Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny‐based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co‐evolved with body size. Multiple independent times smaller, shorter‐lived species changed to having longer telomeres and expressing telomerase. Trade‐offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human‐like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.     

Cellular senescence and longevity of osteophyte‐derived mesenchymal stem cells compared to patient‐matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte‐derived mesenchymal cells (oMSCs) in comparison to patient‐matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell‐cycle‐related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase‐positive cells were found to be located perivascularly and were Stro‐1 positive. Fifteen cell‐cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839–850, 2009. © 2009 Wiley‐Liss, Inc.     

Transient introduction of human telomerase mRNA improves hallmarks of progeria cells

Hutchinson–Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra‐individual heterogeneity of expression of telomere‐associated proteins DNA PKcs/Ku70/Ku80, with low‐expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere‐associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by β‐galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.     

In vitro aging of rat lung cells. Downregulation of telomerase activity and continuous decrease of telomere length are not incompatible with malignant transformation

Most normal mammalian somatic cells cultivated in vitro enter replicative senescence after a finite number of divisions, as a consequence of the progressive shortening of telomeres during proliferation that reflects one aspect of organism/cellular aging. The situation appears more complex in rodent cells due to physiological telomerase expression in most somatic normal tissues, great telomere length, and the difficulties of finding suitable in vitro culture conditions. To study in vitro aging of rat lung epithelial cells, we have developed primary culture conditions adapted to rat fresh lung explants and have studied for 1 year (50 passages) the changes in cellular proliferation and mortality, genetic instability, telomerase activity, telomere length, and tumorigenic potential. We have observed an absence of senescence and/or crisis, a transient genetic instability, the persistence of a differentiated Clara cell phenotype, a steady decrease in telomerase activity followed by a low residual activity together with a continuous decrease in telomere length, a constant rate of proliferation, and the acquisition of tumorigenic potential. The bypass of the growth arrest and the acquisition of long-term growth properties could be explained by the loss of p16(INK4a) expression, the ARF/p53 pathway not being altered. In conclusion, these results clearly indicate that, in rat lung epithelial cells, in vitro transformation and acquisition of tumorigenic properties can occur even if the telomere length is still decreasing and telomerase activity remains downregulated.     

Stabilization of telomere length and karyotypic stability are directly correlated with the level of hTERT gene expression in primary fibroblasts

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. However, telomere length is not maintained in all cell lines, even though in vitro telomerase activity is restored in all of them. Cell lines expressing higher levels of hTERT mRNA do not exhibit telomere erosion or genomic instability. By contrast, fibroblasts expressing lower levels of hTERT do exhibit telomere shortening, although the telomeres eventually stabilize at a shorter length. The shorter telomere lengths and the extent of karyotypic abnormalities are both functions of hTERT expression level. We conclude that telomerase activity is required to bypass senescence but is not sufficient to prevent telomere erosion and genomic instability at lower levels of expression.     

Behaviour of telomere and telomerase during aging and regeneration in zebrafish

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.     

Wild-derived inbred mouse strains have short telomeres

Telomere length and telomerase activity directly affect the replicative capacity of primary human cells. Some have suggested that telomere length influences organismal lifespan. We compared telomere length distributions in a number of inbred and outbred established mouse strains with those of strains recently derived from wild mice. Telomere length was considerably shorter in wild-derived strains than in the established strains. We found no correlation of telomere length with lifespan, even among closely related inbred mouse strains. Thus, while telomere length plays a role in cellular lifespan in cultured human cells, it is not a major factor in determining organismal lifespan.     

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.     

MicroRNA-340-5p increases telomere length by targeting telomere protein POT1 to improve Alzheimer's disease in mice

Alzheimer′s disease (AD) is a chronic neurodegenerative disorder which is the primary cause of dementia in the elderly. Telomere attrition has been proposed as a hallmark of aging. Our study aimed to explore the mechanism of the protection of telomere 1 (POT1) in regulating telomere length and affecting cellular senescence in AD. The AD mouse model was established by d -galactose and aluminum chloride, and the water maze test and dark avoidance test were used to detect the behaviors of mice and confirm the success of AD mouse model. AD cell model was established with HT22 cells induced by Aβ₄₂ oligomers. POT1 expression in the AD model was detected by quantitative real-time polymerase chain reaction. Cellular telomere length in hippocampal tissue was analyzed by telomere restriction fragment. Localization of intracellular POT1, telomerase, and telomeres was analyzed by immunofluorescence and fluorescence in situ hybridization. Dual-luciferase assay was used to validate the targeted binding relationship between microRNA-340-5p (miR-340-5p) and POT1. After inhibiting POT1 expression, the symptoms of AD in mice were improved. Aβ_1–42 deposition was reduced, whereas telomere length and telomerase activity was increased. Dual-luciferase assay verified the binding relationship between miR-340-5p and POT1. An increase in miR-340-5p expression could alleviate cellular senescence and AD symptoms. miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms. This study made a conclusion that miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms in mice.     

Telomere length, stem cells and aging

Telomere shortening occurs concomitant with organismal aging, and it is accelerated in the context of human diseases associated with mutations in telomerase, such as some cases of dyskeratosis congenita, idiopathic pulmonary fibrosis and aplastic anemia. People with these diseases, as well as Terc-deficient mice, show decreased lifespan coincidental with a premature loss of tissue renewal, which suggests that telomerase is rate-limiting for tissue homeostasis and organismal survival. These findings have gained special relevance as they suggest that telomerase activity and telomere length can directly affect the ability of stem cells to regenerate tissues. If this is true, stem cell dysfunction provoked by telomere shortening may be one of the mechanisms responsible for organismal aging in both humans and mice. Here, we will review the current evidence linking telomere shortening to aging and stem cell dysfunction.     

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.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes

Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.     

Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG_(1–3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase‐mediated telomere repeat addition is negatively modulated by the levels of telomere‐bound Rap1‐Rif1‐Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere‐binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady‐state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C‐terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere‐bound Est2 is increased, while telomere‐bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.     

Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation

Although chronological donor age is the most potent predictor of long-term outcome after renal transplantation, it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre-implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.     

Telomerase regulation and progressive telomere shortening of rat hepatic stem-like epithelial cells during in vitro aging

Rat hepatic stem-like epithelial cells, LE/2, LE/6, and WB-F344, share some phenotypic properties with oval cells, observed in the early stages of hepatocarcinogenesis. Here, we describe regulations of telomerase and telomere length during in vitro aging of LEs and WB-F344. These cells displayed no apparent aging phenotypes for over 140 passages. Telomerase activity and telomere length of these cells progressively decreased with the passages, and at the late passages, telomere shortening appeared to be reduced as telomerase activity increased. Regulation of TERT and TR, key components of telomerase, was similar to that of the telomerase activity. LEs possessed weak telomerase activity with a slow rate of proliferation compared to WB-F344, and were not tumorigenic, whereas WB-F344 was transformed in vitro from intermediate passage. In conclusion, LEs and WB-F344 have different biochemical properties, and telomerase activation and short telomeres are unlikely necessary for the transformation of WB-F344. TERT and TR seem to be the regulators of the telomerase activity. The relationship between telomere length and telomerase activity suggests that telomerase contributes to the regulation of telomere length in these cells. Our findings provide a better understanding of mechanisms in neoplastic transformation of rat hepatic stem-like epithelial cells.     

端粒及端粒酶的研究进展

端粒是染色体末端独特的蛋白质-DNA结构,在保护染色体的完整性和维持细胞的复制能力方面起着重要的作用.端粒酶则是由RNA和蛋白质亚基组成的、能够延长端粒的一种特殊反转录酶.端粒长度和端粒酶活性的变化与细胞衰老和癌变密切相关.端粒结合蛋白可能通过调节端粒酶的活性来调节端粒长度,进而控制细胞的衰老、永生化和癌变.研制端粒酶的专一性抑制剂在肿瘤治疗方面有着广阔的前景.