Average Telomere Length Research Articles

Telomere Restriction Fragment (TRF) Analysis.

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Impact of chemotherapy on telomere length in sporadic and familial breast cancer patients.

Recently, we observed that telomeres of BRCA1/2 mutation carriers were shorter than those of controls or sporadic breast cancer patients, suggesting that mutations in these genes might be responsible for this event. Given the contradictory results reported in the literature, we tested whether other parameters, such as chemotherapy, could be modifying telomere length (TL). We performed a cross-sectional study measuring leukocyte TL of 266 sporadic breasts cancer patients treated with first-line chemotherapy, with a median follow-up of 240 days. Additionally, we performed both cross-sectional and longitudinal studies in a series of 236 familial breast cancer patients that included affected and non-affected BRCA1/2 mutation carriers. We have measured in leukocytes from peripheral blood: the TL, percentage of short telomeres (<3 kb), telomerase activity levels and the annual telomere shortening speed. In sporadic cases we found that chemotherapy exerts a transient telomere shortening effect (around 2 years) that varies depending on the drug combination. In familial cases, only patients receiving treatment were associated with telomere shortening but they recovered normal TL after a period of 2 years. Chemotherapy affects TL and should be considered in the studies that correlate TL with disease susceptibility.

Long-Term Outcomes of Accelerated Telomere Shortening in Acquired Aplastic Anemia

Introduction: Accelerated telomere shortening (TS) in blood cells has been well documented in both dyskeratosis congenita and acquired aplastic anemia (AA). The present study investigated the incidence and long-term outcomes of TS in AA patients treated with ATG plus cyclosporine immunosuppressive therapy (IST).Methods: A total of 53 patients (31 M and 22 F, median age 24, 9-62) with moderate (MAA 15), severe (SAA 22) and very severe (vSAA 16) AA were included in cross-sectional study of telomere length (TEL) between 2008 and 2009 and then followed prospectively for 5 years. Treatment-naïve, recovering after IST and refractory AA were in 13, 35 and 5 patients respectively. There were no cases with physical abnormalities or family history of hematological disorders. TEL in granulocytes was assessed by Flow-FISH method with calculation of absolute and age-adjusted (DeltaTEL) values. TS was defined as absolute TEL value below the normal 99% confidence interval in age matched healthy donors (n=71). Outcome measures were response rate in treatment-naïve patients, cumulative incidence (CI) of relapse and clonal evolution, overall (OS) and failure-free survival (FFS). Adult patients or parents of children signed informed consent.Results: TS was detected in 24 patients (45%) with a median DeltaTEL of -1.715 kbp compared to -0.009 kbp in 29 patients with normal telomere length (TN) (Table 1).Table 1Patient and disease characteristics in cross-sectional studyTS (n=24)TN (n=29)PDeltaTEL, kbp, median (range)-1.715 (-5.100 – -0.983)-0.009 (-0.760 – 4.142)<0.001Age, years, median (range)22.5 (9-37)26 (14-62)0.102Gender, M/F, n15/916/130.780Severity, n (%)MAA10 (42%)5 (17%)0.0485SAA/vSAA14 (58%)24 (83%)Response to prior IST, n (%)Treatment-naïve4 (17%)9 (31%)0.338Partial response7 (29%)9 (31%)Complete response8 (33%)11 (38%)Refractory5 (21%)0 (0%)0.047PNH positive, n/n (%)12/24 (50%)13/28 (46%)1.000Interval between AA onset and IST, days, median (range)92 (17-508)102 (26-585)0.848Interval between AA onset and TEL testing, days, median (range)1093 (49-3792)785 (64-3494)0.173TS was associated with MAA (p = 0.049) and refractory disease (p=0.047) in cross-sectional study. No significant differences in gender, median age, baseline blood counts, PNH positivity or interval from AA onset and IST were observed between the two groups.Median follow-up after IST start and TEL testing were 90 and 67 months respectively. Response rates to IST were similar in TS (3/4, 75%) and TN (6/9, 67%) patients evaluated prospectively (p=1.0). TS and TN patients showed similar 10-year CI of relapse (16% vs 14%, p=0.905) and hemolytic PNH (28% vs 20%, p=0.532). However, all 3 cases of MDS /AML occurred in patients with TS with CI of 15.4% vs. 0% in the TN (p = 0.063). Compared patient groups have similar high probability of 10-year OS (TS 86.9% vs TN 92.6%, p=0.571) due to the large proportion of patients responding to treatment. The FFS rates were much lower (38.5% and 57.5% respectively, p=0.510).Conclusions: TS is a relatively common phenomenon in patients with otherwise typical acquired AA and more frequently detected in MAA and refractory to IST cases in cross-sectional study. Our data confirm the previously reported association of TS and increased risk of MDS/AML, which should be considered for a more careful monitoring and treatment planning, including allogeneic BMT as a curative approach. DisclosuresNo relevant conflicts of interest to declare.

Telomere Length in Inherited Bone Marrow Failure Syndromes

Background: Telomeres are short DNA repeats and a protein complex at chromosome ends that are essential for genome integrity. Telomeres are very short in patients with dyskeratosis congenita (DC) due to germline mutations in telomere biology genes. Telomere lengths (TL) were reported to be “short” in a few small studies of patients with other inherited bone marrow failure syndromes (IBMFS), specifically Fanconi anemia (FA), Diamond-Blackfan anemia (DBA), and Shwachman-Diamond syndrome (SDS), although their relation to the very short TL seen in DC was not clear, and various analytic methods were used.Objectives: 1) To compare TL in a cohort of patients with FA, DBA, and SDS, with TL in DC. 2) To determine whether any individuals with non-DC IBMFS had TL as short as in DC.Methods: Blood was obtained from 100 patients with DC, 30 FA, 34 DBA, and 14 SDS enrolled in the NCI IBMFS Cohort Study. TL was measured by flow cytometry with fluorescence in situ hybridization (flow-FISH) in 6 leukocyte subsets: granulocytes, total lymphocytes, CD45RA-positive/CD20-negative naïve T cells, CD45RA-negative memory T cells, CD20-positive B cells, and CD57-positive NK/NKT cells. “Very short” TL was below the 1st percentile for age (defined by 400 normal controls). The diagnosis of DC required very short TL in 3 of 4 lymphocyte lineages including total lymphocytes. Z-scores were used to adjust for age; a Z-score of -2.3 equals the 1st percentile. Canonical Variate Analysis (CVA) including all 6 Z-score measurements was used for group comparisons and outlier identification.Results: Two patients with FA and 1 each with DBA and SDS had TL slightly below the 1st percentile in 3 or more lymphocyte lineages. In univariate analysis, the mean of the DC TL Z-scores was approximately -4, versus approximately -0.5 in FA, DBA, and SDS. Most individual DC Z-score measurements were well below -2.3, and all were below zero (the mean for the control data). Seventy percent of the FA, DBA and SDS lymphocyte TL Z-scores clustered in the bottom half of the normal range (i.e. -2.3 to 0, p <0.001), compared with the expected 50% if the non-DC patients had essentially normal TLs. In addition, short lymphocyte TL correlated with severity of marrow failure in DC; there was a non-significant trend in FA and no trend in DBA or SDS. Further refinement of interpretation utilized the CVA analysis. The first canonical variate captured 89.7% of the total variance, and separated DC widely from the other three IBMFS. The second canonical variate captured 10% of the variance, and separated DBA, FA, and SDS. In the CVA analysis, only 2 patients with FA and 1 each with DBA and SDS had results closer to the mean for DC patients than to the mean for their own disorder.Conclusions: Only 5% of individuals with FA, DBA, or SDS had TL in the DC range, and thus non-DC IBMFS in general do not have “very short” TL. However, 70% of non-DC IBMFS patients did have average TLs below the average for normal individuals. Thus disorders of DNA repair or ribosome biogenesis may result in TL changes, but the deficit in TL is not nearly as extreme as in DC itself. DisclosuresNo relevant conflicts of interest to declare.

Predicting Response to Immunosuppressive Therapy By the Combination of Minor Paroxysmal Nocturnal Hemoglobinuria Clones and Lymphocyte Telomere Length in Children with Aplastic Anemia

Introduction: Acquired aplastic anemia (AA) is an immune mediated disease characterized by severe quantitative defects in stem cell number resulting in a hypocellular marrow and peripheral blood cytopenias. Immunosuppressive therapy (IST) using anti-thymocyte globulin (ATG) and cyclosporine A (CyA) is the standard therapy for patients with AA who lack a human leukocyte antigen (HLA)-matched sibling donor. Previous studies identified minor paroxysmal nocturnal hemoglobinuria (PNH) clones and short telomere length (TL) as predictive markers of the response to IST. However, the likelihood ratio of these markers was insufficient to influence clinical decisions. Here we assessed whether the combination of these markers could more accurately predict the response to IST.Methods: Between July 2001 and November 2013, 116 patients (65 boys and 51 girls) diagnosed with AA were examined to identify a minor population of PNH-type cells and TL of peripheral blood lymphocytes. Fifty-three patients received horse ATG (Lymphoglobulin, 15mg/kg/day for 5 days) and 63 received rabbit ATG (Thymoglobulin, 2.5−3.75 mg/kg/day for 5 days). CyA (6mg/kg/day) was started on day 1 and continued to at least day 180. The dose of CyA was adjusted to maintain trough levels between 100 and 200 ng/ml. Written informed consent was obtained from all patients’ parents. The ethics committee of each participating hospital approved the study. We used a flow cytometric assay to detect PNH-type granulocytes and red blood cells (RBCs). Presence of >0.007% CD13+ CD55− CD59− granulocytes and/or >0.009% glycophorin A+ CD55− CD59−RBCs was defined as PNH positive (PNH+), based on the results from healthy individuals. The average relative TL of peripheral lymphocytes was measured by flow-fluorescence in situ hybridization (flow-FISH) using a Telomere PNA Kit (Dako Cytomation). Based on our previous study, we defined −1.0 SD of age-adjusted controls as the cut-off value.Results: The median age at IST was 9.8 years (0.9−16.0). Disease severity of patients was assessed as moderate in 39, severe in 37, and very severe in 40, respectively. The causes of AA were idiopathic in 104 patients and hepatitis-associated in 12 patients. The median follow-up period from the time of IST was 36 months (range 1−134). Thirty-nine patients (34%) carried a minor PNH clone at diagnosis. Compared with healthy individuals, their median of TL was −1.08 SD (−4.01 to +3.01 SD). Overall, 60 of 116 patients (51.7%) responded to IST at 6 months after administration of ATG. In univariate analysis, the response rate at 6 months was higher in PNH+ patients than in PNH- patients (74.4% vs. 40.3%, respectively, p = 0.001). Longer TLs (≥−1.0 SD of age-adjusted control) were also associated with a favorable response to IST at 6 months (73.2% vs. 31.7%, p<0.001). In multivariate logistic regression analysis, PNH clone presence [odds ratio (OR), 4.41; 95% CI, 1.68−11.60; p = 0.003], longer TLs (OR, 5.59; 95% CI, 2.33−13.40; p < 0.001), and male gender (OR, 2.72; 95% CI, 1.12−6.62; p = 0.028) were found to be independent favorable predictors of response to IST at 6 months. Different types of ATG (horse vs. rabbit) did not affect response rate (OR, 1.94; 95% CI, 0.92−4.10; p = 0.084). According to these findings, the cohort was stratified into two groups: possible poor responders [PPR; PNH− and shorter TLs (<−1.0 SD), 45 patients] and possible good responders (PGR; PNH+ and/or longer TLs, 71 patients). The response rate at 6 months in the PPR group was as low as 24.4%, which was significantly lower than that in the PGR group (69.0%, p < 0.001). There was no significant difference in the incidence of relapse (0.0% vs. 20.8%, p=0.190) and clonal evolution (4.5% vs. 5.7%, p=1.000) between two groups. Five-year probabilities of transplant-free survival (TFS) and failure-free survival (FFS) were significantly lower in the PPR group than in the PGR group (TFS, 49.1% vs. 71.4%, p = 0.008; FFS, 42.6% vs. 57.3%, p = 0.020). However, 5-year overall survival showed no difference between the two groups (PPR, 97.7% vs. PGR, 95.5%, p = 0.910) because of the effective salvage therapies.Conclusion: Combination of the absence of a minor PNH clone and a short TL was an efficient predictor of poor response to IST. These patients had low chances to respond to IST, which challenges the current standard of treatment. Prospective clinical trials are required to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

Protective Cytotoxic Clonal T-Cells in Myeloma Have the Characteristics of Telomere-Independent Senescence Rather Than an Exhausted or Anergic Phenotype: Implications for Immunotherapy

Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function.Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p<0.01) and maintain high cytokine secretion. CFSE proliferation assays of flow sorted T-cell clones demonstrated a significant lack of proliferation after stimulation with MACS iBeads (n=12; p<0.0001). They failed to respond in vitro to cytokines such as IL- 2, IL-12, IL-15 and OX40. In contrast, the T-cell clones were proliferative in a group of long term myeloma survivors in whom such T-cells are universally present suggesting that the impaired proliferation may be reversible. Geneset enrichment analysis of mRNA expression from Affymetrix U133 plus 2.0 arrays demonstrated pleiotropic dysfunction in multiple intracellular signalling pathways involved with proliferation and T cell inactivation. Utilizing phospho-flow techniques we demonstrated reduced phosphorylated ERK levels in clonal vs non-clonal T-cells (n=8; p<0.002) as well as elevated levels of phosphorylated Smad2/3 (n=10; p<0.02) and Bcl-xL (n=7; p<0.04) .These findings suggest that the ERK pathway, associated with proliferation, was suppressed, the TGFβ pathway was activated and that the dysfunctional T-cell clones have a survival advantage due to apoptotic resistance. In contrast, normal SHP-2 and phosphorylated ZAP-70 levels suggested there was no defect in upstream TCR signaling.Clonal T-cells lacked CD28, PD-1, CTLA-4, LAG-3, CD160 and Tim-3 expression and therefore are neither exhausted nor anergic. However, telomere assessment by qPCR demonstrated telomere T/S ratios were normal length for age and not significantly less than autologous CD57+TCR-Vβ- cells. T-cell clones also had normal p38 expression suggesting that the senescence was not due to TNF-α inhibiting the p38/MAPK pathway.The cytotoxic T-cell clones in patients with myeloma have the characteristics of telomere-independent senescence. The normal telomere length suggests that the hypo-responsiveness is induced independent of telomeres, and is therefore potentially reversible, though the pleiotropic nature of this dysfunction might require the targeting of multiple intracellular signalling pathways. The lack of PD-1 and CTLA-4 expression suggests that immune checkpoint blockade in myeloma would be unable to reverse the T cell dysfunction. DisclosuresNo relevant conflicts of interest to declare.

Leukocyte telomere length and hemostatic factors in a South African cohort: the SABPA Study

Incident atherothrombotic disease is predicted by leukocyte telomere length, a marker of biological age, and hemostatic factor levels, indicating a hypercoagulable state. We hypothesized that shorter telomeres are associated with elevated circulating levels of hemostatic factors. We examined 171 South African (black) and 182 Caucasian (white) schoolteachers (mean age ± standard deviation, 48.5 ± 9.0 years; 50.4% women). Levels of fibrinogen, von Willebrand factor antigen (VWF:Ag), D-dimer and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) were measured in plasma, and values were log-transformed before analysis. Relative average telomere length (content of telomere PCR product/content of human β-globin PCR product ratio, i.e. telomere/single-copy gene ratio) was assessed with multiplex quantitative real-time PCRs. Multivariate analyses included demographics, metabolic factors, health behavior, and medication. Africans had shorter mean telomere length (0.82, 95% confidence interval [CI] 0.79-0.86 vs. 1.07, 95% CI 1.04-1.10) and higher fibrinogen (B = 0.085, 95% CI 0.061-0.109) and PAI-1:Ag (B = 0.255, 95% CI 0.206-0.303) levels, but lower VWF:Ag levels (B = - 0.059, 95% CI - 0.089 to - 0.028), than Caucasians. Shorter telomeres were associated with higher fibrinogen (B = - 0.045, 95% CI - 0.088 to - 0.001), VWF:Ag (B = - 0.137, 95% CI - 0.193 to - 0.081) and D-dimer (B = - 0.201, 95% CI - 0.377 to - 0.025) levels, conditional on ethnicity. An interaction emerged between ethnicity and telomere length for VWF:Ag level; that is, shorter telomeres were associated with higher VWF:Ag levels in Caucasians (B = - 0.170, 95% CI - 0.232 to - 0.108) but not in Africans. Shorter telomeres were associated with increased levels of several hemostatic factors after adjustment for confounding variables, whereby ethnicity partially moderated this effect. A relationship between accelerated biological aging and hypercoagulability might contribute to the risk of premature atherothrombotic events.

Examining a scaled dynamical system of telomere shortening

A model of telomere dynamics is proposed and examined. Our model, which extends a previously introduced model that incorporates stem cells as progenitors of new cells, imposes the Hayflick limit, the maximum number of cell divisions that are possible. This new model leads to cell populations for which the average telomere length is not necessarily a monotonically decreasing function of time, in contrast to previously published models. We provide a phase diagram indicating where such results would be expected via the introduction of scaled populations, rate constants and time. The application of this model to available leukocyte baboon data is discussed.

Oligonucleotides and G-quadruplex stabilizers: targeting telomeres and telomerase in cancer therapy.

Cancer is a leading cause of death worldwide and an estimated 1 in 4 deaths in the United States is due to cancer. Despite recent advances in cancer treatment, adverse effects related to cancer therapy remain a limiting factor for many patients. The ideal cancer treatment would selectively target cancerous cells while sparing normal, healthy cells to offer maximal therapeutic benefit while minimizing toxicity. Telomeres are structurally unique DNA sequences at the end of human chromosomes, which play an integral role in the cellular mortality of normal cells. As telomeres shorten with successive cellular divisions, cells develop chromosomal instability and undergo either apoptosis or senescence. In many cancers, this apoptosis or senescence is avoided as normal telomere length is maintained by a ribonucleoprotein reverse transcriptase called telomerase. Telomerase is expressed in more than 85% of all cancers and confers cancerous cells with a replicative immortality, which is a hallmark of malignant tumors. In contrast, telomerase activity is not detectable in the majority of normal somatic cell populations. Therefore, the targeting of telomerase and telomere maintenance mechanisms represent a potentially promising therapeutic approach for various types of cancer. This review evaluates the roles of GRN163L, T-oligo and small molecule G-quadruplex stabilizers as potential anticancer therapies by targeting telomerase and other telomere maintenance mechanisms.

P. berghei telomerase subunit TERT is essential for parasite survival.

Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert − mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to identify telomerase inhibitors to induce parasite cell death.

Enhancement of telomere maintenance using telomerase activation in human cells

Enhancement of telomere maintenance using telomerase activation in human cells

Telomere length in non-neoplastic colonic mucosa in ulcerative colitis (UC) and its relationship to the severe clinical phenotypes.

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. To clarify the clinical importance of telomere shortening in colonic mucosa in ulcerative colitis (UC), we measured average telomere length using quantitative real-time PCR in non-neoplastic colonic mucosa in UC patients and assessed its relationship to various clinical subtypes. Relative telomere length in genomic DNA was measured in colonic biopsies obtained from rectal inflammatory mucosa from 86 UC patients as well as paired non-inflammatory proximal colonic mucosae from 10 patients. Data were correlated with various clinical phenotypes. In paired samples, average relative telomere length of rectal inflammatory mucosa was shortened compared to normal appearing proximal colon in eight out of ten cases (p=0.01). Telomere length shortening was significantly associated with more severe Mayo endoscopic subscore (p<0.0001) and cases needing surgery due to toxic megacolon or cancer occurrence (p=0.043). When the severe clinical phenotype was defined as having at least one of following phenotypes, more than two times of hospitalization, highest Mayo endoscopic subscore, steroid dependent, refractory, or needing operation, average relative telomere length was significantly shortened in the same phenotypes than the others (p=0.003). Telomere shortening is associated with more severe clinical phenotypes of UC, reflecting severe inflammatory state in the colonic mucosa.

Normal telomere length maintenance in Saccharomyces cerevisiae requires nuclear import of the ever shorter telomeres 1 (Est1) protein via the importin alpha pathway.

The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component (TLC1) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening-phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the TLC1 RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin α. The reduction in telomere length observed at the semipermissive temperature in a srp1 mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast.

Telomere length – A cellular aging marker for depression and Post-traumatic Stress Disorder

Telomeres play a central role in cell fate and aging by adjusting the cellular response to both biological and psychological stress. Human telomeres are regions of tandem TTAGGG repeats at chromosomal ends that protect chromosomes from degradation, fusion, and recombination. They are made up of approximately 1000–2500 copies of the repeated DNA sequence. Over time, at each cell division, the telomere ends become shorter. Thus, telomere length (TL) has been considered a cellular marker for age-related diseases. In addition to biochemical stressors such as oxidation and inflammation, psychosocial traumatic stress has also been linked to shorter telomeres. TL is significantly inversely correlated with long-term depression, even after controlling for age. Average TL in depressed subjects, who were above the median of lifetime depression, was 281 base pairs shorter than that in controls, corresponding to approximately 7years of accelerated cell aging. Several recent studies have also demonstrated an inverse relationship between leukocyte telomere length (LTL) and the risk of PTSD. TL was inversely correlated with the duration of caregiving and PTSD. Here, we focus on the discussion of findings in studies of the relationships between stress-related disorders (e.g., depression and PTSD) and telomeres. We also present direct evidence that TL is associated with traumatic stress, depression, and PTSD, and hypothesize that traumatic stress affects not only mental disorders but also cellular aging. The nature of this relationship between stress and TL warrants further evaluation in psychiatry.

Abstract 39: Telomerase Reverse Transcriptase Mediates Vascular Smooth Muscle Cell Proliferation and Cell Cycle Progression Through Epigenetic Regulation of E2F Target Genes

INTRODUCTION: By maintaining the stability of telomeres, DNA-protein complexes that protect the ends of chromosomes, telomerase is rate limiting for tissue renewal and cell proliferation. In addition to this telomere maintenance function, mounting evidence suggests that the telomerase reverse transcriptase (TERT) exhibits a direct regulatory role in the activation of mitotic gene expression without apparent involvement of its well-established function in telomere homeostasis. In the present study, we investigated the role of TERT in vascular remodeling and the hypothesis that TERT regulates vascular smooth muscle cell (VSMC) proliferation through an epigenetic activation of an S Phase gene expression program. METHODS AND RESULTS: We first demonstrate high levels of TERT expression in replicating SMC of the developing neointima of human atherosclerosis. Studies in VSMC isolated from genetically engineered TERT-deficient mice with normal telomere length establish that TERT is necessary for cell proliferation. Conversely, overexpression of TERT induces cell proliferation without extending telomere length. Although TERT-deficient VSMC are not senescent, decreased proliferation is associated with stable and irreversible silencing of S phase gene expression, including target genes of the S Phase transcription factor E2F. Chromatin immunoprecipitation assays confirm that TERT is recruited to E2F target sites and promotes chromatin accessibility by facilitating the acquisition of permissive histone modifications. Finally, using a model of guide-wire induced arterial injury, we demonstrate these observations are applicable in vivo and identify decreased neointima formation in TERT-deficient mice. CONCLUSIONS: These data indicate a previously unrecognized role for TERT in the epigenetic regulation of proliferative gene expression in VSMC.

Relationship between average leukocyte telomere length and aortic pulse wave velocity

Relationship between average leukocyte telomere length and aortic pulse wave velocity

Estimating telomere length from whole genome sequence data

Telomeres play a key role in replicative ageing and undergo age-dependent attrition in vivo. Here, we report a novel method, TelSeq, to measure average telomere length from whole genome or exome shotgun sequence data. In 260 leukocyte samples, we show that TelSeq results correlate with Southern blot measurements of the mean length of terminal restriction fragments (mTRFs) and display age-dependent attrition comparably well as mTRFs.

Abstract C22: Telomere length variation and frequency of short telomeres in blood lymphocytes: Novel biomarkers for lung cancer risk

Abstract Background: Although it is widely recognized that telomere dysfunction plays an important role in cancer, the relationship between telomere function in somatic cells and lung cancer risk is not well defined. In a case-control study of non-small cell lung cancer, we examined relationship between several key telomere features and lung cancer risk. Method: Telomere fluorescent in situ hybridization was used to measure telomere features using short-term cultured blood lymphocytes. Logistic regression was used to estimate the strength of association between telomere features and lung cancer risk. Results: Telomere length variation across all chromosomal ends was significantly associated with lung cancer risk, with adjusted odds ratios of 4.67 [95% confidence interval (CI) = 1.46 – 14.9] and 0.46 (95% CI = 0.25 – 0.84) for young (age &amp;lt; 60) and old (age &amp;gt; 60) individuals, respectively. Similar strength and direction of associations between percentage of short telomeres and lung cancer risk were also observed. Telomere length variation and average telomere length jointly affect lung cancer risk. When comparing individuals with short telomere and high telomere length variation to those with long telomere and low telomere length variation, adjusted odd ratios were 8.21 (95% CI = 1.71 – 39.5) and 0.33 (95% CI = 0.15 – 0.72) for young and old individuals, respectively. Conclusion: Telomere length variation and percentage of short telomeres in blood lymphocytes are significantly associated with lung cancer risk and the associations are modulated by age. Telomere length variation in combination with average telomere length may be a promising tool for lung cancer risk assessment. Citation Format: Bing Sun, Ying Wang, Krishna Kota, Yaru Shi, Salaam Motlek, Kepher Makambi, Christopher A. Loffredo, Peter G. Shields, Qin Yang, Curtis C. Harris, Yun-Ling Zheng. Telomere length variation and frequency of short telomeres in blood lymphocytes: Novel biomarkers for lung cancer risk. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C22.

Chromosomal Instability and Telomere Shortening in Long-Term Culture of Hematopoietic Stem Cells: Insights from a Cell Culture Model of RPS14 Haploinsufficiency

The fate of cultivated primary hematopoietic stem cells (HSCs) with respect to genetic instability and telomere attrition has not yet been described in great detail. Thus, knowledge of the genetic constitution of HSCs is important when interpreting results of HSCs in culture. While establishing a cell culture model for myelodysplastic syndrome with a deletion in 5q by performing RPS14 knockdown, we found surprising data that may be of importance for any CD34+ cell culture experiments. We performed cytogenetic analyses and telomere length measurement on transduced CD34+ cells and untransduced control cells to observe the effects of long-term culturing. Initially, CD34+ cells had a normal median telomere length of about 12 kb and showed no signs of chromosomal instability. During follow-up, the median telomere length seemed to decrease and, simultaneously, increased chromosomal instability could be observed - in modified and control cells. One culture showed a clonal monosomy 7 - independent of prior RPS14 knockdown. During further culturing, it seemed that the telomeres re-elongated, and chromosomes stabilized, while TERT expression was not elevated. In summary, irrespective of our results of RPS14 knockdown in the long-term culture of CD34+ cells, it becomes clear that cell culture artefacts inducing telomere shortening and chromosomal instability have to be taken into account and regular cytogenetic analyses should always be performed.

A non-genetic, epigenetic-like mechanism of telomere length inheritance?

A non-genetic, epigenetic-like mechanism of telomere length inheritance?