Telomerase, cervical cancer, and human papillomavirus

Review of the available data indicates that telomerase is activated in the majority of cervical squamous cell carcinomas as it is in most malignant neoplasms. Telomerase activity can also be detected in some preneoplastic cervical lesions, but the significance of this in unclear, because nonneoplastic, proliferating epithelial cells also can have telomerase activity. The bias introduced by cytologic sampling methods can complicate the interpretation of results. Quantitative telomerase assays may be useful in distinguishing nonmalignant, physiologic activation of telomerase from malignant activation. Studies evaluating telomerase component (hTR or hTERT) expression by evaluation of RNA, mRNA, or antigen have yielded conflicting results, but the observation that many nonmalignant, nontelomerase active cells have detectable hTR and hTERT suggests that many cells express telomerase RNA and catalytic components, but do not have active telomerase. The implication is that a regulatory overlay must exist that controls telomerase activation. Activation of the enzyme in carcinogenesis could conceivably be a physiologic activation that normally accompanies cellular proliferation, a direct appropriation of telomerase activity by the neoplastic process, or both. The presence of inactive telomerase in many cells also raises the possibility of a noncatalytic function for the telomerase complex. An understanding of telomerase interaction with HPV infection in the pathogenesis of cervical neoplasia must await a further elaboration of telomerase regulation. Likewise, application of telomerase detection in cervical cancer screening programs must await a better integration of telomerase regulation in normal and specifically in HPV-infected squamous epithelial cells.     

构建人端粒酶催化亚单位基因慢病毒表达载体转染人骨髓基质细胞

背景：骨髓基质细胞不表达端粒酶,因而在体外传代扩增过程中端粒长度逐渐缩短,导致细胞衰老,这是限制其用于细胞治疗应用的一个重要因素。目的：构建人端粒酶催化亚单位基因慢病毒表达载体,探讨以慢病毒介导的人端粒酶催化亚单位基因修饰人骨髓基质细胞的可行性。方法：以pReceiver-M02-hTERT质粒为模板PCR扩增获得目的基因。用BP重组系统将目的片段重组到载体pDONR221上。然后使用LR重组系统将目的序列重组到载体pLenti6.3/V5-DEST上。将重组载体与包装质粒充分混合,利用脂质体共转染293FT细胞获得慢病毒颗粒。结果与结论：成功构建人端粒酶催化亚单位慢病毒表达载体,病毒的平均物理滴度为1.07×1012LP/L。以之转染人骨髓基质细胞,目的基因表达水平有显著提升,细胞正确表达人端粒酶反转录酶蛋白。说明以慢病毒介导的人端粒酶催化亚单位基因修饰人骨髓基质细胞能强化细胞表达人端粒酶催化亚单位。     

Telomerase activation, cellular immortalization and cancer

The maintenance of specialized nucleoprotein structures termed telomeres is essential for chromosome stability. Without new synthesis of telomeres at chromosome ends the chromosomes shorten with progressive cell division, eventually triggering either replicative senescence or apoptosis when telomere length becomes critically short. The regulation of telomerase activity in human cells plays a significant role in the development of cancer. Telomerase is tightly repressed in the vast majority of normal human somatic cells but becomes activated during cellular immortalization and in cancers. While the mechanisms for telomerase activation in cancers have not been fully defined, they include telomerase catalytic subunit gene (hTERT) amplification and trans-activation of the hTERT promoter by the myc oncogene product. Ectopic expression of hTERT is sufficient to restore telomerase activity in cells that lack the enzyme and can immortalize many cell types. Understanding telomerase biology will eventually lead to several clinically relevant telomerase-based therapies. These applications include inhibiting or targeting telomerase as a novel antineoplastic strategy and using cells immortalized by telomerase for therapeutic applications.     

端粒、端粒酶及hTERT／hTERC基因的研究进展

端粒是真核细胞染色体的末端DNA序列,在维持染色体稳定性中起重要作用。染色体的不完全复制使得端粒随着细胞的分裂而逐渐缩短,快速分裂细胞通过端粒酶合成端粒,以弥补端粒的消耗。端粒酶相关基因突变可导致端粒酶活性的降低和端粒缩短,过短的端粒不再保护基因组稳定性,将引起细胞的老化、凋亡或恶变。端粒酶基因的扩增出现在一些肿瘤细胞中,是癌细胞增殖的重要原因,其扩增的机制及其对端粒酶活性的调节作用尚不完全清楚。近期研究表明,端粒酶基因扩增是基因组不稳定的结果,扩增的hTERT／hTERC基因对端粒酶激活和癌变进展有促进作用。     

端粒酶hTERT促进肿瘤细胞侵袭与转移及其机制研究

目的观察hTERT基因修饰对人骨肉瘤细胞系U-2OS生物学行为的影响。方法采用脂质体法将克隆有人全长cDNAhTERT的真核荧光质粒(pIRES2-EGFP-hTERT)转染端粒酶阴性的人骨肉瘤U-2OS细胞,经G418筛选,免疫组化和Westorn Blot鉴定后,检测其端粒酶活性的改变、生长周期以及生物力学的变化。结果成功转染人真核荧光表达载体的U-2OS细胞能有效抵抗G418,并可有效表达hTERT蛋白,细胞内端粒酶活性明显增强,G1期细胞比例下降,S期细胞比例升高,并且还明显增强了对细胞外基质的粘附力。进一步采用Transwell小孔迁移实验检测发现,hTERT/U2OS侵袭能力明显增强。结论转染hTERT基因后,U-2OS细胞内可同时通过端粒酶途径延长端粒。hTERT基因修饰可促进细胞周期进程,促使细胞从G1期→S期。通过替代途径延长端粒的肿瘤细胞在hTERT基因修饰后,增殖能力及侵袭能力明显增强。     

RNA干扰人端粒酶hTR基因对HeLa细胞增殖的影响

目的:探讨RNA干扰抑制人端粒酶RNA组分human telomerase RNA component,hTR)的表达时宫颈癌细胞(HeLa细胞)增殖的影响.方法:hTR基因的RNA干扰表达重组体用脂质体介导方法稳定转染HeLa细胞.RTPCR法检测hTR的mRNA表达水平,TRAP-PCR法检测端粒酶活性,MTT法检测细胞增殖.结果:RT-PCR结果表明,稳定转染RNA干扰表达重组体的细胞株,其hTR基因的mRNA抑制率较无转染对照组下降了(59.7±3.3)%,较转染空载体组下降了(56.3±4.4)%;实验组细胞端粒酶活性下降,生长速度明显减慢.结论:利用RNA干扰技术抑制hTR基因在HeLa细胞的表达能抑制该细胞端粒酶活性和增殖活性.     

端粒、端粒酶与干细胞

端粒、端粒酶与干细胞密切相关,在维持干细胞自我更新和增殖能力中起重要作用。端粒是真核细胞染色体末端的DNA重复序列和特异结合蛋白的复合体,富含鸟嘌呤,具有保护染色体的作用,端粒长度反映细胞的复制史及复制潜能。影响端粒长度的因素包括：端粒结合蛋白、端粒帽蛋白、端粒酶及DNA复制酶等,其中端粒酶是最主要的因素。端粒酶位于端粒末端,作用是合成端粒DNA序列,以抵消或延缓端粒随细胞分裂的不断缩短。端粒酶活性的丧失及其增殖相关基因表达的改变是造成干细胞体外复制和扩增受限的主要原因。随着组织细胞工程学的兴起,体外定向诱导干细胞分化为各种所需组织细胞已经成为研究的焦点,因此诱导和增加端粒酶的活性,维持干细胞分化、自我更新和增殖能力,延长干细胞的寿命具有重要意义。     

人端粒酶反转录酶小发夹RNA质粒表达载体的构建及鉴定

背景:端粒酶反转录酶是端粒酶的活性亚基,已成为肿瘤研究的热点。RNA干扰技术作为一种基因沉默方法,具有高效、特异等优点,现已广泛应用于肿瘤、病毒等研究领域。目的:构建针对人端粒酶反转录酶的小发夹RNA质粒表达载体,并观察其对乳腺癌T47D细胞人端粒酶反转录酶基因的表达和端粒酶活性的影响。方法:以Genbank中人端粒酶反转录酶基因的mRNA序列为基础,设计人端粒酶反转录酶基因的小干扰RNA序列,将其连接到具有G418抗性的质粒pBAsi-hU6-Neo(BamHⅠ/HindⅢ)中,应用基因测序加以验证,扩增提取质粒,以脂质体转染表达小发夹RNA的质粒到乳腺癌T47D细胞,抗生素G418筛选出转染成功的各组细胞。结果与结论:实验所构建的人端粒酶反转录酶的小发夹RNA质粒表达载体,经测序验证无误。将pBAsi-hU6-Neo重组质粒转染入T47D细胞,经G418筛选获得了转染成功的细胞。经RT-PCR和Western blot检测,转染后的人端粒酶反转录酶基因在mRNA和蛋白水平的表达均明显降低(P<0.01),经TRAP-ELISA法检测实验组细胞端粒酶活性出现显著下降(P<0.01)。结果证实,实验成功构建人端粒酶反转录酶的小发夹RNA质粒表达载体,实验所设计的小干扰RNA能有效抑制肿瘤细胞人端粒酶反转录酶基因的表达,进而降低细胞的端粒酶活性。     

A novel telomerase-specific gene therapy: gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter

Apoptosis is a genetically encoded cell death process and is a pathway that may be disrupted in tumor cells. Therefore, therapies that restore the ability to undergo apoptosis are promising for the treatment of tumor cells. We have demonstrated that the transfer of apoptosis-inducible genes inhibits the growth of tumors in vitro and in vivo through induction of apoptosis. However, to restrict induction of apoptosis to tumor cells, we need to explore a tumor-specific expression system of these genes. In the present study, we developed the telomerase-specific transfer system of apoptosis-inducible genes, utilizing the promoter of the human telomerase catalytic subunit (hTERT) gene. Approximately 90% of tumors have telomerase activity whereas most normal cells do not express the activity. These observations indicate that telomerase is a particularly attractive target for the tumor-specific expression system of vectors. We demonstrate here that by using the hTERT promoter-driven caspase-8 expression vector (hTERT/caspase-8), apoptosis is restricted to telomerase-positive tumor cells of wide range, and is not seen in normal fibroblast cells without telomerase activity. Furthermore, treatment of subcutaneous tumors in nude mice with the hTERT/caspase-8 construct inhibited tumor growth significantly because of induction of apoptosis (p < 0.01). The telomerase-specific expression of apoptosis-inducible genes afforded by the hTERT promoter, therefore, may be a novel and promising targeting approach for the treatment of tumors with telomerase activity.