tumor cells. The mechanism of radiosensitization is likely to be:Inhibit the corresponding sites of serine (S), threonine (T) autophosphorylation and further inhibit the downstream phosphorylation of Artemis.We called the protein transduction domain of the tumor cell targeting domain, select part of HIV1-TAT sequence. Amino acid sequence:YGRKKRRQRRR, a molecular weight of 1560. An interference domain, select part of the ABCDE domain in DNA-PKcs. Include Peptides sequence:FVET (2609) QAS (2612) QGT, QTRT (2620) QEGS (2624) LS and T (2638) QQQ HDFTLT (2647). Fused with HIV 1-TAT, respectively. It facilitate to study radiation sensitivity and the corresponding mechanism. And design random peptides sequence (scrambled sequence) as negative controls. Biotin labeled in the N-terminal peptide (N-terminus of the peptides) facilitate the observation of peptides into the cells. Biotin-TAT (BT) is used to observe the cell penetrating peptide TAT functions itself. Biotin-wtDIP1 (Bw1) is a only Biotin labeled peptides (FVET (2609) QAS (2612) QGT), not connected with the HIV1-TAT, to observe if the interference domain peptides can penetrate cell membrane in the absence of TAT cell penetrating peptide. Biotin-TAT-scDIPl (BTs1) and Biotin-TAT-scDIP2 (BTs2) are random peptides connected with the cell penetrating peptide TAT. Biotin-TAT-wtDIP1 (BTw1), Biotin-TAT-wtDIP2 (BTw2) and Biotin-TAT-wtDIP3 (BTw3) are fusion peptides of HIV1-TAT and FVET (2609) QAS (2612) QGT, QTRT (2620) QEGS (2624) LS and T (2638) QQQHDFTLT (2647) 3 molecules targeted domains.Peptide synthesis is an iterative process of adding amino acids, synthesis sequence is to the N-terminal from C. In order to prevent additional reactions, the side chain of amino acids to participate in the reaction are protected. The carboxyl group is free, and must be activated before the reaction. There are two methods of chemical synthesis, the Fmoc and tBoc.We detected all the actual molecular weight of synthetic peptides are close to the actual respective molecular weights by ESI-MS, this shows that the peptides molecular weights are consistent with the theoretical value, each molecular weight is correct, the sequences are correct. The purity of the samples were more than 95% by HPLC detection. Part II Study of fusion peptide penetrating membrane into thecells, cell kinetics and cell toxicityWe first observed and detected fusion peptides penetrating membrane into the cellular effects in multiple cell lines. Cell lines include human colon cancer cell lines RKO, SW480, and human cervical cancer cell line Hela. AddedlOμM peptides in cultured cells, assessed the peptide orientation by immunofluorescence microscopy in 60 minutes later. Observed the peptide retention time in the cells by confocal microscope. Observed half-life of fusion peptides. Tested and evaluated cytotoxicity of peptides With MTT (3-(4,5-dimethylthiazolyl -2) -2,5-diphenyltetrazolium bromide). DDP was used as a positive control.The results showed that:the synthesis of various peptides (1OμM) added the cells, observed the condition of peptides into the cell by confocal microscopy 1 hour later: The interference domain (peptide molecular target domain) can not enter the cell (cytoplasm or nucleus), The TAT, TAT fusion peptide with a random peptide (BTs1 and BTs1) and the TAT fusion peptides with molecular target domain (BTw1, BTw2 and BTw3) were uptaken significantly by tumor cells, mainly distributed in cytoplasm and nucleus of the cells. The results of cell kinetics show that 4 hours after the fusion peptide uptaking, the fluorescence intensity are significantly decreased. The results of each peptide cytotoxicity (MTT assay) showed that: the concentration set by final concentraton adding peptide (0,5,10,20,40,60,80μM) and the positive control drug cisplatin (0,5,10,20,40,60,80μM), cultured for 48h, the peptides did not show cytotoxicity to the tumor cells. Cisplatin toxicity was significantly increased with increasing concentrations of the drug.Based on MTT cytotoxicity assay, we choose 10μM,20μM,30μM and 40μM concentrations of peptides that were lower than 80μM as the working concentration in the next series of experiments. Part III Radiosensitivity effects of fusion peptide to tumor cellsThis part study radiation sensitivity effects of fusion peptides on the tumor cell lines (mainly RKO cells) by clony-forming assays in vitro. Study Impact of fusion peptides on the formation of tumor cell DNA damage and repair of damage In vitro by measuring y-H2AX foci formation.We found that BTw1 and BTw2 had no effect of radiosensitivity, and BTw3 can significantly reduce radiation-induced cell survival. DO represents the average lethal dose of 37% tumor cell survival. Do value is the indicators of tumor cells intrinsic cellular radiosensitivity. After treatment with BTw3, Do is 1.95Gy, after the scrambled peptide (BTs2) treatment, the Do value 2.46Gy. Radiosensitization ratio (SER) of 1.25. The results suggested that BTw3 has radiosensitivity effect on RKO cells.At time of 0 hour, we did not detecteγ-H2AX focus. After 0.5 hours of irradiation,γ-H2AX focus numbers of each group were significantly increased, and the number of γ-H2AX focus of each group at 0.5h points was no significant difference (P> 0.05).1 hour after irradiation,γ-H2AX focus number of BTw3 group was higher than BTs1 group (P= 0.032), but no significant difference compared with BTs2 group (P= 0.132). At this time.γ-H2AX focus of BTw3 group were significantly greater than the numbers of BTwl and BTw2 group (P= 0.031 and 0.009).2,4,8,24 and 48 hours after irradiation, y-H2AX focus number of BTw3 group was higher than that of the other groups (radiation alone, BTs1, BTs2, BTwl and BTw2 group) (P< 0.00001). According to the results we can speculate that fusion peptide BTw3 plus radiation did not induce or increase theγ-H2AX focus, but prolonged BTw3 DNA double-strand break repair time, thereby extended the existence time ofγ-H2AX focus. The results suggest that BTw3 inhibited DNA double-strand break repair of RKO cells.In short, cloning experiments andγ-H2AX foci results support that the BTw3 (T (2638) QQQHDFTLT (2647)) has the potential radiosensitizing effect. Part IV Radiosensitization molecular biologic mechanism of the fusion peptide (TAT-QQQHDFTLT)Based on these results, we further studied BTw3 effect on its corresponding threonine phosphorylation sites (such as the T2647 phosphorylation sites), and downstream molecule Artemis phosphorylation of DNA-PKcs, and impact of BTw3 on caspase-3 of the key implementation elements of caspase family.The results suggest that:20 minutes irradiation, T2647 phosphorylation of DNA-PKcs did not change significantly, but 60 minutes after irradiation, we observed that T2647 phosphorylation level of DNA-PKcs significantly decreased.We studied the ionizing radiation effects of Artemis protein. We observed effects of the fusion peptide BTw3 (TAT-T (2638) QQQHDFTLT (2647)) combined with radiation on Artemis S516 phosphorylation. To detect and Analysis of protein phosphorylation changes in Artemis with Artemis S516 specific phosphorylation antibodies. The results showed that 30 and 60 minutes after 10 Gy irradiation treatment, the fusion peptide BTw3 inhibited Artemis S516 phosphorylation in RKO cells.Changes of caspase-3 results:BTw3 combined with radiation can significantly increase the expression of cleaved caspase-3 protein compared with radiation alone. This indicats that BTw3 could further enhanced radiation-induced apoptosis.Conclusion:1、MS analysis of the molecular weight determination of peptides are consistent with the theoretical values. Proved correct sequence. To ensure that the synthesized peptide for the purpose of high purity peptide.2、HPLC purity of synthetic peptides analysis showed the purity of all synthetic peptides were more than 95%, in line with requirements of high-purity synthetic peptides.3、TAT or fusion TAT with other peptide can penetrate the cell membrane, and localized in the cytoplasm and nucleus, but also has high efficiency into the cell. None penetrating peptides can not enter the cells.4、BTw1 and BTw2 had no radiosensitivity effect on tumor cells, and BTw3 can significantly reduce RKO cell survival after radiation. BTw3 is potential radiosensitizer.5、BTw3 significantly prolonged existence of radiation inducedγ-H2AX focus.6、Cloning experiments andγ-H2AX foci results support the BTw3 (T (2638) QQQHDFTLT (2647)) the potential radiosensitizing effect on RKO cells. 7、BTw3 (TAT-T (2638) QQQHDFTLT (2647)) could inhibit T2647 auto-phosphorylation in RKO cells.8、BTw3 (TAT-T (2638) QQQHDFTLT (2647)) inhibited the Artemis S516 phosphorylation of RKO cell.9、BTw3 combined with radiotherapy can significantly increase the expression of cleaved caspase-3 protein compared with radiotherapy alone. This indicates that BTw3 could further enhanceradiation-induced apoptosis.更多还原