Telomere length behaves as biomarker of somatic redundancy rather than biological age

Biomarkers of aging are essential to predict mortality and aging‐related diseases. Paradoxically, age itself imposes a limitation on the use of known biomarkers of aging because their associations with mortality generally diminish with age. How this pattern arises is, however, not understood. With meta‐analysis we show that human leucocyte telomere length (TL) predicts mortality, and that this mortality association diminishes with age, as found for other biomarkers of aging. Subsequently, we demonstrate with simulation models that this observation cannot be reconciled with the popular hypothesis that TL is proportional to biological age. Using the reliability theory of aging, we instead propose that TL is a biomarker of somatic redundancy, the body's capacity to absorb damage, which fits the observed pattern well. We discuss to what extent diminishing redundancy with age may also explain the observed diminishing mortality modulation with age of other biomarkers of aging. Considering diminishing somatic redundancy as the causal agent of aging may critically advance our understanding of the aging process, and improve predictions of life expectancy and vulnerability to aging‐related diseases.     

Telomere length regulation in budding yeasts

Telomeres are the nucleoprotein caps of chromosomes. Their length must be tightly regulated in order to maintain the stability of the genome. This is achieved by the intricate network of interactions between different proteins and protein–RNA complexes. Different organisms use various mechanisms for telomere length homeostasis. However, details of these mechanisms are not yet completely understood. In this review we have summarized our latest achievements in the understanding of telomere length regulation in budding yeasts.     

Telomere length provides a new technique for aging animals

Field biologists often work with animals for which there is no prior history. A marker of an animal's age would offer insight into how age and experience affect reproductive success and other life history parameters. Telomere length shortens with age in cultured cells and mouse and human tissues. We found that lengths of telomere restriction fragments cleaved from blood cell DNA shorten predictably with age in the zebra finch (Taeniopygia guttata). If this relationship holds in other species, it should be possible, once the relationship between telomere length and age has been determined for a given species, to use blood samples to estimate ages of free-living animals. This will allow the incorporation of age into estimates of factors affecting life history parameters in cases where previous histories of animals are unknown.     

Telomere length maintenance in stem cell populations

The maintenance of telomere length is essential for upholding the integrity of the genome. There is good evidence to suggest that telomere length maintenance in stem cell populations is important to facilitate the cell division required for tissue homeostasis. This is balanced against the requirement in long lived species for proliferative life span barriers for tumour suppression; the gradual erosion of telomeres provides one such barrier. The dynamics of telomeres in stem cell populations may thus be crucial in the balance between tumour suppression and tissue homeostasis. Here we briefly discuss our current understanding of telomere dynamics in stem cell populations, and provide some data to indicate that telomeres in human embryonic stem cells may be more stable and less prone to large-scale stochastic telomeric deletion.     

Telomere length and age in pinnipeds: The endangered Australian sea lion as a case study

Telomeres are the protective caps at the ends of all eukaryotic chromosomes. Because DNA replication of chromosome ends is incomplete, telomeres undergo sequence loss with each cell division resulting in the progressive shortening of their lengths. Telomere shortening with age is known from terrestrial mammals. We test whether this pattern is shared by marine mammals, by comparing telomere lengths between age classes in a pinniped species, the Australian sea lion (Neophoca cinerea). Telomere lengths were measured using a real‐time quantitative polymerase chain reaction (PCR) method in specimens from three age classes: pup (<1.5 yr), juvenile (1.5–5 yr), and adult (>5 yr). Mean telomere lengths of the adults were significantly shorter than the juvenile and pup classes. However, we were unable to differentiate between pups and juveniles. These findings confirm that the Australian sea lion shares the general pattern of shortening telomere lengths with age as documented in terrestrial mammals. The application of telomere lengths as an age determinant requires considerable development to refine the scale of the age estimates derived, which will require the use of known‐aged individuals. Nonetheless, measures of telomere lengths have the potential to become valuable tools in molecular ecology and forensics for assessing compliance in harvesting situations.     

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy

Recent progress in cytogenetic and biochemical mutation assay technologies has enabled us to detect single gene alterations and gross chromosomal rearrangements, and it became clear that all cancer cells are genetically unstable. In order to detect the genome-wide instability of cancer cells, a new simple method, the DNA-instability test, was developed. The methods to detect genomic instability so far reported have only demonstrated the presence of qualitative and quantitative alterations in certain specific genomic loci. In contrast to these commonly used methods to reveal the genomic instability at certain specific DNA regions, the newly introduced DNA-instability test revealed the presence of physical DNA-instability in the entire DNA molecule of a cancer cell nucleus as revealed by increased liability to denature upon HCl hydrolysis or formamide exposure. When this test was applied to borderline malignancies, cancer clones were detected in all cases at an early-stage of cancer progression. We proposed a new concept of "procancer" clones to define those cancer clones with "functional atypia" showing positivities for various cancer markers, as well as DNA-instability testing, but showing no remarkable ordinary "morphological atypia" which is commonly used as the basis of histopathological diagnosis of malignancy.     

Telomere length and risk of glioma

BackgroundTelomere length in blood or buccal cell DNA has been associated with risk of various cancers. Glioma can be a highly malignant brain tumor and has few known risk factors. Genetic variants in or near RTEL1 and TERT, key components of telomere biology, are associated with glioma risk. Therefore, we evaluated the association between relative telomere length (RTL) and glioma in a prospective study.Materials and methodsWe performed a nested case-control study within the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. RTL was determined by quantitative PCR on blood or buccal cell DNA obtained at least 2 years prior to diagnosis from 101 individuals with glioma cases. Healthy controls (n = 198) were matched to cases (2:1) on age, gender, smoking status, calendar year, and DNA source. Conditional logistic regression was used to investigate the association between RTL and glioma.ResultsAs expected, RTL declined with increasing age in both cases and controls. There was no statistically significant association between RTL and glioma overall. An analysis stratified by gender suggested that short RTL (1st tertile) in males was associated with glioma (odds ratio, [OR] = 2.29, 95% confidence interval [CI] 1.02–5.11); this association was not observed for females (OR = 0.41, 95% CI 0.14–1.17).ConclusionsThis prospective study did not identify significant associations between RTL and glioma risk, but there may be gender-specific differences. Larger, prospective studies are needed to evaluate these findings.     

Telomere length measurement in mouse chromosomes by a modified Q-FISH method

Telomeres are physical ends of mammalian chromosomes that dynamically change during the lifetime of a cell or organism. In order to understand mechanisms responsible for telomere dynamics, it is necessary to develop methods for accurate telomere length measurement. The most sensitive method for measuring telomere length in mouse chromosomes is quantitative fluorescence in situ hybridization (Q-FISH). The usual protocol for Q-FISH requires plasmids with variable numbers of telomeric repeats and fluorescence beads as calibration standards. Here, we describe a Q-FISH protocol in which two mouse lymphoma cell lines with well-defined telomere lengths are used as calibration standards. Using this protocol we demonstrate that reproducible results can be obtained in a set of four different mouse cell lines. This method can be adapted so that any pair of mammalian cell lines can serve as an internal calibration standard.     

Telomere length analysis in residents of a community exposed to arsenic

Differentiated cells telomere length is an indicator of senescence or lifespan; however, in peripheral blood leukocytes the relative shortening of the telomere has been considered as a biological marker of aging, and lengthening telomere as an associated risk to cancer. Individual’s age, type of tissue, lifestyle, and environmental factors make telomere length variable. The presence of environmental carcinogens such as arsenic (As) influence as causal agents of these alterations, the main modes of action for As described are oxidative stress, reduction in DNA repair capacity, overexpression of genes, alteration of telomerase activity, and damage to telomeres. The telomeres of leukocytes resulting a finite capacity of replication due to the low or no activity of the telomerase enzyme, therefore, elongation telomere in this kind of cells is a potential biological marker associated with the development of chronic diseases and carcinogenesis.     

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.     

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.     

Telomere length as a quantitative trait: genome-wide survey and genetic mapping of telomere length-control genes in yeast

Telomere length-variation in deletion strains of Saccharomyces cerevisiae was used to identify genes and pathways that regulate telomere length. We found 72 genes that when deleted confer short telomeres, and 80 genes that confer long telomeres relative to those of wild-type yeast. Among identified genes, 88 have not been previously implicated in telomere length control. Genes that regulate telomere length span a variety of functions that can be broadly separated into telomerase-dependent and telomerase-independent pathways. We also found 39 genes that have an important role in telomere maintenance or cell proliferation in the absence of telomerase, including genes that participate in deoxyribonucleotide biosynthesis, sister chromatid cohesion, and vacuolar protein sorting. Given the large number of loci identified, we investigated telomere lengths in 13 wild yeast strains and found substantial natural variation in telomere length among the isolates. Furthermore, we crossed a wild isolate to a laboratory strain and analyzed telomere length in 122 progeny. Genome-wide linkage analysis among these segregants revealed two loci that account for 30%–35% of telomere length-variation between the strains. These findings support a general model of telomere length-variation in outbred populations that results from polymorphisms at a large number of loci. Furthermore, our results laid the foundation for studying genetic determinants of telomere length-variation and their roles in human disease.     

Telomere rapid deletion regulates telomere length in Arabidopsis thaliana

Telomere length is maintained in species-specific equilibrium primarily through a competition between telomerase-mediated elongation and the loss of terminal DNA through the end-replication problem. Recombinational activities are also capable of both lengthening and shortening telomeres. Here we demonstrate that elongated telomeres in Arabidopsis Ku70 mutants reach a new length set point after three generations. Restoration of wild-type Ku70 in these mutants leads to discrete telomere-shortening events consistent with telomere rapid deletion (TRD). These findings imply that the longer telomere length set point is achieved through competition between overactive telomerase and TRD. Surprisingly, in the absence of telomerase, a subset of elongated telomeres was further lengthened, suggesting that in this background a mechanism of telomerase-independent lengthening of telomeres operates. Unexpectedly, we also found that plants possessing wild-type-length telomeres exhibit TRD when telomerase is inactivated. TRD is stochastic, and all chromosome ends appear to be equally susceptible. The frequency of TRD decreases as telomeres shorten; telomeres less than 2 kb in length are rarely subject to TRD. We conclude that TRD functions as a potent force to regulate telomere length in Arabidopsis.     

Telomere length measurement by a novel monochrome multiplex quantitative PCR method

The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R² = 0.844) than with our original singleplex method (R² = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R² = 0.91).     

Experimental study of umbilical cord length as a marker of fetal alcohol syndrome

Umbilical cord length has long been investigated as a potential marker of intrauterine events that may place the neonate at risk for future adverse developmental sequelae. Experimentally, significantly shortened cords have been reported in association with prenatal exposure to common drugs of abuse. This study in rats reports the time course of effects on umbilical cord length of a daily maternal ethanol gavage (3,200 mg/kg) from gestational day 6 through termination of pregnancy at either day 17, 18, 19, or 20. A total of 786 fetuses derived from 60 litters were examined. Control fetuses demonstrated a linear increase in umbilical cord length and body weight gain during late gestation, findings that support previous studies. The body weights of the ethanol-exposed fetuses were reduced significantly on all gestational days examined, indicating intrauterine growth retardation, a characteristic of fetal alcohol syndrome. Similarly, acute fetal akinesia as well as long-term sequelae stemming from impaired neurological development would result from the elevated blood ethanol levels achieved in this study. The umbilical cords of ethanol-exposed fetuses were significantly shorter on gestational days 19 and 20 in comparison to their controls, while cord lengths on days 17 and 18 were not shortened significantly. A stretch hypothesis has been proposed suggesting that the degree of fetal activity is the main determinant of umbilical cord length. In rats, there is a physiologic diminution of the volume of amniotic fluid (oligohydramnios) in late gestation (day 19 to term), which restricts fetal movements but does not appear to alter the linear relationships between gestational age and cord length in controls, thus arguing against the stretch hypothesis. However, cord lengths in the ethanol-exposed fetuses plateaued in late gestation, suggesting possible adherence to a stretch hypothesis. This dichotomy is discussed emphasizing fetal growth and activity as well as intrauterine space.