Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.     

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.     

Telomere loss: mitotic clock or genetic time bomb?

The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.     

DNA氧化性损伤与端粒缩短

末端复制问题（the end replication problem）不能完全解释端粒在某些细胞分裂过程中迅速缩短的现象.40％的高压氧下细胞传代次数降低,端粒缩短速率增大,细胞出现衰老特征,端粒DNA上单链断裂积累.推测端粒缩短的主要原因在于衰老过程中或氧胁迫下端粒DNA单链断裂增多,使端粒末端单链片段在DNA复制时丢失.端粒酶和活性氧对端粒长度的正负调控作用的准确机制还有待于更深入的研究.     

Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts

Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization. © 1996 Wiley-Liss, Inc.     

DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence.     

Diminished Telomeric 3′ Overhangs Are Associated with Telomere Dysfunction in Hoyeraal-Hreidarsson Syndrome

Background

Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency.

Methodology/Principal Findings

We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3′ overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types.

Conclusions/Significance

Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease.     

Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells

One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.     

Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress

Telomerase inhibition may be a novel anti-cancer strategy that can be used in combination with conventional therapies, such as DNA damaging agents. There are conflicting reports as to whether and to what extent telomerase and telomere length influence the sensitivity of cells to genotoxins. To understand the relationship between telomere length, telomerase expression, and sensitivity to genotoxic stress, we expressed the catalytic subunit of telomerase, hTERT, in human fibroblasts having different telomere lengths. We show that telomerase confers resistance to ionizing radiation, bleomycin, hydrogen peroxide, and etoposide only in cells with short, presumably near-dysfunctional, telomeres. This resistance depended on the ability of telomerase to elongate the short telomeres, and telomerase did not protect cells with long telomeres. Interestingly, although long telomeres had no effect on sensitivity to etoposide and bleomycin, they exacerbated sensitivity to hydrogen peroxide, supporting the idea that, compared to other types of DNA damage, telomeres are particularly vulnerable to oxidative damage. Our findings identify a mechanism and conditions under which telomerase and telomeres affect the response of human cells to genotoxic agents and may have important implications for anti-cancer interventions.     

Identification of telomere-dependent "senescence-like" arrest in mouse embryonic fibroblasts

In contrast to human primary cells, mouse embryonic fibroblasts (MEF) do not show telomere shortening-mediated replicative senescence due to the fact that they have telomerase activity and show sufficiently long telomeres. Instead, it is now generally accepted that the "senescence-like" arrest that occurs in MEF after 5-10 divisions in culture is mediated by telomere-length-independent mechanisms generally referred to as stress. Using telomerase-deficient MEF Terc(-/-), we show here that telomere shortening to a critical length leads to a premature senescence-like arrest in MEF, as well as has a negative effect on spontaneous immortalization. Similarly, elimination of the telomere end-capping protein Ku86 also leads to a premature senescence-like arrest and has a negative effect on spontaneous immortalization. Both Terc(-/-) MEF with short telomeres and Ku86(-/-) MEF show dysfunctional telomeres, as indicated by similarly increased frequencies of end-to-end fusions. These results suggest that loss of telomere function is a general mechanism leading to cell arrest. These observations also indicate that telomere dysfunction is interfering with successful cell division and thus interferes with tumor formation. In summary, we have identified here two different ways to induce a telomere-dependent senescence-like arrest in MEF.     

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.     

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

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

Phosphorylation of H2AX at short telomeres in T cells and fibroblasts

Eukaryotic cells undergo arrest and enter apoptosis in response to short telomeres. T cells from late generation mTR(-/-) mice that lack telomerase show increased apoptosis when stimulated to enter the cell cycle. The increased apoptosis was not inhibited by colcemid, indicating that the response did not result from breakage of dicentric chromosomes at mitosis. The damage response protein gamma-H2AX localized to telomeres in metaphases from T cells and fibroblasts from mTR(-/-) cells with short telomeres. These data suggest that the major mechanism for induction of apoptosis in late generation mTR(-/-) cells is independent of chromosome segregation and that loss of telomere function through progressive telomere shortening in the absence of telomerase leads to recognition of telomeres as DNA breaks.     

Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects

Telomeres are chromosome end structures and are essential for maintenance of genome stability. Highly repetitive telomere sequences appear to be susceptible to oxidative stress-induced damage. Oxidation may therefore have a severe impact on telomere integrity and function. A wide spectrum of oxidative pyrimidine-derivatives has been reported, including thymine glycol (Tg), that are primarily removed by a DNA glycosylase, Endonuclease III-like protein 1 (Nth1). Here, we investigate the effect of Nth1 deficiency on telomere integrity in mice. Nth1 null (Nth1^−/−) mouse tissues and primary MEFs harbor higher levels of Endonuclease III-sensitive DNA lesions at telomeric repeats, in comparison to a non-telomeric locus. Furthermore, oxidative DNA damage induced by acute exposure to an oxidant is repaired slowly at telomeres in Nth1^−/− MEFs. Although telomere length is not affected in the hematopoietic tissues of Nth1^−/− adult mice, telomeres suffer from attrition and increased recombination and DNA damage foci formation in Nth1^−/− bone marrow cells that are stimulated ex vivo in the presence of 20% oxygen. Nth1 deficiency also enhances telomere fragility in mice. Lastly, in a telomerase null background, Nth1^−/− bone marrow cells undergo severe telomere loss at some chromosome ends and cell apoptosis upon replicative stress. These results suggest that Nth1 plays an important role in telomere maintenance and base repair against oxidative stress-induced base modifications. The fact that telomerase deficiency can exacerbate telomere shortening in Nth1 deficient mouse cells supports that base excision repair cooperates with telomerase to maintain telomere integrity.     

Use of exogenous hTERT to immortalize primary human cells

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence. Telomere-controlled senescence is caused by the shortening of telomeres that occurs each time somatic human cells divide. The enzyme telomerase can prevent the erosion of telomeres and block the onset of telomere-controlled senescence, but its expression is restricted to the early stages of embryonic development, and in the adult, to rare cells of the blood, skin and digestive track. However, we and others have shown that the transfer of an exogenous hTERT cDNA, encoding the catalytic subunit of human telomerase, can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells. Most importantly, hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties. Primary human cells that have so far been established by the forced expression of hTERT alone include fibroblasts, retinal pigmented epithelial cells, endothelial cells, oesophageal squamous cells, mammary epithelial cells, keratinocytes, osteoblasts, and Nestin-positive cells of the pancreas. In this article, we discuss the use of hTERT to immortalize of human cells, the properties of hTERT-immortalized cells, and their applications to cancer research and tissue engineering.     

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres. Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20% oxygen. MEFs did not senesce in physiological (3%) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs suggested that DNA damage limits MEF proliferation in 20% oxygen. Indeed, MEFs accumulated more DNA damage in 20% oxygen than 3% oxygen, and more damage than human fibroblasts in 20% oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing.     

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 elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.     

Telomere shortening: The main mechanism of natural and radiation aging

We present evidence that telomere shortening is the main or even sole mechanism of natural and radiation aging. All apparent contradictions, primarily, the absence of exact inverse correlation between residual telomere length and donor age, are explained using telomere theory. We try to explain in how telomere shortening might be the cause of aging and longevity limitation. We also show the inability of oxidative theory to explain a number of indisputable facts easily explained by telomere theory, such as unlimited growth of tumor cells or why a newborn child starts to age from zero level rather than the level reached by the cells of his parents at the moment of conception. We postulate that if oxidative damage were entirely absent, telomeres would nevertheless shorten with each mitotic cycle, because such is the mechanism of DNA replication, and aging would progress, which we invariantly observe in the presence of any antioxidants. However, if telomeres do not shorten, as happens in transformed cells because telomerase works there, aging does cease and the transformed cells show no senescence. We also observe it in spite of the damaging effect of reactive oxygen species. which may be even more than in normal cells.