Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.     

Normal Human Telomeres Are Not Late Replicating

Telomeres in yeast are late replicating. Genes placed next to telomeres in yeast can be repressed (telomere positional effects), leading to the hypothesis that telomeres may be heterochromatic and may control the expression of subtelomeric genes. In addition, yeast telomeres are processed to have a transient long overhang at the end of S phase. The applicability of the yeast data to human biology was examined by determining the timing of telomere replication and processing in normal human diploid fibroblasts. Telomeres were purified from synchronized cells that had been labeled with 5-bromodeoxyuridine (BrdU) at hourly intervals, and the fraction of labeled telomeres was analyzed by retrieval with anti-BrdU antibodies. We determined that normal human telomeres replicate throughout S phase rather than being very late replicating. Furthermore, the overall timing of replication was unaffected by telomere length in young versus old cells or cells whose telomeres had been elongated following transfection with the catalytic subunit of telomerase. Finally, the asymmetry in the length of the G-rich overhang in daughter telomeres produced by leading versus lagging strand synthesis was shown to be established within 1 h of telomere replication, indicating there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs. Therefore, the timings of replication and processing of human telomeres are very different from those of yeast.     

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.     

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.     

Modelling the regulation of telomere length: the effects of telomerase and G-quadruplex stabilising drugs

Telomeres are guanine-rich sequences at the end of chromosomes which shorten during each replication event and trigger cell cycle arrest and/or controlled death (apoptosis) when reaching a threshold length. The enzyme telomerase replenishes the ends of telomeres and thus prolongs the life span of cells, but also causes cellular immortalisation in human cancer. G-quadruplex (G4) stabilising drugs are a potential anticancer treatment which work by changing the molecular structure of telomeres to inhibit the activity of telomerase. We investigate the dynamics of telomere length in different conformational states, namely t-loops, G-quadruplex structures and those being elongated by telomerase. By formulating deterministic differential equation models we study the effects of various levels of both telomerase and concentrations of a G4-stabilising drug on the distribution of telomere lengths, and analyse how these effects evolve over large numbers of cell generations. As well as calculating numerical solutions, we use quasicontinuum methods to approximate the behaviour of the system over time, and predict the shape of the telomere length distribution. We find those telomerase and G4-concentrations where telomere length maintenance is successfully regulated. Excessively high levels of telomerase lead to continuous telomere lengthening, whereas large concentrations of the drug lead to progressive telomere erosion. Furthermore, our models predict a positively skewed distribution of telomere lengths, that is, telomeres accumulate over lengths shorter than the mean telomere length at equilibrium. Our model results for telomere length distributions of telomerase-positive cells in drug-free assays are in good agreement with the limited amount of experimental data available.     

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

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

Targeting telomeres to enforce cancer cells to senesce

The telomeres protect the end of chromosomes from being recognized and processed as an accidental double stranded break. In human somatic cells, telomeres shorten progressively with every round of DNA replication, leading to dysfunctional telomeres that trigger cellular senescence or apoptosis depending on the cell type. This telomere erosion appears to play a role in cell renewal, ageing and cancer. Two recent studies demonstrated in mouse that eroded telomeres in cancer cells blocked for apoptosis limit cancer formation by triggering senescence. These results suggest that provoking senescence may provide a way to cure cancer and point to new therapeutical strategies targeting specific telomeric functions. Nevertheless, an important question remains unanswered: does replicative senescence limit tumor formation in human?     

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.     

Telomerase: Structure,functions, and activity regulation

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.     

Telomere length homeostasis is achieved via a switch between telomerase- extendible and -nonextendible states

Telomerase counteracts telomere erosion that stems from incomplete chromosome end replication and nucleolytic processing. A precise understanding of telomere length homeostasis has been hampered by the lack of assays that delineate the nonuniform telomere extension events of single chromosome molecules. Here, we measure telomere elongation at nucleotide resolution in Saccharomyces cerevisiae. The number of nucleotides added to a telomere in a single cell cycle varies between a few to more than 100 nucleotides and is independent of telomere length. Telomerase does not act on every telomere in each cell cycle, however. Instead, it exhibits an increasing preference for telomeres as their lengths decline. Deletion of the telomeric proteins Rif1 or Rif2 gives rise to longer telomeres by increasing the frequency of elongation events. Thus, by taking a molecular snapshot of a single round of telomere replication, we demonstrate that telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states.     

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.     

Telomerase abrogates aneuploidy‐induced telomere replication stress,senescence and cell depletion

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy‐controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb‐dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase‐deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy‐induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.     

Telomere biology in mammalian germ cells and during development

The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, and in highly proliferative organs, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Telomere length is to a large extent genetically determined and the neonatal telomere length equilibrium is, in fact, a matter of evolution. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that induce replicative senescence in vitro and probably in vivo. Hence, a molecular clock is set during development, which determines the replicative potential of cells during extrauterine life. Telomeres might be directly or indirectly implicated in longevity determination in vivo, and information on telomere length setting in utero and beyond should help elucidate presumed causal connections between early growth and aging disorders later in life. Only limited information exists concerning the mechanisms underlying overall telomere length regulation in the germline and during early development, especially in humans. The intent of this review is to focus on recent advances in our understanding of telomere biology in germline cells as well as during development (pre- and postimplantation periods) in an attempt to summarize our knowledge about telomere length determination and its importance for normal development in utero and the occurrence of the aging and abnormal phenotype later on.     

Telomere dynamics in a human cancer cell line

Telomere maintenance is thought to be essential for immortalization of human cancer cells to compensate for the loss of DNA from the ends of chromosomes and to prevent chromosome fusion. We have investigated telomere dynamics in the telomerase-positive squamous cell carcinoma cell line SCC-61 by marking the ends of chromosomes with integrated plasmid sequences so that changes in the length of individual telomeres could be monitored. Despite having very short telomeres, SCC-61 has a relatively stable genome and few telomere associations. The marked telomeres in different SCC-61 clones have similar mean lengths which show little change with increasing time in culture. Thus, each marked telomere is maintained at a specific length, which we term the equilibrium mean length (EML). The Gaussian distribution in the length of the marked telomeres demonstrates that telomeres continuously fluctuate in length. Consistent with this observation, the mean lengths of the marked telomere in subclones of these cell lines initially differ, but then gradually return to the EML of the original clone with increasing time in culture. The analysis of a clone with two marked telomeres demonstrated that changes in telomere length can occur on each marked telomere independently or coordinately on both telomeres. These results suggest that the short telomeres in many tumor cell lines do not result from an inability to properly maintain telomeres at a specific length.     

Telomere recombination in normal mammalian cells

Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15–20% of tumors lacking active telomerase and,, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the “gold standard” of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed.     

Replication Timing of Human Telomeres Is Chromosome Arm–Specific,Influenced by Subtelomeric Structures and Connected to Nuclear Localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.     

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow-FISH-DDD) to determine telomere length dynamics in proliferating cells.

BACKGROUND: Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. METHODS: Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. RESULTS: In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. CONCLUSIONS: Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.     

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.