Significant Telomere Shortening Research Articles

Genetics of digital osteoarthritis

Genetic factors contribute to the development of digital osteoarthritis, whose heritability has been estimated at 48 to 65%. Among the manifestations of digital osteoarthritis, only Heberden's nodes are transmitted by Mendelian inheritance, as a dominant trait in women and a recessive trait in men. The other forms of digital osteoarthritis are multifactorial, with a major gene and a residual multifactorial component that probably interacts with environmental factors. Hindrances to molecular studies include the absence to date of a universally accepted definition of the phenotype and the late onset of the manifestations. Genetic association studies of selected class I and II HLA genes produced conflicting results. The T303M polymorphism of the MATN3 gene, which was initially described as associated with hand osteoarthritis, may be more closely linked to trapeziometacarpal osteoarthritis than to digital osteoarthritis. Genome-wide scans have identified numerous loci linked to digital osteoarthritis. Replication has been achieved for some of these loci, most notably those located at 2p, 2q, 3p, 4q, and 7p. A recently published genome-wide association study showed that an A2BP1 gene polymorphism was significantly associated with hand osteoarthritis. Many candidate-gene studies found associations with AGC1, ASPN, ENPP1, HFE, KL, VDR, IL-1 cluster, and IL-6, although the results were not consistently reproducible. In one study, women with hand osteoarthritis had significant telomere shortening. Telomere shortening has also been reported in other age-related conditions.

Functional p53 is required for effective execution of telomerase inhibition in BCR-ABL–positive CML cells

In chronic myeloid leukemia (CML), increased cellular turnover of hematopoietic cells driven by the oncogene BCR-ABL leads to accelerated telomere shortening despite increased telomerase activity. It has been postulated that shortened telomeres, particularly in the context of increased telomerase activity, might facilitate accumulation of genetic aberrations and, consequently, disease progression from chronic phase to accelerated phase and blast crisis. Therefore, inhibition of telomerase might be a promising approach in CML therapy. To investigate the therapeutic potential of telomerase inhibition in this model disorder, we used a small molecule telomerase inhibitor, BIBR1532 as well as expression of a dominant-negative mutant of hTERT (DNhTERT-IRES-GFP) in the p53-negative CML blast crisis cell line K562 and characterized the effects in long-term culture. Furthermore, we expressed an inducible p53 construct (vector pBabe-p53ER(tam)) via retroviral transduction in cells with critically short telomeres and in cells with a normal telomere length to explain the role of the tumor suppressor in response to critical telomere shortening in BCR-ABL-positive cells. BIBR1532-treated bulk cultures did not show altered growth kinetics despite significant telomere shortening to a critical length of approximately 5 kb. In comparison, DNhTERT-expressing clones either lost telomere length, leading to a significant but transient slow down in proliferation but eventually all escaped senescence/crisis (group I) or, alternatively, remained virtually unaffected despite measurable telomerase inhibition (group II). Further analyses of group I clones revealed impaired DNA damage response and an accumulation of dicentric chromosomes. However, upon restoration of p53 in telomerase-negative K562 clones with critically short telomeres, immediate reinduction of apoptosis and complete eradication of cells was observed, whereas vector control cells continued to escape from crisis. These results suggest that the success of strategies aimed at telomerase inhibition in CML is highly dependent on the presence of functional p53 and should be explored preferentially in chronic phase CML.

OP20 Hepatocytes and cholangiocytes do not have significant telomere shortening with increasing chronological age in normal livers

IntroductionTelomeres, which cap and protect chromosomal DNA, shorten with each cell division reaching a critical point eventually when the cell is arrested in G1 phase and enters a state of...

3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost' nuclei and very short telomeres

In Epstein–Barr virus (EBV) negative Hodgkin’s cell lines and classical EBV-negative Hodgkin’s lymphoma (HL), Reed–Sternberg cells (RS cells) represent end-stage tumor cells, in which further nuclear division becomes impossible because of sustained telomere loss, shortening and aggregation. However, the three-dimensional (3D) telomere organization in latent membrane protein 1 (LMP1)-expressing RS cells of EBV-associated HL is not known. We performed a 3D telomere analysis after quantitative fluorescent in situ hybridization on 5 μm tissue sections on two LMP1-expressing HL cases and showed highly significant telomere shortening (P<0.0001) and formation of telomere aggregates in RS cells (P<0.0001), when compared with the mononuclear precursor Hodgkin cells (H cells). Telomere-poor or telomere-free ‘ghost' nuclei were a regular finding in these RS cells. These nuclei and their telomere content strongly contrasted with the corona of surrounding lymphocytes showing numerous midsized telomere hybridization signals. Both H cells and RS cells of two EBV-negative HL cases analyzed in parallel showed 3D telomere patterns identical to those of LMP1-expressing cases. As a major advance, our 3D nuclear imaging approach allows the visualization of hitherto unknown profound changes in the 3D nuclear telomere organization associated with the transition from LMP1-positive H cells to LMP1-positive RS cells. We conclude that RS cells irrespective of LMP1 expression are end-stage tumor cells in which the extent of their inability to divide further is proportional to the increase of very short telomeres, telomere loss, aggregate formation and the generation of ‘ghost' nuclei.

The effects of telomerase inhibition on prostate tumor‐initiating cells

Prostate cancer is the most common malignancy in men, and patients with metastatic disease have poor outcome even with the most advanced therapeutic approaches. Most cancer therapies target the bulk tumor cells, but may leave intact a small population of tumor-initiating cells (TICs), which are believed to be responsible for the subsequent relapse and metastasis. Using specific surface markers (CD44, integrin alpha(2)beta(1) and CD133), Hoechst 33342 dye exclusion, and holoclone formation, we isolated TICs from a panel of prostate cancer cell lines (DU145, C4-2 and LNCaP). We have found that prostate TICs have significant telomerase activity which is inhibited by imetelstat sodium (GRN163L), a new telomerase antagonist that is currently in Phase I/II clinical trials for several hematological and solid tumor malignancies. Prostate TICs telomeres were of similar average length to the telomeres of the main population of cells and significant telomere shortening was detected in prostate TICs as a result of imetelstat treatment. These findings suggest that telomerase inhibition therapy may be able to efficiently target the prostate TICs in addition to the bulk tumor cells, providing new opportunities for combination therapies.

Screening of Telomere Length in Peripheral Blood Lymphocytes From Patients with Aplastic Anemia by Flow-Fluorescence in Situ Hybridization.

Abstract 3205Poster Board III-142Telomeres in peripheral blood cells from patients with aplastic anemia (AA) are shorter than those from normal individuals. One possible explanation is that telomere shortening reflects the extent and duration of damage to hematopoietic stem cells and the number of compensatory stem cell divisions. Patients with such stressed hematopoiesis probably do not respond to immunosuppressive therapy (IST). Cryptic forms of dyskeratosis congenita (DC) have been recognized in patients with AA who do not have physical abnormalities, but who harbor the gene mutation associated with telomere maintenance. To identify such patients is important because those who have these mutations might respond poorly to IST. The present study evaluates the usefulness of screening telomere length in peripheral blood lymphocytes from patients at the time of diagnosis with AA by flow-fluorescence in situ hybridization (flow-FISH) using Telomere PNA kits (Dako Cytomation, Glostrup, Denmark). After gating diploid cells based on propidium iodine staining, lymphocytes were isolated according to size and granularity. Relative telomere length (RTL) was calculated as the ratio between the telomere signal of each sample and the telomere signal of the control cell line. Because telomeres shorten with age, we obtained age-adjusted measurements for comparison. We used flow-FISH to measure the length of telomeres in peripheral blood lymphocytes from five and two patients with classical and cryptic DC who had the TERT mutation, respectively, to determine whether these two forms of DC can be screened. Significant telomere shortening was detected in all seven patients compared with age-matched normal individuals. To determine the association between telomere length and response to IST, we compared telomere lengths in peripheral blood lymphocytes from 61 normal controls and 27 patients with AA (very severe, severe and non-severe, n = 9 each) at diagnosis who received IST with antithymocyte globulin (ATG) and cyclosporine. The median age of the patients was 9.0 years (range, 1.5 to 14.8). The causes of AA were unknown etiology in 25 patients and hepatitis in 2. In total, 17 of the 27 patients (63%) responded to IST at 6 months after administration of ATG. Age-adjusted delta RTLs did not significantly differ between normal controls and responders (p = 0.181), but those in non-responders were significantly lower than those in normal individuals (p = 0.000). Our findings suggest that screening telomere length in patients with AA at diagnosis is useful for detecting cryptic DC and for predicting responses to IST. DisclosuresNo relevant conflicts of interest to declare.

Telomere shortening may be associated with human keloids

BackgroundKeloids are benign skin tumors that are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are inherited with an autosomal dominant mode with incomplete clinical penetrance and variable expression. Keloids are characterized by formation of excess scar tissue beyond the boundaries of the wound. The exact etiology is still unknown and there is currently no appropriate treatment for keloid disease.MethodsWe analyzed sample tissues were obtained from 20 patients with keloid skin lesions and normal skin was obtained from 20 healthy donors. The telomeres were measured by Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay. Quantitative Real-Time RT-PCR analysis of hTERT gene expression was performed and intracellular ROS generation was measured.ResultsIn this study, we determined whether telomeric shortening and the expression of human telomerase reverse transcriptase (hTERT) occurs in keloid patients. Using Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay, we detected a significant telomere shortening of 30% in keloid specimens compared to normal skin. Using quantitative Real-Time RT-PCR, telomerase activity was found absent in the keloid tissues. Moreover, an increase in ROS generation was detected in fibroblasts cell cultures from keloid specimens as more time elapsed compared to fibroblasts from normal skin.ConclusionTelomere shortening has been reported in several metabolic and cardiovascular diseases. We found that telomere shortening can also be associated with human keloids. Chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases. Here we found increased ROS generation in fibroblasts from keloid fibroblasts cell cultures when compared to normal skin fibroblasts. Hence we conclude that oxidative stress might be an important modulator of telomere loss in keloid because of the absence of active telomerase that counteracts telomere shortening.

Nanoparticle mediated delivery of 2′-O-methyl-RNA leads to efficient telomerase inhibition and telomere shortening in human lung cancer cells

A promising approach for treatment of non-small cell lung cancer (NSCLC) is based on the inhibition of telomerase in cancer cells. The antisense oligonucleotide 2′-O-methyl-RNA binding to the RNA component of telomerase acts as a selective telomerase inhibitor. We developed chitosan-coated polylactide-coglycolide (PLGA) nanoparticles to mediate efficient delivery of 2′-O-methyl-RNA into human lung cancer cells. Cellular uptake of the inhibitor mediated by chitosan-coated PLGA nanoparticles was greatly enhanced compared to the uptake of antisense oligonucleotide alone as shown by flow cytometry analysis. Confocal laser scanning microscopy clearly demonstrated internalization of 2′-O-methyl-RNA. 2′-O-methyl-RNA-nanoparticle complexes exhibited nearly no acute cytotoxicity in human lung cancer cells and did not influence the viability of primary tumor lung fibroblasts. Human NSCLC A549 cells treated with 2′-O-methyl-RNA-nanoparticle complexes showed 87% viability compared to untreated control cells. 2′-O-methyl-RNA delivered by nanoparticle complexes inhibited telomerase activity in a sequence-specific manner. During long-term treatment (15 weeks) telomerase activity was continuously reduced by approximately 80%. Furthermore, nanoparticle mediated delivery of 2′-O-methyl-RNA resulted in significant telomere shortening from 5.9 kb to 4 kb ( p = 0.008) in A549 cells. In summary, our data demonstrate that nanoparticle mediated delivery of 2′-O-methyl-RNA induces effective telomerase inhibition and telomere shortening in human lung cancer cells and therefore represents a novel and promising strategy for the treatment of lung cancer.

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

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

Apoptosis and telomeres shortening related to HIV-1 induced oxidative stress in an astrocytoma cell line

BackgroundOxidative stress plays a key role in the neuropathogenesis of Human Immunodeficiency Virus-1 (HIV-1) infection causing apoptosis of astroglia cells and neurons. Recent data have shown that oxidative stress is also responsible for the acceleration of human fibroblast telomere shortening in vitro. In the present study we analyzed the potential relations occurring between free radicals formation and telomere length during HIV-1 mediated astroglial death.ResultsTo this end, U373 human astrocytoma cells have been directly exposed to X4-using HIV-1IIIB strain, for 1, 3 or 5 days and treated (where requested) with N-acetylcysteine (NAC), a cysteine donor involved in the synthesis of glutathione (GSH, a cellular antioxidant) and apoptosis has been evaluated by FACS analysis. Quantitative-FISH (Q-FISH) has been employed for studying the telomere length while intracellular reduced/oxidized glutathione (GSH/GSSG) ratio has been determined by High-Performance Liquid Chromatography (HPLC). Incubation of U373 with HIV-1IIIB led to significant induction of cellular apoptosis that was reduced in the presence of 1 mM NAC. Moreover, NAC improved the GSH/GSSG, a sensitive indicator of oxidative stress, that significantly decreased after HIV-1IIIB exposure in U373. Analysis of telomere length in HIV-1 exposed U373 showed a statistically significant telomere shortening, that was completely reverted in NAC-treated U373.ConclusionOur results support the role of HIV-1-mediated oxidative stress in astrocytic death and the importance of antioxidant compounds in preventing these cellular damages. Moreover, these data indicate that the telomere structure, target for oxidative damage, could be the key sensor of cell apoptosis induced by oxidative stress after HIV infection.

Topographical analysis of telomere length and correlation with genomic instability in whole mount prostatectomies

11107 Background: Many critical events in prostatic carcinogenesis appear to relate to the emergence of chromosomal instability and acquisition of genomic rearrangements. Characteristic abnormalities such as 8p loss, 8q gain, trisomy 7, PTEN microdeletions and TMPRSS2-ERG gene fusions appear to mediate mechanisms to increase neoplastic transformation in prostate cancer. Current evidence suggests that telomere dysfunction is a likely causative factor for some of these abnormalities on the basis of its relationship to mechanisms such as the break-fusion-bridge cycle that can lead to the onset of chromosomal instability. Methods: In this study, we correlated telomere length in various prostatic histologies by quantitative FISH with genomic markers of chromosomal instability by standard FISH and immunohistochemical measures of proliferation in 3 whole mount prostatectomies. Results: After analysing approximately 25,000 cells, we found that telomere shortening was correlated with an increase in the number of cells with abnormalities on chromosome 8, such as an increase in the average number of c-myc signals (r∼0.35, p∼0.02). However, there were no significant correlations with abnormalities such as trisomy 7 or abnormalities of the PTEN locus in any sample. Additional findings included; associations found with the probability of C-MYC aberrations in stroma with greater proximity to cancer (&lt;1,000 um), a correlation between telomere length in a number of prostatic histologies (normal, atrophy, HPIN and cancer) with the adjacent stroma, and a lack of correlation between the Ki67 index of various histologies and their telomere length - all suggesting the importance of microenvironmental effects on telomere maintenance in the prostate. Finally, we also report significant telomere shortening in BPH in 2 cases, a phenomenon that has not been noted previously. Conclusions: This is the first study to directly link a mechanism of chromosomal instability with specific chromosomal abnormalities in prostatic carcinogenesis and also suggests that the microenvironmental milieu is of critical importance in the evolution of in vivo telomere homeostasis. No significant financial relationships to disclose.

Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri

Age research in vertebrates is often limited by the longevity of available models. The teleost fish Nothobranchius furzeri has an exceptionally short lifespan with 3.5 months for the laboratory strain GRZ and about 6 months for the wild-derived strain MZM-0403. Here we have investigated telomere length in muscle and skin tissue of young and old fish of both strains using different methods. We found age-dependent telomere shortening in the MZM-0403 strain with the longer lifespan, whereas the short-lived GRZ strain showed no significant telomere shortening with advanced age. Sequencing of the two main telomerase genes Tert and Terc revealed that both genes are highly conserved between the N. furzeri strains while there is little conservation to other fish species and humans. Both genes are ubiquitously expressed in N. furzeri and expression levels of Tert and Terc correlate with telomerase activity in a tissue-specific manner. Unexpectedly, the expression level of Tert is increased in aged muscle and skin tissue of MZM-0403 suggesting that telomeres shorten upon ageing despite increased Tert expression and hence high telomerase activity. We further conclude that the extremely short lifespan of the GRZ strain is not caused by diminished telomerase activity or accelerated telomere shortening.

Granulocyte colony-stimulating factor mobilized CFU-F can be found in the peripheral blood but have limited expansion potential

Bone marrow mesenchymal stem cells are multipotent cells found lining the bone marrow cavity supporting the growth and differentiation of hematologic progenitors. There is growing evidence that these cells can, under the right circumstances, enter the peripheral circulation. We show that granulocyte colony-stimulating factor mobilized peripheral blood contains cells which form colonies and have a similar fibroblastic morphology (termed CFU-F) to bone marrow mesenchymal stem cells. These cells were found at a very low incidence (0.0002%). Mobilized peripheral blood CFU-F were successfully differentiated into osteogenic and adipogenic lineages. FACS analysis showed that the cells had a similar profile to bone marrow mesenchymal stem cells. Importantly, mobilized peripheral blood CFU-F had limited expansion potential and became senescent 20-25 days after isolation. Mobilized peripheral blood CFU-F also did not have any telomerase activity and displayed significant telomere shortening. The rarity of CFU-F in mobilized peripheral blood and the subsequent pressure to divide in cell culture probably contribute to early cellular senescence. Their potential for use in transplant or gene therapy is, therefore, limited.

Telomere length shortening in Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a clonal, proliferative disorder of phenotypically immature CD1a(+) Langerhans cells (LC). The aetiology of LCH is unknown and data supporting an immune dysregulatory disorder as well as a clonal neoplasm have been reported. Telomere shortening has been associated with cancers and premalignant lesions as well as promoting chromosomal instability. To determine whether LCH LC have altered telomere lengths, we used dual detection of CD1a expression by immunofluorescence and telomere length by fluorescence in situ hybridization of LCH LC and lymphocytes in local, multisystem and systemic LCH and compared these with telomere lengths of LC and lymphocytes in reactive lymph nodes. LCH LC showed significantly shorter telomere lengths than LC from reactive lymph nodes or unaffected skin. Lymphocyte telomere lengths showed similar profiles among the different samples. These data show a significant telomere shortening in LCH LC in all stages of disease involvement compared with LC from reactive lymph nodes, suggesting that LCH may share mechanisms of telomere shortening and survival with clonal preneoplastic disorders and cancer, although an initiating infectious or immune event is still possible.

Telomere length analysis in monocytes and lymphocytes from patients with systemic lupus erythematosus using multi-color flow-FISH

In order to analyse telomere length in subsets of human peripheral blood lymphocytes and monocytes, we modified a recently developed multicolor flow- fluorescent in situ hybridization (FISH) methodology that combines flow-FISH and antibody staining for cell surface antigens. We analysed telomere length of peripheral blood mononuclear cells in a group of 22 patients with systemic lupus erythematosus (SLE) and 20 age-matched healthy donors. We found that neither CD4+, CD8+, CD19+ cells nor CD14+ monocytes have significantly shorter telomeres compared with their healthy counterparts. On the basis of these findings, we then used monocyte telomere length as internal reference in order to control for intra-individual variability in telomere length. By using this approach, we could demonstrate significant telomere shortening in all three lymphocyte subsets (in all cases P < 0.05) compared with monocytes. However, these differences did not vary significantly between SLE patients and controls. In summary, telomere lengths in subpopulations of hematopoietic cells can be monitored in patients with SLE using multicolor flow-FISH. While confirming data by other groups on telomere length in lymphocyte subpopulations, our data argue against an increased proliferation rate of peripheral blood monocytes reflected by accelerated telomere shortening in patients with SLE.

Telomere Length Shortening in Langerhans Cell Histiocytosis.

The purpose of this study was to analyze telomere lengths in Langerhans Cell Histiocytosis (LCH). LCH is a clonally derived, proliferative disorder of phenotypically immature CD1a+ Langerhans cells (LCs). The etiology of LCH is unknown and data supporting an immune dysregulatory disorder as well as a clonal neoplasm have been reported. Telomere shortening has been associated with cancers and premalignant lesions as well as being important in promoting chromosomal instability. We therefore conducted a study to quantitatively determine the telomere lengths in LCH. Tissue arrays of reactive lymph nodes, local, multisystem, and systemic LCH containing 9, 7, 12, and 10 specimens respectively were used. These tissue arrays included 2 specimens that had unaffected skin represented, providing an opportunity to examine non-pathologic LCs. The tissue arrays were first hybridized with a N-terminal covalently linked Cy3-labeled, telomere specific peptide nucleic acid (PNA) probe to stain telomeres. The slides were then processed for indirect immunofluorescence to identify LCs using an anti-CD1a primary antibody followed by an anti-mouse IgG fraction Alexa Fluor 488. Finally, slides were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Digital fluorescent telomere signals were visualized using a Nikon Eclipse E400 epifluorescence microscope and telomere lengths were analyzed with ImageJ software. A total of 1039 measurements were made from 38 individuals. A significant shortening of telomeres was demonstrated in LCH LCs of local, multisystem and systemic LCH in comparison to telomere lengths of reactive lymph node LCs and normal skin, which were not significantly different. To investigate whether telomere length shortening of LCH LCs is selective, we analyzed the telomere length in lymphocytes in LCH specimens and in the reactive lymph node specimens. There was no significant evidence of telomere shortening of lymphocytes in LCH compared to lymphocytes in reactive lymph nodes (p=0.1862). These data show significant telomere shortening in the LCs in all stages of disease involvement with more variability among local cases. These observations suggest that in most cases LCH may share important mechanisms of telomere shortening and survival with clonal pre-neoplastic disorders and cancer.

Premature Aging of Hematopoietic Cells in Long Term Survivors after HSCT with Chronic GVHD and a Female Donor.

The establishment of donor-derived hematopoiesis in recipients of hematopoietic stem cell (HSC) transplantation (HSCT) involves extensive proliferation of HSCs and might lead to “premature aging” of hematopoietic cells. Telomere shortening as indicator of cell proliferation has been described after HSCT. The telomere attrition has mainly been observed during the first year after allogeneic HSCT. Thereafter, telomere length dynamics of recipients appeared not to differ from their donors. The aim of our cross-sectional study was to evaluate the telomere attrition in leukocyte subsets of very long term survivors (LTS) after HSCT in relation to donor/recipient age, sex, cell counts in peripheral blood, number of transplanted nucleated cells (NTNC), and occurrence of acute and chronic graft versus host disease (GVHD). Automated multicolour flow-FISH was used to measure the telomere length in granulocytes, naive T-cells, B-cells and NK/NKT-cells of 44 LTS and their HLA-identical sibling donors. At HSCT the median age of donors and recipients was 25.8 (2–46) and 26.8 years (5–50). The median follow-up after HSCT was 17.5 years (11–26). Four patients received HSCT for aplastic anemia, 40 for hematological malignancies. All patients received bone marrow as source of HSC and TBI was part of the conditioning in 39 (89%). Acute GVHD was observed in 31 (70%), and chronic GVHD in 22 (50%) patients. The age-matched and cell-type specific absolute telomere length values for recipients and donors fell between the 1st-99th percentile of the normal distribution. The telomere length (mean ± std) was significantly shorter in recipients as compared to their donors (p<0.01) for granulocytes (6.5±0.9 vs 7.1±0.9), T-cells (5.7±1.2 vs 6.6±1.2), B-cells (7.1±1.1 vs 7.8±1.1) and NK/NKT-cells (4.8±1.0 vs 5.6±1.3). The mean difference between recipients and donors for each subset of cells was between 0.6–0.9 kb. The mean telomere length of all subsets of cells was significantly shorter for males compared to females even though this difference was small. Age, sex of recipient, NTNC, and acute GVHD had no impact on telomere attrition. In all cell types except NK/NKT-cells we found a significant telomere shortening in recipients transplanted with a female donor (p<0.04) and in those with chronic GVHD (p<0.04). The telomere length difference between donor/recipient was most pronounced for recipients with the combination of a female donor and chronic GVHD (Figure 1). The loss in telomere length is more than twice for patients with a female donor and chronic GVHD compared to those with a male donor without chronic GVHD. In summary, our data reveal chronic GVHD and a female donor to be predictors for higher telomere attrition in LTS after HSCT. The more pronounced telomere attrition with a female donor and chronic GVHD corresponds to approximately 30–60 years of cell aging and raises once more the question of cellular immuno-senescence and its consequences in LTS of HSCT. [Display omitted]

Effective Telomerase Inhibition Via Expression of Dominant-Negative HTERT Leads to Altered Growth Kinetics, Elevated Apoptosis and Increased Radiosensitivity of Bcr-Abl-Positive K562 Cells In Vitro.

Disease progression in CML is associated with accelerated telomere shortening caused by increased turnover of Bcr-Abl positive cells. We have recently proposed a model in which continuous telomere shortening is correlated with increasing genetic instability in Bcr-Abl-positive cells thus facilitating the acquisition of secondary genetic abnormalities and, as a consequence clonal succession and eventually clinical progression of the disease. Based on this hypothesis, telomerase upregulation in late stage disease is a prerequisite for prevention of replication-induced senescence in Bcr-Abl positive cells. As a consequence, treatment of CML with Telomerase inhibitors (TI) represents an attractive strategy aiming at the potential eradication of cycling CML stem cells. Therefore, we have exploited both pharmacological (small molecule inhibitor) and genetic strategies (dominant negative hTERT mutants, DN-hTERT) of telomerase inhibition in CML cells in vitro We first treated K562 cells with the pharmacological telomerase inhibitor BIBR1532 in vitro. After around 400 population doublings (PD), no differences in growth kinetics nor signs of senescence or apoptosis were observed in BIBR1532 treated cells despite of significant telomere shortening (22 base pairs (bp) per PD) compared to control cells. Furthermore, neither significant differences in mRNA expression of telomere/telomerase-associated proteins, nor accumulation of double strand breaks (DSBs) under irradiation was observed in treated cells with short telomeres as opposed to untreated control cells. The very slow shortening rate of 22bp/PD plus the lack of stigmata pointing to induction of senescence in K562 cells lead to the assumption that telomerase activity is not complete inhibited by the compound. In order to verify the potency of telomerase directed treatments in CML, we therefore expressed DN-hTERT in K562 cells. Integration of DN-hTERT led to a significant decrease in telomerase activity (measured by RQ-TRAP). Furthermore, DN-hTERT expressing cells underwent accelerated telomere shortening at a substantially higher rate (>100 bp/PD) from 15kb to around 4kb within 110 days of culture. In contrast to BIBR1532-treated cells, DN-hTERT expressing K562 cells slowed down growth kinetic in comparison to control cells after 80 days of culture. By using Annexin 5 staining, 25% of apoptotic cells could be detected in cells with critically short telomeres as compared to control cells (<3%). Finally, a significantly increased accumulation of double strand breaks (DSBs) detected by gammaH2AX foci after exposure to irradiation was observed in DN-hTERT K562 cells as compared to control cells pointing to an impaired DNA repair machinery in Bcr-Abl positive cells with disrupted telomere maintenance. In summary, the data suggest that pharmacological telomerase inhibition by BIBR1532 is insufficient to induce telomere-mediated senescence in Bcr-Abl-positive cells. However, accelerated telomere shortening, slowing down of growth kinetics, elevated apoptosis and increased radiosensitivity induced by expression of DN-hTERT indicate a therapeutic potential for telomerase-directed treatment strategies in CML.

Abstract 3354: Increase of Telomere Gap between Granulocytes and Lymphocytes in Patients with Previous Myocardial Infarction: A Novel Measure for Accelerated Telomere Erosion

Individuals with age-corrected reduction of total leukocyte telomere length have been shown to exhibit a remarkable increase in mortality from cardiovascular and infectious complications. Given the considerable interindividual variability of telomere length in healthy adults due to genetic differences already present at birth and the heterogeneity in composition of the total leukocyte compartment, extensive study populations are required to detect accelerated telomere shortening between age-matched groups. With this study, we intended to identify a parameter to improve prediction of individual telomere erosion in patients (pts) with coronary artery disease and previous myocardial infarction (CAD). Methods: Mean telomere length (mTL) was measured in 63 CAD pts (mean age 65±4 years, mean ejection fraction 31±9%) and 23 age-matched healthy controls (65±4 years). Using the flow-FISH method, we were able to distinguish between myeloid and lymphoid cell populations in peripheral blood cells as well as in bone marrow mononuclear cells (BMC). Results: The difference between mTL of the granulocyte and lymphocyte population (ΔTEL Gr-Ly ) was significantly increased from 742 ± 396 base pairs (bp) in the control group to 1055±570 bp in CAD pts (p=0.018). Age was the only independent predictor for telomere length of BMCs. Lymphocytes, but not granulocytes, demonstrated significant telomere shortening between BMCs and peripheral blood in CAD patients (−444 ± 624 bp) as opposed to an increase in a young and healthy control group (+307 ± 372 bp) (p&lt;0.001). Using immunomagnetic cell sorting, telomerase activity (TA) was determined in freshly isolated white blood cell subpopulations. Surprisingly, TA could not be detected in peripheral blood granulocytes (CD15 + granulocytes) from CAD pts. In contrast, CD4 + T-lymphocytes contained the highest amount of TA, compared to CD8 + cells and B-lymphocytes. Conclusions: Our results suggest that primarily peripheral blood lymphocytes - and not granulocytes - can develop stress-induced telomere erosion in CAD. Therefore the base pair length difference between the two populations provides a valuable parameter to detect telomere erosion in patients with CAD, largely independent of the hereditary influence on mTL.

Telomere Shortening and Chromosomal Abnormalities in Intestinal Metaplasia of the Urinary Bladder

Although intestinal metaplasia is often found in association with adenocarcinoma of the urinary bladder, it is unclear whether intestinal metaplasia of the bladder is a premalignant lesion. Telomere shortening has recently been implicated in epithelial carcinogenesis. We used quantitative fluorescent in situ hybridization (FISH) to measure telomere length and UroVysion FISH to detect cytogenetic abnormalities in urinary bladder specimens with intestinal metaplasia. Paraffin-embedded tissue blocks from 34 patients with intestinal metaplasia of the urinary bladder were evaluated. Twelve of the 34 patients had coexistent cystitis glandularis, and telomere length was measured in these lesions for comparison. Tissue sections were prepared and hybridized with a telomere-specific peptide nucleic acid probe. Quantitative FISH on interphase nuclei was used to assess telomere signal intensity. Additional sections were hybridized with centromeric probes for chromosomes 3, 7, and 17 and a locus-specific probe 9p21. Multicolor FISH was used to analyze for cytogenic abnormalities in the interphase nuclei of intestinal metaplasia. In all 34 cases, reduced average telomere signal intensity was observed in the nuclei of intestinal metaplasia cells compared with adjacent control nuclei to produce a mean relative intensity of 48.5% (P < 0.0001). When cystitis glandularis was present, significant differences in the telomere-specific signal intensity existed between cystitis glandularis and normal cells (P = 0.0005) and between cystitis glandularis and intestinal metaplasia cells (P = 0.0015). Three of the 34 cases showed chromosomal gains in the UroVysion FISH assay. Our findings indicate that intestinal metaplasia in the urinary bladder is associated with significant telomere shortening relative to telomere length in adjacent normal urothelial cells. These lesions also occasionally showed cytogenetic abnormalities associated with telomere shortening. Our findings support the hypothesis that intestinal metaplasia of the urinary bladder is a precursor lesion to and could be a marker in the development of adenocarcinoma of the urinary bladder.