Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.     

Stochastic mechanism of cellular aging--abrupt telomere shortening as a model for stochastic nature of cellular aging

A strong stochastic component has been described for the appearance of senescent cells in cultures that have not completed their in vitro lifespan. The proliferative potential of individual clones show a bimodal distribution. Additionally, two cells arising from a single mitotic event can exhibit large differences in their doubling capacities. In this report we present a model and a computer simulation of the model that explains the observed stochastic phenomena. The model is based on both gradual and abrupt telomere shortening.Gradual telomere shortening (GTS) occurs during each cell division as a consequence of the inability of DNA polymerase to replicate the very ends of chromosomal DNA. It is responsible for the gradual decline in proliferative potential of a cell culture, but does not explain the stochastic aspects of cellular aging. Abrupt telomere shortening (ATS) occurs either through DNA recombination or nuclease digestion at the subtelomeric/telomeric border region of the chromosome. Recombination involves the invasion of a telomere single-strand three-prime protruding end at this border in the telomere of the same chromosome or in another subtelomeric/telomeric region. Shortening of one or more telomeres in the cell causes a sudden onset of cell senescence, referred to as sudden senescence syndrome (SSS). This is manifested as a stochastic and abrupt transition of cells from the larger to the smaller proliferative potential pool and can cause cell cycle arrest within one cell division. The computer simulation matches well with experimental data supporting the prediction that abrupt telomere shortening underlies the stochastic onset of cell senescence. Sudden senescence syndrome appears to be the most important mechanism in the control of the extent of proliferation of a cell culture because it prevents virtually every cell in the culture from reaching its maximum doubling capacity, that would otherwise be allowed by telomere shortening via the end-replication mechanism alone.     

Decreasing initial telomere length in humans intergenerationally understates age‐associated telomere shortening

Telomere length shortens with aging, and short telomeres have been linked to a wide variety of pathologies. Previous studies suggested a discrepancy in age‐associated telomere shortening rate estimated by cross‐sectional studies versus the rate measured in longitudinal studies, indicating a potential bias in cross‐sectional estimates. Intergenerational changes in initial telomere length, such as that predicted by the previously described effect of a father's age at birth of his offspring (FAB), could explain the discrepancy in shortening rate measurements. We evaluated whether changes occur in initial telomere length over multiple generations in three large datasets and identified paternal birth year (PBY) as a variable that reconciles the difference between longitudinal and cross‐sectional measurements. We also clarify the association between FAB and offspring telomere length, demonstrating that this effect is substantially larger than reported in the past. These results indicate the presence of a downward secular trend in telomere length at birth over generational time with potential public health implications.     

Renal failure induces telomere shortening in the rat heart

BackgroundRenal failure aggravates pathological cardiac remodelling induced by myocardial infarction (MI). Cardiac remodelling is associated with telomere shortening, a marker for biological ageing. We investigated whether mild and severe renal failure shorten cardiac telomeres and excessively shorten telomeres after MI. MethodsRats were subjected to sham, unilateral (UNX) or 5/6th nephrectomy (5/6NX) to induce none, mild or severe renal failure. MI was induced by left coronary artery ligation. Renal function parameters and blood pressure were measured. DNA was isolated from non-infarcted cardiac tissue. Telomere length was assessed by quantitative polymerase chain reaction (PCR). ResultsProteinuria was unchanged in UNX and MI compared with control, but strongly increased in 5/6NX, UNX+MI and 5/6NX+MI. Serum creatinine levels were increased fourfold in 5/6NX and tenfold in 5/6NX+MI. 5/6NX and groups with both renal failure and MI showed an approximate 20% reduction of telomere length, similar to the MI group. No excess telomere shortening was observed in hearts from rats with renal ablation after MI. ConclusionSevere renal failure, but not mild renal failure, leads to shortening of cardiac telomeres to a similar extent as found after MI. Renal failure did not induce excessive telomere shortening after MI. (Neth Heart J 2009;17:190–4.)     

Accelerated telomere shortening in the human inactive X chromosome

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.     

Accelerated telomere shortening in Fanconi anemia fibroblasts--a longitudinal study

Fanconi anemia (FA) is a fatal inherited disease displaying chromosomal instability, disturbances in oxygen metabolism and a high burden of intracellular radical oxygen species. Oxygen radicals can damage DNA including telomeric regions. Insufficient repair results in single strand breaks that can induce accelerated telomere shortening. In a longitudinal study we demonstrate that telomeric DNA is continuously lost at a higher rate in FA fibroblasts compared to healthy controls. Furthermore, we show that this loss is caused rather by an increased shortening per cell division in regularly replicating cells than by apoptosis.     

A kinetic model of telomere shortening in infants and adults

We have previously demonstrated that telomeres shorten more rapidly in peripheral mononuclear cells (PBMC) of infants than in adults (Zeichner et al., Blood 93 (1999) 2824). Here we describe a mathematical model that allows quantification of telomere dynamics both in infants and in adults. In this model the dependence of the telomere dynamics on age is accounted by assuming proportionality between the body growth, as approximated by the Gompertz equation, and the increase in the number of PBMCs. The model also assumes the existence of two subpopulations of PBMC with significantly different rates of division. This assumption is based on the results from a previous analysis of in vitro data for telomere dynamics in presence of telomerase inhibitors and our recent data obtained by measurements of BrdU incorporation in T lymphocytes in humans (Kovacs et al., J. Exp. Med. 194 (2001) 1731). The average telomere length of PBMC was calculated as the average length of these two subpopulations. The model fitted our experimental data well and allowed to derive a characteristic time of conversion of the rapidly proliferating cells to slowly proliferating cells on the order of 20 days. The half-life of the slowly proliferating cells was estimated to be about 6 months, which is in good agreement with data obtained by independent methodologies. Comparison of the one-population and two-subpopulations models demonstrated that one population model cannot explain the observed parameters of the terminal restriction fragment (TRF) dynamics while two-subpopulations model does. These results suggest that the rapid telomere shortening in infants is largely determined by the faster PBMC turnover compared to adults. This may have major implications for elucidation of the HIV pathogenesis in infants. One can speculate that the more rapid course of the HIV disease in infants is due to the existence of rapidly dividing cells, which are susceptible to HIV infection. In addition, these results could have implications for understanding of mechanisms of aging.     

Divergent patterns of telomere shortening in tropical compared to temperate stonechats

Telomeres have emerged as important biomarkers of health and senescence as they predict chances of survival in various species. Tropical birds live in more benign environments with lower extrinsic mortality and higher juvenile and adult survival than temperate birds. Therefore, telomere biology may play a more important role in tropical compared to temperate birds. We measured mean telomere length of male stonechats (Saxicola spp.) at four age classes from tropical African and temperate European breeding regions. Tropical and temperate stonechats had similarly long telomeres as nestlings. However, while in tropical stonechats pre‐breeding first‐years had longer telomeres than nestlings, in temperate stonechats pre‐breeding first‐years had shorter telomeres than nestlings. During their first breeding season, telomere length was again similar between tropical and temperate stonechats. These patterns may indicate differential survival of high‐quality juveniles in tropical environments. Alternatively, more favorable environmental conditions, that is, extended parental care, may enable tropical juveniles to minimize telomere shortening. As suggested by previous studies, our results imply that variation in life history and life span may be reflected in different patterns of telomere shortening rather than telomere length. Our data provide first evidence that distinct selective pressures in tropical and temperate environments may be reflected in diverging patterns of telomere loss in birds.     

Lack of telomere shortening with age in mouse resting zone chondrocytes

BACKGROUND AND AIM: Telomeres are hexameric repeat sequences that flank eukaryotic chromosomes. The telomere hypothesis of cellular aging proposes that replication of normal somatic cells leads to progressive telomere shortening which induces replicative senescence. Previous studies suggest that growth plate chondrocytes have a finite proliferative capacity in vivo. We therefore hypothesized that telomere shortening in resting zone chondrocytes leads to replicative senescence. METHOD: To test this hypothesis we compared the telomere restriction fragment (TRF) length of Mus casteneus at 1, 4, 8, and 56 weeks of age. RESULTS AND CONCLUSIONS: We found that TRF length did not diminish measurably with age, suggesting that telomere shortening in resting zone chondrocytes is not the mechanism that limits proliferation of growth plate chondrocytes in vivo.     

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.     

L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts

Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. L-carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine.     

Lung alveolar integrity is compromised by telomere shortening in telomerase-null mice

Shortened telomeres are a normal consequence of cell division. However, telomere shortening past a critical point results in cellular senescence and death. To determine the effect of telomere shortening on lung, four generations of B6.Cg-Terc(tm1Rdp) mice, null for the terc component of telomerase, the holoenzyme that maintains telomeres, were bred and analyzed. Generational inbreeding of terc-/- mice caused sequential shortening of telomeres. Lung histology from the generation with the shortest telomeres (terc-/- F4) showed alveolar wall thinning and increased alveolar size. Morphometric analysis confirmed a significant increase in mean linear intercept (MLI). terc-/- F4 lung showed normal elastin deposition but had significantly decreased collagen content. Both airway and alveolar epithelial type 1 cells (AEC1) appeared normal by immunohistochemistry, and the percentage of alveolar epithelial type 2 cells (AEC2) per total cell number was similar to wild type. However, because of a decrease in distal lung cellularity, the absolute number of AEC2 in terc-/- F4 lung was significantly reduced. In contrast to wild type, terc-/- F4 distal lung epithelium from normoxia-maintained mice exhibited DNA damage by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) and 8-oxoguanine immunohistochemistry. Western blotting of freshly isolated AEC2 lysates for stress signaling kinases confirmed that the stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) stress response pathway is stimulated in telomerase-null AEC2 even under normoxic conditions. Expression of downstream apoptotic/stress markers, including caspase-3, caspase-6, Bax, and HSP-25, was also observed in telomerase-null, but not wild-type, AEC2. TUNEL analysis of freshly isolated normoxic AEC2 showed that DNA strand breaks, essentially absent in wild-type cells, increased with each successive terc-/- generation and correlated strongly with telomere length (R(2) = 0.9631). Thus lung alveolar integrity, particularly in the distal epithelial compartment, depends on proper telomere maintenance.     

Genetic anticipation is associated with telomere shortening in hereditary breast cancer

There is increasing evidence suggesting that short telomeres and subsequent genomic instability contribute to malignant transformation. Telomere shortening has been described as a mechanism to explain genetic anticipation in dyskeratosis congenita and Li-Fraumeni syndrome. Since genetic anticipation has been observed in familial breast cancer, we aimed to study telomere length in familial breast cancer patients and hypothesized that genetic defects causing this disease would affect telomere maintenance resulting in shortened telomeres. Here, we first investigated age anticipation in mother-daughter pairs with breast cancer in 623 breast cancer families, classified as BRCA1, BRCA2, and BRCAX. Moreover, we analyzed telomere length in DNA from peripheral blood leukocytes by quantitative PCR in a set of 198 hereditary breast cancer patients, and compared them with 267 control samples and 71 sporadic breast cancer patients. Changes in telomere length in mother-daughter pairs from breast cancer families and controls were also evaluated to address differences through generations. We demonstrated that short telomeres characterize hereditary but not sporadic breast cancer. We have defined a group of BRCAX families with short telomeres, suggesting that telomere maintenance genes might be susceptibility genes for breast cancer. Significantly, we described that progressive telomere shortening is associated with earlier onset of breast cancer in successive generations of affected families. Our results provide evidence that telomere shortening is associated with earlier age of cancer onset in successive generations, suggesting that it might be a mechanism of genetic anticipation in hereditary breast cancer.     

Nestling telomere shortening,but not telomere length,reflects developmental stress and predicts survival in wild birds

Developmental stressors often have long-term fitness consequences, but linking offspring traits to fitness prospects has remained a challenge. Telomere length predicts mortality in adult birds, and may provide a link between developmental conditions and fitness prospects. Here, we examine the effects of manipulated brood size on growth, telomere dynamics and post-fledging survival in free-living jackdaws. Nestlings in enlarged broods achieved lower mass and lost 21% more telomere repeats relative to nestlings in reduced broods, showing that developmental stress accelerates telomere shortening. Adult telomere length was positively correlated with their telomere length as nestling (r = 0.83). Thus, an advantage of long telomeres in nestlings is carried through to adulthood. Nestling telomere shortening predicted post-fledging survival and recruitment independent of manipulation and fledgling mass. This effect was strong, with a threefold difference in recruitment probability over the telomere shortening range. By contrast, absolute telomere length was neither affected by brood size manipulation nor related to survival. We conclude that telomere loss, but not absolute telomere length, links developmental conditions to subsequent survival and suggest that telomere shortening may provide a key to unravelling the physiological causes of developmental effects on fitness.     

Stochastic models of kleptoparasitism

In this paper, we consider a model of kleptoparasitism amongst a small group of individuals, where the state of the population is described by the distribution of its individuals over three specific types of behaviour (handling, searching for or fighting over, food). The model used is based upon earlier work which considered an equivalent deterministic model relating to large, effectively infinite, populations. We find explicit equations for the probability of the population being in each state. For any reasonably sized population, the number of possible states, and hence the number of equations, is large. These equations are used to find a set of equations for the means, variances, covariances and higher moments for the number of individuals performing each type of behaviour. Given the fixed population size, there are five moments of order one or two (two means, two variances and a covariance). A normal approximation is used to find a set of equations for these five principal moments. The results of our model are then analysed numerically, with the exact solutions, the normal approximation and the deterministic infinite population model compared. It is found that the original deterministic models approximate the stochastic model well in most situations, but that the normal approximations are better, proving to be good approximations to the exact distribution, which can greatly reduce computing time.     

Ageing and telomeres: a study into organ- and gender-specific telomere shortening

Telomeres, the non-coding sequences at the ends of chromosomes, in the absence of telomerase, progressively shorten with each cell division. Shortening of telomeres can induce cell cycle arrest and apoptosis. The aim of this study was to investigate age- and gender-related changes in telomere length in the rat and to detect possible tissue- specific rates of telomere shortening. Changes with age in telomere lengths were assessed by Southern blotting in the kidney, pancreas, liver, lung and brain of male and female rats. We determined the percentage of telomeres in various molecular size regions rather than measuring the average telomere length. The latter was unable to detect telomere shortening in the tissues. The percentage of short telomeres increased with age in the kidney, liver, pancreas and lung of both males and females, but not in the brain. Males had shorter telomeres than females in all organs analysed except the brain, where the lengths were similar. These findings indicate that telomeres shorten in the rat kidney, liver, pancreas and the lung in an age-dependent manner. These data also provide a novel mechanism for the gender-related differences in lifespan and suggest a tissue-specific regulation of telomere length during development and ageing in the rat.