【作者】 陈伟；

【导师】 陶泽璋；

【作者基本信息】 武汉大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 背景与目的：恶性肿瘤主要表现为细胞失去控制无限制地增长和／或凋亡通路受阻导致细胞堆积,而端粒酶的活化是使细胞获得无限增殖能力成为永生化细胞的关键因素之一。80-96%原发性喉鳞状细胞癌有端粒酶活性表达。端粒酶延长端粒使端粒结构维持相对稳定的机制是：端粒酶—RNA依赖的DNA多聚酶,催化TTAGGG加在染色体末端,从而维持端粒的长度,使细胞保持旺盛的分裂增殖能力。在各国学者的努力下,端粒酶途径维持端粒长度的机制已经被阐明,其关键蛋白是人端粒酶逆转录酶(human telomerase reverse transcriptase, hTERT)。虽然端粒酶是保持恶性肿瘤细胞端粒DNA长度稳定的最主要途径,但它不是唯一的途径。研究发现某些缺乏端粒酶活性的肿瘤细胞也能够永生,而且具有长短不一的端粒DNA,这类肿瘤细胞保持端粒DNA长度稳定的机制被称为端粒延长替代机制(alternative lengthening of telomeres, ALT)。大约有15%的恶性肿瘤细胞依赖ALT机制维持端粒结构稳定。ALT机制在同一恶性肿瘤细胞中可以与端粒酶机制共存或者互换,即在某些肿瘤细胞中端粒结构稳定可能是由端粒酶机制和ALT机制共同维持,而抑制端粒酶途径后肿瘤细胞有可能依赖ALT机制而维持端粒结构稳定。前期研究中,我们根据RNAi原理设计并构建了能表达短发夹RNA (short hairpin, shRNA)的质粒pshRNA-TERT,其靶基因为hTERT mRNA。将该质粒转染体外培养的喉鳞癌Hep-2细胞,观察到癌细胞hTERT基因表达显著下降、端粒酶失活,细胞生长增殖明显受到抑制。端粒酶活性受抑制初期发现每天均有许多凋亡、死亡细胞出现,存活细胞逐渐减少。但是从治疗后8天起凋亡、死亡细胞很少出现,存活细胞维持相对稳定,约占癌细胞生长增殖高峰期的10%。实验得出了一个重要的结论：端粒酶活性受抑制后仍有少量喉癌细胞可以继续存活。该结果很好地解释了迄今为止为何各种抑制端粒酶活性的基因治疗方法都只能抑制部分癌细胞的生长增殖、未能达到完全治愈肿瘤的困惑：端粒酶被抑制后一段时间,其它端粒维持机制可能已激活或将被激活,因此端粒结构继续得到维持,癌细胞继续存活并增殖。喉癌是头颈部第二位原发于上皮的常见恶性肿瘤。同其它肿瘤一样,其发病率有明显上升趋势。功能性喉癌外科的开展,使患者得以获得较高的五年生存率及满意的生存质量。但仍有不少患者,虽经根治手术和放化疗,因局部复发或转移而难以救治以致死亡。当术后复发和出现转移时,肿瘤对放、化疗多不敏感,预后很差。因此,寻找出一种新的、有效的、特异性的治疗途径挽救或延长这类患者的生命具有十分重要的意义。因此,本研究中我们对端粒酶活性受抑制后的喉癌细胞进行深入探讨：从生物学行为变化入手研究治疗后存活细胞的转归；从端粒维持机制变化入手研究其转归的机制,从维持端粒关键物质入手研究其起作用的核心蛋白。目的：运用RNA干扰技术抑制喉癌细胞端粒酶活性,收集并研究存活喉癌细胞的特性及转归。通过端粒动力学和肿瘤特性的研究,明确存活细胞在端粒维持机制及肿瘤特性上的转变。通过比较两种细胞中差异蛋白的表达,筛选出端粒维持机制中的关键蛋白,为喉癌细胞的基因治疗提供新的靶点和方向。方法：构建靶向端粒酶逆转录酶的质粒pshRNA,导入喉癌细胞后用流式细胞仪分选和收集干扰成功的细胞。继续培养该细胞,检测其生长情况,稳定传代后,分别检测端粒酶活性、hTERT的表达及APBs小体表达情况,明确存活细胞的端粒维持特性。FLOW-FISH技术检测喉癌细胞和存活细胞端粒长度变化,CO-FISH法检测细胞中T-SCE水平,Transwell实验和裸鼠成瘤实验比较两种细胞在侵袭力和成瘤性上的差异。运用二维电泳(2-DE)和质谱技术(MALDI-TOF-MS)技术,筛选与鉴定出喉癌细胞和存活细胞核中的差异蛋白,并运用Westernblot法验证差异蛋白的表达。结果：喉癌细胞端粒酶活性被抑制后,流式细胞仪分选出有绿色荧光表达的干扰成功细胞。干扰初期,该细胞在增殖的同时伴有大量的衰老和凋亡,但仍有少量细胞可以存活,15天左右该细胞开始稳定传代生长。检测存活细胞的特性发现,该细胞没有端粒酶活性,hTERT表达比喉癌细胞明显减弱,同时伴有APBs小体表达增高,其差异有统计学意义。存活细胞的端粒长度比喉癌细胞长,而且端粒结构分析发现存活细胞具有ALT机制特异性的T-SCE结构。Transwell实验和裸鼠成瘤实验证实存活细胞侵袭力和成瘤性比喉癌细胞弱,差异有统计学意义。运用二维电泳和质谱技术并通过Westernblot验证,我们发现同喉癌细胞相比,存活细胞中SAGE1、GAS8、PML、SYCE1和NBS1表达增高(P<0.05),提示这些蛋白可能为ALT机制的关键蛋白。相反,在喉癌细胞中存在TERT和：SMC1β蛋白的高表达(P<0.05),提示这些蛋白可能为端粒酶延长机制的关键蛋白。结论：RNA干扰端粒酶活性后喉癌细胞仍有部分存活,该细胞能够正常增殖而无端粒酶活性,且端粒延长替代机制中关键物质APBs小体表达增高,因此推测存活细胞利用非端粒酶途径来维持端粒的稳定。稳定传代的存活细胞具有相对较长的端粒,端粒延长可能依赖于T-SCE为基础的端粒延长替代机制,进一步证实存活细胞具有ALT细胞的特性。侵袭力和成瘤性的改变提示存活的喉癌细胞可能发生了肿瘤特性的转变。差异蛋白组学研究发现两种细胞中存在的差异蛋白可能为端粒酶途径和端粒延长替代机制中起作用的关键蛋白。这些关键蛋白的发现为以端粒为靶点的肿瘤基因治疗提供新的靶点和方向。综合以上实验结果,本研究抑制喉癌细胞的端粒酶活性,收集存活的喉癌细胞进行分析发现,细胞没有端粒酶活性,hTERT表达下降,同时伴有APBs小体表达增高。端粒动力学研究也发现存活细胞具有特异性的端粒姐妹染色体重组现象,端粒长度增加,这些都证实存活细胞具有ALT细胞的特征。存活细胞也可能发生了肿瘤特性的减弱。此外,蛋白组学研究鉴别出喉癌细胞和存活细胞存在数种差异蛋白,这些蛋白可能是端粒酶机制以及端粒延长替代机制中起作用的关键蛋白。研究的结果为今后以端粒为靶点的肿瘤基因治疗提供新的靶点和方向。更多还原

【Abstract】 Telomere length is associated with the proliferative capacity of cells. Whereas normal cells with reduced proliferative capacity exhibit low telomerase activity and shortened telomeres, cancer cells maintain telomere length primarily by reactivation of telomerase. The telomerase enzyme which adds specific DNA sequences to the ends of chromosomes is composed of three elements:human telomerase reverse transcriptase (hTERT), telomerase RNA, and dyskerin. While the majority of tumors show high telomerase activity, some cancer cells may utilize the alternative lengthening of telomeres (ALT) mechanism. The ALT-mediated elongation of telomeres has been associated with homologous recombination events between sister chromatids at telomeres, known as telomeric sister-chromatid exchange (T-SCE). ALT cells are usually characterized by a remarkable heterogeneous telomere length within a given cell and the presence of promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins. While these ALT-associated PML bodies (APBs) may be used to identify tumors which use the ALT pathway, their function is still unknown.It has been hypothesized that cancer cells may activate the ALT pathway when telomerase is inhibited or otherwise rendered non-functional. For example, when cells with active ALT were fused with normal somatic cells or telomerase-positive cancer cells, the ALT activity was suppressed, suggesting that it is normally repressed. Another study demonstrated that human fibroblasts with spontaneous inactivation of telomerase had sustained proliferation in vitro, although these cells did not express APBs or telomere length patterns typical of ALT. When treated with the demethylating agent 5-aza-20-deoxycytidine, these cells reactivated telomerase. Still, other studies suggest that ALT and telomerase can occur together in human cancer cells. Therefore, the environmental conditions which trigger or maintain the ALT pathway are yet to be precisely defined. In a previous study, we reported that some laryngeal cancer cells could still replicate after RNAi-hTERT-based anti-telomerase treatment, but whether the survival of these cells depended on the ALT pathway upon inhibition of telomerase was not clear. In the present study, we collected cells surviving RNAi-hTERT treatment and analyzed their hTERT levels, telomerase activity and APBs expression in order to explore the telomere maintenance mechanisms (TMM) in laryngeal squamous carcinoma (Hep-2) cells when telomerase is inhibited. We also evaluated differences in invasive ability, tumorigenicity, telomere length and T-SCE between Hep-2 cells and the surviving cells. We further performed a proteomic survey of proteins differentially-expressed between the two cell lines by two-dimensional gel electrophoresis (2-DE) and ESI-MS/MS and validated the differentially expressed proteins by immunoblot analysis. These ALT specific proteins may be candidates for further studies as diagnostic, prognostic or therapeutic tools for cancer.Objective:Using RNA interference to inhibit telomerase activity in laryngeal carcinoma cells, collect and study the prognosis and characteristics of surviving cells. Identify the telomere maintenance mechanism and the changes of the tumor characteristics through the study for the telomere dynamics and tumor characteristics. To compare differences proteins between two cell and selected the key protein in telomere maintenance mechanisms, that provide a new target and direction for gene therapy for laryngeal cancer cells.Methods:A plasmid termed pEGFP-shTERT was constructed.After transfected with the plasmids, cells was analyzed and sorted by flow cytometry. Continue to cultivate the cells to detect growth. After stabile passage, we measure the telomerase activity and detect the expression hTERT and APBs bodies to clear the characteristics of surviving cells.To detect the telomere length between untreated laryngeal carcinoma cells and surviving cells by FLOW-FISH and T-SCE levels by CO-FISH. To compare the invasion and tumorigenicity by transwell experiments and tumor formation in vivo. Using two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS) technology to screen and identify the differences protein between the untreated laryngeal cells and the surviving cells in the cell nucleus, and used between the nucleus Westernblot method validation expression.Results:Flow cytometer had sorted the transfected laryngeal carcinoma cells that expressed green fluorescent successfully. Interference early, the proliferation of the cells accompanied by a large number of aging and apoptosis, but there are still a few cells can survive about 15 days, the cells begin to stabilize the growth of passage. Compared with untreated laryngeal carcinoma cells, telomerase activity in surviving cells was inhibited, hTERT expression significantly decreased accompanied by increased expression of APBs bodies, the difference was statistically significant(P<0.05). The result showed that the surviving cells has long telomere length, telomere structure analysis revealed that the ALT mechanism specific T-SCE structure exist in surviving cells. Transwell experiments and tumor formation in vivo confirmed the tumor growth and invasion in surviving cells is more weaker than the untreated cells.Using two-dimensional electrophoresis and mass spectrometry and verified by Westernblot, we found that compared with untreated cells, the proteins of SAGE1, GAS8, PML, SYCE1, NBS1 high express in surviving cells (P<0.05), suggesting that these proteins may be the key proteins for the ALT mechanism. On the contrary, the proteins TERT and SMC1βthat increased expressed in untreated cells (P<0.05) may be the key proteins in the telomerase mechanism.Conclusion:In the surviving cells, telomerase activity was eliminated. Additionally, these cells were enriched for the APBs bodies, suggesting an alternative mechanism for telomere maintenance following telomerase inhibition. These results could have major impacts in designing new cancer treatments.The surviving cells has long telomeres. Similar to ALT cells, the surviving cells showed evidence of ALT telomere homeostasis. Invasiveness and tumorigenicity changes in surviving cells suggest possible changes in the characteristics of the tumor.The proteomics study found that the difference proteins between two cells may be the key protein in telomere maintenance mechanisms. The discovery of these key proteins provide a new target to improve telomere-based therapy for cancer.SummaryHere, we report that surviving Hep-2 cells that survived anti-telomerase treatments showed sustained proliferation in culture with down-regulated hTERT expression and the greatly raised levels of alternative lengthening of telomeres specific promyelocytic leukaemia bodies. Analysis of the telomere length kinetics also demonstrated elevated telomeric sister chromatid exchange (T-SCE) in surviving Hep-2 cells, consistent with their long telomeres. Similar to ALT cells, the surviving cells showed evidence of ALT telomere homeostasis. Furthermore, proteomic analysis identified several proteins differentially expressed between the original Hep-2 cells and surviving cells that may provide new insight for understanding these two telomere maintenance mechanisms. Thus, the findings in this study may help to improve telomerase-based therapy for cancer.更多还原