Telomere stability and telomerase in mesenchymal stem cells

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important. In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall telomere length and the telomere length of individual chromosomes.     

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.     

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.     

Telomere regulation in pluripotent stem cells

Pluripotent stem cells (PSCs) have the potential to produce any types of cells from all three basic germ layers and the capacity to self-renew and proliferate indefinitely in vitro. The two main types of PSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), share common features such as colony morphology, high expression of Oct4 and Nanog, and strong alkaline phosphatase activity. In recent years, increasing evidences suggest that telomere length represents another important internal factor in maintaining stem cell pluripotency. Telomere length homeostasis and its structural integrity help to protect chromosome ends from recombination, end fusion, and DNA damage responses, ensuring the divisional ability of mammalian cells. PSCs generally exhibit high telomerase activity to maintain their extremely long and stable telomeres, and emerging data indicate the alternative lengthening of telomeres (ALT) pathway may play an important role in telomere functions too. Such characteristics are likely key to their abilities to differentiate into diverse cell types in vivo. In this review,we will focus on the function and regulation of telomeres in ESCs and iPSCs, thereby shedding light on the importance of telomere length to pluripotency and the mechanisms that regulate telomeres in PSCs.     

Characterization of tree shrew telomeres and telomerase

The use of tree shrews as experimental animals for biomedical research is a new practice. Several recent studies suggest that tree shrews are suitable for studying cancers, including breast cancer, glioblastoma,lung cancer, and hepatocellular carcinoma. However, the telomeres and the telomerase of tree shrews have not been studied to date. Here, we characterize telomeres and telomerase in tree shrews. The telomere length of tree shrews is approximately 23 kb, which is longer than that of primates and shorter than that of mice, and it is extended in breast tumor tissues according to Southern blot and flow-fluorescence in situ hybridization(FISH) analyses. Tree shrew spleen, bone marrow, testis, ovary, and uterus show high telomerase activities, which are increased in breast tumor tissues by telomeric repeat amplification protocol assays. The telomere length becomes shorter, and telomerase activity decreases with age. The tree shrew TERT and TERC are more highly similar to primates than to rodents. These findings lay a solid foundation for using tree shrews to study aging and cancers.     

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.     

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.     

Tankyrase 2 poly(ADP-ribose) polymerase domain-deleted mice exhibit growth defects but have normal telomere length and capping

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardation phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement for Tnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together, these results suggest that Tnks2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.     

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.     

利用流式荧光原位杂交法测定细胞端粒长度

目的建立利用流式荧光原位杂交法检测细胞端粒长度的技术方法。方法以端粒酶敲除的G3小鼠和同龄野生型小鼠为检测对象,分离其外周血中的单个核细胞后与肽核酸荧光探针杂交,用流式细胞仪采集和分析其端粒长度,分别用荧光原位杂交方法和SYBR Green荧光定量PCR方法验证其准确性。结果流式荧光原位杂交法测定G3小鼠细胞端粒相对长度与C57BJ/6野生型小鼠相比为0.5345,荧光定量PCR测定端粒相对长度为0.5717,结果基本一致。结论流式细胞术与原位杂交方法结合起来检测细胞端粒的平均长度可靠易行,对单个核细胞端粒平均长度的检测有较高的实用性。     

Telomerase Activity and Telomere Length in Daphnia

Telomeres, comprised of short repetitive sequences, are essential for genome stability and have been studied in relation to cellular senescence and aging. Telomerase, the enzyme that adds telomeric repeats to chromosome ends, is essential for maintaining the overall telomere length. A lack of telomerase activity in mammalian somatic cells results in progressive shortening of telomeres with each cellular replication event. Mammals exhibit high rates of cell proliferation during embryonic and juvenile stages but very little somatic cell proliferation occurs during adult and senescent stages. The telomere hypothesis of cellular aging states that telomeres serve as an internal mitotic clock and telomere length erosion leads to cellular senescence and eventual cell death. In this report, we have examined telomerase activity, processivity, and telomere length in Daphnia, an organism that grows continuously throughout its life. Similar to insects, Daphnia telomeric repeat sequence was determined to be TTAGG and telomerase products with five-nucleotide periodicity were generated in the telomerase activity assay. We investigated telomerase function and telomere lengths in two closely related ecotypes of Daphnia with divergent lifespans, short-lived D. pulex and long-lived D. pulicaria. Our results indicate that there is no age-dependent decline in telomere length, telomerase activity, or processivity in short-lived D. pulex. On the contrary, a significant age dependent decline in telomere length, telomerase activity and processivity is observed during life span in long-lived D. pulicaria. While providing the first report on characterization of Daphnia telomeres and telomerase activity, our results also indicate that mechanisms other than telomere shortening may be responsible for the strikingly short life span of D. pulex.     

Telomeres in cancer and ageing

Telomeres protect the chromosome ends from unscheduled DNA repair and degradation. Telomeres are heterochromatic domains composed of repetitive DNA (TTAGGG repeats) bound to an array of specialized proteins. The length of telomere repeats and the integrity of telomere-binding proteins are both important for telomere protection. Furthermore, telomere length and integrity are regulated by a number of epigenetic modifications, thus pointing to higher order control of telomere function. In this regard, we have recently discovered that telomeres are transcribed generating long, non-coding RNAs, which remain associated with the telomeric chromatin and are likely to have important roles in telomere regulation. In the past, we showed that telomere length and the catalytic component of telomerase, Tert, are critical determinants for the mobilization of stem cells. These effects of telomerase and telomere length on stem cell behaviour anticipate the premature ageing and cancer phenotypes of telomerase mutant mice. Recently, we have demonstrated the anti-ageing activity of telomerase by forcing telomerase expression in mice with augmented cancer resistance. Shelterin is the major protein complex bound to mammalian telomeres; however, its potential relevance for cancer and ageing remained unaddressed to date. To this end, we have generated mice conditionally deleted for the shelterin proteins TRF1, TPP1 and Rap1. The study of these mice demonstrates that telomere dysfunction, even if telomeres are of a normal length, is sufficient to produce premature tissue degeneration, acquisition of chromosomal aberrations and initiation of neoplastic lesions. These new mouse models, together with the telomerase-deficient mouse model, are valuable tools for understanding human pathologies produced by telomere dysfunction.     

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.     

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.     

Human telomerase can immortalize Indian muntjac cells

The mechanisms of replicative senescence by telomere shortening are not fully understood. The Indian muntjac has the fewest chromosomes of all mammals, greatly simplifying the analysis of each telomere over time. In this study, telomere shortening was observed throughout the life span of cultured normal muntjac cells by quantitative fluorescence in situ hybridization and terminal restriction fragment analysis. Ectopic expression of the human telomerase catalytic subunit in these cells reconstituted telomerase activity, extended telomere lengths, and immortalized the cells, demonstrating that the Indian muntjac cells can serve as a telomere-based replicative senescence model for human cells. In one strain, two chromosome ends had significantly shorter telomeres than the other ends, which led to a variety of chromosome abnormalities. Near senescence, additional ends became telomere signal free, and chromosome aberrancies increased dramatically. Interstitial telomere sequences coincided with fragile sites, suggesting that these remnants of chromosome fusion events might contribute to genome instability. One SV40-immortalized cell line lacked telomerase, and its genetic instability was corrected by the ectopic expression of telomerase, confirming that too-short telomeres were the source of abnormalities. Indian muntjac cells provide an excellent system for understanding the mechanism of replicative senescence and the role of telomerase in the elongation of individual telomeres.