Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells

Chromosome aberrations such as loss of chromosome 13 were frequently observed in human endothelial cells from umbilical cord veins (HUVEC). A recent study showed that the length of telomeric single-stranded 3'-overhangs (G-tails) is more important as an essential structure for chromosome maintenance than the net telomere length in telomere t-loop formation. Here, we have examined G-tail length using G-tail telomere HPA in normal and hTERT-transduced HUVECs. We found that forced expression of hTERT in HUVEC induced G-tail as well as total telomere length elongation. G-tail length was well correlated with total telomere length. However, hTERT introduction did not prevent chromosome aberrations such as loss of chromosome 13. Normal characteristics such as morphology, up-regulation of vWF, and tube formation were observed in hTERT-HUVEC as in young normal HUVEC. These results show that chromosome aberrations in HUVEC are independent of telomere G-tail and total telomere attrition.     

Survival advantage of cultured human vascular endothelial cells that lost chromosome 13

We explored the nature of chromosome 13 loss in cultured human vascular endothelial cells (HUVECs). Chromosome 13 loss detected by metaphase and interphase analysis was noted in earlier passages of HUVEC strains with no relation to telomere length or replicative senescence. Ectopic expression of telomerase did not influence the loss of chromosome 13. HUVECs losing chromosome 13 demonstrated increased migratory potential and loss of heterozygosity. Collectively, these observations suggest that the loss of chromosome 13 gives cultured HUVECs a replicative advantage.Communicated by E.A. Nigg     

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

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 length in fibroblasts and blood cells from healthy centenarians

Several lines of evidence indicate that telomere shortening during in vitro aging of human somatic cells plays a causal role in cellular senescence. A critical telomere length seems to be associated with the replicative block characterizing senescent cells. In this paper we analyzed the mean length of the terminal restriction fragments (TRF) in fibroblast strains from 4 healthy centenarians, that is, in cells aged in vivo, and from 11 individuals of different ages. No correlation between mean TRF length and donor age was found. As expected, telomere shortening was detected during in vitro propagation of centenarian fibroblasts, suggesting that in fibroblasts aged in vivo telomeres can be far from reaching a critical length. Accordingly, chromosome analysis did not show the presence of telomeric associations in early passage centenarian fibroblasts. In blood cells from various individuals, the expected inverse correlation between mean TRF length and donor age was found. In particular, a substantial difference (about 2 kb) between telomere length in the two cell types was observed in the same centenarian. Expression analysis of three senescence-induced genes, i.e., fibronectin, apolipoprotein J, and p21, revealed for only the fibronectin expression levels a clear positive correlation with donor age. Our results suggest that (1) telomere shortening could play a different role in the aging of different cell types and (2) the characteristics of fibroblasts aged in vitro might not be representative of what occurs in vivo.     

TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation

A cancer is a robustly evolving cell population originating from a normal diploid cell. Improper chromosome segregation causes aneuploidy, a driving force of cancer development and malignant progression. Telomeric repeat binding factor 1 (TRF1) has been established as a telomeric protein that negatively regulates telomere elongation by telomerase and promotes efficient DNA replication at telomeres. Intriguingly, overexpression of a mitotic kinase, Aurora-A, compromises efficient microtubule-kinetochore attachment in a TRF1-dependent manner. However, the precise role of TRF1 in mitosis remains elusive. Here we demonstrate that TRF1 is required for the centromeric function of Aurora-B, which ensures proper chromosome segregation. TRF1 depletion abolishes centromeric recruitment of Aurora-B and loosens sister centromere cohesion, resulting in the induction of merotelic kinetochore attachments, lagging chromosomes, and micronuclei. Accordingly, an absence of TRF1 in human and mouse diploid cells induces aneuploidy. These phenomena seem to be telomere independent, because a telomere-unbound TRF1 mutant can suppress the TRF1 knockdown phenotype. These observations indicate that TRF1 regulates the rigidity of the microtubule-kinetochore attachment, contributing to proper chromosome segregation and the maintenance of genomic integrity.     

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.     

Loss of hPot1 function leads to telomere instability and a cut-like phenotype

The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2. Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation. However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death. Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans. Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability. In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation. However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation. We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.     

The role of the poly(ADP-ribose) polymerase tankyrase1 in telomere length control by the TRF1 component of the shelterin complex

Tankyrase1 is a multifunctional poly(ADP-ribose) polymerase that can localize to telomeres through its interaction with the shelterin component TRF1. Tankyrase1 poly(ADP-ribosyl)ates TRF1 in vitro, and its nuclear overexpression leads to loss of TRF1 and telomere elongation, suggesting that tankyrase1 is a positive regulator of telomere length. In agreement with this proposal, we show that tankyrase1 RNA interference results in telomere shortening proportional to the level of knockdown. Furthermore, we show that a tankyrase1-resistant form of TRF1 enforced normal telomere length control, indicating that tankyrase1 is not required downstream of TRF1 in this pathway. Thus, in human cells, tankyrase1 appears to act upstream of TRF1, promoting telomere elongation through the removal of TRF1. This pathway appears absent from mouse cells. We show that murine TRF1, which lacks the canonical tankyrase1-binding site, is not a substrate for tankyrase1 poly(ADP-ribosyl)sylation in vitro. Furthermore, overexpression of tankyrase1 in mouse nuclei did not remove TRF1 from telomeres and had no detectable effect on other components of mouse shelterin. We propose that the tankyrase1-controlled telomere extension is a human-specific elaboration that allows additional control over telomere length in telomerase positive cells.     

Functional interaction between poly(ADP-Ribose) polymerase 2 (PARP-2) and TRF2: PARP activity negatively regulates TRF2

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T(2)AG(3) repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2(-/-) primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T(2)AG(3) repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity.     

TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons

Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.     

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway

The DNA‐binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/β‐catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, β‐catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced β‐catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/β‐catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.     

Telomere instability in a human tumor cell line expressing NBS1 with mutations at sites phosphorylated by ATM

Nijmegen breakage syndrome (NBS) is an autosomal genetic disease demonstrating a variety of phenotypic abnormalities, including premature aging, increased cancer incidence, chromosome instability, and sensitivity to ionizing radiation. The gene involved in NBS, NBS1, is part of the MRE11/RAD50/NBS1 (MRN) complex that also includes MRE11 and RAD50, which is involved in DNA repair and cell cycle regulation in response to DNA damage. The MRN complex is also involved in telomere maintenance, as demonstrated by the shortened telomeres in NBS primary human fibroblasts and the association of NBS1 with the telomere-binding protein TRF2. To learn more about how a deficiency in telomere maintenance might contribute to chromosome instability in NBS, we have investigated the stability of telomeres in two telomerase-positive human tumor cell clones, BNmt-On and BNmt-Off, expressing an inducible NBS1(S278A/S343A) gene containing mutations at serines 278 and 343 phosphorylated by ATM. The results demonstrate an increased rate of telomere loss in both clones following expression of NBS1(S278A/S343A). The absence of detectable changes in average telomere length suggests that NBS1-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. The recombination events associated with telomere loss were found to be similar to those shown previously to result in breakage/fusion/bridge cycles, suggesting that telomere loss can contribute to chromosome instability in NBS1-deficient cells. Telomere loss showed no correlation with radiosensitivity or radioresistant DNA synthesis, demonstrating that NBS1(S278A/S343A) promotes telomere loss through a separate pathway from these other phenotypes associated with NBS.     

Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function

Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction ( approximately 73%) has binding dynamics similar to TRF1 (residence time of approximately 44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of approximately 11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.     

Telomere dynamics in an immortal human cell line.

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines.     

Telomere shortening in Fanconi anaemia demonstrated by a direct FISH approach

Analysis of telomere status in patients with Fanconi anaemia (FA) has previously been carried out by measurement of telomere restriction fragment (TRF) length by Southern blotting and densitometry. Results from these studies indicated that FA patients had significant reduction in telomere length compared with age-matched controls. This paper confirms and extends these findings using a direct FISH technique, which showed that 15 out of 16 FA patients had increased loss of telomere signals compared with controls. In 12 out of the 16 patients, decrease in telomere signal intensity could also be detected using a Q-FISH approach.     

TIN2 mediates functions of TRF2 at human telomeres

Telomeres are protective structures at chromosome ends and are crucial for genomic stability. Mammalian TRF1 and TRF2 bind the double-stranded telomeric repeat sequence and in turn are bound by TIN2, TANK1, TANK2, and hRAP1. TRF1 is a negative regulator of telomere length in telomerase-positive cells, whereas TRF2 is important for telomere capping. TIN2 was identified as a TRF1-interacting protein that mediates TRF1 function. We show here that TIN2 also interacts with TRF2 in vitro and in yeast and mammalian cells. TIN2 mutants defective in binding of TRF1 or TRF2 induce a DNA damage response and destabilize TRF1 and TRF2 at telomeres in human cells. Our findings suggest that the functions of TRF1 and TRF2 are linked by TIN2.