Progressive Telomere Erosion Research Articles

Erythrocyte membrane-camouflaged nanodelivery strategy enhances gene editing efficiency of Cas9 RNP for boosting tumor senescence

CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ). However, the inefficient precise editing of genome sequences remains a challenge for clinical treatment. Herein, an erythrocyte membrane (EM)-camouflaged and tumor microenvironment (TME)-responsive nanosystem was constructed to achieve synergistic combination of enhanced Cas9 ribonucleoprotein (RNP) gene editing efficiency and telomere dysfunction, and thus precise induced tumor inhibition. The EM-camouflaged nanosystem escaped from the reticuloendothelial system (RES) and was endocytosed by tumor cells. Azidothymidine (AZT) was gradually released as the erythrocyte shell ablation, which competitively prevented the binding of single nucleotides to the telomere replication template TR, terminate the extension of the telomerase DNA chain. Additionally, Cas9 RNP was released through disulfide bond cleavage and entered into the nucleus to realize genome editing of telomerase reverse transcriptase (TERT) gene. Simultaneous, AZT enhanced NHEJ pathway to improve the gene editing efficiency of Cas9 RNP, which further promoted the progressive telomere erosion and thus induced tumor inhibition. Overall, this biomembrane-camouflaged nanosystem realizes enhanced Cas9 RNP gene editing efficiency with high-level biosafety and promotes tumor senescence by triggering telomere disorder, which provides strategy for improving the gene editing efficiency to induce senescence of tumor cells.

Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline.

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

Transgenerational telomere erosion in the monogametic sex: human telomeres progressively erode in the female germline and do not lengthen in aged testes

Long telomeres, the protective caps of eukaryotic chromosomes, which erode during aging, have been the symbol of youth and regenerative potential. It therefore came as a surprise, when several cross-sectional studies reported that telomeres in sperm cells of old men are longer than in young men and that paternal age is positively linked to telomere length of children. To explain the puzzling data, several theories have been put forward, from Darwinian selection to high telomerase activity or alternative telomere lengthening in sperms of geriatrics. Unfortunately, the idea of a birth-cohort effect has been ignored, despite existing theoretical models and despite findings of progressive telomere erosion between human generations. The old theoretical model of progressive telomere erosion in the female germline is discussed here and updated with the hypothesis that progressive telomere erosion is tied to the monogametic sex in all higher animals. Longitudinal studies of germline telomere length in humans are much needed, since a limited regenerative capacity of somatic tissues will most likely result in an increase in and earlier onset of the so-called age-associated diseases.

Telomere Damage Maintains Hematopoietic Stem Cells (HSCs) in an Activated Metabolic State, Which Compromises Their Self-Renewal Capability

DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown.Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit+Sca+Lin-CD34-flk2-CD150+CD48-CD41-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability.To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERTER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERTER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p<10-3) that was associated with a decreased number of the lymphoid-biased MPP4 cells, and an increased number of both megakaryocyte-biased MPP2 and myeloid-biased MPP3 cells. HSC exhaustion and increased myeloid-to lymphoid output were reminiscent of stressed hematopoiesis and premature aging. G5/G6 HSCs exhibited a significant accumulation of telomere dysfunction-induced foci (p<10-5) but did not display increased levels of apoptosis in steady-state conditions.HSC exhaustion could result from apoptosis and/or senescence induced by telomere damage in HSCs entering the cell cycle or from an altered balance between self-renewal and differentiation. To distinguish between these two possibilities, we first investigated the effect of inducing young G5/G6 HSCs out of a homeostatic quiescent state. By tracking the real-time changes in the expression level of annexin V on HSCs induced to differentiate towards the myeloid lineage, we found that apoptosis was not the primary fate of G5/G6 HSCs upon entry into the cell cycle. Similarly, in vivo treatment with poly I:C induced the G5/G6 HSCs to enter into the cell cycle at the same rate as that of the G0 mice without inducing apoptosis. Transcriptomic analysis of poly I:C-treated G0 and G5/G6 HSCs, compared with vehicle-treated controls, revealed a significant enrichment of genes involved in the regulation of the cell cycle and platelet production, which is consistent with previous findings showing that megakaryocyte differentiation of HSCs occurs in response to poly I:C to replenish platelets that are lost during inflammatory insult. Importantly, we did not observe any significant change in gene expression between G0 and G5/G6 HSCs isolated from poly I:C-treated mice, which confirmed that telomeric damage did not limit HSCs' proliferation potential by activating programs of senescence or apoptosis.Next, we evaluated the capability of single HSCs isolated from G0 or G5/G6 mice to either self-renew or differentiate. An evaluation of Numb inheritance and expression in G0 and G5/G6 HSCs (n=133 and n=113, respectively) induced to proliferate in vitro showed that G5/G6 HSCs had a 2-fold lower frequency of symmetric self-renewal division (p<10-3) and a concomitant 2-fold higher frequency of symmetric commitment (p<10-4). Accordingly, PB analysis revealed that the CD45.2-derived reconstitution was severely compromised in mice competitively transplanted with G5/G6 HSCs (0.26% vs 77%; p<10-3). Single cell RNA-seq analysis of G0 and G5/G6 HSCs followed by the differential analysis of the clusters showed that 40% of G5/G6 HSCs were in an activated metabolic state associated with hyperactive OXPHOS and ROS signaling pathways, which are directly involved in HSC functional decline. Finally, we reactivated telomerase to investigate the possibility of restoring normal HSC function upon elimination of damage. Single cell RNA-seq and functional studies are ongoing to evaluate whether HSCs' activated metabolic state and compromised self-renewal capability are reversible processes.This study challenges the concept that telomeric damage limits HSC's proliferative potential and offers unparalleled opportunities for unraveling regenerative strategies to ameliorate their decline. DisclosuresColla:Abbvie: Research Funding.

Leukocyte telomere length in mild cognitive impairment and Alzheimer's disease patients

Numerous studies have reported an association between shortened leukocyte telomere length (LTL) and increased risk of Alzheimer's disease (AD). In this study we investigated the relationship between LTL and AD development, including in the analysis patients with amnestic mild cognitive impairment (aMCI), a clinical entity considered prodromal of AD. LTL (T/S ratio) was measured in patients with AD (n=61) or aMCI (n=46), and compared with LTL of age-matched controls (n=56). Significant LTL differences were observed between controls, aMCI and AD patients (p<0.0001), with mean LTL values (±s.d) in the order: AD patients (0.70±0.15)<aMCI patients (0.80±0.14)<controls (0.88±0.15). A positive relationship (linear regression p=0.004) was observed between LTL and cognitive performance (measured by Mini Mental State Examination score). LTL did not differ by APOE genotype. The shortened LTL observed in AD patients appears to stem from progressive telomere erosion possibly correlated with the cognitive decline characterizing conversion from aMCI to AD. LTL reduction, indicating active cell proliferation, may reflect immune system involvement in AD pathogenesis.

Enhancement of telomere maintenance using telomerase activation in human cells

Enhancement of telomere maintenance using telomerase activation in human cells

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

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

Progressive Telomere Shortening Is Part of the Natural History of Chronic Lymphocytic Leukemia (CLL) and Impacts Clinical Outcome

Abstract 2845 Introduction:Telomere length (TL) at diagnosis has been established as an independent outcome predictor in CLL (Rossi et al Leukemia 2009). However data on TL dynamics over time are scant and anedoctal. Aim of this study was to evaluate telomere dynamics in the natural history of CLL. This issue has been here addressed on a series of 88 CLL patients (pts). Methods:25 pts were assessed for TL at diagnosis and at relapse and 63 pts had two determinations during the “watch and wait” (WW) phase. The series was fully characterized in terms of Binet stage, ALC, CD38, ZAP-70, IGHV mutational status (IGHV-MS), stereotyped receptors, cytogenetics and detailed clinical history. LDH, B2-microglobulin, p53 mutations and CD49d were available in more than 70% of pts. Treatment-free survival (TFS) analysis was performed exclusively in pts undergoing kinetic evaluation during the WW phase. This population had a median follow-up of 73 months and a median TFS of 130 months. TL was analyzed as previously described (Rossi et al Leukemia 2009; Ladetto et al Blood 2004). Median time between TL determinations was 44 months (range 12–231). Telomere loss was calculated in terms of both absolute loss (AL) and yearly loss (YL). Continuous variables were compared by the Mann-Whitney test, while TFS by the stratified Kaplan-Meyer method. Results:Telomeres were shorter at follow-up compared to baseline with a median loss of 651bp (range +493bp, −5874bp; p<0.001) (Fig 1A). AL and YL were greater in cases with higher baseline TL while those with short telomeres at diagnosis had only modest additional erosion (p=ns for pts in the 25th lowest percentile) (Fig 1B). Telomere loss over time was noticeable both in pts assessed at diagnosis and at relapse as well as in those assessed during the WW phase, but clearly inferior in the former subgroup (YL of −61bp, p<0.05 and −210bp, p<0.01, respectively), possibly due to the higher number of patients with short telomeres. AL and YL did not correlate with any available clinical or biological parameter, with the exception of a positive association with IGHV-MS (p<0.05). Pts with baseline TL shorter than the validated cut-off value of 5000bp (Rossi et al Leukemia 2009) were associated to an inferior TFS (median TFS 41 months vs 182 months; p<0.0001) as expected. Moreover also Binet status and IGVH-MS were predictive for TFS in this series. Surprisingly, also an YL above the median value (-210bp) appeared to be predictive for an inferior TFS (median TFS 82 months vs 182 months; p<0.05) (Fig 1C), despite being more common in pts with longer telomeres and VH-mutated IgH genes. Following stratification of pts according to baseline TL (< or > 5000bp), YL was predictive for TFS in both pts subgroups (Fig 1D i.e. baseline TL >5000bp; YL ≥ −210bp vs YL <-210bp: TFS 88 months vs not reached p<0.01. Figure 1E i.e. baseline TL <5000bp; YL ≥-210bp vs TL <210bp: median TFS 36 months vs 50 months, p<0.01). Conclusions:The results of the first systematic analysis on TL dynamics in CLL indicate the following: i) progressive telomere erosion occurs as part of the natural history of CLL; ii) telomere loss is more pronounced when baseline TL is higher; iii) accelerated telomeric loss associates to an inferior TFS. The results described in the present analysis corroborate basic studies suggesting that telomere disruption represents a critical step associated to CLL progression. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Telomere sister chromatid exchange and the process of aging

Telomeres are a hotspot for sister chromatid exchange (T-SCE). Any biological consequence of this form of instability remained obscure until quantitative modeling revealed a link between elevated T-SCE rates and accelerated cellular replicative senescence. This work strongly suggests that progressive telomere erosion is not the only determinant of replicative capacity; instead, T-SCE need to be considered as an independent factor controlling colony growth and senescence. Additionally high T-SCE rates have been observed in cells with deficiencies in WRN and BLM, the genes that are defective in Werner's and Bloom's syndromes, implying a connection to premature aging. In this Research Perspective we will explore some of the implications this recent work has for human health.

Telomerase Immortalized Human Amnion- and Adipose-Derived Mesenchymal Stem Cells: Maintenance of Differentiation and Immunomodulatory Characteristics

Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increased the feasibility of SC-based therapies. Sources such as adipose tissue and amnion offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scaleable production and long-term analysis of these cells. Here we report the establishment and characterization of two human amnion-derived and two human adipose-derived SC lines immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT). hTERT overexpression resulted in continuously growing SC lines that were largely unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differentiation potential for up to 87 population doublings. While all generated lines showed equal immunomodulation compared to the parental cells, one of the amnion-derived immortalized lines resulted in significantly increased immunogenicity. Although telomerase proves as important tool for immortalizing cells, our data emphasize the need for careful and standardized characterization of each individual cell population for cell banks.

Matrix Metalloproteinase-7 and the 20 S Proteasome Contribute to Cellular SenescenceA presentation from the 11th Joint Meeting of the Signal Transduction Society (STS), Signal Transduction: Receptors, Mediators and Genes, Weimar, Germany, 1 to 3 November 2007.

A detailed understanding of aging and senescence is limited by the complex interplay of the effects of extracellular and environmental stimuli on cellular metabolic, mutational, and epigenetic phenomena. For example, STASIS (stress or aberrant signaling-induced senescence) is affected by the exposure to free radicals and conditions that cause an increased cellular production of reactive oxygen species (ROS) during normal life span. In addition, progressive telomere erosion and telomeric dysfunction contribute to a cellular feature termed replicative or cellular senescence. To focus on distinct cellular pathways that contribute to these different forms of senescence, we investigated the reversible differentiation and aging process of the human U937 leukemia cell line. This was compared to cellular senescence that occurred in normal primary human mammary epithelial cells (HMECs). These two cell systems revealed an important role of the proteolytic activity of the 20S proteasome and its activation by the nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) during "retrodifferentiation" and rejuvenation of the leukemic cells. Moreover, reduced extracellular proteolytic activity of certain matrix metalloproteinases-for example, MMP-7-is associated with accelerated aging and senescence in normal HMECs.

Identification of a quinoxaline derivative that is a potent telomerase inhibitor leading to cellular senescence of human cancer cells.

Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity allows cells to maintain their telomeric DNA and contributes to the indefinite replicative capacity of cancer cells. Telomerase is expressed in most cancer cells, but not in normal somatic cells, suggesting that telomerase is an attractive target for cancer chemotherapy. Here we screened a chemical library for inhibition of human telomerase, and identified 2,3,7-trichloro-5-nitroquinoxaline (TNQX) as a potent inhibitor. TNQX showed a potent inhibitory effect, with 50% inhibition at approximately 1.4 microM, and did not inhibit DNA and RNA polymerases, including retroviral reverse trancriptase. A series of enzyme kinetic experiments suggested that TNQX is a mixed-type non-competitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate (TS) primer and the dNTPs. Long-term cultivation of the MCF7 cell line with a drug concentration that did not cause acute cytotoxicity resulted in progressive telomere erosion followed by an increased incidence of chromosome abnormalities and induction of the senescence phenotype. The results presented here indicate that TNQX is a highly potent and selective anti-telomerase agent with good potential for further development as a promising anti-cancer agent.

Potent inhibition of human telomerase by nitrostyrene derivatives.

Telomerase activity is expressed in most types of cancer cells but not in normal somatic cells, suggesting that telomerase may be an important target for cancer chemotherapy. Inhibition of telomerase results in telomere erosion, leading to the subsequent growth arrest of cancer cells followed by senescence or cell death. In this study, we screened a chemical library for the inhibition of human telomerase, identifying three inhibitors. All compounds contained a common nitrostyrene moiety conjugated to different side chains. One of these compounds, 3-(3,5-dichlorophenoxy)-nitrostyrene (DPNS), showed the most potent inhibitory effect, with 50% inhibition at approximately 0.4 microM and did not inhibit DNA and RNA polymerases, including retroviral reverse transcriptase. A series of enzyme kinetic experiments suggests that DPNS is a mixed-type noncompetitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate primer and the deoxynucleoside-5'-triphosphates. Extensive propagation of cancer cell line in the presence of DPNS resulted in progressive telomere erosion followed by the induction of senescence phenotype. The results presented here demonstrate that DPNS is a highly selective, small-molecule telomerase inhibitor in vitro and could be useful as a lead molecule for the further development of inhibitors with an improved potential for efficacy in vivo.

Progressive Telomere Shortening in Aplastic Anemia

Improved survival in aplastic anemia (AA) has shown a high incidence of late clonal marrow disorders. To investigate whether accelerated senescence of hematopoietic stem cells might underlie the pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal controls. TRF lengths in the patient group were significantly shorter for age than normals (P &lt; .0001). Telomere shortening was apparent in both granulocyte and mononuclear cell fractions, suggesting loss at the level of the hematopoietic stem cell. In patients with acquired AA with persistent cytopenias (n = 40), there was significant correlation between telomere loss and disease duration (r = −.685; P &lt; .0001), equivalent to progressive telomere erosion at 216 bp/yr, in addition to the normal age-related loss. In patients who had achieved normal full blood counts (n = 20), the rate of telomere loss had apparently stabilised. There was no apparent association between telomere loss and secondary PNH (n = 13). However, of the 5 patients in the study with TRF less than 5.0 kb, 3 had acquired cytogenetic abnormalities, suggesting that telomere erosion may be relevant to the pathogenesis of MDS in aplastic anemia.

Progressive Telomere Shortening in Aplastic Anemia

AbstractImproved survival in aplastic anemia (AA) has shown a high incidence of late clonal marrow disorders. To investigate whether accelerated senescence of hematopoietic stem cells might underlie the pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal controls. TRF lengths in the patient group were significantly shorter for age than normals (P < .0001). Telomere shortening was apparent in both granulocyte and mononuclear cell fractions, suggesting loss at the level of the hematopoietic stem cell. In patients with acquired AA with persistent cytopenias (n = 40), there was significant correlation between telomere loss and disease duration (r = −.685; P < .0001), equivalent to progressive telomere erosion at 216 bp/yr, in addition to the normal age-related loss. In patients who had achieved normal full blood counts (n = 20), the rate of telomere loss had apparently stabilised. There was no apparent association between telomere loss and secondary PNH (n = 13). However, of the 5 patients in the study with TRF less than 5.0 kb, 3 had acquired cytogenetic abnormalities, suggesting that telomere erosion may be relevant to the pathogenesis of MDS in aplastic anemia.

Telomerase activity in human proliferative breast lesions.

Telomerase, a cellular reverse transcriptase, has been detected in the majority of human malignant tumors, where it provides an escape mechanism from proliferative limitations due to progressive telomere erosion with each cell division. In this study, we used a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase assay standard for the detection and semiquantitative analysis of 98 single frozen sections of normal breast tissue and benign and malignant breast lesions on an automated laser-fluorescence sequencer. Telomerase activity was detected in 36 of 40 (90%) infiltrating breast carcinomas, whereas no activity was found in nonmalignant breast tissues including blunt duct adenosis, papilloma, ductal hyperplasia and atypical ductal hyperplasia. However, telomerase activity was detected in 59% of ductal in situ carcinomas, suggesting that telomerase reactivation is an early event in breast carcinogenesis. We found a positive correlation between telomerase activity levels and cell proliferation determined by MIB1 immunostaining. No correlation, however, could be demonstrated between telomerase activity and other known breast cancer prognostic indicators. Telomerase activity was also detected in 60% of fibroadenomas indicating that careful interpretation of analysis of telomerase activity in fine needle aspirates is required, since low telomerase activity may not necessarily be an indicator of malignancy in breast tissue.