Telomere Dysfunctional Research Articles

Telomere dysfunction alters intestinal stem cell dynamics to promote cancer

Telomere dynamics are linked to aging hallmarks, and age-associated telomere loss fuels the development of epithelial cancers. In Apc-mutant mice, the onset of DNA damage associated with telomere dysfunction has been shown to accelerate adenoma initiation via unknown mechanisms. Here, we observed that Apc-mutant mice engineered to experience telomere dysfunction show accelerated adenoma formation resulting from augmented cell competition and clonal expansion. Mechanistically, telomere dysfunction induces the repression of EZH2, resulting in the derepression of Wnt antagonists, which causes the differentiation of adjacent stem cells and a relative growth advantage to Apc-deficient telomere dysfunctional cells. Correspondingly, in this mouse model, GSK3β inhibition countered the actions of Wnt antagonists on intestinal stem cells, resulting in impaired adenoma formation of telomere dysfunctional Apc-mutant cells. Thus, telomere dysfunction contributes to cancer initiation through altered stem cell dynamics, identifying an interception strategy for human APC-mutant cancers with shortened telomeres.

Telomere Dysfunction Drives Aberrant Hematopoietic Differentiation and Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

A Mouse Model of Telomere Dysfunction Recapitulates Hallmark Features of Human Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic neoplastic disorders that are characterized by ineffective myeloid differentiation and dysplasia as well as telomere shortening and accumulated DNA damage in progenitor cells. Less understood is whether DNA damage is the instigator of impaired progenitor cell differentiation and MDS development. Telomerase deficient mice have served as a model system to demonstrate the adverse effects of wide-spread endogenous DNA damage signaling on stem cell function in vivo. In recent studies, we sought to determine whether persistent physiological DNA damage can impair the function of specific hematopoietic lineages by employing the 4-hydroxytamoxifen (OHT)-inducible telomerase reverse transcriptase-estrogen receptor (TERTER) model. For the first time, we demonstrate that late generation TERTER/ER mice with dysfunctional telomeres exhibit hallmark features of MDS, including peripheral blood cytopenias, bone marrow (BM) hyper-cellularity, and an increased myeloid-to-erythroid progenitor ratio in the absence of increased apoptosis. Severe tri-lineage myelodysplasia, and an increase of immature, morphologically abnormal myeloid blasts frequently with pronounced monocytic differentiation were consistent with refractory anemia with excess of blasts (RAEB) or chronic myelo-monocytic leukemia (CMML), a specific sub-group of MDS that is characterized by a high propensity to develop acute myeloid leukemia (AML). Accordingly, approximately 5% of aged TERTER/ER mice progressed to AML, as demonstrated by a marked increase of BM myeloid blasts, and infiltration of myeloid precursors into the splenic white-red pulp architecture, resulting in myeloid sarcoma with the complete effacement of lymphoid follicles. Compared to control mice with intact telomeres, the progenitor compartment of telomere dysfunctional mice shows a significant increase in the number of granulocyte-macrophage progenitors (GMP) with a concomitant loss of the megakaryocyte-erythroid progenitors (MEP) and slight reduction in the number of common myeloid progenitors (CMP), which is consistent with the condition of skewed myeloid differentiation occurring in MDS patients with higher risk of leukemic transformation. Transplantation experiments of long-term hematopoietic stem cells isolated from telomere dysfunctional mice into wild type congenic recipients revealed that the level of donor-derived skewed myeloid differentiation was comparable to that observed at steady state in the same telomere dysfunctional mice before transplantation, suggesting that impaired progenitor differentiation occurred as a result of cell intrinsic defects of telomere dysfunctional hematopoietic cells. In the setting of telomere dysfunction, somatic in vivo and in vitro telomerase reactivation reduced DNA damage signaling and specifically reversed defective differentiation and MDS phenotypes. Unbiased transcriptomic network analyses of CMP with telomere dysfunction revealed profound down-regulation of genes in the mRNA splicing and processing pathways which was rescued by telomerase reactivation, indicating that telomere dysfunction-induced DNA damage response can impact on the expression of genes involved in splicing regulation. RNA-seq analysis of telomere dysfunctional CMP suggested altered splicing activity at the level of exon usage and identified aberrantly spliced variants of genes implicated in chromatin remodeling, and histone modifications. The prominence of aberrantly spliced epigenetic regulators prompted us to hypothesize that there was a link between impaired myeloid differentiation and aberrant splicing patterns as a result of telomere dysfunction-induced repression of splicing components. In conclusion, our studies have revealed an unanticipated link between telomere biology, RNA splicing, and MDS pathogenesis and support the development of strategies designed to modulate the downstream targets of splicing alterations in specific hematopoietic populations. DisclosuresNo relevant conflicts of interest to declare.

Glucose substitution prolongs maintenance of energy homeostasis and lifespan of telomere dysfunctional mice.

DNA damage and telomere dysfunction shorten organismal lifespan. Here we show that oral glucose administration at advanced age increases health and lifespan of telomere dysfunctional mice. The study reveals that energy consumption increases in telomere dysfunctional cells resulting in enhanced glucose metabolism both in glycolysis and in the tricarboxylic acid cycle at organismal level. In ageing telomere dysfunctional mice, normal diet provides insufficient amounts of glucose thus leading to impaired energy homeostasis, catabolism, suppression of IGF-1/mTOR signalling, suppression of mitochondrial biogenesis and tissue atrophy. A glucose-enriched diet reverts these defects by activating glycolysis, mitochondrial biogenesis and oxidative glucose metabolism. The beneficial effects of glucose substitution on mitochondrial function and glucose metabolism are blocked by mTOR inhibition but mimicked by IGF-1 application. Together, these results provide the first experimental evidence that telomere dysfunction enhances the requirement of glucose substitution for the maintenance of energy homeostasis and IGF-1/mTOR-dependent mitochondrial biogenesis in ageing tissues.

Increased expression of stathmin and elongation factor 1α in precancerous nodules with telomere dysfunction in hepatitis B viral cirrhotic patients

BackgroundTelomere dysfunction is important in carcinogenesis, and recently, stathmin and elongation factor 1α (EF1α) were reported to be up-regulated in telomere dysfunctional mice.MethodsIn the present study, the expression levels of stathmin and EF1α in relation to telomere length, telomere dysfunction-induced foci (TIF), γ-H2AX, and p21WAF1/CIP1 expression were assessed in specimens of hepatitis B virus (HBV)-related multistep hepatocarcinogenesis, including 13 liver cirrhosis specimens, 14 low-grade dysplastic nodules (DN), 17 high-grade DNs, and 14 hepatocellular carcinomas (HCC). Five normal liver specimens were used as controls. TIF were analyzed by telomere fluorescent in situ hybridization (FISH) combined with immunostaining, while the protein expressions of stathmin, EF1α, γ-H2AX, and p21WAF1/CIP1 were detected by immunohistochemistry.ResultThe expressions of stathmin and EF1α gradually increased as multistep hepatocarcinogenesis progressed, showing the highest levels in HCC. Stathmin mRNA levels were higher in high-grade DNs than normal liver and liver cirrhosis, whereas EF1α mRNA expression did not show such a difference. The protein expressions of stathmin and EF1α were found in DNs of precancerous lesions, whereas they were absent or present at very low levels in normal liver and liver cirrhosis. Stathmin histoscores were higher in high-grade DNs and low-grade DNs than in normal liver (all, P < 0.05). EF1α histoscores were higher in high-grade DNs than in normal liver and liver cirrhosis (all, P < 0.05). Stathmin mRNA levels and histoscores, as well as EF1α histoscores (but not mRNA levels), were positively correlated with telomere shortening and γ-H2AX labeling index (all, P < 0.05). EF1α histoscores were also positively correlated with TIF (P < 0.001). Significantly greater inactivation of p21WAF1/CIP1 was observed in low-grade DNs, high-grade DNs, and HCC, compared to liver cirrhosis (all, P < 0.05). p21WAF1/CIP1 labeling index was inversely correlated with TIF, stathmin mRNA level, and EF1α histoscore (all, P < 0.05).ConclusionStathmin and EF1α are suggested to be closely related to telomere dysfunction, DNA damage, and inactivation of p21WAF1/CIP1 in HBV-related multistep hepatocarcinogenesis. Accordingly, assessment of stathmin and EF1α levels as a reflection of telomere dysfunction may be helpful in evaluating the biological characteristics of precancerous hepatic nodules in hepatitis B viral cirrhotic patients.

Telomere dysfunctional environment induces loss of quiescence and inherent impairments of hematopoietic stem cell function

Previous studies have shown that telomere dysfunction induces alteration in the systemic (circulatory) environment impairing the differentiation of hematopoietic stem cells (HSCs) but these defects can be reverted by re-exposing HSCs to an environment with functional telomeres. In contrast, HSC intrinsic telomere dysfunction induces permanent and irreversible limitations in the repopulation capacity partially depending on the induction of checkpoints such as cell cycle arrest, differentiation, or apoptosis. It is currently unknown whether telomere dysfunctional environment can induce irreversible, cell intrinsic defects impairing the function of HSCs. Here, we analyzed the functional reserves of murine, wild-type HSCs with intact telomeres that were transiently exposed to a telomere dysfunctional environment (late generation telomerase knockout mice) or to an environment with functional telomeres (wild-type mice). The study shows that the telomere dysfunctional environment leads to irreversible impairments in the repopulation capacity of wild-type HSCs. The telomere dysfunctional environment impaired the maintenance of HSC quiescent. Moreover, the study shows that alterations in the systemic (circulatory) environment rather than the bone stromal niche induce loss of stem cell quiescence and irreversible deficiencies of HSCs exposed to a telomere dysfunctional environment.

Telomere Shortening Impairs Regeneration of the Olfactory Epithelium in Response to Injury but Not Under Homeostatic Conditions

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc−/−) with short telomeres compared to wild type mice (mTerc+/+) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc−/− mice compared to mTerc+/+ mice. Seven days after chemical induced damage, G3 mTerc−/− mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc+/+ mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people.

CHK2‐independent induction of telomere dysfunction checkpoints in stem and progenitor cells

Telomere shortening limits the proliferation of primary human fibroblasts by the induction of senescence, which is mediated by ataxia telangiectasia mutated-dependent activation of p53. Here, we show that CHK2 deletion impairs the induction of senescence in mouse and human fibroblasts. By contrast, CHK2 deletion did not improve the stem-cell function, organ maintenance and lifespan of telomere dysfunctional mice and did not prevent the induction of p53/p21, apoptosis and cell-cycle arrest in telomere dysfunctional progenitor cells. Together, these results indicate that CHK2 mediates the induction of senescence in fibroblasts, but is dispensable for the induction of telomere dysfunction checkpoints at the stem and progenitor cell level in vivo.

Abstract 2972: Intestinal p53 deletion leads to accumulation of chromosomal instability without promoting tumor formation

Abstract Background: Colorectal cancer is after lung cancer the second biggest cancer threat for humans and the tumor burden is rising steadily during human ageing. One important issue during human ageing is the accumulation of DNA damage inducing a p53 dependent response leading to p21-dependent senescence or apoptosis. It has been shown that deletion of DNA repair proteins as well as other components of the DNA damage pathway leads to tumor formation. Methods: In this approach we investigated the role of the tumor suppressor p53 with and without telomere dysfunction in intestinal tumor formation. Late generation of iG4 mTerc−/−; p53lox/lox and iF1 mTerc+/− p53lox/lox mice were crossed to Villin-Cre-ERT2 transgenic mouse model. In 1 month old mice Tamoxifen was injected on 5 consecutive days leading to functional deletion of the Trp53 gene. Results: In aging IF1 Terc+/− p53 deletion did not induce an obvious intestinal phenotype including tumor formation until 24 months. In contrast to p21 deletion the deletion of p53 significantly shortened the lifespan of iG4 Terc−/− Trp53−/− animals compared to iG4 Terc−/− Trp53+/+. This reduction correlated with earlier appearance of crypt atrophy and weight loss but was not associated with tumor formation. Deletion of the Trp53 gene resulted in an abrogation of the p21-dependent senescence and accumulation of DNA damage in the intestinal epithelium of both p53 deleted cohorts. Moreover this correlated with an accumulation of intestinal stem cells carrying high levels of DNA damage. On contrary to p21 deletion the deletion of p53 impaired the depletion of chromosomal instable intestinal stem cells in aging telomere dysfunctional mice. Conclusion: These results provide first proof that deletion of p53 is not able to promote tumorigenesis in the intestinal epithelium of mice and that p53 induction has a protective role preventing accumulation of damaged intestinal stem cells in aging telomere dysfunctional mice. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2972.

ELISA for Aging Biomarkers Induced by Telomere Dysfunction in Human Plasma

Background. We identified cathelicidin related antimicrobial protein (CRAMP) secreted from telomere dysfunctional bone marrow cells of late generation telomerase knockout mice (G4mTerc−/−), increased in blood and various tissues. It can represented human aging and disease. The main aim of this study is to investigate the sensitive direct enzyme-linked immunosorbent assay (ELISA) method to analyze the human aging and disease in plasma and the detailed methods to quantify the direct ELISA of these aging biomarkers. Methods. Telomere lengths of 50 healthy persons are measured with real-time PCR in blood cells. Plasma samples from all subjects are analyzed using direct ELISA. Results. From 25 years old person to 78 years, the telomere length becomes shorter during aging. In blood plasma, the expression levels of CRAMP increases during human aging. There is the reverse correspondence between the telomere length and the plasma CRAMP level. We also find that the fresh plasma, the frozen plasma which thawed less than 3 times, and the plasma kept in the room temperature less than 3 hours are better for the ELISA analyze of CRAMP in the plasma. Conclusion. This CRAMP ELISA could become a powerful tool for investigating the relationship between human aging and telomere length shortening.

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Keeping your senescent cells under control

Keeping your senescent cells under control

Sod2 haploinsufficiency does not accelerate aging of telomere dysfunctional mice

Telomere shortening represents a causal factor of cellular senescence. At the same time, several lines of evidence indicate a pivotal role of oxidative DNA damage for the aging process in vivo. A causal connection between the two observations was suggested by experiments showing accelerated telomere shorting under conditions of oxidative stress in cultured cells, but has never been studied in vivo. We therefore have analysed whether an increase in mitochondrial derived oxidative stress in response to heterozygous deletion of superoxide dismutase (Sod2(+/-)) would exacerbate aging phenotypes in telomere dysfunctional (mTerc(-/-)) mice. Heterozygous deletion of Sod2 resulted in reduced SOD2 protein levels and increased oxidative stress in aging telomere dysfunctional mice, but this did not lead to an increase in basal levels of oxidative nuclear DNA damage, an accumulation of nuclear DNA breaks, or an increased rate of telomere shortening in the mice. Moreover, heterozygous deletion of Sod2 did not accelerate the depletion of stem cells and the impairment in organ maintenance in aging mTerc(-/-) mice. In agreement with these observations, Sod2 haploinsufficiency did not lead to a further reduction in lifespan of mTerc(-/-) mice. Together, these results indicate that a decrease in SOD2-dependent antioxidant defence does not exacerbate aging in the context of telomere dysfunction.

Diminished lifespan and acute stress-induced death in DNA-PKcs-deficient mice with limiting telomeres

An adequate and appropriate response to physiological and pathophysiological stresses is critical for long-term homeostasis and viability of the aging organism. Previous work has pointed to the immune system, telomeres and DNA repair pathways as important and distinct determinants of a normal healthy lifespan. In this study, we explored the genetic interactions of telomeres and DNA-PKcs, a protein involved in non-homologous end-joining (NHEJ) and immune responses, in the context of a key aspect of aging and lifespan--the capacity to mount an acute and appropriate immune-mediated stress response. We observed that the combination of DNA-PKcs deficiency and telomere dysfunction resulted in a shortened lifespan that was reduced further following viral infection or experimental activation of the innate immune response. Analysis of the innate immune response in the DNA-PKcs-deficient mice with short dysfunctional telomeres revealed high basal serum levels of tumor necrosis factor alpha (TNFalpha) and hyper-active cytokine responses upon challenge with polyinosinic-polycytidylic acid (poly-IC). We further show that serum cytokine levels become elevated in telomere dysfunctional mice as a function of age. These results raise speculation that these genetic factors may contribute to misdirected immune responses of the aged under conditions of acute and chronic stress.

Telomere dysfunction alters the chemotherapeutic profile of transformed cells

Telomerase inhibition has been touted as a novel cancer-selective therapeutic goal based on the observation of high telomerase levels in most cancers and the importance of telomere maintenance in long-term cellular growth and survival. Here, the impact of telomere dysfunction on chemotherapeutic responses was assessed in normal and neoplastic cells derived from telomerase RNA null (mTERC(-/-)) mice. Telomere dysfunction, rather than telomerase per se, was found to be the principal determinant governing chemosensitivity specifically to agents that induced double-stranded DNA breaks (DSB). Enhanced chemosensitivity in telomere dysfunctional cells was linked to therapy-induced fragmentation and multichromosomal fusions, whereas telomerase reconstitution restored genomic integrity and chemoresistance. Loss of p53 function muted the cytotoxic effects of DSB-inducing agents in cells with telomere dysfunction. Together, these results point to the combined use of DSB-inducing agents and telomere maintenance inhibition as an effective anticancer therapeutic approach particularly in cells with intact p53-dependent checkpoint responses.

Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation

Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.