Association of telomere instability with senescence of porcine cells

Telomeres are essential for the maintenance of genomic stability, and telomere dysfunction leads to cellular senescence, carcinogenesis, aging, and age-related diseases in humans. Pigs have become increasingly important large animal models for preclinical tests and study of human diseases, and also may provide xeno-transplantation sources. Thus far, Southern blot analysis has been used to estimate average telomere lengths in pigs. Telomere quantitative fluorescence in situ hybridization (Q-FISH), however, can reveal status of individual telomeres in fewer cells, in addition to quantifying relative telomere lengths, and has been commonly used for study of telomere function of mouse and human cells. We attempted to investigate telomere characteristics of porcine cells using telomere Q-FISH method. The average telomere lengths in porcine cells measured by Q-FISH correlated with those of quantitative real-time PCR method (qPCR) or telomere restriction fragments (TRFs) by Southern blot analysis. Unexpectedly, we found that porcine cells exhibited high incidence of telomere doublets revealed by Q-FISH method, coincided with increased frequency of cellular senescence. Also, telomeres shortened during subculture of various porcine primary cell types. Interestingly, the high frequency of porcine telomere doublets and telomere loss was associated with telomere dysfunction-induced foci (TIFs). The incidence of TIFs, telomere doublets and telomere loss increased with telomere shortening and cellular senescence during subculture. Q-FISH method using telomere PNA probe is particularly useful for characterization of porcine telomeres. Porcine cells exhibit high frequency of telomere instability and are susceptible to telomere damage and replicative senescence.     

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.     

Developmental stress and telomere dynamics in a genetically polymorphic species

A central objective of evolutionary biology is understanding variation in life‐history trajectories and the rate of aging, or senescence. Senescence can be affected by trade‐offs and behavioural strategies in adults but may also be affected by developmental stress. Developmental stress can accelerate telomere degradation, with long‐term longevity and fitness consequences. Little is known regarding whether variation in developmental stress and telomere dynamics contributes to patterns of senescence during adulthood. We investigated this question in the dimorphic white‐throated sparrow (Zonotrichia albicollis), a species in which adults of the two morphs exhibit established differences in behavioural strategy and patterns of senescence, and also evaluated the relationship between oxidative stress and telomere length. Tan morph females, which exhibit high levels of unassisted parental care, display faster reproductive senescence than white females, and faster actuarial senescence than all of the other morph–sex classes. We hypothesized that high oxidative stress and telomere attrition in tan female nestlings could contribute to this pattern, since tan females are small and potentially at a competitive disadvantage even as nestlings. Nestlings that were smaller than nest mates had higher oxidative stress, and nestlings with high oxidative stress and fast growth rates displayed shorter telomeres. However, we found no consistent morph–sex differences in oxidative stress or telomere length. Results suggest that oxidative stress and fast growth contribute to developmental telomere attrition, with potential ramifications for adults, but that developmental oxidative stress and telomere dynamics do not account for morph–sex differences in senescence during adulthood.     

The Length of the Shortest Telomere as the Major Determinant of the Onset of Replicative Senescence

The absence of telomerase in many eukaryotes leads to the gradual shortening of telomeres, causing replicative senescence. In humans, this proliferation barrier constitutes a tumor suppressor mechanism and may be involved in cellular aging. Yet the heterogeneity of the senescence phenotype has hindered the understanding of its onset. Here we investigated the regulation of telomere length and its control of senescence heterogeneity. Because the length of the shortest telomeres can potentially regulate cell fate, we focus on their dynamics in Saccharomyces cerevisiae. We developed a stochastic model of telomere dynamics built on the protein-counting model, where an increasing number of protein-bound telomeric repeats shift telomeres into a nonextendable state by telomerase. Using numerical simulations, we found that the length of the shortest telomere is well separated from the length of the others, suggesting a prominent role in triggering senescence. We evaluated this possibility using classical genetic analyses of tetrads, combined with a quantitative and sensitive assay for senescence. In contrast to mitosis of telomerase-negative cells, which produces two cells with identical senescence onset, meiosis is able to segregate a determinant of senescence onset among the telomerase-negative spores. The frequency of such segregation is in accordance with this determinant being the length of the shortest telomere. Taken together, our results substantiate the length of the shortest telomere as being the key genetic marker determining senescence onset in S. cerevisiae.     

Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However, the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.     

Telomeres, crisis and cancer

Eukaryotic chromosomes terminate in specialized nucleic acid-protein complexes known as telomeres. Disruption of telomere structure by erosion of telomeric DNA or loss of telomere binding protein function activates a signal transduction program that closely resembles the cellular responses generated upon DNA damage. Telomere dysfunction in turn induces a permanent proliferation arrest known as senescence. Senescence is postulated to perform a tumor suppressor function by limiting cellular proliferative capacity, thus imposing a barrier to cellular immortalization. Genetic or epigenetic silencing of components of the DNA damage pathway, allows cells to proliferate beyond senescence limits. However, these cells eventually reach a stage of extreme telomere dysfunction known as crisis that is characterized by cell death and the concomitant appearance of cytogenetic abnormalities. Telomeric crisis produces significant chromosomal instability, a hallmark of human cancer, and may thus be relevant to carcinogenesis by increasing the occurrence of genetic alterations that would favor neoplastic transformation. The following review examines the relationship of telomere function during crisis in accelerating chromosomal instability and cancer.     

Neutrophils induce paracrine telomere dysfunction and senescence in ROS‐dependent manner

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro‐inflammatory phenotype, thought to contribute to aging and age‐related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age‐related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non‐immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS‐dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil‐induced senescence may be beneficial during aging and age‐related disease.     

Role of telomere in endothelial dysfunction in atherosclerosis

PURPOSE OF REVIEW: Telomeres consist of repeats of G-rich sequence at the end of chromosomes. These DNA repeats are synthesized by enzymatic activity associated with an RNA protein complex called telomerase. In most somatic cells, telomerase activity is insufficient, and telomere length decreases with increasing cell division, resulting in an irreversible cell growth arrest, termed cellular senescence. Cellular senescence is associated with an array of phenotypic changes suggestive of aging. Until recently, cellular senescence has largely been studied as an in-vitro phenomenon; however, there is accumulating evidence that indicates a critical role of telomere function in the pathogenesis of human atherosclerosis. This review attempts to summarize recent work in vascular biology that supports the "telomere hypothesis". We discuss the possible relevance of telomere function to vascular aging and the therapeutic potential of telomere manipulation. RECENT FINDINGS: It has been reported that many of the changes in senescent vascular cell behavior are consistent with known changes seen in age-related vascular diseases. Introduction of telomere malfunction has been shown to lead to endothelial dysfunction that promotes atherogenesis, whereas telomere lengthening extends cell lifespan and protects against endothelial dysfunction associated with senescence. Indeed, recent studies have demonstrated that telomere attrition and cellular senescence occur in the blood vessels and are associated with human atherosclerosis. SUMMARY: Recent findings suggest that vascular cell senescence induced by telomere shortening may contribute to atherogenesis and may provide insights into a novel treatment of antisenescence to prevent atherosclerosis.     

Mating type influences chromosome loss and replicative senescence in telomerase-deficient budding yeast by Dnl4-dependent telomere fusion

As we age, the majority of our cells gradually lose the capacity to divide because of replicative senescence that results from the inability to replicate the ends of chromosomes. The timing of senescence is dependent on the length of telomeric DNA, which elicits a checkpoint signal when critically short. Critically short telomeres also become vulnerable to deleterious rearrangements, end-degradation and telomere–telomere fusions. Here we report a novel role of non-homologous end-joining (NHEJ), a pathway of double-strand break repair in influencing both the kinetics of replicative senescence and the rate of chromosome loss in telomerase-deficient Saccharomyces cerevisiae . In telomerase-deficient cells, the absence of NHEJ delays replicative senescence, decreases loss of viability during senescence, and suppresses senescence-associated chromosome loss and telomere–telomere fusion. Differences in mating-type gene expression in haploid and diploid cells affect NHEJ function, resulting in distinct kinetics of replicative senescence. These results suggest that the differences in the kinetics of replicative senescence in haploid and diploid telomerase-deficient yeast are determined by changes in NHEJ-dependent telomere fusion, perhaps through the initiation of the breakage-fusion-bridge cycle.     

Sexual signals reflect telomere dynamics in a wild bird

Telomere dynamics in natural populations have been linked to survival, reproduction, and energetic investment. Given their putative role in mediating life‐history trade‐offs, telomeres are also a likely candidate for maintaining honesty in sexually selected signals; few studies to date, however, have demonstrated a correlation between sexual signals and telomere dynamics. Here, we show that plumage coloration in male common yellowthroats (Geothlypis trichas) is correlated with both relative telomere length and with the rate of telomere loss between years. Elevated antioxidant capacity is also associated with reduced telomere loss, but only among older males. Previous work in this population has demonstrated that males with brighter plumage are in better condition, have higher reproductive success, and are more likely to survive over winter. Thus, the signal attribute associated with mate choice in this system also conveys reliable information about telomere dynamics. At present, it is unclear whether telomere maintenance plays a causal role in maintaining signal honesty or whether the correlation arises due to underlying variation in individual resources or genotypes. We suggest that subsequent work should consider the possibility that fundamental trade‐offs between signal investment and cell‐level processes that influence aging and reproductive senescence may provide a foundation for understanding the maintenance of sexual signal honesty.     

A mathematical model of cellular apoptosis and senescence through the dynamics of telomere loss

The shortening of telomeric repeats as a cell replicates has long been implicated as a determinant of cell viability. However, recent studies have indicated that it is not telomere length, but rather whether telomeres have bound a telomere-related protein, which in mammals is TTAGGG repeat binding factor-2 (TRF2), that determines whether a cell undergoes apoptosis (programmed cell death), enters senescence (a quiescent, non-replicative state), or continues to proliferate. When bound to a telomere, TRF2 allows a cell to recognize the telomere as the point where a chromosome ends rather than a break in DNA. When telomeres are not bound by TRF2, the cell can either immediately trigger senescence or apoptosis via the DNA damage response pathway, or indirectly trigger it by attempting to repair the chromosome, which results in chromosomal end joining. We model the ability of telomeres to bind TRF2 as a function of telomere length and apply the resulting binding probability to a model of cellular replication that assumes a homogeneous cell population. The model fits data from cultured human fibroblasts and human embryonic kidney cells for two free parameters well. We extract values for the percent of telomere loss at which cell proliferation ceases. We show, in agreement with previous experiments, that overexpression of TRF2 allows a cell to delay the senescence setpoint. We explore the effect of oxidative stress, which increases the rate of telomere loss, on cell viability and show that cells in the presence of oxidative stress have reduced lifespans. We also show that the addition of telomerase, an enzyme that maintains telomere length, is sufficient to result in cell immortality. We conclude that the increasing inability of TRF2 to bind telomeres as they shorten is a quantitatively reasonable model for a cause of either cellular apoptosis or senescence.     

Mitochondrial dysfunction leads to telomere attrition and genomic instability

Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging-associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging-associated pathologies.     

Chromosome positional effects of gene expressions after cellular senescence

Normal human fibroblasts stop dividing after a limited number of cell divisions termed cellular senescence. Telomere shortening has been shown to be the main factor that causes cellular senescence, however, the molecular mechanism of how telomere shortening causes cellular senescence is unclear. Here we analyze the relationship between gene expressions and their chromosomal locations during cellular senescence. It appears that the expression of genes located in chromosome 4 is preferentially altered after senescence. Moreover, we identify four chromosomal loci in which gene expressions are affected by senescence. Finally, we show that there is no preferential alteration of telomere-proximal genes during cellular senescence, implying that cellular senescence is not caused by derepression of telomere-proximal genes.     

Proliferation dynamics in cultured skin fibroblasts from Down syndrome subjects

With a view to better understanding the role of oxidant/antioxidant variables in proliferation dynamics of somatic cells, we explored the relationships among superoxide dismutase (SOD) activity, glutathione peroxidase (Gpx) activity, reactive oxygen intermediates (ROI), and indices of cellular proliferation and senescence in cultured fibroblasts from Down syndrome and normal donors. We found that Down syndrome cells had a significantly slower proliferative rate, but attain replicative senescence at similar population doubling (PD) as control cells. Irrespective of donor origin, the number of PD until replicative senescence was positively correlated with Gpx activity (r = 0.784, P = 0.007). In addition, the presence of exogenous catalase in the growth medium significantly extended the number of PD until replicative senescence (P = 0.011). The loss of telomere repeats per PD was not different between Down syndrome cells and controls. However, SOD activity was inversely correlated with the loss of telomere repeats per PD. Collectively, these findings suggest that replicative senescence ultimately relates to mechanisms downstream to SOD (i.e., Gpx and catalase) and confirmed previous observations about inverse relationships between SOD activity and telomere repeat loss per cellular replication.     

Uncoupling of longevity and telomere length in C. elegans

The nematode Caenorhabditis elegans, after completing its developmental stages and a brief reproductive period, spends the remainder of its adult life as an organism consisting exclusively of post-mitotic cells. Here we show that telomere length varies considerably in clonal populations of wild-type worms, and that these length differences are conserved over at least ten generations, suggesting a length regulation mechanism in cis. This observation is strengthened by the finding that the bulk telomere length in different worm strains varies considerably. Despite the close correlation of telomere length and clonal cellular senescence in mammalian cells, nematodes with long telomeres were neither long lived, nor did worm populations with comparably short telomeres exhibit a shorter life span. Conversely, long-lived daf-2 and short-lived daf-16 mutant animals can have either long or short telomeres. Telomere length of post-mitotic cells did not change during the aging process, and the response of animals to stress was found independent of telomere length. Collectively, our data indicate that telomere length and life span can be uncoupled in a post-mitotic setting, suggesting separate pathways for replication-dependent and -independent aging.     

Maize grain concentrations and above-ground shoot acquisition of micronutrients as affected by intercropping with turnip, faba bean, chickpea, and soybean

Most research on micronutrients in maize has focused on maize grown as a monocrop. The aim of this study was to determine the effects of intercropping on the concentrations of micronutrients in maize grain and their acquisition via the shoot. We conducted field experiments to investigate the effects of intercropping with turnip (Brassica campestris L.), faba bean (Vicia faba L.), chickpea (Cicer arietinum L.), and soybean (Glycine max L.) on the iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) concentrations in the grain and their acquisition via the above-ground shoots of maize (Zea mays L.). Compared with monocropped maize grain, the grain of maize intercropped with legumes showed lower concentrations of Fe, Mn, Cu, and Zn and lower values of their corresponding harvest indexes. The micronutrient concentrations and harvest indexes in grain of maize intercropped with turnip were the same as those in monocropped maize grain. Intercropping stimulated the above-ground maize shoot acquisition of Fe, Mn, Cu and Zn, when averaged over different phosphorus (P) application rates. To our knowledge, this is the first report on the effects of intercropping on micronutrient concentrations in maize grain and on micronutrients acquisition via maize shoots (straw+grain). The maize grain Fe and Cu concentrations, but not Mn and Zn concentrations, were negatively correlated with maize grain yields. The concentrations of Fe, Mn, Cu, and Zn in maize grain were positively correlated with their corresponding harvest indexes. The decreased Fe, Mn, Cu, and Zn concentrations in grain of maize intercropped with legumes were attributed to reduced translocation of Fe, Mn, Cu, and Zn from vegetative tissues to grains. This may also be related to the delayed senescence of maize plants intercropped with legumes. We conclude that turnip/maize intercropping is beneficial to obtain high maize grain yield without decreased concentrations of Fe, Mn, Cu, and Zn in the grain. Further research is required to clarify the mechanisms underlying the changes in micronutrient concentrations in grain of intercropped maize.     

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.