Loss Of Telomere Length Research Articles

Analysis of the Hematopoietic System in Long-Term Survivors of Allogeneic Stem Cell Transplantation and Their Respective Donors

For a variety of malignant and non-malignant hematopoietic disorders, allogeneic stem cell transplantation (HSCT) is the only curative treatment option. Over the past decades, the outcome of patients undergoing HSCT continuously improved. However, there is limited knowledge about the hematopoietic system of long-time survivors of HSCT in comparison to their respective donors. Telomere length shortening reflects the proliferation history of cells. With respect to HSCT, seminal studies have demonstrated that accelerated telomere length shortening occurs during the early post-transplant phase but the rate of telomere shortening then slows down and becomes indistinguishable between recipients and donors thereafter, leading to a parallel decline. This results in an overall pre-mature ageing of the hematopoietic system in HSCT recipients. We here analyzed blood counts and telomere length of 45 long-time survivors of HSCT and their respective donors.Blood counts were analyzed at the University Hospital Zurich. Telomere length was measured by Flow-FISH at RWTH Aachen. The study was approved by the local ethics committee. Patients and donors gave informed consent.Of the 45 donor-recipient pairs enrolled in this study, three recipients showed hematological diseases (donor-derived Thalassemia minor, MDS, relapsed Multiple Myeloma) and were thus excluded from this analysis. All remaining 42 analyzed patients were considered to be cured from their underlying hematological disease. The sex ratio was 1:1 in donors and 1:1.1 in recipients in favor of male recipients. The median age of donors and recipients at the time of HSCT was 36 and 35 years, and the age at presentation for this study was 59 and 56 years, respectively. The median time since HSCT was 16 years (range 9-33 years). When comparing donors and recipients, we did not detect any relevant differences in hemoglobin concentration (donors: 139±12g/L, recipients: 143±15g/l, p=0.54) or erythrocyte counts (donors: 4.73±0.49T/L, recipients 4.55±0.47T/L, p=0.32). Recipients showed significantly higher MCV values (donors: 91.6±4.4fL, recipients 94.1±3.8fL, p=0.03) and significantly higher MCH (donors 30.5±1.3pg, recipients 31.3±1.3pg, p<0.001), while MCHC did not differ (donors: 329±11g/L, recipients 333±9g/L, p=0.20). Recipients showed a trend to higher relative but not absolute reticulocyte counts (donor: 1.02±0.38%, 50±17G/L, recipients: 1.23±0.40%, 56±18G/L, p=0.08 and p=0.18). Also, we did not observe significant alterations in platelet counts (donors: 248±62G/L, recipients 234±70G/L, p=0.31). In the leukocyte compartment, neutrophil counts were not altered (donors: 3.8±1.5G/L, recipients 4.2±1.6G/L, p=0.87), while recipients had significantly more monocytes (donors: 0.52±0.17G/L, recipients 0.62±0.20G/L, p<0.001). Eosinophil and basophil counts did not show differences, while recipients had higher lymphocyte counts (donors: 1.7±0.6G/L, recipients 2.2±5G/L, p=0.007).In 28 of 42 pairs, telomere length analysis was logistically possible. Telomere length was measured separately in lymphocytes as well as in granulocytes. The median telomere length of granulocytes was 8.00±1.30kb in donors and 7.46±1.16kb in recipients, respectively (p=0.043). The median difference in granulocyte telomere length between donors and recipients was 0.38±0.82kb reflecting a hypothetical age difference of 14.2 years assuming a loss in telomere length of 27bp per year. In lymphocytes, the median telomere length was 7.13±1.06kb in donors and 6.43±1.02kb in recipients (p<0.001). The median difference in telomere length between donors' and recipients' lymphocytes was 0.48±0.99kb corresponding to a longer proliferation history of approximately 17.8 years in the recipients.Peripheral blood counts are stable and widely inconspicuous in long-time HSCT-survivors. Telomere length is significantly shorter in recipients' lymphocytes and granulocytes. This telomere shortening is most likely due to increased HSC proliferation (reflected by granulocyte telomere length) and inflammatory stimuli (affecting both HSCs and lymphocytes) in HSCT recipients. However, it does not have a clinically relevant effect on blood counts in this small group of long-term HSCT-survivors. In how far the increased proliferative history of donor HSCs in allo-HSCT recipients is of biologic relevance needs further investigation. DisclosuresBrümmendorf:Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Heritable variation in telomere length predicts mortality in Soay sheep

Telomere length (TL) is considered an important biomarker of whole-organism health and aging. Across humans and other vertebrates, short telomeres are associated with increased subsequent mortality risk, but the processes responsible for this correlation remain uncertain. A key unanswered question is whether TL-mortality associations arise due to positive effects of genes or early-life environment on both an individual's average lifetime TL and their longevity, or due to more immediate effects of environmental stressors on within-individual TL loss and increased mortality risk. Addressing this question requires longitudinal TL and life history data across the entire lifetimes of many individuals, which are difficult to obtain for long-lived species like humans. Using longitudinal data and samples collected over nearly two decades, as part of a long-term study of wild Soay sheep, we dissected an observed positive association between TL and subsequent survival using multivariate quantitative genetic models. We found no evidence that telomere attrition was associated with increased mortality risk, suggesting that TL is not an important marker of biological aging or exposure to environmental stress in our study system. Instead, we find that among-individual differences in average TL are associated with increased lifespan. Our analyses suggest that this correlation between an individual's average TL and lifespan has a genetic basis. This demonstrates that TL has the potential to evolve under natural conditions, and suggests an important role of genetics underlying the widespread observation that short telomeres predict mortality.

Relative Telomere Length in Peripheral Blood Cells and Hypertension Risk among Mine Workers: A Case-Control Study in Chinese Coal Miners.

Hypertension is a common chronic disease in middle-aged and elderly people and is an important risk factor for many cardiovascular diseases. Its pathogenesis remains unclear. Epidemiological studies have found that the loss of telomere length in peripheral blood cells can increase the risk of coronary heart disease, myocardial infarction, and other diseases. However, a correlation between loss of telomere length and hypertension has not been established. In this study, we aimed to explore the association between telomere length and the risk of essential hypertension (EH) in Chinese coal miners. A case-control study was performed with 215 EH patients and 222 healthy controls in a large coal mining group located in North China. Face-to-face interviews were conducted by trained staff with the necessary medical knowledge. Relative telomere length (RTL) was measured by a quantitative real-time PCR assay using DNA extracted from peripheral blood. In the control group, the age-adjusted RTL was statistically significantly lower in miners performing hard physical labour compared with nonphysical labour (P = 0.043). A significantly shorter age-adjusted RTL was found in the control group of participants who consumed alcohol regularly compared with those who do not consume alcohol (P = 0.024). Age-adjusted RTL was negatively correlated with body mass index (BMI) and alcohol consumption. Hypertension was also found to be significantly correlated with factors such as age, BMI, alcohol consumption, smoking, and tea consumption. Our results suggest that RTL is associated with hypertension in coal miners.

Correlation of Telomere Length in Adipose Tissue and Leukocytes and its Association with Postsurgical Weight Loss.

The aim of this study was to determine the relationship between telomere length (TL) in subcutaneous adipose tissue (SAT), visceral adipose tissues (VAT), and leukocytes, as well as to examine the associations of TL in these tissues with postsurgical weight loss in Asians with severe obesity. Presurgery TL was measured in leukocytes, SAT, and VAT of 91 patients who underwent weight loss surgery. Correlation between TL in multiple tissues was assessed using Pearson correlation. The association of presurgery TL and postsurgical weight loss at 6 or 12 months, expressed as a percentage of weight loss, was determined using linear regression in 70 patients. Telomeres were longer in VAT compared with those in leukocytes and SAT (P < 0.001) but were highly correlated between tissues. The strongest correlation was observed between TL in VAT and leukocytes (r = 0.739, P = 6.22 × 10-17 ). Compared with individuals in the highest tertile, those in the lowest tertile of VAT TL showed greater weight loss (β = 6.23, SE = 3.10, P = 0.044) independent of age, sex, ethnicity, types of surgery, diabetes condition, preoperative BMI, and follow-up period. Among patients with severe obesity, TL in leukocytes and adipose tissue was highly correlated. However, there was variability in the association of TL in these tissues with weight loss after surgery.

New somatic TERT promoter variants enhance the Telomerase activity in Glioblastoma

The catalytic activity of human Telomerase Reverse Transcriptase (TERT) compensates for the loss of telomere length, eroded during each cell cycle, to ensure a correct division of stem and germinal cells. In human tumors, ectopic TERT reactivation, most frequently due to hotspot mutations in the promoter region (TERTp), i.e. c.1-124 C > T, c.1-146 C > T, confers a proliferative advantage to neoplastic cells. In gliomas, TERTp mutations (TERTpmut) mainly occur in oligodendroglioma and glioblastoma. We screened, for TERTp hotspot mutations, 301 adult patients with gliomas and identified heterozygous mutations in 239 cases: 94% of oligodendroglioma, 85% of glioblastoma, and 37.5% of diffuse/anaplastic astrocytoma. Besides the recurrent c.1-124 C > T and c.1-146 C > T, two cases of glioblastoma harbored novel somatic TERTp variants, which consisted of a tandem duplications of 22 nucleotides, i.e. a TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, both located downstream c.1-124 C > T and c.1-146 C > T. In silico analysis predicted the formation of 119 and 108 new transcription factor’s recognition sites for TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, respectively. TERTp duplications (TERTpdup) mainly affected the binding capacity of two transcription factors’ families, i.e. the members of the E-twenty-six and the Specificity Protein/Krüppel-Like Factor groups. In fact, these new TERTpdup significantly enhanced the E-twenty-six transcription factors’ binding capacity, which is also typically increased by the two c.1-124 C > T/c.1-146 C > T hotspot TERTpmut. On the other hand, they were distinguished by enhanced affinity for the Krüppel proteins. The luciferase assay confirmed that TERTpdup behaved as gain-of-function mutations causing a 2,3-2,5 fold increase of TERT transcription. The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors, with the identification of new recurrent somatic gain-of-function mutations, occurring in 0.8% of glioblastoma IDH-wildtype.

Shorter telomere length following lung transplantation is associated with clinically significant leukopenia and decreased chronic lung allograft dysfunction-free survival.

Patients with short telomeres and interstitial lung disease may have decreased chronic lung allograft dysfunction (CLAD)-free survival following lung transplantation. The relationship between post-transplant telomere length and outcomes following lung transplantation has not been characterised among all recipients, regardless of native lung disease.This was a single-centre prospective cohort study. Consenting transplant recipients had their telomere length measured using quantitative real-time PCR assays on peripheral blood collected at the time of surveillance bronchoscopy. We assessed the association between early post-transplant telomere length (as measured in the first 100 days) and CLAD-free survival, time to clinically significant leukopenia, cytomegalovirus (CMV) viraemia, chronic kidney disease, and acute cellular rejection. We also assessed the association between rate of telomere shortening and CLAD-free survival.Telomere lengths were available for 98 out of 215 (45.6%) recipients who underwent lung transplant during the study period (median measurement per patient=2 (interquartile range, 1–3)). Shorter telomere length was associated with decreased CLAD-free survival (hazard ratio (HR)=1.24; 95% CI=1.03–1.48; p=0.02), leukopenia requiring granulocyte colony-stimulating factor (HR=1.17, 95% CI=1.01–1.35, p=0.03), and CMV viraemia among CMV-mismatch recipients (HR=4.04, 95% CI=1.05–15.5, p=0.04). Telomere length was not associated with acute cellular rejection or chronic kidney disease. Recipients with more rapid loss in telomere length (defined as the highest tertile of telomere shortening) did not have worse subsequent CLAD-free survival than those without rapid loss (HR=1.38, 95% CI=0.27–7.01, p=0.70).Shorter early post-transplant telomere length is associated with decreased CLAD-free survival and clinically significant leukopenia in lung transplant recipients, regardless of native lung disease.

THE LONGEVITY ASSOCIATED ALLELE OF FOXO3 PROTECTS AGAINST TELOMERE ATTRITION DURING AGING

Telomere attrition in proliferative tissues is a hallmark feature of human aging. To date, the genetic influence on the rate of telomere attrition is poorly understood. Previously we discovered a variant of the FOXO3 gene that is strongly associated with human longevity, an observation that has been now reproduced in over a dozen independent studies. In the present study, we sought to assess the effect of the longevity associated variant of FOXO3 (rs2802292 - G allele) on the rate of telomere attrition during aging. The results from a cohort of Okinawan-Japanese (N=121), ranging in age from 25 – 94 years, demonstrates carriers of 1 or 2 copies of the longevity-associated G allele of FOXO3 showed markedly reduced rates of telomere loss in peripheral blood leucocytes as compared to carriers of the more common FOXO3 variant (TT – common genotype, m= -33bp/year, P=0.008). Interestingly, telomere shortening was not observed as a function of age for G allele carriers (m= -2bp/year, P>0.1). In an independent study of women (N=6,565) from the Nurses’ Health Study cohort, ranging in age from 40 to 70 years, a similar observation was found. Notably, carriers of the TT or GT FOXO3 genotype showed a significant decline in telomere length with age (m= -15.5 bp/year, P0.1). These results mark the first validated longevity gene variant showing an association with negligible loss of telomere length with age in humans.

Obesity, weight loss, and influence on telomere length: New insights for personalized nutrition.

Telomeres are structures located at the ends of chromosomes associated with proteins, from the shelterin complex, which are responsible for the protection and preservation of the genetic material. The telomere length (TL) progressively decreases with each cell division, and recent evidence suggests that lifestyle can lead to telomere shortening. In individuals with obesity, excess adipose tissue plays a key role in inducing a chronic and systemic inflammatory state, which can cause TL shortening. Thus, the aim of the present review was to show the relationship between obesity and TL in addition to the possible risk factors for its shortening and how the different strategies for weight loss can modulate TL. As the crucial result, we can consider the association between TL and weight loss, and adiposity changes after different interventions, showing that TL may be used as a biomarker of responses to obesity treatment.

Mouse Fanconi Anemia (FA) Fancd2-/- Bone Marrow Stromal Cells Demonstrate Ionizing Irradiation Induced Senescence

Senescent cells accumulate in tissues affected by aging, ionizing irradiation, and loss of telomere length and other sources of DNA damage signaling. FA mice deficient in the Fancd2 protein display reduced homologous recombination and increased nonhomologous end joining for repair of DNA double strand breaks. Studies also show a role for Fancd2 in the ALT pathway of telomere maintenance. Fancd2-/- bone marrow stromal cell lines are radiosensitive compared to Fancd2+/+ marrow stromal cells (Do = 1.4 ± 0.1 Gy vs 1.6 ± 0.1 Gy, p = 0.0124), and by comet assay where Fancd2-/- cells display slower DNA repair after irradiation (p < 0.0001) (Berhane, et. al., Radiation Research 181:76-89, 2014). We investigated whether Fancd2-/- cells were more susceptible to induction of senescence. Fancd2+/+ control and Fancd2-/- murine bone marrow stromal cell lines were analyzed for irradiation induced senescence. Bone marrow stromal cell lines derived from long term bone marrow cultures of Fancd2+/+, and Fancd2-/- marrow (129/Sv) were plated in 6 well tissue culture plates and irradiated to 5 or 10 Gy. At 24, or 48 hr after irradiation, cells were stained for senescence biomarkers B-Gal and p21. Fancd2+/+ and Fancd2-/- bone marrow stromal cells stained for percent B-Gal positive cells following 0, 5 or 10 Gy at 24 and 48 hr after irradiation showed that Fancd2-/- cells had increased B-Gal staining compared to Fancd2+/+ cells at 0 Gy (10 ± 1% vs 1 ± 1%, p<0.0001). At 24 and 48 hr after 5 and 10 Gy Fancd2-/- cells had increased B-Gal staining (14 ± 1% at 24 hr after 5 Gy, 18 ± 1% at 24 hr after 10 Gy, 18 ± 1% at 48 hr after 5 Gy and 23 ± 1% at 48 hr after 10 Gy, p < 0.0123 compared to 0 Gy). In contrast increased B-Gal staining was delayed in FancA+/+ cells to 48 hr after both 5 and 10 Gy (7 ± 4% at 24 hr after 5 Gy, 3 ± 1% at 48 hr after 10 Gy, 3 ± 1% at 48 hr after 5 Gy, 4 ± 1% at 48 hr after 10 Gy, p = 0.0027 and < 0.0001 comparing 5 and 10 Gy at 48 hr to 0 Gy, respectively). The percent of nonirradiated Fancd2-/- cells positive for p21 was statistically increased compared to Fancd2+/+ cells (16 ± 2% vs 4 ± 1%, p = 0.0007). Both control Fancd2+/+ and Fancd2-/- cells had increased p21 positive cells at 24 and 48 hr following 5 or 10 Gy (p < 0.05) was always greater in Fancd2-/- cells . At 24 and 48 hrs after 5 Gy, Fancd2-/- cells had 45 ± 5% and 39 ± 1% positive p21 cells compared to 9 ± 1% and 5 ± 1% for Fancd2+/+ cells (p < 0.0001). At 24 and 48 hours after 10 Gy the percent of Fancd2-/- cells positive for p21 was 58 ± 3% and 48 ± 5%, respectively, compared to 11 ± 1% and 7 ± 1%, respectively, for Fancd2+/+ cells (p < 0.0001). The radiosensitivity of Fancd2-/- bone marrow stromal cells increases senescence in vitro as measured by both B-Gal and p21. Study of FA tissues in vivo after TBI should reveal organ specific acceleration of senescence and provide insight into late irradiation effects in marrow transplanted FA patients.

Abstract 15911: Reduced Senescence and Enhanced Proliferation of Human Cardiac Progenitor Cells Isolated and Expanded Under Reduced Oxygen Tension

Background: Oxygen availability at the cellular level in vivo is significantly lower (~1-7%) than that used for typical ambient cell culture conditions (21%). Here we investigated whether prolonged culture at reduced O2 concentrations affected proliferation, senescence and oxidative stress of human cardiac progenitor cells (hCPCs). Methods: hCPCs positive for c-kit and negative for lineage markers (ckit+/Lin-) were isolated from right atrial tissues obtained from five infants during repair of congenital heart defects and expanded for a maximum of 10 passages in varying O2 concentrations (1, 5, and 21%); all manipulations were performed at the target O2 in a hypoxic chamber. Cellular phenotype was confirmed by ICC staining and flow cytometry. Doubling time, oxidative stress (8-OH-deoxyguanosine [8OHdG], protein carbonyl formation) and senescence markers (telomere length, telomerase activity, P16ink4a staining) were measured. Results: Reducing ambient O2 from 21% to 1% did not alter cell surface marker expression. Culture and expansion at 21% O2 markedly accelerated hCPC senescence compared to 1% or 5% O2, as indicated by increased P16ink4a positive hCPCs and greater loss of telomere length and telomerase activity; much of this damage appeared to occur during early passage and expansion. Both protein carbonyl and 8OHdG formation progressively increased in 21% O2, whereas these oxidative injury markers showed little change at 1 and 5% O2 concentrations. hCPCs that were cultured at either 5% or 1% O2 demonstrated shorter doubling times with resultant higher cell yields during in vitro expansion. Conclusion: Culturing ckit+/Lin- hCPCs at lower oxygen tension minimizes oxidative damage, reduces senescence, and enhances proliferative potential during long-term culture; expansion at 1% ambient O2 appeared to be most effective. This relatively straightforward modification may further understanding of the biology of CPCs and their regenerative potential.

Association between telomere length and chromosome 21 nondisjunction in the oocyte.

Chromosome 21 nondisjunction in oocytes is the most common cause of trisomy 21, the primary chromosomal abnormality responsible for Down syndrome (DS). This specific type of error is estimated to account for over 90 % of live births with DS, with maternal age being the best known risk factor for chromosome 21 nondisjunction. The loss of telomere length and the concomitant shortening of chromosomes are considered a biological marker for aging. Thus, we tested the hypothesis that mothers who had a maternal nondisjunction error leading to a live birth with DS (n = 404) have shorter telomeres than mothers with live births without DS (n = 42). In effect, our hypothesis suggests that mothers of children with DS will appear "biologically older" as compared to the mothers of euploid children. We applied a quantitative PCR assay to measure the genome-wide relative telomere length to test this hypothesis. The results of our study support the hypothesis that young mothers of DS babies are "biologically older" than mothers of euploid babies in the same age group and supports telomere length as a biomarker of age and hence risk for chromosome nondisjunction.

Models to explore genetics of human aging.

Genetic studies have bestowed insight into the biological mechanisms underlying inter-individual differences in susceptibility to (or resistance to) organisms’ aging. Recent advances in molecular and genetic epidemiology provide tools to explore the genetic sources of the variability in biological aging in humans. To be successful, the genetic study of a complex condition such as aging requires the clear definition of essential traits that can characterize the aging process phenotypically. Phenotypes of human aging have long relied on mortality rate or exceptional longevity. Genome-wide association studies (GWAS) have been shown to present an unbiased approach to the identification of new candidate genes for human diseases. The GWAS approach can also be used for positive health phenotypes such as longevity or a delay in age-related chronic disease, as well as for other age related changes such as loss of telomere length or lens transparency. Sequencing, either in targeted regions or across the whole genome can further identify rare variation that may contribute to the biological aging mechanisms. To date, the results of the GWAS for longevity are rather disappointing, possibly in part due to the small number of individuals with GWAS data who have reached advanced old age.Human aging phenotypes are needed that can be assessed prior to death, and should be both heritable and validated as predictors of longevity. Potentially, phenotypes that focus on “successful” or “healthy” aging will be more powerful as they can be measured in large numbers of people and also are clinically relevant.We postulate that construction of an integrated phenotype of aging can be achieved capitalizing on multiple traits that may have weak correlations, but a shared underlying genetic architecture. This is based on a hypothesis that convergent results from multiple individual aging-related traits will point out the pleiotropic signals responsible for the overall rate of aging of the organism. An approach would be to investigate traits that are linked to the state of many vital functions, disability, and ultimately survival rates, to identify common biological pathways that govern aging processes in humans. New composite aging phenotypes must be validated by predicting all-cause mortality, major chronic disease and disability late in life.

Age, sex, and telomere dynamics in a long-lived seabird with male-biased parental care.

The examination of telomere dynamics is a recent technique in ecology for assessing physiological state and age-related traits from individuals of unknown age. Telomeres shorten with age in most species and are expected to reflect physiological state, reproductive investment, and chronological age. Loss of telomere length is used as an indicator of biological aging, as this detrimental deterioration is associated with lowered survival. Lifespan dimorphism and more rapid senescence in the larger, shorter-lived sex are predicted in species with sexual size dimorphism, however, little is known about the effects of behavioral dimorphism on senescence and life history traits in species with sexual monomorphism. Here we compare telomere dynamics of thick-billed murres ( Uria lomvia ), a species with male-biased parental care, in two ways: 1) cross-sectionally in birds of known-age (0-28 years) from one colony and 2) longitudinally in birds from four colonies. Telomere dynamics are compared using three measures: the telomere restriction fragment (TRF), a lower window of TRF (TOE), and qPCR. All showed age-related shortening of telomeres, but the TRF measure also indicated that adult female murres have shorter telomere length than adult males, consistent with sex-specific patterns of ageing. Adult males had longer telomeres than adult females on all colonies examined, but chick telomere length did not differ by sex. Additionally, inter-annual telomere changes may be related to environmental conditions; birds from a potentially low quality colony lost telomeres, while those at more hospitable colonies maintained telomere length. We conclude that sex-specific patterns of telomere loss exist in the sexually monomorphic thick-billed murre but are likely to occur between fledging and recruitment. Longer telomeres in males may be related to their homogamous sex chromosomes (ZZ) or to selection for longer life in the care-giving sex. Environmental conditions appeared to be the primary drivers of annual changes in adult birds.

Early and Permanent Telomere Shortening in Bone Marrow-Derived Cells Following Chemotherapy: A Parallel Study In Vivo in Lymphoma Patients and In Vitro in Cultured Mesenchymal Stem Cells

Abstract 1620 IntroductionPatients with lymphoproliferative disease very often require treatments with chemotherapy. Several recent reports suggest that the exposure of hematopoietic cells to DNA-damaging substances, such as chemotherapeutic drugs, may trigger the ageing process and induce premature cell ageing. A good indicator of cell replication history and thus of cell ageing is the length of telomeres. Indeed, telomere shortening and/or telomere dysfunction have been documented in patients receiving chemotherapy. The loss of telomere sequences has been linked to the increased risk of developing secondary malignancy in subjects previously treated with chemotherapy. However, it is still unexplained whether telomere loss is secondary to direct damage of bone marrow (BM) cells by cytotoxic drugs, or merely the consequence of increased BM cell proliferation after exposure to chemotherapy. Moreover, the time course for the onset of chemotherapy-induced telomere shortening and the cell types mainly involved in the premature cell aging induced by chemotherapy remain to be elucidated. In the present study changes in telomere length (TL) following chemotherapy were evaluated both in vivo and in vitro. Main aims of the study were: i. to define whether TL shortening following chemotherapy is reversible or permanent; ii. to verify whether TL shortening is a phenomenon induced by extensive chemotherapy treatments or it may occur even after minimal drug exposures; iii. to identify possible cell populations that are particularly susceptible to drug-induced telomere loss. Patients and methods Mononuclear Cells (MNC) or granulocytes were obtained from BM and/or Peripheral blood (PB) cells from 31 lymphoma patients treated with high-dose chemotherapy and autograft and from 14 lymphoma patients undergoing chemotherapy (6 CHOP, 1 CVP, 1 MINE, 1 DHAP and 5 ABVD). Overall, 27 patients of the autograft and 13 patients of the conventional chemotherapy groups were at their first treatment line. Median age of patients was 45 years. TL was assessed on granulocytes and MNC by Southern Blotting, as previously reported (Ricca et al, Leukemia 2005). In vitro studies were performed on cultured mesenchymal stem cells (MSCs). Briefly, MNC were obtained from BM of normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium and 10% platelet lysates and fed at 3–4 day intervals; cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and then assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed, Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times. TL was evaluated both by flow-fish and southern-blot analysis. Results A marked reduction in TL was detected in all patients undergoing autograft in PB granulocytes and in BM cells, compared to age-matched controls; the degree of TL loss remained detectable even in patients up to 10 yrs. since autograft. For patients receiving conventional chemotherapy, a significant TL shortening could be detected in granulocyte obtained after chemotherapy compared to pre-treatment values (p=0.029), while no significant variations could be documented in MNC. TL shortening was detectable already after the first chemotherapy course in six patients and at the second in four patients. A marked TL shortening occurred in cultured MSC after exposure to sub-lethal doses of Doxo and Eto. Initial TL shortening was detectable already at 5 days after drug exposure, with progressive reduction compared with untreated cells at 7, 14, 21, and 28 days in culture. Following a single exposure, MSCs were unable to regain the lost telomere sequences for up to 28 days in culture. Conclusions The results indicate that TL shortening: i. is a permanent signature of the previous DNA damage in BM cells exposed to chemotherapy; ii. is a phenomenon that can be detected early following chemotherapy exposure, even with low drug dosages; iii. can be most easily detectable in myeloid cells, in particular in granulocytes, although also BM mesenchymal cells may be susceptible to drug-induced telomere loss. Disclosures:No relevant conflicts of interest to declare.

The aging effect of chemotherapy on cultured human mesenchymal stem cells

Various agents, including chemotherapeutic drugs, can induce cell senescence. However, the mechanisms involved in the aging pathway, particularly the stress that chemotherapy imposes on telomeres, are still undefined. To address these issues, human mesenchymal stem cells (MSCs) were assessed as target cells to investigate the initiation of the aging process by chemotherapy. The MSCs were obtained from bone marrow (BM) cells from normal adults and grown in the presence of platelet lysates. Cultured MSCs were identified for immunophenotype, and for growth and differentiation properties. The MSCs were exposed to 10 nM doxorubicin and 500 ng/mL etoposide, sublethal doses that induce DNA double-stranded breaks. Telomere length (TL) was assessed by flow-fluorescence in situ hybridization and Southern blotting. Initial TL shortening was detectable in MSCs at 5 days after drug exposure, with progressive reduction compared with untreated cells at 7, 14, 21, and 28 days in culture. After a single exposure, MSCs were unable to regain the lost telomere sequences for up to 28 days in culture. The ATM phosphorylation was documented early after drug exposure, while no telomerase activation was observed. Chemotherapy-induced TL shortening was associated with reduced clonogenic activity invitro and accelerated adipose differentiation. Analogous behavior in the differentiation pattern was observed in naturally aged MSCs. These results indicate that cultured MSCs represent a useful cellular model to investigate novel drugs that may favor or, conversely, might prevent TL loss in human stem cells. The TL shortening is a permanent signature of previous chemotherapy-mediated DNAdamage, and predicts impaired proliferative and differentiation potential.

Ku circles the telomere?

Ku circles the telomere?

Telomere Length In Ph - Negative Chronic Myeloproliferative Neoplasms: It Is Reduced According to JAK2 V617F Mutation Allele Burden and It Is Not Affected by Cytoreductive Treatment with Hydroxyurea

AbstractAbstract 1975 Introduction:Telomeres are reliable indicators of previous cell proliferation and cell ageing. Moreover, a marked though variable loss of telomere length (TL) has been observed in several hematological malignancies. In particular, some recent studies have reported a marked TL reduction in patients with Ph-negative Chronic Myeloproliferative Neoplasms (Ph-neg-CMNs) (Ferraris AM et al, Br J Haematol 2005; Bernard L et al, Leukemia 2009). This supports the possible influence of TL in the development of CMNs. Moreover, TL might be of prognostic relevance in these disorders. The present study reports the analysis of TL in a large series of patients with Ph-neg-CMNs, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF), and in a few cases of Secondary Erythrocytosis (SE), as well. Aims of the study were: i. to verify the rate of TL reduction in Ph-neg-CMNs; ii. to compare TL in ET, PV and MF; iii. to investigate the role of TL as a marker of proliferation, evaluating differences in TL compared to healthy subjects and subjects with Secondary Erythrocytosis (SE); iv. to verify telomere dynamics according to the treatment received. Methods:Peripheral blood (PB) samples were obtained from 239 Ph-neg-CMNs patients (median age 68 yrs, range 10–91): 78 had PV, 100 had ET and 61 MF. Most patients were analyzed for JAK2 mutations: among 72 evaluated PV patients, a JAK2V617F mutation was found in 66 (92%) and an exon 12 mutation was identified in 2 patients; a JAK2 V617F mutation was detected in 50 (50%) ET patients and in 44 (73%) MF patients. Samples were obtained either at diagnosis or during follow-up. More than a half of CMNs patients received before TL analysis at least 1 year of cytoreduction (129 patients); 90% received Hydroxyurea (HU) and 26% other cytoreductive drugs with or without HU. As control, PB samples from 202 healthy age-matched subjects and from 14 SE subjects were analyzed. TL was assessed by Southern blot analysis, according to standard procedures (TeloTAGGG Telomere Length Assay Kit, Roche Diagnostic, Mannheim, Germany). JAK2V617F mutation analysis was performed by ASO-PCR and digestion with BSAXI (Guerini et al, Leukemia 2008). Results:PV, ET and MF patients showed individual progressive TL shortening correlated with age as observed in the healthy population. However, CMNs patients had TL significantly shortened (5,890 bp, ± 1,305) compared to healthy age-matched individuals (median: 7,330 bp, ± 1574) (p<.0001). PV and MF showed the most pronounced TL loss among CMNs, significantly different from healthy controls, with TL values respectively of 5,621 bp (± 1,446) (p<.0001) and 5,361 bp (± 1,335 bp) (p<.0001). ET patients had a median TL of 6,422 bp, (± 1131bp). SE patients had a median TL of 6,972 bp (± 1355 bp) with no difference from healthy subjects (p= 1.00). At ANCOVA multivariate analysis of disease characteristics, short TL correlated significantly with JAK2V617F mutation allele burden >50% (p=.0025), age (p=.0132) and diagnosis of PV (p=.0122). No significant correlation was found with disease duration, history of thrombosis and treatment with cytoreductive or anti-aggregants agents. Conclusions:i. Ph-negative CMNs are confirmed to present with TL reduction compared to the age-matched healthy population; ii. PV and MF show higher degree of TL loss than ET; ii. high JAK2V617F mutation burden is strongly correlated to TL loss; iii. SE patients and healthy subjects do not differ in TL, suggesting the possible use of TL as a parameter to distinguish SE from PV, particularly in the few cases lacking JAK2 mutations. iv. no evident influence on accelerated cell ageing is observed in patients receiving cytoreductive treatment with Hydroxyurea. The absence of a marked telomere loss in patients receiving Hydroxyurea, reduces the harm of using this drug when clinically indicated. Disclosures:No relevant conflicts of interest to declare.

Exposure of Cultured Human Mesenchymal Stem Cells to Chemotherapy Induces An Early and Permanent Telomere Loss: Biological and Clinical Implications

AbstractAbstract 4776 Introduction.A good indicator of the rate of cell ageing is the length of telomeres, the long tandem repeat sequences at the end of chromosomes. Telomere length (TL) in humans decreases at each somatic cell division, and undergoes progressive shortening with increasing age. Indeed, cell proliferation has been considered the primary factor responsible of TL shortening and ultimately of the cell ageing process. Exposure to DNA-damaging substances has been proposed as an alternative and relevant factor involved in the cell ageing process. In spite of the number of studies in both human and animal models, few information is available on the role of both cell proliferation and DNA damage in normal human stem cells. The present study addressed this issue by investigating the ageing process in cultured Bone-Marrow (BM) human Mesenchymal Stem Cells (MSCs). Aim of the study has been to investigate whether and how exposure to toxic agents may affect the rate of TL shortening in cultured MSCs. Methods.1. MSCs were cultured from human mononuclear BM cells, obtained from normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium containing heparin and 10% platelet lysates and fed at 3–4 day intervals, until they reached confluence; they were then detached with trypsin-EDTA solution and reseeded for the experimental tests. Cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and negativity for CD14, CD34 and CD45 by flow cytometry; 2. cultured MSC were assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed to induce DNA damage: Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times; 3. telomere was evaluate in MSCs before (day 0 or T0) and after exposure to chemotherapeutic drugs, i.e. at 7 – 14 –21 and 28 days of culture since drug exposure. TL was determined by the cytofluorimetric Flow-fish assay and by southern-blot analysis; 4. MSC were also cultured in differentiating medium in order to induce osteogenic and adipogenic differentiation, evaluated by Alizarin red and Oil red staining, respectively; 5. lastly, both ATM phosphorilation and telomerase activation were assessed, by Western blot analysis and the TRAP assay, respectively. Results.1. as expected, TL progressively decreased after 7 days in culture in untreated (NT) MSC (88,8% compared to T0); TL was further shortened as long as the cells were maintained in culture; 2. a marked TL shortening occurred in MSC already at day 7 after exposure to sub-lethal doses of Doxo and Eto, with a mean of 81,7% and 79,6% compared to T0, respectively. Indeed, a minimum of 5 days in culture was required to identify the earliest signs of telomere loss. Again, further TL shortening was observed later on and the difference in TL between NT and chemotherapy-exposed cells progressively increased: compared to T0, TL resulted of 82 % and 74% in NT cells, whereas TL was 68% and 54% in Doxo-treated, and 70% and 62% in Eto-treated, at week 3 and 4, respectively; 3. a single 2-hr exposure to Doxo or Eto induced a TL loss compared to NT cells, that was maintained unchanged at long-term in culture; 4. when MSC were assayed in differentiation inducing medium, cells exposed to chemotherapy displayed enhanced adipogenic and osteogenic differentiation compared to NT cells; 5. ATM was strongly phosphorilated following exposure to Doxo and Eto compared to NT MSC; ATM phosphorilation preceded TL loss; by contrast, telomerase was unaffected by chemotherapy exposure. Conclusions.i. human cultured MSCs exposed to chemotherapeutic drugs offer a simple and reliable means to investigate the correlation between DNA damage and cell ageing; ii. telomere loss is clearly detectable in MSCs already after 7 days following exposure to Doxo and Eto and remains detectable at long-term in culture as a permanent signature of the previous DNA damage; iii. the in vitro observation suggests that not only hematopoietic cells but also stromal cells, including MSCs, may experience relevant DNA damages following in vivo treatments with chemotherapeutic drugs. Disclosures:No relevant conflicts of interest to declare.

The Risk of Secondary Myelodysplastic Syndrome/Acute Leukemia Following High-Dose Yttrium-90 Ibritumomab Tiuxetan Is Analogous to That Observed Following High-Dose Chemotherapy: a Matched-Pair Analysis In Non-Hodgkin Lymphoma Patients.

AbstractAbstract 1289 Introduction:Myeloablative doses of Yttrium-90 (90Y)-Ibritumomab Tiuxetan combined with tandem autologous peripheral blood stem cell (PBSC) support can be safely administered to patients aged up to 76 years (Devizzi L et al, J Clin Oncol 2008). Whether marrow exposure to myeloablative doses of radiolabelled antibodies (hd-radio-immunotherapy) could increase the risk of secondary myelodysplastic syndrome and acute leukemia (sMDS/AL) remains a matter of concern. Therefore, we prospectively investigated by means of a longitudinal study the incidence of sMDS/AL in a cohort of high-risk non-Hodgkin lymphoma (NHL) patients receiving hd-radio-immunotherapy. The cumulative incidence of sMDS/AL in the study group was compared with that observed in a historical group of patients autografted following chemotherapy-based myeloablative conditioning. Additionally, to further investigate marrow damage induced by hd-radio-immunotherapy, hematopoietic and stromal progenitors, as well as telomere length (TL) were assessed. Patients and Methods:From July 2004 through December 2007, 53 relapsed/refractory (n= 36) or de novo high-risk (n= 17) NHL patients (median age, 64 years; range, 26 – 76 years) received a high-dose sequential (HDS) chemotherapy program, including a myeloablative phase with 90Y-Ibritumomab Tiuxetan (1.2 mCi/kg body weight). Patients were monitored for frequency of colony-forming cells (CFCs), cytogenetic and TL analysis. Bone marrow (BM) samples were collected prior to hd-radio-immunotherapy, every six months for two years and annually thereafter. The study was approved by the Institutional Ethical Committee and written informed consent was obtained from each patient. The study group included 29 patients with aggressive lymphoma (diffuse large B-cell lymphoma, n = 19; mantle cell lymphoma, n = 8; Richter syndrome, n = 2) and 24 with indolent NHL subtypes (follicular lymphoma, n = 20; marginal zone lymphoma, n = 3; lymphocytic lymphoma, n = 1). Thirty patients were relapsed after one (n=15) or more (n=15) previous chemotherapy regimens, 6 patients were refractory and 17 patients were previously untreated with high-risk prognostic index. Pre-exposure to alkylating agent involved 68% of the patients. A historical group of 55 NHL patients who received an analogous HDS program with final chemotherapy-based autografting was used as control group. Study and control groups were pair-matched for age, gender, histological subtype, disease status at transplant and type of grafted PBSC. Results:At a median follow-up of 49 months (range, 32 – 74), 4 out of 53 patients of the study group developed sMDS/AL at 6, 12, 27 and 36 months from transplant. According to WHO criteria, refractory anemia with excess of blasts 1 (RAEB-1) was diagnosed in one patient, whereas refractory cytopenia with unilineage dysplasia (RCUD) was diagnosed in the other three cases, with acute leukemia transformation being observed in two patients at 12 and 18 months after initial detection of abnormal cytogenetics, respectively. The cumulative incidence of sMDS/AL resulted of 8.29% at 5-years. An analogous 5-year cumulative incidence of 8.05% was observed in the historical control group autografted following a chemotherapy-based conditioning. Radioimmunotherapy-treated patients showed a significant but transient decrease in granulocyte-macrophage colony forming unit (CFU-GM) at 6 months after transplant, with recovery to baseline values at 12 months. Multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and fibroblast CFU (CFU-F) were not show significantly affected over time. In contrast, a progressive and significant decrease in BM TL was evident from 12 months after transplant. Conclusion:In our series of elderly NHL patients receiving hd-radio-immunotherapy, the incidence of sMDS/AL was found increased compared to recently reported series of younger lymphoma patients receiving chemotherapy-based conditioning. However, the increased risk of sMDS/AL was not associated with the type of myeloablative conditioning, i.e. chemotherapy vs radio-immunotherapy. Other factors may have favored the development of sMDS/AL, including advanced age at transplant, extensive pre-transplant therapy and type of reinfused PBSC. The observed progressive TL loss gives support to the concept that TL loss might be involved in sMDS/AL development. Disclosures:No relevant conflicts of interest to declare.

RNA interference-mediated hTERT inhibition enhances TRAIL-induced apoptosis in resistant hepatocellular carcinoma cells

TRAIL has been reported to induce apoptosis in a variety of tumor cell types including hepato-cellular carcinoma (HCC) cell lines. However, considerable numbers of HCC cells, especially some highly malignant tumors, show resistance to TRAIL-induced apoptosis. The molecular mechanisms that regulate sensitivity versus resistance of tumor cells to TRAIL-induced apoptosis remain poorly defined. It has been shown that human telomerase catalytic subunit (hTERT) is overexpressed in human HCCs. In this study, we investigated the effects and the mechanisms of hTERT RNAi on the TRAIL-induced apoptosis of HCC cells that exhibit resistance to TRAIL. Our results indicate that hTERT RNAi sensitizes TRAIL-resistant HCC cells to TRAIL-induced apoptosis. hTERT RNAi-mediated sensitization to TRAIL-induced apoptosis is accompanied up-regulation of procaspases-8 and -9, inhibition of telomerase activity and loss of telomere length. Our results suggest that hTERT RNAi overcame the resistance of the HCC cells against TRAIL, at least in part, via the mitochondrial type II apoptosis pathway and telomerase-dependent pathway.