【作者】 周彩红；

【导师】 薛京伦；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 DNA损伤修复与肿瘤的发生、发展密切相关,其中的关键分子和信号通路是抗肿瘤药物研究和开发的重要靶点。TTRAP (TRAF and TNF receptor-associated protein)是一个新鉴定的5’-酪氨酰DNA磷酸二酯酶,在细胞发生DNA损伤时,可将双链断裂缺口处5’端的磷酯酪氨酰键水解转化为5’磷酸,促进DNA双链断裂修复。本论文旨在研究TTRAP对肿瘤细胞生长和化疗药物敏感性的影响,探讨其能否作为肿瘤辅助治疗的靶标。为确定TTRAP是否与肿瘤发生有关,我们检索了TTRAP在基因表达综合库中的数据,发现在早期发病的结直肠癌患者结肠黏膜和恶性转化的前列腺癌患者肿瘤内TTRAP表达量均较低。检测肺癌、乳腺癌、前列腺癌及骨肉瘤等多种肿瘤细胞株的TTRAP蛋白表达谱,发现TTRAP在所有细胞中普遍存在,但其表达形式和表达丰度在各细胞株中存在较大差异。在内源TTRAP表达量相对较低的人骨肉瘤细胞株U20S中,过表达野生型TTRAP (TTRAP wild type, TTRAPwt)显著抑制U20S细胞的克隆形成。稳定转染TTRAP的细胞株在持续6天的生长曲线中显示生长变缓。表达TTRAP酶活性的无功能突变体TTRAP (E152A)及TTRAP (D262A)可部分缓解这种抑制作用。TTRAPwt的过表达对NF-κB的转录活性有抑制作用,该作用可被突变体TTRAP (E152A)及TTRAP (D262A)的表达所抵消。TTRAP也可抑制U20S细胞迁移,但TTRAP酶活性的突变对这一作用没有影响。上述结果提示TTRAP对肿瘤细胞有生长抑制作用,这种抑制作用依赖于其5’-酪氨酰DNA磷酸二酯酶活性,可能与NF-κB信号通路相关。TP53是DNA损伤反应中决定细胞命运的关键蛋白。为探讨TTRAP影响肿瘤细胞生长的分子机制,我们应用酵母双杂交鉴定了TTRAP与TP53的直接相互作用。TP53的截短突变体与TTRAP的酵母配对实验结果表明,TP53的DNA结合域与两者间的相互作用有关。免疫共沉淀实验证实293T细胞内源表达的TP53和TTRAP存在相互作用。荧光蛋白融合体EGFP-p53和TTRAP-DsRed可共定位于细胞核内,表明TTRAP和TP53在空间上存在相互作用的可能。应用萤光素酶报告基因检测TP53对下游靶基因的转录调控作用,在低水平表达TP53的情况下,TTRAP促进TP53对下游基因的转录激活作用。依托泊苷是临床常用的抗肿瘤药物,通过干扰拓扑异构酶2(topisomerase2, Top2)介导的DNA再连接反应使DNA双链断裂。体外药物敏感性研究表明,U20S细胞过表达TTRAPwt后,依托泊苜处理使其克隆存活率显著高于空载体对照；细胞内源TTRAP用siRNA沉默表达,药物处理后的克隆存活率低于对照。在细胞增殖实验中,U20S细胞过表达TTRAPwt后对依托泊苷的细胞毒效应低于空载体对照,而过表达TTRAP (E152A)和TTRAP (D262A)后其效应高于对照。这表明TTRAP的表达量与U20S细胞对依托泊苷的药物敏感性呈负相关。为进一步探讨TTRAP与依托泊苷细胞杀伤效应的相关性,我们检测了药物处理情况下TTRAP转录和蛋白表达的动态变化,发现依托泊苷处理1-6小时内TTRAP的转录没有显著提高,但其蛋白量有所增加。以亚胺环己酮(cycloheximide, CHX)抑制细胞内蛋白质合成时,TTRAP的蛋白表达量在1-9小时内逐渐减少,而同时加CHX和依托泊苷处理,细胞内TTRAP蛋白降解速度减缓,表明依托泊苷处理提高了TTRAP蛋白的稳定性,减缓了蛋白的降解过程。因翻译后修饰(post-translational modifications, PTM),如泛素化和SOMO化情况的变化会影响蛋白稳定性,依托泊苷对’TTRAP蛋白稳定性的影响可能涉及蛋白的泛素化抑制和SOMO化激活,其具体机理还需要进一步实验验证。SUMO非共价结合也属于蛋白翻译后修饰的一种。将TTRAP基因中的SUMO蛋白非共价结合位点突变后与红荧光基因融合后构建成融合蛋白表达质粒pTTRAP(SIMm)-DsRed,该质粒在细胞内的表达量显著下降,且不再呈现PML核体典型的点状分布特征,说明SUMO蛋白的结合对TTRAP蛋白在细胞内的稳定表达和亚细胞定位起着重要作用。综上所述,本论文发现了TTRAP对U20S肿瘤细胞的生长有抑制作用,U20S细胞对抗肿瘤药物依托泊苷的敏感性与TTRAP的表达量呈负有关。上述结果表明对TTRAP表达和活性的调节有助于改善肿瘤细胞对依托泊苷的药物敏感性,为TTRAP应用于肿瘤的辅助治疗提供了初步的实验和理论依据。更多还原

【Abstract】 The repair of DNA damage is related to the occurrence and development of tumors. The key molecules and signal pathways in this process will be important targets for the development of new anti-cancer agents. TTRAP (TRAF and TNF receptor-associated protein) is a newly defined5’tyrosyl-DNA phosphodiesterase. Upon DNA damage, it will cleave the5’-phosphotyrosyl bonds, helping to release the free5’-phosphate termini and to repair the double-strand DNA breaks. The object of our research will focus on the effects of TTRAP on the growth of tumor cells and their chemosensitivity for anti-cancer drugs.In order to know if TTRAP is related to tumor occurrence, we surveyed the expression of TTRAP in Gene Expression Omnibus. The expression of TTRAP was relatively lower in normal-appearing colonic mucosa of early onset colorectal cancer patients and in metastatic prostate cancer tumors that refractory to hormones. We also evaluated the endogenous expression of TTRAP protein with Western blot assay in different tumor cell lines, including lung cancer, breast cancer, prostate cancer and osteosarcoma cell lines. The results indicated that TTRAP ubiquitously expressed in all cell lines, but its expression profile was different depending on individual cell line.U2OS is a human osteosarcoma cell line with relatively lower expression of TTRAP protein. Both in transient transfection assay and in lentivirus transduction process, TTRAP significantly inhibited the colony formation in U2OS cells. The stable transfectant of wild type TTRAP (TTRAPwt) gene showed decreased growth speed when cultured continuously for6days. Two dominant negative mutations for TTRAP’s tyrosyl DNA phosphodiesterase activity, TTRAP (E152A) and TTRAP (D262A), could partially block this inhibition. These two dominant negative mutants of TTRAP also rescued the inhibition of wild TTRAP on the transactivation activity of NF-κB. TTRAP also inhibited the migration of U2OS cells in a cell wound healing assay, but these two mutants could not. These results indicated that TTRAP could inhibit the growth of tumor cells, which depends on its5’tyrosyl-DNA phosphodiesterase activity. This inhibitory effect might be related to the NF-κB signaling pathway.In the DNA damage response, TP53is a key mediator for DNA repair, cell apoptosis and senescence. In order to find TTRAP’s effects on signal pathways relating to tumor cell growth, we employed yeast two hybridization assay to detect the direct interaction between TTRAP and TP53. Deletion analysis revealed that the DNA-binding motif of TP53is responsible for the interaction with TTRAP. TTRAP could be co-immunoprecipitated with TP53in293T cells. The fusion protein EGFP-p53and TTRAP-DsRed could also be co-localized in the nucleus, indicating the possibility of interaction of TTRAP and TP53in space. With luciferase-based reporter assay, we found that the transactivation activity of TP53could be weakly promoted by TTRAP when the expression level of TP53was relatively low.Etoposide is a wide-employed anti-tumor drug in the clinic. It can increase topoisemerase2-mediated DNA breakage primarily by inhibiting the ability of the enzyme to religate cleaved nucleic acid molecules and result in cell death. With in vitro chemosnsitivity assay, we detected that the overexpression of TTRAPwt increased the survival of cell colonies than vector control in U2OS cells. When the endogeneous TTRAP was knock down by SiRNA, the number of survived colonies decreased. The cytotoxicity of etoposide in TTRAPwt overexpressed U2OS cells was lower than the vector control in a cell proliferation assay, while in cells expressing the two mutants, TTRAP (E152A) and TTRAP (D262A), the cytotoxicity was higher. These results indicated that the expression of TTRAP could influent the etoposide chemosensitivity of U2OS cells.We further tested the time-dependent profile of the transcription and translation of TTRAP after the application of TTRAP. There was no significant up-regulation of TTRAP transcription1-6hours later after etoposide treatment, but the protein level of TTRAP increased. Cycloheximide could inhibit new protein synthesis. TTRAP protein was degraded slowly upon cycloheximide treatment. When cells were treated with cycloheximide and etoposide together, the degradation of TTRAP protein speed down. The stability of a protein could be influenced by post-translational modifications (PTM), such as ubiquitinylation and sumoylation, we presumed that the effects of etoposide on the stability of TTRAP might relate to these two PMT mechanisms. It needed to be tested by appropriate experiments. Non-covalent SUMO interaction is also one kind of PMT. The expression vector for TTRAP(SIMm), in which the SUMO-interacting motif was mutated, was constructed to test the effects of SUMO-binding on TTRAP’s stability. As compared with wild-type TTRAP, the expression level of TTRAP (SIMm)-DsRed was reduced significantly. The mutant’s localization in the cell also changed from the classical dot-like profile of PML-NBs to irregular distribution in the nucleus. This result suggested that SUMO interaction is quite important for the stability and localization of TTRAP protein.The preliminary study here indicated for the first time that TTRAP had inhibitory effects on tumor cell growth, and etoposide chemosensitivity of U2OS cells was correlated negatively with the expression of TTRAP. These results supplied information for the potential application of TTRAP in the adjuvant therapy for solid tumors.更多还原