Telomere dysfunction puts the brakes on oncogene-induced cancers

EMBO J 31 13, 2839–2851 (2012); published online May082012Senescence represents a major tumour suppressor checkpoint activated by telomere dysfunction or cellular stress factors such as oncogene activation. In this issue of The EMBO Journal, Suram et al (2012) reveal a surprising interconnection between oncogene activation and telomere dysfunction induced senescence. The study supports an alternative model of tumour suppression, indicating that oncogene-induced accumulation of telomeric DNA damage contributes to the induction of senescence in telomerase-negative tumours.Telomere shortening limits the proliferative capacity of primary human cells after 50–70 cell divisions by induction of replicative senescence activated by critically short, dysfunctional telomeres. Different mechanisms were thought to initiate senescence in response to oncogene activation, which occurs abruptly within a few cell doublings (Serrano et al, 1997). Oncogene-induced senescence (OIS) involves an activation of DNA damage signals at stalled replication forks induced by DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006). Replication fork stalling in response to oncogene activation preferentially affects common fragile sites of the DNA (Tsantoulis et al, 2008). The ends of eukaryotic chromosomes—the telomeres–represent common fragile sites that are sensitive to replication fork stalling (Sfeir et al, 2009). These data made it tempting to speculate whether replication fork stalling at telomeres was causatively involved in OIS. Studies on replicative senescence in human fibroblast also supported this possibility showing that mitogenic signals amplify DNA damage responses in senescent cells (Satyanarayana et al, 2004).Multiple studies revealed experimental evidences that senescence suppresses tumour progression in mouse models and early human tumours (for review see Collado and Serrano, 2010). The relative contribution of OIS and telomere dysfunction induced senescence (TDIS) to tumour suppression and possible interconnections between the two pathways at the level of checkpoint induction were not investigated in previous studies. In this issue of The EMBO Journal, Suram et al (2012) describe the presence of TDIS in human precursor lesions but not in the corresponding malignant tumours. Mechanistically, the study shows that oncogenic signals cause replication fork stalling, resulting in telomeric DNA damage accumulation and activation of DNA damage checkpoints reminiscent to TDIS. Telomerase expression does not rescue replication fork stalling but prevents the accumulation of DNA damage at telomeres allowing a bypass of OIS.The study has several important implications for molecular pathways and therapeutic approaches in cancer that need to be further explored (Figure 1):Open in a separate windowFigure 1Traditional and new models of senescence in tumour suppression. (A) Traditional model of replicative senescence: Telomerase-negative tumour cell clones experience telomere shortening as a consequence of cell division. After a lack period depending on the initial telomere length, tumour cells accumulate telomere dysfunction and activation of senescence impairs tumour growth. Telomerase activation represents a late event allowing tumour progression. (B) New model of oncogene induced, telomere-dependent senescence: Oncogene activation leads to abrupt accumulation of DNA damage at telomeres resulting in senescence and tumour suppression. Telomerase-positive stem cells could be resistant to OIS and may be selected as the cell type of origin of tumour development.(i) Telomere length independent roles of telomeres in tumour suppressionThe classical model of telomere-dependent tumour suppression indicates that proliferation-dependent telomere shortening leads to telomere dysfunction, activation of DNA damage checkpoints, and induction of senescence suppressing the growth of telomerase-negative tumour clones. Studies on mouse models supported this concept showing that telomere shortening impairs the progression of initiated tumours in a telomere length-dependent manner (Feldser and Greider, 2007). The new data from Suram et al (2012) indicate that oncogene-induced replication fork stalling activates a telomere-dependent senescence checkpoint, which is independent of telomere length. The study shows that replication forks stall in response to oncogene activation throughout the genome. However, stalled replication forks are resolved in non-telomeric regions, whereas fork stalling inside telomeres leads to un-repairable DNA damage in telomerase-negative cells. These findings are in line with recent publication showing accumulation of un-repairable DNA damage in telomeric DNA in response to aging and stress-induced DNA damage (Fumagalli et al, 2012).(ii) Telomere length independent roles of telomerase in tumour progressionFollowing the classical model telomeres in tumour suppression (Figure 1A), telomerase re-activation is required for tumour progression by limiting telomere dysfunction and the induction of DNA damage checkpoints in response to telomere shortening. The new data from Suram et al (2012) indicate that telomerase has an additional telomere length independent role in tumour progression. The study shows that catalytically active telomerase prevents the activation of DNA damage signals originating from stalled replication forks inside telomeres in response to oncogene activation (Figure 1B). The exact mechanisms of telomerase-dependent healing of stalled replication forks at telomeres remain to be elucidated. It is also unclear whether telomerase activity can prevent any type of DNA damage at telomeres as an over-expression of TERT could not suppress irradiation-induced cellular senescence or the persistence of telomeric DDR following irradiation, H₂O₂, or chemotherapy induced DNA damage (Hewitt et al, 2012).The data could provide a plausible explanation for the increased tumorigenesis in telomerase transgenic mice—a finding which is difficult to explain by telomere length dependent effects of telomerase given the long telomere reserves in mouse tissues (Gonzalez-Suarez et al, 2001). According to the findings of Suram et al (2012), anti-telomerase therapies could have immediate anti-cancer effects in tumours depending on telomerase-mediated healing of stalled replication forks at telomeres. Specific markers for this dependency could be of clinical value. In addition, the data support the concept that somatic stem cells could represent the cell type of origin of cancers. In contrast to differentiated somatic cells, tissues stem cells are often telomerase-positive, indicating that stem cells might be less sensitive to OIS.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

Oxidative stress shortens telomeres

Telomeres in most human cells shorten with each round of DNA replication, because they lack the enzyme telomerase. This is not, however, the only determinant of the rate of loss of telomeric DNA. Oxidative damage is repaired less well in telomeric DNA than elsewhere in the chromosome, and oxidative stress accelerates telomere loss, whereas antioxidants decelerate it. I suggest here that oxidative stress is an important modulator of telomere loss and that telomere-driven replicative senescence is primarily a stress response. This might have evolved to block the growth of cells that have been exposed to a high risk of mutation.     

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.     

Diet, nutrition and telomere length

The ends of human chromosomes are protected by DNA–protein complexes termed telomeres, which prevent the chromosomes from fusing with each other and from being recognized as a double-strand break by DNA repair proteins. Due to the incomplete replication of linear chromosomes by DNA polymerase, telomeric DNA shortens with repeated cell divisions until the telomeres reach a critical length, at which point the cells enter senescence. Telomere length is an indicator of biological aging, and dysfunction of telomeres is linked to age-related pathologies like cardiovascular disease, Parkinson disease, Alzheimer disease and cancer. Telomere length has been shown to be positively associated with nutritional status in human and animal studies. Various nutrients influence telomere length potentially through mechanisms that reflect their role in cellular functions including inflammation, oxidative stress, DNA integrity, DNA methylation and activity of telomerase, the enzyme that adds the telomeric repeats to the ends of the newly synthesized DNA.     

Cancer and aging: the importance of telomeres in genome maintenance

Telomeres are the specialized DNA-protein structures that cap the ends of linear chromosomes, thereby protecting them from degradation and fusion by cellular DNA repair processes. In vertebrate cells, telomeres consist of several kilobase pairs of DNA having the sequence TTAGGG, a few hundred base pairs of single-stranded DNA at the 3' end of the telomeric DNA tract, and a host of proteins that organize the telomeric double and single-stranded DNA into a protective structure. Functional telomeres are essential for maintaining the integrity and stability of genomes. When combined with loss of cell cycle checkpoint controls, telomere dysfunction can lead to genomic instability, a common cause and hallmark of cancer. Consequently, normal mammalian cells respond to dysfunctional telomeres by undergoing apoptosis (programmed cell death) or cellular senescence (permanent cell cycle arrest), two cellular tumor suppressor mechanisms. These tumor suppressor mechanisms are potent suppressors of cancer, but recent evidence suggests that they can antagonistically also contribute to aging phenotypes. Here, we review what is known about the structure and function of telomeres in mammalian cells, particularly human cells, and how telomere dysfunction may arise and contribute to cancer and aging phenotypes.     

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.     

G-quadruplex stabilizer 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide induces accelerated senescence and inhibits tumorigenic properties in cancer cells

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13 degrees C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 mumol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated beta-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways.     

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.     

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?     

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.     

Telomeric injury by KML001 in human T cells induces mitochondrial dysfunction through the p53-PGC-1α pathway

Telomere erosion and mitochondrial dysfunction are prominent features of aging cells with progressive declines of cellular functions. Whether telomere injury induces mitochondrial dysfunction in human T lymphocytes, the major component of adaptive host immunity against infection and malignancy, remains unclear. We have recently shown that disruption of telomere integrity by KML001, a telomere-targeting drug, induces T cell senescence and apoptosis via the telomeric DNA damage response (DDR). In this study, we used KML001 to further investigate the role and mechanism of telomere injury in mitochondrial dysregulation in aging T cells. We demonstrate that targeting telomeres by KML001 induces mitochondrial dysfunction, as evidenced by increased mitochondrial swelling and decreased mitochondrial membrane potential, oxidative phosphorylation, mitochondrial DNA content, mitochondrial respiration, oxygen consumption, glycolysis, and ATP energy production. Mechanistically, we found that the KML001-induced telomeric DDR activated p53 signaling, which in turn repressed the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1), leading to T cell mitochondrial dysfunction. These results, forging a direct link between telomeric and mitochondrial biology, shed new light on the human T cell aging network, and demonstrate that the p53-PGC-1α-NRF-1 axis contributes to mitochondrial dysfunction in the setting of telomeric DDR. This study suggests that targeting this axis may offer an alternative, novel approach to prevent telomere damage-mediated mitochondrial and T cell dysfunctions to combat a wide range of immune aging-associated human diseases.Subject terms: Immunology, Diseases     

Endless quest

The replication of linear chromosome DNA by DNA polymerase leads to the loss of terminal sequences, in the absence of a special mechanism to maintain ends or telomeres. This mechanism is known to consist of short terminal repeats and the enzyme telomerase, which contains RNA complementary to the DNA repeats. There is evidence that telomeric DNA continually decreases in size in the absence of telomerase, and this is followed by cellular senescence. Immortalisation of somatic cells is accompanied, at least in some cases, by acquisition of telomerase activity. The cloning of DNA coding for the RNA component of telomerase has opened up some new experimental approaches, including the study of telomerases with mutant RNA^(1,2). The telomere theory of cellular senescence appears to provide a molecular basis for the ‘Hayflick limit’ to human fibroblast growth. However the telomeres and behaviour of primary mouse cells are anomolous⁽³⁾, and many immortalised human cell lines lack normal telomerase activity⁽⁴⁾. These exceptions are not easily accommodated in the telomere theory.     

Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G‐quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2‐deficient mice develop aneuploidy‐associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.     

Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.     

Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.     

Aging of the cells: Insight into cellular senescence and detection Methods

Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means “growing old,” is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.     

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.     

Telomere attrition and accumulation of senescent cells in cultured human endothelial cells

The human umbilical vein endothelial cell (HUVEC) is an important model of the human endothelium that is widely used in vascular research. HUVECs and the adult endothelium share many characteristics including progression into senescence as the cells age. Despite this, the shortening of telomeres and its relationship to the progression into senescence are poorly defined in HUVECs. In this study of several HUVEC lines we show notable consistency in their growth curves. There is a steady decline in the growth rate of HUVECs grown continually in culture and we estimate complete cessation of growth after approximately 70 population doublings. The HUVECs lose telomeric DNA at a consistent rate of 90 base pairs/population doubling and show a progressive accumulation of shortened telomeres (below 5 kilobases). This telomeric loss correlates with the accumulation of senescent HUVECs in culture as assessed by staining for beta-galactosidase activity at pH 6. Although the telomere length of a large population of cells is a relatively crude measure, we suggest that in HUVECs a mean telomere length (as measured by terminal restriction fragment length) of 5 kilobases is associated with entry into senescence. These data demonstrate the strong relationship between telomere attrition and cell senescence in HUVECs. They suggest that DNA damage and subsequent telomere attrition are likely to be key mechanisms driving the development of endothelial senescence in the pathogenesis of vascular disease.     

Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2

The ends of linear chromosomes are capped by protein–DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2.