Telomeres and telomerase in leukaemia and lymphoma.

Telomeres are DNA structures which serve to stabilize chromosomes. In human cells telomeres progressively shorten with each cell division leading to eventual chromosome instability and cell death. Telomerase is a DNA polymerase which is required for the maintenance of telomeres. Therefore, telomeres and telomerase play a role in the regulation of the life span of the cell. Human cells express low levels of telomerase, however when telomere length reaches a critical level abnormal activation of telomerase can lead to immortalization and uncontrolled proliferation. This process has been associated with the development of many leukaemias and lymphomas. Understanding these processes in normal and malignant cells could lead to therapies which target the telomere/telomerase complex.     

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.     

The role of telomeres in musculoskeletal diseases

The telomeric region of repetitive DNA sequences at the end of chromosomes prevents end-to-end fusion of chromosome terminals and deterioration of the doublestrand free ends. Because of the 'end-replication problem', telomeres shorten with each round of cell division, resulting in cell senescence. The enzyme telomerase compensates for telomere shortening by elongating telomeric sequences, thereby prolonging the lifespan of the cell. Studies of articular cartilage and bone tissues have indicated that telomere shortening limits normal cell function and proliferation, while the telomere maintenance mechanisms of osteosarcoma cells facilitate escape from cell death and promote immortality. This article reviews the literature on this topic and provides an extensive discussion of the basic molecular biology and roles of telomeres and telomerase in musculoskeletal diseases such as osteoarthritis, osteoporosis and osteosarcoma. Findings to date suggest that telomeres and telomerase may become novel therapeutic targets for the diagnosis, treatment and prevention of musculoskeletal disorders.     

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。     

Plumbagin triggers DNA damage response,telomere dysfunction and genome instability of human breast cancer cells

AimNatural plant products are increasingly being used in cancer therapeutic studies due to their reduced normal cell toxicity. In this study, the anti-cancer properties of plumbagin, a naphthoquinone derivative extracted from the roots of Plumbago, were evaluated in breast cancer cells.MethodsTo evaluate the effects of plumbagin on breast cancer cell types, we employed a variety of techniques comprising cell viability, cell cycle assay, comet assay, western blotting, immunocytochemistry, measurement of telomerase activity, telomere restriction fragment length, quantitative fluorescence in situ hybridisation, along with gene expression analysis of untreated cells.ResultsPlumbagin treatment induced cytotoxicity in human breast cancer cells along with cell cycle arrest, DNA damage and cell death leading to apoptosis. Plumbagin was also found to suppress the telomerase activity in cancer cells accompanied by telomere attrition. Telomere shortening was corroborated by reduced telomere fluorescence on chromosome ends and genome instability.ConclusionTogether, these findings may suggest the application of plumbagin as adjuvant modality in breast cancer therapeutics.     

Telomerase targeted therapy in cancer and cancer stem cells

Telomerase plays a key role in cell fate: loss of telomerase in normal differentiated cells heralds senescence and limits cell division, whereas reactivation of telomerase sustains proliferation and potentiates mutagenesis and transformation. Given this pivotal role, telomerase has been the subject of intense investigation in the field of developmental cancer therapeutics. To date, a broad spectrum of therapeutic strategies has been developed, ranging from direct targeting or reprogramming of the enzyme, to immune or virus-mediated targeting of cells expressing telomerase, to strategies focusing on the telomeres themselves. The recent discovery and growing interest in cancer stem cells has thrust telomerase therapy into new relief as an approach that may be uniquely suited to neutralizing this treatment-resistant subpopulation of cancer cells. Here we will review the mechanistic rationale and preclinical and clinical state of development of the various telomerase-based therapeutic approaches, with emphasis on the role of telomerase in cancer stem cell biology and its implications for therapeutic efforts.     

Status,challenges, and future prospects of stem cell therapy in pelvic floor disorders

Pelvic floor disorders(PFDs)represent a group of common and frequentlyoccurring diseases that seriously affect the life quality of women,generally including stress urinary incontinence and pelvic organ prolapse.Surgery has been used as a treatment for PFD,but almost 30%of patients require subsequent surgery due to a high incidence of postoperative complications and high recurrence rates.Therefore,investigations of new therapeutic strategies are urgently needed.Stem cells possess strong multi-differentiation,self-renewal,immunomodulation,and angiogenesis abilities and they are able to differentiate into various cell types of pelvic floor tissues and thus provide a potential therapeutic approach for PFD.Recently,various studies using different autologous stem cells have achieved promising results by improving the pelvic ligament and muscle regeneration and conferring the tissue elasticity and strength to the damaged tissue in PFD,as well as reduced inflammatory reactions,collagen deposition,and foreign body reaction.However,with relatively high rates of complications such as bladder stone formation and wound infections,further studies are necessary to investigate the role of stem cells as maintainers of tissue homeostasis and modulators in early interventions including therapies using new stem cell sources,exosomes,and tissueengineering combined with stem cell-based implants,among others.This review describes the types of stem cells and the possible interaction mechanisms in PFD treatment,with the hope of providing more promising stem cell treatment strategies for PFD in the future.     

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。     

Telomere stability and carcinogenesis: an off-again, on-again relationship

Previous studies in mice have demonstrated antagonistic effects of telomerase loss on carcinogenesis. Telomere attrition can promote genome instability, thereby stimulating initiation of early-stage cancers, but can also inhibit tumorigenesis by promoting permanent cell growth arrest or death. Human cancers likely develop in cell lineages with low levels of telomerase, leading to telomere losses in early lesions, followed by subsequent activation of telomerase. Mouse models constitutively lacking telomerase have thus not addressed how telomere losses within telomerase-proficient cells have an impact on carcinogenesis. Using a novel transgenic mouse model, Begus-Nahrmann et al. demonstrate in this issue of the JCI that transient telomere dysfunction in telomerase-proficient animals is a potent stimulus of tumor formation.     

三氧化二砷、人参皂甙和β榄香烯对K562细胞株端粒-端粒酶系统作用机制的研究

本研究目的是探索三氧化二砷、人参皂甙及β榄香烯等几种药物在不同浓度作用时对K562细胞株端粒长度和端粒酶活性的调节及抑制作用，并研究上述药物抗白血病的作用机制，为寻求治疗急性白血病的新方法奠定基础。用3组不同浓度的三氧化二砷、人参皂甙及β榄香烯与人类红白血病细胞株K562共培养，采用PCR-ELISA法检测端粒酶活性，Southem blot法检测端粒长度。观察上述药物对K562细胞端粒酶活性及端粒长度的影响。结果表明：①经人参皂甙、三氧化二砷及β榄香烯作用后，K562细胞株端粒酶活性下降，下降程度呈浓度、时间依赖性，在一定浓度及作用时间后端粒酶呈阴性。②三氧化二砷、人参皂甙、β-榄香烯作用后K562细胞的存活率下降，且抑制作用呈浓度、时间依赖性。③三氧化二砷、人参皂甙及β-榄香烯作用于K562细胞72小时后，端粒长度略有延长。结论：①三氧化二砷、人参皂甙及β-榄香烯均能抑制K562细胞的端粒酶活性，抑制作用呈浓度、时间依赖性。抑制端粒酶活性可能是其抗肿瘤作用的机制之一。②三氧化二砷、人参皂甙及β-榄香烯均能抑制K562细胞的生长，抑制作用呈浓度、时间依赖性。③抑制端粒酶活性后，K562细胞的端粒长度略有延长。本研究结果提示除了端粒酶以外，白血病细胞可能存在其他的端粒长度调节机制。     

Telomere/telomerase interplay in virus‐driven and virus‐independent lymphomagenesis: pathogenic and clinical implications

Telomerase is a ribonucleoprotein complex critically involved in extending and maintaining telomeres. Unlike the majority of somatic cells, in which hTERT and telomerase activity are generally silent, normal lymphocytes show transient physiological hTERT expression and telomerase activity according to their differentiation/activation status. During lymphomagenesis, induction of persistent telomerase expression and activity may occur before or after telomere shortening, as a consequence of the different mechanisms through which transforming factors/agents may activate telomerase. Available data indicate that the timing of telomerase activation may allow the distinction of two different lymphomagenetic models: (i) an early activation of telomerase via exogenous regulators of hTERT, along with an increased lymphocyte growth and a subsequent selection of cells with increased transforming potential may characterize several virus‐related lymphoid malignancies; (ii) a progressive shortening of telomeres, leading to genetic instability which favors a subsequent activation of telomerase via endogenous regulators may occur in most virus‐unrelated lymphoid tumors. These models may have clinically relevant implications, particularly for the tailoring of therapeutic strategies targeting telomerase. © 2010 Wiley Periodicals, Inc. Med Res Rev     

Detection of telomerase activity by the TRAP assay and its variants and alternatives

Telomerase activity is closely connected to problems of cellular immortality, proliferative capacity, differentiation, cancer and aging. Correspondingly, techniques for its detection have been essential for progress in telomere biology and are of still increasing importance in molecular diagnostics and therapy of cancer. This article reviews the development of the telomere repeat amplification protocol (TRAP) and its various modifications as the most widespread assay to detect and measure telomerase activity. Alternative possibilities of telomerase activity detection are also discussed which make it possible to omit the PCR-mediated amplification of telomerase products. These approaches are based on recent advances in highly sensitive detection systems.     

Persistence of tumor infiltrating lymphocytes in adoptive immunotherapy correlates with telomere length

Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.     

端粒和端粒酶与肝细胞的永生化

目的：端粒是位于真核细胞染色体末端的蛋白-DNA复合体，保护染色体完整性和稳定性。端粒酶是一种特殊的细胞核蛋白反转录酶，能以自身的RNA为模板，反转录合成端粒DNA序列，添加到染色体来端。端粒酶介导的端粒延长作为细胞中端粒缩短的主要补偿机制，对正常细胞以及肿瘤细胞延长分裂代数起着重要作用、分化良好并具有肝脏正常合成代谢功能的永生化肝细胞在生物医学研究，尤其是肝细胞移植、生物人工肝支持治疗以及药理学和毒理学研究方面具有广泛的应用前景。 资料来源：应用计算机检索Pubmed数据库1990—01／2005—12有关端粒。端粒酶和肝细胞永生化的文章，检索词“telomere，telomerase，hepatocyte，immortalization”，限定文章语言种类为English。同时计算机检索中国期刊全文数据库、万方数据库1994-01／2005—12期间的相关文章，检索词“端粒、端粒酶、永生化、肝细胞”，限定文章语言种类为中文。 资料选择：纳入标准：（1）有关端粒和端粒酶结构和功能的研究文献：②有关端粒、端粒酶与肝细胞永生化相关性的文献：排除标准：重复研究。 资料提炼：共收集到32篇有关端粒、端粒酶与肝细胞永生化相关性的文献，查找全文，从中选取14篇作为主要参考文献。 资料综合：将所选文献资料按照以下顺序归纳总结：①端粒的结构和功能：端粒是指位于真核细胞染色体末端的一种特殊结构，由富含G的DNA重复序列与端粒结合蛋白所构成的一种蛋白-DNA复合体，它既可保护染色体不受核酸酶的破坏，又避免了因DNA粘性末端的裸露而发生的染色体融合．②端粒酶的结构和功能：端粒酶是一种RNA依赖的DNA聚合酶，能以自身的RNA为模板，反转录合成端粒DNA序列，添加到染色体末端，以补偿细胞分裂时端粒DNA缩短，使细胞克服危机期，?     

三氧化二砷、人参皂甙、β-榄香烯联合环磷酰胺对K562细胞株端粒-端粒酶系统的作用

本研究探索三氧化二砷、人参皂甙、B榄香烯单独及联合环磷酰胺应用对K562细胞株端粒长度和端粒酶活性的调节作用，探讨上述药物抗白血病的作用机制。用不同浓度的三氧化二砷、人参皂甙及B榄香烯单独及联合环磷酰胺与人类红白血病细胞株K562共培养，采用PCR-ELISA法检测端粒酶活性、Southern杂交法检测端粒长度。观察上述药物对K562细胞端粒酶活性及端粒长度的影响。结果表明：①经人参皂甙、三氧化二砷、B榄香烯及环磷酰胺作用后，K562细胞株端粒酶活性下降，下降程度呈浓度时间依赖性。当与环磷酰胺联合应用后，下降程度更明显。②三氧化二砷、人参皂甙、β-榄香烯作用后K562细胞的存活率下降，且抑制作用呈浓度、时间依赖性。当与环磷酰胺联合应用后，抑制作用更明显。③三氧化二砷、人参皂甙、β榄香烯及环磷酰胺作用于K562细胞株72小时后，端粒长度略有延长。结论：三氧化二砷、人参皂甙、β榄香烯及环磷酰胺均能抑制K562细胞株的生长及端粒酶活性，抑制端粒酶活性可能是其抗肿瘤作用的机制之一；三氧化二砷、人参皂甙、β榄香烯与环磷酰胺联合应用后抑制作用增强；联合环磷酰胺可望降低上述药物的给药浓度。抑制端粒酶活性后，K562细胞株的端粒长度略有延长，提示除了端粒酶以外，白血病细胞可能存在其他的端粒长度调节机制。     

Telomere erosion in memory T cells induced by telomerase inhibition at the site of antigenic challenge in vivo

The extent of human memory T cell proliferation, differentiation, and telomere erosion that occurs after a single episode of immune challenge in vivo is unclear. To investigate this, we injected tuberculin purified protein derivative (PPD) into the skin of immune individuals and isolated responsive T cells from the site of antigenic challenge at different times. PPD-specific CD4+ T cells proliferated and differentiated extensively in the skin during this secondary response. Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals. Tissue fluid obtained from the site of PPD challenge in the skin inhibited the induction of the enzyme telomerase in T cells in vitro. Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition. Furthermore, the addition of IFN-alpha to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro. Therefore, these results suggest that the rate of telomere erosion in proliferating, antigen-specific CD4+ T cells may be accelerated by type I IFN during a secondary response in vivo.