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.     

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.     

Telomere lengthening early in development

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.     

Targeting the telomere and shelterin complex for cancer therapy: current views and future perspectives

Aberrant telomere homeostasis is essential for cell immortality, enabling cells to evade telomere dependent senescence. Disruption of telomere structure and function in cancer cells is highly toxic as shown by detailed pre-clinical evaluation of telomerase inhibitors. Under telomerase inhibition, cells must divide sufficiently frequently to allow one or more telomeres to shorten to an unprotected length. Functioning telomeres are disguised from the DNA damage machinery by DNA remodelling and other activities of the telomere binding complex shelterin. Direct interference with shelterin has been shown to result in cell killing and small molecules directly targeting telomere DNA also have anti-tumour effects partially dependent on shelterin disruption. However, shelterin components have not generally been regarded as therapeutic targets in their own right. In this review, we explore the possibilities for therapeutic targeting of the shelterin complex.     

Alternative lengthening of telomeres (ALT) and chromatin: is there a connection?

The acquisition of cellular immortality is a critical step in the tumorigenic process that requires stabilization of the telomeres, nucleoprotein structures at the termini of chromosomes. While the majority of human tumors stabilize their telomeres through activation of telomerase (hTERT), a significant portion (10-15%) utilize a poorly understood alternative mechanism of telomere maintenance referred to as ALT (Alternative Lengthening of Telomeres). Strikingly, the ALT mechanism is more prevalent in tumors arising from tissues of mesenchymal origin than in those of epithelial origin. This observation suggests that cell type specific mechanisms favor the activation of the ALT mechanism versus telomerase in human tumorigenesis. In addition, the presence of an alternative mechanism of telomere maintenance raises the possibility that telomerase-positive tumors undergoing anti-telomerase therapies might escape by activating the ALT pathway. For these reasons, delineating the ALT mechanism is critical for our understanding of the tumorigenic process and the development of ALT-specific anti-neoplastic therapies. Recent studies have demonstrated that epigenetic modifications at telomeres have a profound effect on telomere length, and may also be linked to the ALT mechanism. In this review we focus on these recent advances and their implications in telomere maintenance.     

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.     

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.     

Telomerase: Structure,functions, and activity regulation

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.     

Short Telomeres are Sufficient to Cause the Degenerative Defects Associated with Aging

Telomerase function is critical for telomere maintenance. Mutations in telomerase components lead to telomere shortening and progressive bone marrow failure in the premature aging syndrome dyskeratosis congenita. Short telomeres are also acquired with aging, yet the role that they play in mediating age-related disease is not fully known. We generated wild-type mice that have short telomeres. In these mice, we identified hematopoietic and immune defects that resembled those present in dyskeratosis congenita patients. When mice with short telomeres were interbred, telomere length was only incrementally restored, and even several generations later, wild-type mice with short telomeres still displayed degenerative defects. Our findings implicate telomere length as a unique heritable trait that, when short, is sufficient to mediate the degenerative defects of aging, even when telomerase is wild-type.     

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。     

Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines

The ribonucleoprotein, telomerase, is responsible for the maintenance of telomere length in most immortal and cancer cells. Telomerase appears to be a marker of human malignancy with at least 85% of human cancers expressing its activity. In the present study, we examined a series of tumor-derived and in vitro immortalized cell lines for telomerase activity levels, telomere lengths, and expression levels of the RNA and catalytic components of telomerase. We found significant variability in both telomere lengths and telomerase activity in clones from tumor cells. In addition, the levels of telomerase components or telomerase activity were not predictive of telomere length. Data from clonally derived cells suggest that critically shortened telomeres in these tumor-derived cell lines may signal activation of telomerase activity through an increase in the expression of the catalytic subunit of telomerase. Although clones with low telomerase shorten their telomeres over time, their subclones all have high levels of telomerase activity with no telomere shortening. In addition, analysis of early clones for telomerase activity indicates substantial variability, which suggests that activity levels fluctuate in individual cells. Our data imply that cell populations exhibit a cyclic expression of telomerase activity, which may be partially regulated by telomere shortening.     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

Methods of studying telomere damage induced by quadruplex-ligand complexes

The burgeoning knowledge about the structure of telomeres and the roles of various factors involved in telomere maintenance provides several possible targets for pharmacological intervention. To date the area that has received major attention regarding drug discovery is the targeting the telomeric G-quadruplex (G4) structure. G4 ligands were initially designed to counteract telomerase action at telomeres. Surprisingly, their antiproliferative effects can occur in telomerase negative cells and follow kinetics, which cannot be merely explained by telomere shortening, suggesting that these compounds affect other pathways, not necessarily related to telomere biology. Impressively, it has been shown that polyaromatic compounds featuring end-stacking binding properties trigger a strong DNA damage response at telomeres. This is typical of the telomere deprotection occurring during cellular senescence or upon telomere injury. It emerged that the G4-interacting agents are more than simple telomerase inhibitors and that their direct target is rather telomere than telomerase. This review summarizes the most valid experimental approaches for studying the pharmacological telomere damage induced by G4-ligand complexes.     

Cancer treatment by telomerase inhibitors: predictions by a kinetic model

The inhibition of telomerase activity in actively dividing cells leads to shortening of their telomeres and suppression of cell growth when the telomere lengths become smaller than a certain threshold value (typically about 1-2 kb of DNA). We evaluated the time (efficacy delay) required to reach the threshold telomeric DNA size after initiation of treatment, which is of critical importance for the efficacy of telomerase inhibitors. A model based on the solution of a system of differential equations was developed to analyze the efficacy delay and dynamics of tumor growth. The efficacy delay was strongly dependent on the size distribution of telomere lengths at the treatment initiation. An increase in the heterogeneity of telomere size resulted in shortening of the delay. However, the long-term dynamics of tumors with homogeneous populations of telomeres were more significantly affected by telomerase inhibitors compared to tumors with heterogeneous size distribution of telomeres. Size distribution of telomeres and tumor doubling times are of critical importance for the dynamics of tumor growth in presence of telomerase inhibitors.     

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.