Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation

Using quantitative fluorescence in situ hybridization and flow cytometry, the telomere length of telomere repeat sequences after stem cell transplantation (SCT) were measured. The study included the telomeres of peripheral blood monocytes that should reflect the length of telomeres in stem cells and the telomeres of T lymphocytes that could shorten as a result of peripheral expansion. The loss of telomeres in monocytes and in memory T cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. In addition, the telomere length in the naive T cells that were produced by the thymus was comparable to the telomere length in the naive T cells of the donor. Compared to the total length of telomeres available, the loss of telomere repeats in leukocytes after SCT resembles the accelerated shortening seen in early childhood and remains, therefore, relatively insignificant.     

Homeostasis of telomere length rather than telomere shortening after allogeneic peripheral blood stem cell transplantation

Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell-depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.     

Telomerase activity is maintained throughout the lifespan of long-lived birds

Telomerase is an enzyme capable of elongating telomeres, the caps at the ends of chromosomes associated with aging, lifespan and survival. We investigated tissue-level variation in telomerase across different ages in four bird species that vary widely in their life history. Telomerase activity in bone marrow may be associated with the rate of erythrocyte telomere shortening; birds with lower rates of telomere shortening and longer lifespans have higher bone marrow telomerase activity throughout life. Telomerase activity in all of the species appears to be tightly correlated with the proliferative potential of specific organs, and it is also highest in the hatchling age-class, when the proliferative demands of most organs are the highest. This study offers an alternative view to the commonly held hypothesis that telomerase activity is down-regulated in all post-mitotic somatic tissues in long-lived organisms as a tumor-protective mechanism. This highlights the need for more comparative analyses of telomerase, lifespan and the incidence of tumor formation.     

Accelerated telomere shortening following allogeneic transplantation is independent of the cell source and occurs within the first year post transplant

Telomere shortening has been documented in the blood cells of recipients of allogeneic bone marrow transplants compared with their donors. Allogeneic peripheral blood progenitor cells (PBPCs) have been increasingly used as an alternative to bone marrow. Their advantages include earlier engraftment and immune reconstitution following transplantation. We have measured telomere length of neutrophils and T cells in fully engrafted recipients of allogeneic bone marrow (n = 19) and allogeneic PBPC (n = 17) and also measured sequential telomere length in four patients after transplantation. Overall, significant telomere shortening occurred in recipients in neutrophils (0.3 kb, P < 0.001) and T cells (0.2 kb, P = 0.045). The data demonstrate that first, the degree of shortening was the same for BM and PBPC transplants and was not related to the time taken to engraft neutrophils and platelets and second, telomere shortening occurs in the first year post transplant without further shortening during the period of observation. These data suggest that the superiority of engraftment seen in PBPC transplants is independent of telomere shortening and other mechanisms such as homing or seeding may be more important.     

Lack of telomere shortening during senescence in Paramecium.

Paramecium tetraurelia cells have a limited clonallife span and die after approximately 200 fissions if they do not undergo theprocess of autogamy or conjugation. To test the possibility that cellularsenescence of this species is caused by telomere shortening, we analyzed thegenomic DNA of the macronucleus during the clonal life span of P. tetraurelia.We found that telomeric DNA sequences were not shortened during the interval ofdecreased fission rate and cellular death, defined as senescence in these cells.However, the mean size of the macronuclear DNA was markedly decreased during theclonal life span. We present a model that expands upon previous proposals thataccumulated DNA damage causes cellular senescence in P.tetraurelia.     

rDNA array length is a major determinant of replicative lifespan in budding yeast

The complex processes and interactions that regulate aging and determine lifespan are not fully defined for any organism. Here, taking advantage of recent technological advances in studying aging in budding yeast, we discovered a previously unappreciated relationship between the number of copies of the ribosomal RNA gene present in its chromosomal array and replicative lifespan (RLS). Specifically, the chromosomal ribosomal DNA (rDNA) copy number (rDNA CN) positively correlated with RLS and this interaction explained over 70% of variability in RLS among a series of wild-type strains. In strains with low rDNA CN, SIR2 expression was attenuated and extrachromosomal rDNA circle (ERC) accumulation was increased, leading to shorter lifespan. Suppressing ERC formation by deletion of FOB1 eliminated the relationship between rDNA CN and RLS. These data suggest that previously identified rDNA CN regulatory mechanisms limit lifespan. Importantly, the RLSs of reported lifespan-enhancing mutations were significantly impacted by rDNA CN, suggesting that changes in rDNA CN might explain the magnitude of some of those reported effects. We propose that because rDNA CN is modulated by environmental, genetic, and stochastic factors, considering rDNA CN is a prerequisite for accurate interpretation of lifespan data.

Budding yeast, Saccharomyces cerevisiae, has been a foundational model organism for the study of cellular aging. Cells divide asymmetrically and the mother cell undergoes a limited number of divisions, which defines the cell’s replicative lifespan (RLS). Measuring RLS is technically challenging and only recent advances have enabled more efficient screening for lifespan-modulating factors (1–5).A number of processes, pathways, and mechanisms have been implicated in yeast aging (reviewed in ref. 6), with regulators of the ribosomal RNA gene array (rDNA) representing perhaps the best-characterized group of lifespan modulators. Proteins that act at the rDNA locus modify rDNA stability and the formation of extrachromosomal rDNA circles (ERCs), a known aging factor in S. cerevisiae (7). Sir2, the defining member of the Sirtuin family of histone deacetylases, silences the rDNA locus and suppresses formation of ERCs (8). Conversely, the protein Fob1 binds to a replication fork barrier site in the rDNA locus, decreases rDNA stability, and thus promotes the production of ERCs (9, 10). Since the accumulation of ERCs in the mother cell limits its RLS, sir2Δ and fob1Δ strains are short and long lived, respectively (8, 11).The rDNA locus is highly repetitive and dynamic. While 150 repeats are considered a normal copy number (CN) for the strain background used in this study, the number of repeats commonly ranges from 100 to 250 copies (12). And while the size of the rDNA array is relatively stable for a limited number of divisions, rDNA CN can change on a timescale faster than the estimated mutation rate (13). Thus, it may be considered a type of “contingency locus,” which is characterized by environmentally responsive genetic variation that results in distinct phenotypic outcomes (14–16).The rDNA copy number varies significantly in strains found in the wild and those used in the laboratory (12, 13, 17–19). This variation can occur spontaneously, but can also be introduced by standard laboratory DNA transformation protocols or changes in growth environment (12, 13, 20). In addition, there are Sir2- and Fob1-dependent feedback mechanisms in place by which a “normal” rDNA CN is maintained through modulation of Sir2 expression levels (21). Interestingly, rDNA array size is anticorrelated with ERC abundance in young cells, suggesting that chromosomal and extrachromosomal rDNA are in an equilibrium (13). Despite this known connection between rDNA array size and Sir2 and ERC levels, no evidence for array size impacting lifespan has been found (12, 15, 18).Yet, significant variability exists within reported lifespans of S. cerevisiae. A metastudy found that the RLS of the same wild-type strains varied between 20 and 40, depending on the study in which it was reported (22). This variability was attributed to a reporting bias and small sample sizes. However, these findings are also consistent with uncontrolled genetic or environmental factors contributing to the variability. In addition, a genome-wide screen measuring the RLS of deleted nonessential genes found a significant discrepancy between strains with opposing mating types carrying the same gene deletion (23). This discrepancy was mostly attributed to statistical error due to a low number of cells analyzed, but was not fully explored. Given this large degree of variability, which likely impacts the interpretation of any lifespan measurement, we wanted to address the underlying cause. Here we show that the chromosomal rDNA copy number is an important determinant of replicative lifespan in yeast and can explain a large part of the reported variability in lifespan.     

Yeast YPK9 deficiency results in shortened replicative lifespan and sensitivity to hydrogen peroxide

YPK9/YOR291W of Saccharomyces cerevisiae encodes a vacuolar membrane protein. Previous research has suggested that Ypk9p is similar to the yeast P5-type ATPase Spf1p and that it plays a role in the sequestration of heavy metals. In addition, bioinformatics analysis has suggested that Ypk9p is a homolog of human ATP13A2, which encodes a protein of the subfamily of P5 ATPases. However, no specific function of Ypk9p has been described to date. In this study, we found, for the first time, that YPK9 is involved in the oxidative stress response and modulation of the replicative lifespan (RLS). We found that YPK9 deficiency confers sensitivity to the oxidative stress inducer hydrogen peroxide accompanied by increased intracellular ROS levels, decreased mitochondrial membrane potential, abnormal mitochondrial function, and increased incidence of early apoptosis in budding yeast. More importantly, YPK9 deficiency can lead to a shortened RLS. In addition, we found that overexpression of the catalase-encoding gene CTA1 can reverse the phenotypic abnormalities of the ypk9Δ yeast strain. Collectively, these findings highlight the involvement of Ypk9p in the oxidative stress response and modulation of RLS.

     

Dynamics of telomere shortening in neutrophils and T lymphocytes during ageing and the relationship to skewed X chromosome inactivation patterns

Human haemopoiesis undergoes profound changes throughout life, resulting in compromised regenerative capacity of haemopoietic stem cells. It has been suggested that telomere shortening results in senescence of haemopoietic stem cell subsets and may influence the balance between stem cell renewal and proliferation. Telomere length and telomerase activity was measured in whole blood leucocytes, neutrophils and T cells from cord blood and individuals aged from 1 year to 96 years. Rapid telomere shortening [700 base pairs (bp)] was demonstrated in the first year of life, followed by a gradual decline of 31 bp/year. T cells were shown to have longer telomeres than neutrophils (mean difference 372 bp, P = < 0.001) but demonstrated similar rates of shortening (20 +/- 0.3 bp/year vs. 22 +/- 0.3 bp/year). Telomerase was detectable in T cells but not in neutrophils, suggesting that telomerase is not the rate-limiting step for regulation of telomere length in haemopoietic cells. Stem cell utilization as measured by X chromosome inactivation patterns was found to be independent of telomere length. This supports the concept that age-dependent skewed haemopoiesis is the result of random stem cell loss or X-allelic exclusion rather than telomeric senescence. These studies provide insight into the ageing process and a reference point for evaluating replicative stress in individuals of different age groups.     

Telomerase is not required for experimental tumorigenesis of human and bovine adrenocortical cells

Telomerase has often been thought to be essential for tumorigenesis of human cells. Adrenocortical cancers, like other cancers, typically have telomerase activity. We reinvestigated the requirement for telomerase in the conversion of normal human and bovine adrenocortical cells to cancer cells. When primary adrenocortical cells were transduced with retroviruses encoding SV40 large T antigen and Ha-RasG12V and immediately transplanted into immunodeficient mice they produced invasive and metastatic tumors. Cells had negligible telomerase activity before transplantation and after recovery from tumors. However, these tumors were not immortal and cells entered crisis, limiting further growth of the tumor as well as invasion and metastasis. Infection of these tumor cells with a retrovirus encoding hTERT restored growth in culture and restored the malignant properties of the cells in immunodeficient animals. These experiments differ from previous studies in which telomerase was found to be essential for tumorigenicity: 1) we used tissue reconstruction techniques for introduction of cells into host animals and 2) we infected primary cells with retroviruses and immediately transplanted them without drug selection.     

Premature telomere shortening in polymorphonuclear neutrophils from patients with systemic lupus erythematosus is related to the lupus disease activity

We investigated whether premature telomeric loss occurred in peripheral polymorphonuclear neutrophils (PMN) as well as mononuclear cells (MNC) from patients with systemic lupus erythematosus (SLE). We measured the telomere length of MNC and PMN in 60 SLE patients and 26 sex-, race- and age-matched healthy volunteers by Southern blotting with chemiluminescence method. The possible predisposing factors associated with telomere change were also analysed. We found the telomere length of MNC and PMN shortened with age in different degrees in both SLE and control groups. Compared to the control group, the telomere length was shortened in both SLE-MNC (6.08 kb in SLE versus 6.71 kb in control, P = 0.0008) and PMN (6.24 kb in SLE versus 6.75 kb in control, P = 0.0025). The average reduction in telomere length in SLE patients was equivalent to a premature senescence of 16.5 years in MNC and 13.4 years in PMN. In addition, the accelerated telomere shortening was more prominent in SLE patients younger than 45 years old. SLE disease activity (SLEDAI) contributed remarkably to the accelerated telomere erosion, at least in PMN. Moreover, the telomere length of MNC was significantly shorter than PMN in the same SLE patients with leukopenia and lymphopenia. These data suggested that MNC and PMN from patients with SLE displayed premature and accelerated telomere shortening that SLE is an independent factor for it.     

Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro

Widespread application of beta-cell replacement strategies for diabetes is dependent upon the availability of an unlimited supply of cells exhibiting appropriate glucose-responsive insulin secretion. Therefore, a great deal of effort has been focused on understanding the factors that control beta-cell growth. Previously, we found that human beta-cell-enriched islet cultures can be stimulated to proliferate, but expansion was limited by growth arrest after 10-15 cell divisions. Here, we have investigated the mechanism behind the growth arrest. Our studies, including analyses of the expression of senescence-associated beta-galactosidase, p16(INK4a) levels, and telomere lengths, indicate that cellular senescence is responsible for limiting the number of cell divisions that human beta-cells can undergo. The senescent phenotype was not prevented by retroviral transduction of the hTERT gene, although telomerase activity was induced. These results have implications for the use of primary human islet cells in cell transplantation therapies for diabetes.     

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2'-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.