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.

Inhibition of TRF2 Leads to Ferroptosis, Autophagic Death, and Apoptosis by Causing Telomere Dysfunction.

Background: Gastric cancer (GC) is an aggressive malignancy with a high mortality rate and poor prognosis. Telomeric repeat-binding factor 2 (TRF2) is a critical telomere protection protein. Emerging evidence indicates that TRF2 may be an essential treatment option for GC; however, the exact mechanism remains largely unknown. Objective: We aimed to explore the role of TRF2 in GC cells. The function and molecular mechanisms of TRF2 in the pathogenesis of GC were mainly discussed in this study. Methods: Relevant data from GEPIA and TCGA databases regarding TRF2 gene expression and its prognostic significance in GC samples were analyzed. Analysis of 53BP1 foci at telomeres by immunofluorescence, metaphase spreads, and telomere-specific FISH analysis was carried out to explore telomere damage and dysfunction after TRF2 depletion. CCK8 cell proliferation, trypan blue staining, and colony formation assay were performed to evaluate cell survival. Apoptosis and cell migration were determined with flow cytometry and scratch-wound healing assay, respectively. qRT-PCR and Western blotting were carried out to analyze the mRNA and protein expression levels after TRF2 depletion on apoptosis, autophagic death, and ferroptosis. Results: By searching with GEPIA and TCGA databases, the results showed that the expression levels of TRF2 were obviously elevated in the samples of GC patients, which was associated with adverse prognosis. Knockdown of TRF2 suppressed the cell growth, proliferation, and migration in GC cells, causing significant telomere dysfunction. Apoptosis, autophagic death, and ferroptosis were also triggered in this process. The pretreatment of chloroquine (autophagy inhibitor) and ferrostatin-1 (ferroptosis inhibitor) improved the survival phenotypes of GC cells. Conclusion: Our data suggest that TRF2 depletion can inhibit cell growth, proliferation, and migration through the combined action of ferroptosis, autophagic death, and apoptosis in GC cells. The results indicate that TRF2 might be used as a potential target to develop therapeutic strategies for treating GC.

Homology directed telomere clustering, ultrabright telomere formation and nuclear envelope rupture in cells lacking TRF2B and RAP1.

Double-strand breaks (DSBs) due to genotoxic stress represent potential threats to genome stability. Dysfunctional telomeres are recognized as DSBs and are repaired by distinct DNA repair mechanisms. RAP1 and TRF2 are telomere binding proteins essential to protect telomeres from engaging in homology directed repair (HDR), but how this occurs remains unclear. In this study, we examined how the basic domain of TRF2 (TRF2B) and RAP1 cooperate to repress HDR at telomeres. Telomeres lacking TRF2B and RAP1 cluster into structures termed ultrabright telomeres (UTs). HDR factors localize to UTs, and UT formation is abolished by RNaseH1, DDX21 and ADAR1p110, suggesting that they contain DNA-RNA hybrids. Interaction between the BRCT domain of RAP1 and KU70/KU80 is also required to repress UT formation. Expressing TRF2∆B in Rap1-/- cells resulted in aberrant lamin A localization in the nuclear envelope and dramatically increased UT formation. Expressing lamin A phosphomimetic mutants induced nuclear envelope rupturing and aberrant HDR-mediated UT formation. Our results highlight the importance of shelterin and proteins in the nuclear envelope in repressing aberrant telomere-telomere recombination to maintain telomere homeostasis.

Selective pericentromeric heterochromatin dismantling caused by TP53 activation during senescence.

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53-TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.

TRF2 inhibition rather than telomerase disruption drives CD4T cell dysfunction during chronic viral infection.

We investigated the role of telomerase and telomere repeat-binding factor 2 (TRF2 or TERF2) in T-cell dysfunction in chronic viral infection. We found that the expression and activity of telomerase in CD4+ T (CD4T) cells from patients with hepatitis C virus (HCV) infections or people living with HIV (PLWH) were intact, but TRF2 expression was significantly inhibited at the post-transcriptional level, suggesting that TRF2 inhibition is responsible for the CD4T cell dysfunction observed during chronic viral infection. Silencing TRF2 expression in CD4T cells derived from healthy subjects induced telomeric DNA damage and CD4T cell dysfunction without affecting telomerase activity or translocation - similar to what we observed in CD4T cells from HCV patients and PLWH. These findings indicate that premature T-cell aging and dysfunction during chronic HCV or HIV infection are primarily caused by chronic immune stimulation and T-cell overactivation and/or proliferation that induce telomeric DNA damage due to TRF2 inhibition, rather than telomerase disruption. This study suggests that restoring TRF2 presents a novel approach to prevent telomeric DNA damage and premature T-cell aging, thus rejuvenating T-cell functions during chronic viral infection.

Ultrastructure and nuclear architecture of telomeric chromatin revealed by correlative light and electron microscopy.

Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.

Compartmentalization of telomeres through DNA-scaffolded phase separation.

Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

TRF2 promotes dynamic and stepwise looping of POT1 bound telomeric overhang.

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.

Molecular Consequences of Depression Treatment: A Potential In Vitro Mechanism for Antidepressants-Induced Reprotoxic Side Effects.

The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants' and neuroleptic drugs' toxicity and points to the need for further research in this topic with the use of in vivo models and human samples.

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress.

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

Estrogen Replacement Therapy Induces Antioxidant and Longevity-Related Genes in Women after Medically Induced Menopause.

Females live longer than males in many species, including humans, and estrogens are in part responsible for this protection against aging. We reported previously that estrogens can protect rats against oxidative stress, by inducing antioxidant and longevity-related genes. Thus, this study was aimed at confirming the ability of estrogens to upregulate antioxidant and longevity-related genes in humans. For this purpose, we selected 16 women of reproductive age (18-42 years old) undergoing a fertility treatment that includes a medically induced menopause, at the Valencian Infertility Institute. We took blood samples at each time point of the treatment (basal, induced menopause, estrogen, and estrogen plus progesterone replacement therapy). mRNA expression of antioxidant and longevity-related genes in peripheral blood mononuclear cells (PBMC) was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Determination of reduced glutathione (GSH) in total blood was carried out using high-performance liquid chromatography (HPLC). As expected, we found that medically induced menopause significantly decreased sexual hormone (estrogens and progesterone) levels. It also lowered glutathione peroxidase (GPx), 16S rRNA, P21, and TERF2 mRNA expression and blood GSH levels. Estrogen replacement therapy significantly restored estrogen levels and induced mRNA expression of manganese superoxide dismutase (MnSOD), GPx, 16S rRNA, P53, P21, and TERF2 and restored blood GSH levels. Progesterone replacement therapy induced a significant increase in MnSOD, P53, sestrin 2 (SENS2), and TERF2 mRNA expression when compared to basal conditions. These findings provide evidence for estrogen beneficial effects in upregulating antioxidant and longevity-related genes in women.

The Intra- and Extra-Telomeric Role of TRF2 in the DNA Damage Response.

Telomere repeat binding factor 2 (TRF2) has a well-known function at the telomeres, which acts to protect the telomere end from being recognized as a DNA break or from unwanted recombination. This protection mechanism prevents DNA instability from mutation and subsequent severe diseases caused by the changes in DNA, such as cancer. Since TRF2 actively inhibits the DNA damage response factors from recognizing the telomere end as a DNA break, many more studies have also shown its interactions outside of the telomeres. However, very little has been discovered on the mechanisms involved in these interactions. This review aims to discuss the known function of TRF2 and its interaction with the DNA damage response (DDR) factors at both telomeric and non-telomeric regions. In this review, we will summarize recent progress and findings on the interactions between TRF2 and DDR factors at telomeres and outside of telomeres.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Abnormal function of telomere protein TRF2 induces cell mutation and the effects of environmental tumor­promoting factors (Review).

Recent studies have found that somatic gene mutations and environmental tumor­promoting factors are both indispensable for tumor formation. Telomeric repeat­binding factor (TRF)2 is the core component of the telomere shelterin complex, which plays an important role in chromosome stability and the maintenance of normal cell physiological states. In recent years, TRF2 and its role in tumor formation have gradually become a research hot topic, which has promoted in­depth discussions into tumorigenesis and treatment strategies, and has achieved promising results. Some cells bypass elimination, due to either aging, apoptosis via mutations or abnormal prolongation of the mitotic cycle, and enter the telomere crisis period, where large­scale DNA reorganization occurs repeatedly, which manifests as the precancerous cell cycle. Finally, at the end of the crisis cycle, the mutation activates either the expression level of telomerase or activates the alternative lengthening of telomere mechanism to extend the local telomeres. Under the protection of TRF2, chromosomes are gradually stabilized, immortal cells are formed and the stagewise mutation­driven transformation of normal cells to cancer cells is completed. In addition, TRF2 also shares the characteristics of environmental tumor­promoting factors. It acts on multiple signal transduction pathway­related proteins associated with cell proliferation, and affects peripheral angiogenesis, inhibits the immune recognition and killing ability of the microenvironment, and maintains the stemness characteristics of tumor cells. TRF2 levels are abnormally elevated by a variety of tumor control proteins, which are more conducive to the protection of telomeres and the survival of tumor cells. In brief, the various regulatory mechanisms which tumor cells rely on to survive are organically integrated around TRF2, forming a regulatory network, which is conducive to the optimization of the survival direction of heterogeneous tumor cells, and promotes their survival and adaptability. In terms of clinical application, TRF2 is expected to become a new type of cancer prognostic marker and a new tumor treatment target. Inhibition of TRF2 overexpression could effectively cut off the core network regulating tumor cell survival, reduce drug resistance, or bypass the mutation under the pressure of tumor treatment selection, which may represent a promising therapeutic strategy for the complete eradication of tumors in the clinical setting. Based on recent research, the aim of the present review was to systematically elaborate on the basic structure and functional characteristics of TRF2 and its role in tumor formation, and to analyze the findings indicating that TRF2 deficiency or overexpression could cause severe damage to telomere function and telomere shortening, and induce DNA damage response and chromosomal instability.

Cellular senescence promotes endothelial activation through epigenetic alteration, and consequently accelerates atherosclerosis.

Senescent vascular cells are detected in atherosclerotic lesion, and its involvement in the development of atherosclerosis has been revealed; however, whether and the mechanism by which endothelial cell (EC) senescence is causally implicated in atherosclerosis remains unclear. We here investigate a role of EC senescence in atherosclerosis by utilizing EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat-binding factor 2 under the control of the Tie2 or vascular endothelial cadherin promoter. EC-specific progeria accelerated atherosclerosis in mice with target deletion of ApoE. Mechanistically, senescent ECs were markedly sensitive for inflammation-mediated VCAM-1 induction, leading to enhanced monocyte adhesion. Inhibition of NF-κB signaling abolished the enhanced inflammatory responses in senescent ECs, while NF-κB nuclear translocation in response to TNF-α were similar between young and senescent ECs. We found a higher association of VCAM-1 gene with active histone H3 trimethylated on lysine 4, leading to increased NF-κB accessibility in senescent ECs. Our data revealed that EC cellular senescence causes endothelial hyper-inflammability through epigenetic alteration, which consequently accelerates atherosclerosis. Therefore, EC senescence is a promising therapeutic target for the prevention and/or treatment of atherosclerotic disease in elderly population.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment.

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214-218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

Telomere shortening and expression of TRF1 and TRF2 in uterine leiomyoma.

Uterine leiomyoma is a benign smooth muscle tumor of the uterus that can exhibit histopathological traits that mimic malignancy. Telomere shortening is an early event in tumorigenesis and telomerase activation facilitates tumor progression later in the course of carcinogenesis. Telomeric repeat­binding factor (TRF)1 and TRF2 protect telomeres, and their gene expression levels are dysregulated in various cancer types. However, the roles of telomeres and telomere protection proteins in uterine leiomyoma remain largely unknown. In this study, telomere length and the mRNA levels of various telomere­related genes in normal tissues and leiomyoma were determined, and their relationships were evaluated. Uterine leiomyoma and normal myometrium were surgically obtained from 18 and 13 patients, respectively. Telomere length and gene expression were determined by Southern blot analysis and reverse transcription­quantitative PCR, respectively. In matched samples, telomeres were consistently shorter in leiomyoma tissue than in adjacent normal tissue. TRF1, TRF2, PIN2­interacting telomerase inhibitor 1 (PINX1), and telomerase RNA component were expressed at comparable levels in both leiomyoma and normal tissues. None of these genes were associated with telomere length in leiomyoma. All tested tissues were negative for telomerase reverse transcriptase, which encodes the catalytic component of telomerase, indicating that cells in uterine leiomyoma were not immortalized. In summary, telomere erosion, which reflects active proliferation during tumor evolution, was evident in uterine leiomyoma. Steady­state expression of TRF1, TRF2 and PINX1 may be important for maintenance of telomere integrity in leiomyoma, where telomere length is shortened.

Immune Activation Induces Telomeric DNA Damage and Promotes Short-Lived Effector T Cell Differentiation in Chronic HCV Infection.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive. APPROACH AND RESULTS: Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+ . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. CONCLUSIONS: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.