Studies of the molecular mechanisms in the regulation of telomerase activity.

Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, is repressed in normal human somatic cells but is activated during development and upon neoplasia. Whereas activation is involved in immortalization of neoplastic cells, repression of telomerase permits consecutive shortening of telomeres in a chromosome replication-dependent fashion. This cell cycle-dependent, unidirectional catabolism of telomeres constitutes a mechanism for cells to record the extent of DNA loss and cell division number; when telomeres become critically short, the cells terminate chromosome replication and enter cellular senescence. Although neither the telomere signaling mechanisms nor the mechanisms whereby telomerase is repressed in normal cells and activated in neoplastic cells have been established, inhibition of telomerase has been shown to compromise the growth of cancer cells in culture; conversely, forced expression of the enzyme in senescent human cells extends their life span to one typical of young cells. Thus, to switch telomerase on and off has potentially important implications in anti-aging and anti-cancer therapy. There is abundant evidence that the regulation of telomerase is multifactorial in mammalian cells, involving telomerase gene expression, post-translational protein-protein interactions, and protein phosphorylation. Several proto-oncogenes and tumor suppressor genes have been implicated in the regulation of telomerase activity, both directly and indirectly; these include c-Myc, Bcl-2, p21(WAF1), Rb, p53, PKC, Akt/PKB, and protein phosphatase 2A. These findings are evidence for the complexity of telomerase control mechanisms and constitute a point of departure for piecing together an integrated picture of telomerase structure, function, and regulation in aging and tumor development-Liu, J.-P. Studies of the molecular mechanisms in the regulation of telomerase activity.     

Targeting the telosome: Therapeutic implications

Since telomere integrity is required to guarantee the unlimited replicative potential of cancer cells, telomerase, the enzyme responsible for telomere length maintenance in most human tumors, and lately also telomeres themselves have become extremely attractive targets for new anticancer interventions. At the current status of knowledge, it is still not possible to define the best therapeutic target between telomerase and telomeres. It is noteworthy that interfering with telomeres, through direct targeting of telomeric DNA or proteins involved in the telosome complex, could negatively affect the proliferative potential not only of tumors expressing telomerase activity but also of those that maintain their telomeres through alternative lengthening or still unknown mechanisms. This review presents the different therapeutic approaches proposed thus far and developed in preclinical tumor models and discusses the perspectives for their use in the clinical setting.     

Telomeric recombination induced by dysfunctional telomeres

Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.     

Inhibition of telomerase enhances apoptosis induced by sodium butyrate via mitochondrial pathway

Telomerase activation represents an early step in carcinogenesis. Increased telomerase activity in cervical cancer suggests a potential target for the development of novel therapeutic drugs. The aim of this study is to investigate the impact of telomerase activity on the biological features of HeLa cells and the possible mechanisms of enhanced apoptosis rate induced by sodium butyrate after telomerase inhibition. We introduced vectors encoding dominate negative (DN)-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistance marker into HeLa cells. Thus we assessed the biological effects of telomerase activity on telomere length, cell proliferation, chemosensitivity and radiosensitivity. In order to understand the mechanisms in which DN-hTERT enhances the apoptosis induced by sodium butyrate, we detected the release status of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Ectopic expression of DN-hTERT resulted in inhibition of telomerase activity, reduction of telomere length, decreased colony formation ability, and loss of tumorigenicity in nude mice. Moreover, DN-hTERT transfected HeLa cells with shortened telomeres were more susceptible to multiple chemotherapeutic agents and radiation. WT-hTERT transfected HeLa cells with longer telomeres exhibited resistance to radiation and chemotherapeutic agents. Our data demonstrate that elevated release level of cytochrome c and AIF from mitochondria might contribute to the enhanced apoptosis in DN-hTERT transfected HeLa cells after treatment with sodium butyrate. Inhibition of telomerase might serve as a promising adjunctive therapy combined with conventional therapy in cervical cancer. Both of them contributed equally to this work.     

Pharmacological intervention strategies for affecting telomerase activity: future prospects to treat cancer and degenerative disease

Telomerase enzyme is a ribonucleoprotein maintaining the length of the telomeres by adding G-rich repeats to the end of the eukaryotic chromosomes. Normal human somatic cells, cultured in vitro, have a strictly limited proliferative potential undergoing senescence after about 50-70 population doublings. In contrast, most of the tumor cells have unlimited replicative potential. Although the mechanisms of immortalization are not understood completely at a genetic level, the key role of the telomere/telomerase system in the process is clear. The DNA replication machinery is not able to replicate fully the DNA at the very end of the chromosomes; therefore, about 50-200 nucleotides are lost during each of the replication cycles resulting in a gradual decrease of telomere length. Critically short telomere induces senescence, subsequent crisis and cell death. In tumor cells, however, the telomerase enzyme prevents the formation of critically short telomeres, adding GGTTAG repeats to the 3' end of the chromosomes immortalizing the cells. Immortality is one of the hallmarks of cancer. Besides the catalytic activity dependent telomere maintenance, catalytic activity-independent effects of telomerase may also be involved in the regulation of cell cycle. The telomere/telomerase system offers two possibilities to intervene the proliferative activity of the cell: (1) inhibition the telomere maintenance by inhibiting the telomerase activity; (2) activating the residual telomerase enzyme or inducing telomerase expression. Whilst the former approach could abolish the limitless replicative potential of malignant cells, the activation of telomerase might be utilized for treating degenerative diseases. Here, we review the current status of telomerase therapeutics, summarizing the activities of those pharmacological agents which either inhibit or activate the enzyme. We also discuss the future opportunities and challenges of research on pharmacological intervention of telomerase activity.     

Telomere Length Influences Cancer Cell Differentiation In Vivo

Limitless reproductive potential is one of the hallmarks of cancer cells. This ability is due to the maintenance of telomeres, erosion of which causes cellular senescence or death. While most cancer cells activate telomerase, a telomere-elongating enzyme, it remains elusive as to why cancer cells often maintain shorter telomeres than the cells in the surrounding normal tissues. Here, we show that forced telomere elongation in cancer cells promotes their differentiation in vivo. We elongated the telomeres of human prostate cancer cells that possess short telomeres by enhancing their telomerase activity. The resulting cells had long telomeres and retained the ability to form tumors in nude mice. Strikingly, these tumors exhibited many duct-like structures and reduced N-cadherin expression, reminiscent of well-differentiated adenocarcinoma. These changes were caused by telomere elongation and not by enhanced telomerase activity. Gene expression profiling revealed that tumor formation was accompanied by the expression of innate immune system-related genes, which have been implicated in maintaining tumor cells in an undifferentiated state and poor-prognosis cancers. In tumors derived from the telomere-elongated cells, upregulation of such gene sets is not observed. Our observations suggest a functional contribution of short telomeres to tumor malignancy by regulation of cancer cell differentiation.     

端粒酶是干扰素抗肿瘤的新靶点

端粒酶(telomerase)是一种具有逆转录活性的核糖核蛋白酶.端粒酶的异常活化是细胞永生化和肿瘤形成的关键步骤. 端粒酶活性与细胞周期及细胞凋亡调控密切相关;端粒酶由端粒酶逆转录酶、端粒酶RNA、端粒酶相关蛋白质组成,端粒酶逆转录酶是端粒酶活性的决定性组分.干扰素(interferon)是一种具有抗病毒、抗增殖、抗肿瘤和免疫调节等功能的细胞因子;近年研究表明,干扰素通过相关信号转导途径而调节端粒酶活性,诱导细胞凋亡,为肿瘤的生物治疗提供了新思路;但干扰素与端粒酶活性相关的抗肿瘤机制研究尚不充分. 本文综述干扰素通过调节端粒酶逆转录酶转录因子的表达和相互作用而抑制端粒酶活性、调节细胞周期并诱导细胞凋亡等抗肿瘤作用机制.     

Telomerase in endocrine and endocrine-dependent tumors

Telomerase, the ribonucleoprotein enzyme that elongates chromosomal ends, or telomeres, is repressed in most normal somatic cells but reactivated in transformed cells to compensate for the progressive erosion of the telomeres during cell divisions. In accordance with this hypothesis, the presence of telomerase activity has been reported in more than 90% of human cancers, whereas most normal tissues or benign tumors contain low or undetectable telomerase activity. Reactivation of telomerase has also been widely reported in endocrine neoplasms and in hormone-related cancers. In the present study, we review the most recent publications on telomerase in these types of tumors. The hormonal regulation of telomerase activity and the possible strategies for cancer therapy based on the inhibition of telomerase has also been discussed.     

How will telomeric complex be further contributed to our longevity? — The potential novel biomarkers of telomere complex counteracting both aging and cancer

With the smooth move towards the coming expected clinical reports of anticancer pharmaceutical molecules targeting telomeres and telomerase, and also with the exciting success in the extension of lifespan by regulating telomerase activity without increased onset of oncogenesis in laboratory mouse models (Garcia-Cao et al., 2006; Jaskelioff et al., 2011), we are convinced that targeting telomeres based on telomerase will be a potential approach to conquer both aging and cancer and the idea of longevity seems to be no more mysterious. More interestingly, emerging evidences from clinical research reveal that other telomeric factors, like specifi c telomeric binding proteins and nonspecific telomere associated proteins also show crucial importance in aging and oncogenesis. This stems from their roles in the stability of telomere structure and in the inhibition of DNA damage response at telomeres. Uncapping these proteins from chromosome ends leads to dramatic telomere loss and telomere dysfunction which is more abrupt than those induced by telomerase inactivation. Abnormal expression of these factors results in developmental failure, aging and even oncogenesis evidenced by several experimental models and clinical cases, indicating telomere specifi c proteins and its associated proteins have complimentary roles to telomerase in telomere protection and controlling cellular fate. Thus, these telomeric factors might be potential clinical biomarkers for early detection or even therapeutic targets of aging and cancer. Future studies to elucidate how these proteins function in telomere protection might benefit patients suffering aging or cancer who are not sensitive to telomerase mediation.     

Differential regulation of MRN (Mre11-Rad50-Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy.     

端粒、端粒酶与肿瘤的相关研究进展

端粒是真核细胞染色体末端的DNA序列,在维持染色体的稳定中起着重要的作用。快速生长的细胞通过端粒酶来合成端 粒重复序列以弥补其损耗。在人类恶性肿瘤细胞中,85%以上能检测到端粒酶的活性,使其成为一个几乎普遍的癌标志物,而在大 多数正常体细胞中,端粒酶是阴性的。端粒酶与肿瘤之间的最新研究已经在肿瘤生物学领域开辟了新的途径,可能会彻底改变抗 癌疗法。在这篇文章中,我们将会总结端粒和端粒酶在癌细胞中的作用。随着科技的发展,端粒和端粒酶拥有巨大的潜力,必将能 够为肿瘤的治疗带来更多的方法。     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT’s enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.     

The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins

Most cancer cells activate telomerase to elongate telomeres and achieve unlimited replicative potential. Some cancer cells cannot activate telomerase and use telomere homologous recombination (HR) to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). A hallmark of ALT cells is the recruitment of telomeres to PML bodies (termed APBs). Here, we show that the SMC5/6 complex localizes to APBs in ALT cells and is required for targeting telomeres to APBs. The MMS21 SUMO ligase of the SMC5/6 complex SUMOylates multiple telomere-binding proteins, including TRF1 and TRF2. Inhibition of TRF1 or TRF2 SUMOylation prevents APB formation. Depletion of SMC5/6 subunits by RNA interference inhibits telomere HR, causing telomere shortening and senescence in ALT cells. Thus, the SMC5/6 complex facilitates telomere HR and elongation in ALT cells by promoting APB formation through SUMOylation of telomere-binding proteins.     

Telomere dysfunction and telomerase activation in cancer--a pathological paradox?

Telomerase is expressed in more than 90% of human cancers. Telomere maintenance by this enzyme is believed to safeguard genomic integrity in neoplastic cells. Nevertheless, many telomerase-expressing tumours exhibit chromosomal instability triggered by short, dysfunctional telomeres, implying that active telomerase is not sufficient for preserving a functional telosomic nucleoprotein complex in cancer cells. We here examine three possible solutions to this ostensible paradox. First, prior to telomerase activation, telomere erosion may have evolved to a level where telomeric repeat sequences are too short to provide a functional substrate for telomerase enzyme activity. Second, mechanisms other than the continuous telomere erosion counteracted by telomerase may contribute to rapid shortening of telomere repeats. Third, dysfunction of telomere-regulating proteins may result in direct telomere uncapping. Moreover, telomerase may contribute to tumour development also through mechanisms unrelated to telomere length maintenance. Taken together, the available data on the role of telomerase in cancer strongly support that inhibition of this enzyme is a feasible strategy for cancer therapy.     

Preferential extension of short telomeres induced by low extracellular pH

The majority of tumor cells overcome proliferative limit by expressing telomerase. Whether or not telomerase preferentially extends the shortest telomeres is still under debate. When human cancer cells are cultured at neutral pH, telomerase extends telomeres in telomere length-independent manner. However, the microenvironment of tumor is slightly acidic, and it is not yet known how this influences telomerase action. Here, we examine telomere length homeostasis in tumor cells cultured at pHe 6.8. The results indicate that telomerase preferentially extends short telomeres, such that telomere length distribution narrows and telomeres become nearly uniform in size. After growth at pHe 6.8, the expression of telomerase, TRF1, TRF2 and TIN2 decreases, and the abundance of Cajal bodies decreases. Therefore, telomerase are insufficient for extending every telomere and shorter telomeres bearing less shelterin proteins are more accessible for telomerase recruitment. The findings support the ‘protein-counting mechanism’ in which extended and unextended state of telomere is determined by the number of associated shelterin proteins and the abundance of telomerase. Decreased expression of telomerase and preferential extension of short telomeres have important implications for tumor cell viability, and generate a strong rationale for research on telomerase-targeted anti-cancer therapeutics.