Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just as important for causing cellular senescence, cannot be measured properly using these methods. Stress-induced telomere shortening caused by, e.g. oxidative damage happens in a stochastic manner leaving just a few single telomeres critically short. It is now possible to visualize these few ultra-short telomeres due to the advantages of the newly developed Universal single telomere length assay (STELA), and we therefore believe that this method should be considered the method of choice when measuring the length of telomeres after exposure to oxidative stress. In order to test our hypothesis, cultured human mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells estimated by senescence-associated β-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage of senescent cells was absent. Based on the findings in the present study, it seems reasonable to conclude that Universal STELA is superior to TRF in detecting telomere damage caused by exposure to oxidative stress. The choice of method should therefore be considered carefully in studies examining stress-related telomere shortening as well as in the emerging field of lifestyle studies involving telomere length measurements.     

Paternal age is positively linked to telomere length of children

Telomere length is linked to age-associated diseases, with shorter telomeres in blood associated with an increased probability of mortality from infection or heart disease. Little is known about how human telomere length is regulated despite convincing data from twins that telomere length is largely heritable, uniform in various tissues during development until birth and variable between individuals. As sperm cells show increasing telomere length with age, we investigated whether age of fathers at conception correlated with telomere length of their offspring. Telomere length in blood from 125 random subjects was shown to be positively associated with paternal age (+22 bp yr -1, 95% confidence interval 5.2-38.3, P = 0.010), and paternal age was calculated to affect telomere length by up to 20% of average telomere length per generation. Males lose telomeric sequence faster than females (31 bp yr -1, 17.6-43.8, P < 0.0001 vs. 14 bp yr -1, 3.5-24.8, P < 0.01) and the rate of telomere loss slows throughout the human lifespan. These data indicate that paternal age plays a role in the vertical transmission of telomere length and may contribute significantly to the variability of telomere length seen in the human population, particularly if effects are cumulative through generations.     

Telomeres and longevity: testing an evolutionary hypothesis

Identifying mechanisms that underlie variation in adult survivorship provide insight into the evolution of life history strategies and phenotypic variation in longevity. There is accumulating evidence that shortening telomeres, the protective caps at the ends of chromosomes, play an important role in individual variation in longevity. Given that telomeres generally shorten with age, it was surprising to find that in a population of a long-lived seabird, Leach's storm petrel, telomeres appear to lengthen with age. This unique finding suggested that the longest lived individuals are able to elongate telomeres, an interpretation we call the "elongation hypothesis." Alternatively, the "selection hypothesis" states that the longest lived individuals start with the longest telomeres and variation in telomere length decreases with age due to the selective disappearance of individuals with short telomeres. In the same population in which evidence supporting both hypotheses was uncovered, we tested mutually exclusive predictions from the elongation and selection hypotheses by measuring telomere length with the telomere restriction fragment assay in hatchling and old, adult storm petrels. As previously found, adult birds had longer telomeres on average compared with hatchlings. We also found that 3 hatchlings had mean telomere lengths exceeding that of the most extreme old bird, old birds on average had longer initial telomere lengths than hatchlings, and the variance in mean telomere length was significantly greater for hatchlings than for old birds, all predicted by the selection hypothesis. Perhaps more surprisingly, the oldest adults also show little or no accumulation of short telomeres over time, a pattern unknown in other species. Long telomeres are thought to provide a buffer against cellular senescence and be generally indicative of genome stability and overall cell health. In storm petrels, because the progressive accumulation of short telomeres appears negligible, variation in telomere length at birth may be linked to individual variation in longevity.     

Shared environmental factors associated with telomere length maintenance in elderly male twins

During aging, chromosome ends, or telomeres, gradually erode or shorten with each somatic cell division. Loss of telomere length homeostasis has been linked to age-related disease. Remarkably, specific environmental assaults, both physical and psychological, have been shown to correlate with shortened telomeres. However, the extent that genetic and/or environmental factors may influence telomere length during later stages of lifespan is not known. Telomere length was measured in 686 male US World War II and Korean War veteran monozygotic (MZ) and dizygotic (DZ) twins (including 181 MZ and 125 DZ complete pairs) with a mean age of 77.5 years (range 73-85 years). During the entire process of telomere length measurement, participant age and twin status were completely blinded. White blood cell mean telomere length shortened in this elderly population by 71 base pairs per year (P < 0.0001). We observed no evidence of heritable effects in this elderly population on telomere length maintenance, but rather find that telomere length was largely associated with shared environmental factors (P < 0.0001). Additionally, we found that individuals with hypertension and cardiovascular disease had significantly shorter telomeres (P = 0.0025 and 0.002, respectively). Our results emphasize that shared environmental factors can have a primary impact on telomere length maintenance in elderly humans.     

Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans

Terminal restriction fragment analysis is the only method currently available for measuring telomere length in Caenorhabditis elegans. Its limitations include low sensitivity and interference by the presence of interstitial telomeric sequences in the C.elegans genome. Here we report the adaptation of single telomere length analysis (STELA) to measure the length of telomeric repeats on the left arm of chromosome V in C.elegans. This highly sensitive PCR-based method allows telomere length measurement from as few as a single worm. The application of STELA to eight wild-type C.elegans strains revealed considerable strain-specific differences in telomere length. Within individual strains, short outlying telomeres were observed that were clearly distinct from the bulk telomere length distributions, suggesting that processes other than end-replication losses and telomerase-mediated lengthening may generate telomere length heterogeneity in C.elegans. The utility of this method was further demonstrated by the characterization of telomere shortening in mrt-2 mutants. We conclude that STELA appears to be a valuable tool for studying telomere biology in C.elegans.     

Blood cell telomere length is a dynamic feature

There is a considerable heterogeneity in blood cell telomere length (TL) for individuals of similar age and recent studies have revealed that TL changes by time are dependent on TL at baseline. TL is partly inherited, but results from several studies indicate that e.g. life style and/or environmental factors can affect TL during life. Collectively, these studies imply that blood cell TL might fluctuate during a life time and that the actual TL at a defined time point is the result of potential regulatory mechanism(s) and environmental factors. We analyzed relative TL (RTL) in subsequent blood samples taken six months apart from 50 individuals and found significant associations between RTL changes and RTL at baseline. Individual RTL changes per month were more pronounced than the changes recorded in a previously studied population analyzed after 10 years' follow up. The data argues for an oscillating TL pattern which levels out at longer follow up times. In a separate group of five blood donors, a marked telomere loss was demonstrated within a six month period for one donor where after TL was stabilized. PCR determined RTL changes were verified by Southern blotting and STELA (single telomere elongation length analysis). The STELA demonstrated that for the donor with a marked telomere loss, the heterogeneity of the telomere distribution decreased considerably, with a noteworthy loss of the largest telomeres. In summary, the collected data support the concept that individual blood cell telomere length is a dynamic feature and this will be important to recognize in future studies of human telomere biology.     

The relative lengths of individual telomeres are defined in the zygote and strictly maintained during life

Previous studies have indicated that average telomere length is partly inherited ( Slagboom et al., 1994 ; Rufer et al., 1999 ) and that there is an inherited telomere pattern in each cell ( Graakjaer et al., 2003 ); ( Londoño‐Vallejo et al., 2001 ). In this study, we quantify the importance of the initially inherited telomere lengths within cells, in relation to other factors that influence telomere length during life. We have estimated the inheritance by measuring telomere length in monozygotic (MZ) twins using Q‐FISH with a telomere specific peptide nucleic acid (PNA)‐probe. Homologous chromosomes were identified using subtelomeric polymorphic markers. We found that identical homologous telomeres from two aged MZ twins show significantly less differences in relative telomere length than when comparing the two homologues within one individual. This result means that towards the end of life, individual telomeres retain the characteristic relative length they had at the outset of life and that any length alteration during the lifespan impacts equally on genetically identical homologues. As the result applies across independent individuals, we conclude that, at least in lymphocytes, epigenetic/environmental effects on relative telomere length are relatively minor during life.     

The load of short telomeres,estimated by a new method,Universal STELA,correlates with number of senescent cells

Short telomeres are thought to trigger senescence, most likely through a single – or a group of few – critically shortened telomeres. Such short telomeres are thought to result from a combination of gradual linear shortening resulting from the end replication problem, reflecting the division history of the cell, superimposed by a more stochastic mechanism, suddenly causing a significant shortening of a single telomere. Previously, studies that have tried to explore the role of critically shortened telomeres have been hampered by methodological problems. With the method presented here, Universal STELA, we have a tool that can directly investigate the relationship between senescence and the load of short telomeres. The method is a variant of the chromosome‐specific STELA method but has the advantage that it can demonstrate short telomeres regardless of chromosome. With Universal STELA, we find a strong correlation between the load of short telomeres and cellular senescence. Further we show that the load of short telomeres is higher in senescent cells compared to proliferating cells at the same passage, offering an explanation of premature cell senescence. This new method, Universal STELA, offers some advantages compared to existing methods and can be used to explore many of the unanswered questions in telomere biology including the role that telomeres play in cancer and aging.     

Telomere length and dynamics predict mortality in a wild longitudinal study

Explaining variation in life expectancy between individuals of the same age is fundamental to our understanding of population ecology and life history evolution. Variation in the length and rate of loss of the protective telomere chromosome caps has been linked to cellular lifespan. Yet, the extent to which telomere length and dynamics predict organismal lifespan in nature is still contentious. Using longitudinal samples taken from a closed population of Acrocephalus sechellensis (Seychelles warblers) studied for over 20 years, we describe the first study into life‐long adult telomere dynamics (1–17 years) and their relationship to mortality under natural conditions (n = 204 individuals). We show that telomeres shorten with increasing age and body mass, and that shorter telomeres and greater rates of telomere shortening predicted future mortality. Our results provide the first clear and unambiguous evidence of a relationship between telomere length and mortality in the wild, and substantiate the prediction that telomere length and shortening rate can act as an indicator of biological age further to chronological age when exploring life history questions in natural conditions.     

Short telomeres in short‐lived males: what are the molecular and evolutionary causes?

Telomere length regulation is an important aspect of cell maintenance in eukaryotes, since shortened telomeres can lead to a number of defects, including impaired cell division. Although telomere length is correlated with lifespan in some bird species, its possible role in aging and lifespan determination is still poorly understood. Here we investigate telomere dynamics (changes in telomere length and attrition rate) and telomerase activity in the ant Lasius niger, a species in which different groups of individuals have evolved extraordinarily different lifespans. We found that somatic tissues of the short-lived males had dramatically shorter telomeres than those of the much longer-lived queens and workers. These differences were established early during larval development, most likely through faster telomere shortening in males compared with females. Workers did not, however, have shorter telomeres than the longer-lived queens. We discuss various molecular mechanisms that are likely to cause the observed sex-specific telomere dynamics in ants, including cell division, oxidative stress and telomerase activity. In addition, we discuss the evolutionary causes of such patterns in ants and in other species.     

The effects of social status on biological aging as measured by white-blood-cell telomere length

Low socio-economic status (SES) is associated with a shortened life expectancy, but its effect on aging is unknown. The rate of white-blood-cell (WBC) telomere attrition may be a biological indicator of human aging. We tested the hypothesis that SES is associated with telomere attrition independent of known risk factors influencing the aging process. We studied 1552 female twins. A venous blood sample was taken from each twin and isolated WBCs used for extraction of DNA. Terminal restriction fragment length (TRFL) was measured. Questionnaire data were collected on occupation, education, income, smoking, exercise, height and weight. Standard multiple linear regression and multivariate analyses of variance tested for associations between SES and TRFL, adjusting for covariates. A discordant twin analysis was conducted on a subset to verify findings. WBC telomere length was highly variable but significantly shorter in lower SES groups. The mean difference in TRFL between nonmanual and manual SES groups was 163.2 base pairs (bp) of which 22.9 bp (approximately 14%) was accounted for by body mass index, smoking and exercise. Comparison of TRFL in the 17 most discordant SES twin pairs confirmed this difference. Low SES, in addition to the harmful effects of smoking, obesity and lack of exercise, appears to have an impact on telomere length.     

Telomere shortening and survival in free-living corvids

Evidence accumulates that telomere shortening reflects lifestyle and predicts remaining lifespan, but little is known of telomere dynamics and their relation to survival under natural conditions. We present longitudinal telomere data in free-living jackdaws (Corvus monedula) and test hypotheses on telomere shortening and survival. Telomeres in erythrocytes were measured using pulsed-field gel electrophoresis. Telomere shortening rates within individuals were twice as high as the population level slope, demonstrating that individuals with short telomeres are less likely to survive. Further analysis showed that shortening rate in particular predicted survival, because telomere shortening was much accelerated during a bird''s last year in the colony. Telomere shortening was also faster early in life, even after growth was completed. It was previously shown that the lengths of the shortest telomeres best predict cellular senescence, suggesting that shorter telomeres should be better protected. We test the latter hypothesis and show that, within individuals, long telomeres shorten faster than short telomeres in adults and nestlings, a result not previously shown in vivo. Moreover, survival selection in adults was most conspicuous on relatively long telomeres. In conclusion, our longitudinal data indicate that the shortening rate of long telomeres may be a measure of ‘life stress’ and hence holds promise as a biomarker of remaining lifespan.     

The distribution pattern of critically short telomeres in human osteoarthritic knees

Introduction  

Telomere shortening is associated with a number of common age-related diseases. A role of telomere shortening in osteoarthritis (OA) has been suggested, mainly based on the assessment of mean telomere length in ex vivo expanded chondrocytes. We addressed this role directly in vivo by using a newly developed assay, which measures specifically the load of ultra-short single telomeres (below 1,500 base pairs), that is, the telomere subpopulation believed to promote cellular senescence.     

Telomere length in male germ cells is inversely correlated with telomerase activity

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.     

Telomere length measurement-caveats and a critical assessment of the available technologies and tools

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.     

Work-related exhaustion and telomere length: a population-based study

Background

Psychological stress is suggested to accelerate the rate of biological aging. We investigated whether work-related exhaustion, an indicator of prolonged work stress, is associated with accelerated biological aging, as indicated by shorter leukocyte telomeres, that is, the DNA-protein complexes that cap chromosomal ends in cells.

Methods

We used data from a representative sample of the Finnish working-age population, the Health 2000 Study. Our sample consisted of 2911 men and women aged 30–64. Work-related exhaustion was assessed using the Maslach Burnout Inventory - General Survey. We determined relative leukocyte telomere length using a quantitative real-time polymerase chain reaction (PCR) -based method.

Results

After adjustment for age and sex, individuals with severe exhaustion had leukocyte telomeres on average 0.043 relative units shorter (standard error of the mean 0.016) than those with no exhaustion (p = 0.009). The association between exhaustion and relative telomere length remained significant after additional adjustment for marital and socioeconomic status, smoking, body mass index, and morbidities (adjusted difference 0.044 relative units, standard error of the mean 0.017, p = 0.008).

Conclusions

These data suggest that work-related exhaustion is related to the acceleration of the rate of biological aging. This hypothesis awaits confirmation in a prospective study measuring changes in relative telomere length over time.     

Telomere length in fibroblasts and blood cells from healthy centenarians

Several lines of evidence indicate that telomere shortening during in vitro aging of human somatic cells plays a causal role in cellular senescence. A critical telomere length seems to be associated with the replicative block characterizing senescent cells. In this paper we analyzed the mean length of the terminal restriction fragments (TRF) in fibroblast strains from 4 healthy centenarians, that is, in cells aged in vivo, and from 11 individuals of different ages. No correlation between mean TRF length and donor age was found. As expected, telomere shortening was detected during in vitro propagation of centenarian fibroblasts, suggesting that in fibroblasts aged in vivo telomeres can be far from reaching a critical length. Accordingly, chromosome analysis did not show the presence of telomeric associations in early passage centenarian fibroblasts. In blood cells from various individuals, the expected inverse correlation between mean TRF length and donor age was found. In particular, a substantial difference (about 2 kb) between telomere length in the two cell types was observed in the same centenarian. Expression analysis of three senescence-induced genes, i.e., fibronectin, apolipoprotein J, and p21, revealed for only the fibronectin expression levels a clear positive correlation with donor age. Our results suggest that (1) telomere shortening could play a different role in the aging of different cell types and (2) the characteristics of fibroblasts aged in vitro might not be representative of what occurs in vivo.     

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.     

Short Sleep Duration Is Associated with Shorter Telomere Length in Healthy Men: Findings from the Whitehall II Cohort Study

Background

Shorter telomere length and poor sleep are more prevalent at older ages, but their relationship is uncertain. This study explored associations between sleep duration and telomere length in a sample of healthy middle and early old age people.

Methods

Participants were 434 men and women aged 63.3 years on average drawn from the Whitehall II cohort study. Sleep duration was measured by self-report.

Results

There was a linear association between sleep duration and leukocyte telomere length in men but not in women (P = 0.035). Men reporting shorter sleep duration had shorter telomeres, independently of age, body mass index, smoking, educational attainment, current employment, cynical hostility scores and depressive symptoms. Telomeres were on average 6% shorter in men sleeping 5 hours or fewer compared with those sleeping more than 7 hours per night.

Conclusion

This study adds to the growing literature relating sleep duration with biomarkers of aging, and suggests that shortening of telomeres might reflect mechanisms through which short sleep contributes to pathological conditions in older men.     

Telomere lengths at birth in trisomies 18 and 21 measured by Q-FISH

Trisomies 18 and 21 are genetic disorders in which cells possess an extra copy of each of the relevant chromosomes. Individuals with these disorders who survive birth generally have a shortened life expectancy. As telomeres are known to play an important role in the maintenance of genomic integrity by protecting the chromosomal ends, we conducted a study to determine whether there are differences in telomere length at birth between individuals with trisomy and diploidy, and between trisomic chromosomes and normal chromosomes. We examined samples of peripheral blood lymphocytes (PBLs) from 31 live neonates (diploidy: 10, trisomy 18: 10, trisomy 21: 11) and estimated the telomere length of each chromosome arm using Q-FISH. We observed that the telomeres of trisomic chromosomes were neither shorter nor longer than the mean telomere length of chromosomes as a whole among subjects with trisomies 18 and 21 (intra-cell comparison), and we were unable to conclude that there were differences in telomere length between 18 trisomy and diploid subjects, or between 21 trisomy and diploid subjects (inter-individual comparison). Although it has been reported that telomeres are shorter in older individuals with trisomy 21 and show accelerated telomere shortening with age, our data suggest that patients with trisomies 18 and 21 may have comparably sized telomeres. Therefore, it would be advisable for them to avoid lifestyle habits and characteristics such as obesity, cigarette smoking, chronic stress, and alcohol intake, which lead to marked telomere shortening.