Loss Of Telomeric Repeats Research Articles

Conserved satellite DNA motif and lack of interstitial telomeric sites in highly rearranged African Nothobranchius killifish karyotypes.

Using African annual killifishes of the genus Nothobranchius from temporary savannah pools with rapid karyotype and sex chromosome evolution, we analysed the chromosomal distribution of telomeric (TTAGGG)n repeat and Nfu-SatC satellite DNA (satDNA; isolated from Nothobranchius furzeri) in 15 species across the Nothobranchius killifish phylogeny, and with Fundulosoma thierryi as an out-group. Our fluorescence in situ hybridization experiments revealed that all analysed taxa share the presence of Nfu-SatC repeat but with diverse organization and distribution on chromosomes. Nfu-SatC landscape was similar in conspecific populations of Nothobranchius guentheri and Nothobranchius melanospilus but slightly-to-moderately differed between populations of Nothobranchius pienaari, and between closely related Nothobranchius kuhntae and Nothobranchius orthonotus. Inter-individual variability in Nfu-SatC patterns was found in N. orthonotus and Nothobranchius krysanovi. We revealed mostly no sex-linked patterns of studied repetitive DNA distribution. Only in Nothobranchius brieni, possessing multiple sex chromosomes, Nfu-SatC repeat occupied a substantial portion of the neo-Y chromosome, similarly as formerly found in the XY sex chromosome system of turquoise killifish N. furzeri and its sister species Nothobranchius kadleci-representatives not closely related to N. brieni. All studied species further shared patterns of expected telomeric repeats at the ends of all chromosomes and no additional interstitial telomeric sites. In summary, we revealed (i) the presence of conserved satDNA class in Nothobranchius clades (a rare pattern among ray-finned fishes); (ii) independent trajectories of Nothobranchius sex chromosome differentiation, with recurrent and convergent accumulation of Nfu-SatC on the Y chromosome in some species; and (iii) genus-wide shared tendency to loss of telomeric repeats during interchromosomal rearrangements. Collectively, our findings advance our understanding of genome structure, mechanisms of karyotype reshuffling, and sex chromosome differentiation in Nothobranchius killifishes from the genus-wide perspective.

Telomere Length Regulation.

The number of (TTAGGG)n repeats at the ends of chromosomes is highly variable between individual chromosomes, between different cells and between species. Progressive loss of telomere repeats limits the proliferation of pre-malignant human cells but also contributes to aging by inducing apoptosis and senescence in normal cells. Despite enormous progress in understanding distinct pathways that result in loss and gain of telomeric DNA in different cell types, many questions remain. Further studies are needed to delineate the role of damage to telomeric DNA, replication errors, chromatin structure, liquid-liquid phase transition, telomeric transcripts (TERRA) and secondary DNA structures such as guanine quadruplex structures, R-loops and T-loops in inducing gains and losses of telomere repeats in different cell types. Limitations of current telomere length measurements techniques and differences in telomere biology between species and different cell types complicate generalizations about the role of telomeres in aging and cancer. Here some of the factors regulating the telomere length in embryonic and adult cells in mammals are discussed from a mechanistic and evolutionary perspective.

TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction

SUMMARYThe telomeric shelterin protein telomeric repeat-binding factor 2 (TRF2) recruits origin recognition complex (ORC) proteins, the foundational building blocks of DNA replication origins, to telomeres. We seek to determine whether TRF2-recruited ORC proteins give rise to functional origins in telomere repeat tracts. We find that reduction of telomeric recruitment of ORC2 by expression of an ORC interaction-defective TRF2 mutant significantly reduces telomeric initiation events in human cells. This reduction in initiation events is accompanied by telomere repeat loss, telomere aberrations and dysfunction. We demonstrate that telomeric origins are activated by induced replication stress to provide a key rescue mechanism for completing compromised telomere replication. Importantly, our studies also indicate that the chromatin remodeler SNF2H promotes telomeric initiation events by providing access for ORC2. Collectively, our findings reveal that active recruitment of ORC by TRF2 leads to formation of functional origins, providing an important mechanism for avoiding telomere dysfunction and rescuing challenged telomere replication.

Telomere damage induces internal loops that generate telomeric circles

Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops) that occur in the proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage to the accumulation of extrachromosomal telomeric circles and to telomere erosion.

Telomere Dynamics Throughout Spermatogenesis.

Telomeres are repeat regions of DNA that cap either end of each chromosome, thereby providing stability and protection from the degradation of gene-rich regions. Each cell replication causes the loss of telomeric repeats due to incomplete DNA replication, though it is well-established that progressive telomere shortening is evaded in male germ cells by the maintenance of active telomerase. However, germ cell telomeres are still susceptible to disruption or insult by oxidative stress, toxicant exposure, and aging. Our aim was to examine the relative telomere length (rTL) in an outbred Sprague Dawley (SD) and an inbred Brown Norway (BN) rat model for paternal aging. No significant differences were found when comparing pachytene spermatocytes (PS), round spermatids (RS), and sperm obtained from the caput and cauda of the epididymis of young and aged SD rats; this is likely due to the high variance observed among individuals. A significant age-dependent decrease in rTL was observed from 115.6 (±6.5) to 93.3 (±6.3) in caput sperm and from 142.4 (±14.6) to 105.3 (±2.5) in cauda sperm from BN rats. Additionally, an increase in rTL during epididymal maturation was observed in both strains, most strikingly from 115.6 (±6.5) to 142 (±14.6) in young BN rats. These results confirm the decrease in rTL in rodents, but only when an inbred strain is used, and represent the first demonstration that rTL changes as sperm transit through the epididymis.

Mre11 and Blm-Dependent Formation of ALT-Like Telomeres in Ku-Deficient Ustilago maydis.

A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR). These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs). The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and “trapped” the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure.

Telomeres, atherosclerosis, and human longevity: a causal hypothesis.

Human telomere length, as expressed in leukocytes, is strongly fashioned by heritability.1–5 A number of genes that explain some of the inter-individual variation in leukocyte telomere length have already been identified.6–9 Leukocyte telomere length is longer in women than in men3,10,11 and in African Americans than in whites of European descent.12,13 In addition, offspring of older fathers display a longer leukocyte telomere length, a finding that may reflect longer telomeres in sperm of older men.14–17 In contrast to the male germline, telomeres in replicative somatic tissues, including the hematopoietic system, display age-dependent shortening. A shorter leukocyte telomere length is associated with aging-related diseases, principally atherosclerosis18,19 and reduced longevity.20,21 The prevailing interpretation is that a shorter leukocyte telomere length is the outcome of processes, mainly the age-dependent accruing burden of oxidative stress and inflammation22 that ultimately shorten the human lifespan. As such, leukocyte telomere length is considered to be a non-causal biomarker, ie, a telomeric “clock,” of human aging. We propose that telomere length is not only associated with atherosclerosis and longevity but is also a causal determinant of both. This hypothesis is based on a model of leukocyte telomere length dynamics (leukocyte telomere length and its attrition rate) that incorporates two key features. First, the model differentiates the findings of studies of telomere length dynamics in vitro from those in vivo. Second, the model distinguishes two phases of human leukocyte telomere length dynamics in vivo: growth and adulthood.

Telomeres, cardiovascular aging, and potential intervention for cellular senescence.

A consistent association has been observed between leukocyte telomere length (LTL) and atherosclerosis, but the mechanisms underlying these associations are still not well understood. Premature biology aging was evident in atherosclerotic plaques, characterized by reduced cell proliferation, irreversible growth arrest and apoptosis, and telomere attrition. As atherosclerosis is a state of chronic low-grade inflammation and increased oxidative stress, shortened LTL in patients with atherosclerosis might stem from the two sources, one is an accelerated rate in hematopoietic stem cells (HSCs) replication to replace leukocytes consumed in the inflammatory process, and another is the increase in the loss of telomere repeats per replication. Thus, diminished HSC reserves at birth and age-dependent telomere attrition afterward are mirrored in shortened LTL during the adulthood. In addition, the inter-individual variation of LTL in the general population can be partly explained by genetic factors regulating telomere maintenance and the rate of HSCs replication. Atherosclerosis is an aging-related disease, and practically all humans develop atherosclerosis if they live long enough. Here we overview the potential roles of LTL dynamics in the imbalance between injurious oxidative stress/inflammation and endothelial repair during the pathogenesis of age-related atherosclerosis, and discuss the possibility that preventing accelerated cellular senescence is a potential target in prevention of atherosclerosis.

Phenotypic Rescue Of Induced Pluripotent Stem Cells From Dyskeratosis Congenita Patients By Ectopic Expression Of DKC1 But Not TERC

Telomerase is a ribonucleoprotein that adds telomeric repeats onto the chromosome ends, preventing the replication-dependent loss of telomere repeats and cellular senescence in highly proliferative germ-line cells and in stem cells and cancer cells. Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome, which affects tissues that need constant renewal by stem cell activity. So far 8 genes have been found whose mutation causes DC and they all encode products that play a role in telomere maintenance. About 35% of DC patients show X-linked-recessive inheritance due to mutations in the DKC1gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Mutant dyskerin can destabilize telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known and some results suggest dyskerin may play a more direct role in telomerase action. So far studies of the cell biology of DC stem cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in both telomere biology and hematopoiesis between mouse and human.In this study, to investigate disease pathogenesis of X-linked DC, we generated induced pluripotent stem cells (iPSC) from patients' skin fibroblast cell carrying DKC1 mutations Q31E, Δ37 and A353V. The recurrent A353V mutation accounts for about 40% of DKC1 mutations and usually causes a severe clinical phenotype while patients carrying Q31E and Δ37 mutations show a milder phenotype. We found that dyskerin protein expression in all of these dyskerin mutant iPS cells is decreased in agreement with our mouse studies that show mutant proteins are relatively unstable. These iPS cells show dramatically decreased TERC RNA levels and telomerase activity. Telomere length measurement revealed that mutant iPS cells lose the ability to elongate telomeres during the reprogramming processing; telomere erosion was particularly rapid in A353V cells.To further investigate whether dyskerin protein could play direct role in regulating telomerase activity other than stabilization of TERC RNA during the processing of assembly, we tested if the defect in telomerase function in these iPS cells could be rescued by expressing wild type dyskerin or TERC RNA. We expressed the rescuing genes by using the zinc-finger nuclease-mediated method to insert them into the safe harbor AAVs1 site, initially of DKC1Δ37 cells. After testing the telomerase function, we found that expressing WT-dyskerin protein in Δ37 iPS cells fully restores the mature Terc RNA expression level and the telomerase activity to normal levels. However, although Δ37 iPS with over-expressed WT TERC RNA can accumulate normal level of TERC RNA, these cells fail to increase telomerase activity. These results suggest that defective telomerase activity cause by DKC1 mutations can only reversed by expressing WT dyskerin but not by TERC RNA. These data suggest that, as one of three core components of the telomerase complex, dyskerin may play a direct role in telomerase activity. Disclosures:No relevant conflicts of interest to declare.

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.

Imetelstat, A Potent Telomerase Inhibitor, Inhibits the Spontaneous Growth of CFU-Meg In Vitro From Essential Thrombocythemia Patients but Not From Healthy Individuals,

Abstract 3843Background/Aims: Essential thrombocythemia (ET), a clonal myeloproliferative neoplasia, is characterized by elevation of platelets in peripheral blood (PB) and excessive proliferation of megakaryocytes (megs) in bone marrow. Most ET patients exhibit spontaneous growth of megakaryocytic colony-forming units (CFU-Meg) in vitro. One of the most important pathways involved in clonal expansion is reactivation of telomerase, an enzyme complex which is able to compensate for the loss of telomere repeats due to cell divisions. Over 90% of malignancies demonstrate up-regulation of telomerase, which is an essential component for their immortalization. Imetelstat sodium (GRN163L) is a potent and specific telomerase inhibitor that has demonstrated in vitro and in vivo activity against various tumor types and cancer stem cells. Our aims were to study the growth of CFU-Meg and their levels of telomerase activity (TA) from cord blood (CB), mononuclear cells (MNC) from healthy individuals and ET patients, as well as to test whether TA inhibition by imetelstat could suppress CFU-Meg formation. Agents which inhibit clonal expansion in vitro may have promising clinical activity. Methods:CB cells were enriched for CD34 expressing cells using a negative cell separation system. Cells were incubated with imetelstat (1–15μM) in serum free liquid medium, StemSpan® SFEM containing a cytokine formulation designed for the development of meg progenitor cells. CB cells were cultured for a total of 17 days; at various time point cells were enumerated, assessed by flow cytometry for differentiation markers (CD41) and for TA. Mononuclear cells (MNC) from healthy individuals and from 11 ET patients (WHO 2009 criteria) were isolated from PB and suspended in IMDM or plated into collagen ± cytokines (TPO, IL3, IL6, SCF, EPO) and treated with 0, 0.1, 1 and 10 μM imetelstat or a mismatch control, and incubated for several hours (cell suspensions) or 10–12 days (collagen plus 5% CO2) at 37° C. Megs were stained and the number of CFU-Meg was scored. In addition, TA was measured in CD34+ cells, megs and MNC by TRAP assay. Results:TA is low in CD34+ cells from CB of healthy individuals, but increases up to 9 times during early megakaryocytic differentiation (CD41a+CD34-) and peaks at day 3, followed by a decrease as the cells differentiate. Imetelstat (1–15 μM) significantly inhibited TA in a concentration-dependent manner in these progenitor cells (65% to 99%). Despite considerable TA inhibition, no significant inhibition in cellular growth and differentiation of megs was found in these CD34+ cell cultures from healthy CB donors. TA is low in MNC isolated from the PB of healthy individuals and of patients with ET. Imetelstat did not inhibit CFU-Megs in healthy individuals. In contrast, imetelstat significantly (p < 0.001) and concentration-dependently inhibited the spontaneous growth of CFU-Meg in each patient with ET (n=11, 7 JAKV617F-positive and 4 JAKV617F-negative). The growth of CFU-Meg (% of control) with various imetelstat concentrations in μM was: 100% (0 μM), 107% ± 8.6% (0.1 μM), 79% ± 11.8% (1 μM) and 33% ± 9.4% (10 μM). No correlation was found with the JAKV617F mutational status, other laboratory and clinical parameters, or with cytoreductive therapies. Conclusions:In summary, we can demonstrate telomerase activation with megakaryocytic proliferation and differentiation which precedes the decrease in cellular proliferation. Furthermore, we demonstrated concentration-dependent inhibition of TA by imetelstat in cells from healthy donors, but without inhibiting megakaryocytic proliferation. In contrast, TA inhibition by imetelstat results in a dose-dependent inhibition of spontaneous growth of CFU-Meg in ET patients. Inhibition was demonstrated at clinically relevant concentrations. This in vitro CFU-Meg inhibition is independent of the JAKV617F mutational status and of cytoreductive therapy. It appears that the transient inhibition of TA in normal megakaryocytic cells by imetelstat does not inhibit cellular proliferation, whereas TA inhibition by imetelstat in malignant cells significantly inhibited CFU-Meg proliferation. These findings suggest a specificity of imetelstat for malignant megakaryocytic cells. The impact of imetelstat's clinical activity is being explored in an ongoing phase 2 study in ET patients who have failed at least one prior therapy or who refuse standard of care. Disclosures:Go:Geron Corporation: Employment. Ninomoto:Geron Corporation: Employment. Kashani:Geron Corporation: Employment. Stuart:Geron Corporation: Employment. Oppliger Leibundgut:Geron Corporation: Service Contract; Novartis: Membership on an entity's Board of Directors or advisory committees, Service Contract; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Baerlocher:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Geron Corporation: Research Funding, Service Contract; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Genetic Variations in Telomere Maintenance, with Implications on Tissue Renewal Capacity and Chronic Disease Pathologies

Premature loss of telomere repeats underlies the pathologies of inherited bone marrow failure syndromes. Over the past decade, researchers have mapped genetic lesions responsible for the accelerated loss of telomere repeats. Haploinsufficiencies in the catalytic core components of the telomere maintenance enzyme telomerase, as well as genetic defects in telomerase holoenzyme components responsible for enzyme stability, have been linked to hematopoietic failure pathologies. Frequencies of these disease-associated alleles in human populations are low. Accordingly, the diseases themselves are rare. On the other hand, single nucleotide polymorphisms of telomerase enzyme components are found with much higher frequencies, with several non-synonymous SNP alleles observed in 2-4% of the general population. Importantly, recent advents of molecular diagnostic techniques have uncovered links between telomere length maintenance deficiencies and an increasing number of pathologies unrelated to the hematopoietic system. In these cases, short telomere length correlates to tissue renewal capacities and predicts clinical progression and disease severity. To the authors of this review, these new discoveries imply that even minor genetic defects in telomere maintenance can culminate in the premature failure of tissue compartments with high renewal rates. In this review, we discuss the biology and molecules of telomere maintenance, and the pathologies associated with an accelerated loss of telomeres, along with their etiologies. We also discuss single nucleotide polymorphisms of key telomerase components and their association with tissue renewal deficiency syndromes and other pathologies. We suggest that inter-individual variability in telomere maintenance capacity could play a significant role in chronic inflammatory diseases, and that this is not yet fully appreciated in the translational research of pharmacogenomics and personalized medicine.

Telomere Dysfunction in Bone Marrow Failure.

Abstract SCI-12In order to distinguish a normal telomere from a double strand break, a minimum number of telomere repeats must “cap” each chromosome end. The length of each repeat array will reflect a unique history of addition and losses. Telomere losses are known to occur sporadic as well as with every replication cycle. Losses of telomeric DNA are countered by the telomerase enzyme containing telomerase RNA (encoded by the TERC gene) and a reverse transcriptase protein (encoded by TERT gene) as minimal components. Telomerase levels are high in cells of the germline and immortal cellines and the telomere length is typically maintained in such cells. In contrast, telomerase activity is limiting in most human somatic (stem) cells and as a result the average length of telomere repeats in most somatic cells shows a highly significant decline with age. The hypothesis that loss of telomere repeats acts as a “mitotic clock” and a tumor suppressor mechanism in stem cells is strongly supported by recent studies of patients with mild telomerase deficiency resulting from haplo-insufficiency for either the TERC or TERT gene. Such genetic defects can give rise to various disorders including autosomal dominant Dyskeratosis Congenita (DKC), aplastic anemia, liver fibrosis and pulmonary fibrosis. Other recent studies have revealed that amplification of the hTERT gene is one of the most common genetic abnormalities in various cancers. Paradoxically, it is becoming clear that SNPs within the TERT locus are among the most reproducible risk factors for the development of different types of cancer including lung cancer, acute myeloid leukemia and chronic lymphocytic leukemia. The links between hypo- and hyperproliferative consequences of inborn telomerase deficiencies and SNP's in the TERT gene are poorly understood. It seems plausible that the increased risk of leukemia development in aplastic anemia, myelodysplastic syndrome and Dyskeratosis Congenita, results from stem cell failure. Could reduced stem cell numbers by itself provide a risk factor for tumor development? More direct measures of stem cell numbers in vivo are needed to examine this possibility and the relationship between stem cell numbers and tumor development in patients with defective telomere maintenance as well as in normal individuals as a function of age. Measurements of the average telomere length as well as the length of telomere repeats at individual chromosome ends in specific cells and tissues will further calrify the involvement of telomeres in bone marrow failure, normal aging and tumor biology. DisclosuresLansdorp:Repeat Diagnostics Inc.: Equity Ownership.

Telomeres and telomerase in chronic myeloid leukaemia: impact for pathogenesis, disease progression and targeted therapy

Telomeres are specialized structures localized at the end of human chromosomes. Due to the end replication problem, each cell division results in a loss of telomeric repeats in normal somatic cells. In germ line and stem cells, the multicomponent enzyme telomerase maintains the length of telomere repeats. However, elevated telomerase activity has also been reported in the majority of solid tumours as well as in acute and chronic leukaemia. Chronic myeloid leukaemia (CML) serves as a model disease to study telomere biology in clonal myeloproliferative disorders. In CML, telomere shortening correlates with disease stage, duration of chronic phase (CP), prognosis measured by the Hasford risk score and the response to disease-modifying therapeutics such as the tyrosine kinase inhibitor Imatinib. In addition, telomerase activity (TA) is already increased in CP CML and further upregulated with disease progression to accelerated phase and blast crisis (BC). Furthermore, a correlation of TA with increased genetic instability as well as a shorter survival of the patients has been reported. Here, we review the current state of knowledge of the role of telomere and telomerase biology in CML and discuss the possible impact of novel treatment approaches.

Complex rearrangements are involved in Cephalanthera (Orchidaceae) chromosome evolution

The genus Cephalanthera is an excellent plant group for karyotype evolution studies because it exhibits a dysploid series and bimodal karyotypes. With the aim of understanding their chromosomal and phylogenetic relationships, rRNA genes and the Arabidopsis-type telomeric sequence were mapped by fluorescence in-situ hybridization (FISH), and the rDNA intergenic spacer (ITS) was sequenced for the first time in three European species: C. longifolia (2n = 4x = 32), C. damasonium (2n = 4x = 36) and C. rubra (2n = 4x = 44). One 45S and three 5S rDNA sites are observed in C. longifolia, one 45S and two 5S sites in C. damasonium, and two 45S and one 5S site in C. rubra. Telomeric signals were observed at every chromosome end in all three species and C. damasonium also displays interstitial signals on three chromosome pairs. In agreement with chromosome data, molecular analyses support C. longifolia and C. damasonium as closely related taxa, while C. rubra stands apart. Possible pathways of karyotype evolution are discussed in reference to a previous hypothesis. The results indicate that complex chromosomal rearrangements, possibly involving Robertsonian fusions and fissions, loss of telomeric repeats, gain or loss of rDNA sites and other heterochromatic sequences and inversions, may have contributed to generating the present-day karyotypes.

Telomeres in the haemopoietic system.

The limited life span of most blood cells requires the continuous production of cells, which in adults exceeds 10(12) cells/day. This impressive production of cells (approximately 4 x 10(16) cells over a lifetime) is achieved by the proliferation and differentiation of committed progenitor cells, which themselves are derived from a population of pluripotent stem cells with self-renewal potential. Paradoxically, the large majority of stem cells in adult bone marrow are quiescent cells. One possibility is that stem cells, like other somatic cells, have only a limited replicative potential (< 100 divisions). This hypothesis is supported by two key observations and the consideration that, in theory, 55 divisions can yield 4 x 10(16) cells. First, it was shown that 'candidate' stem cells purified from fetal and adult tissue showed dramatic functional differences in turn-over time and the ability to produce cells with stem cell properties, Second, these functional differences were found to correlate with a measurable loss of telomere repeats despite the presence of low but readily detectable levels of telomerase in all purified cell fractions. In order to address questions about the role of telomeres in normal and malignant haemopoiesis, we developed a quantitative fluorescence in situ hybridization technique. Here we review the characteristics of this novel tool to assess the number of telomere repeats at the end of individual chromosomes and provide an overview of recent observations.

Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres

Mammalian telomeres have epigenetic marks of constitutive heterochromatin. Here, we study the impact of telomere length on the maintenance of heterochromatin domains at telomeres. Telomerase-deficient Terc(-/-) mice with short telomeres show decreased trimethylation of histone 3 at Lys9 (H3K9) and histone 4 at Lys20 (H4K20) in telomeric and subtelomeric chromatin as well as decreased CBX3 binding accompanied by increased H3 and H4 acetylation at these regions. Subtelomeric DNA methylation is also decreased in conjunction with telomere shortening in Terc(-/-) mice. In contrast, telomere repeat factors 1 and 2 show normal binding to telomeres independent of telomere length. These results indicate that loss of telomeric repeats leads to a change in the architecture of telomeric and subtelomeric chromatin consisting of loss of heterochromatic features leading to a more 'open' chromatin state. These observations highlight the importance of telomere repeats in the establishment of constitutive heterochromatin at mammalian telomeres and subtelomeres and point to histone modifications as important in counting telomere repeats.

T-Cells with Extremely Short Telomeres and High Telomerase Activity in T-Cell Prolymphocytic Leukemia (T-PLL): The Ideal Target for Telomerase Inhibition.

T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive lymphoproliferative disease characterized by the expansion of a T-cell clone derived from immuno-competent post-thymic T-lymphocytes. Important mechanisms involved in expansion of human malignant cells are reactivation of telomerase, an enzyme complex, which is able to compensate the loss of telomere repeats by cell division, and maintenance or elongation of telomere length.To investigate the role of telomeres and telomerase we measured telomere length by automated multicolor flow-FISH and telomerase activity by telomeric repeat amplification protocol in subsets of peripheral blood leukocytes from 11 newly diagnosed or relapsed patients with sporadic T-PLL. In addition, we analyzed the effect of the selective telomerase inhibitor BIBR1532 on T-PLL cells in short-term culture assays.The average telomere length in the clonal T-cells of all samples analyzed was extremely short (mean ± std: 1.53 kb ± 0.65 kb) compared to the non-clonal T-cells (5.03 kb ± 0.71 kb; p=0.012). The average telomere length for B-cells in these patients was 6.37 kb ± 0.71 kb (n=6). Telomere length values of the clonal T-cells were all below the 1st percentile of telomere length values observed in T-cells from healthy aged-matched controls whereas non-clonal T-cells and B-cells fell between the 1st and 99th percentile of the normal distribution. Interestingly, telomere length in the clonal T-cells remained stably short at 1.0 kb ± 0.6 kb without further telomere loss in one patient over a period of 18 months. No cell doublets indicative of fused or bridged chromosomes and telomere dysfunction were observed. Clonal T-cells exhibited high levels of telomerase activity almost comparable to levels of the positive control K562 whereas there was no measurable telomerase activity in normal, unstimulated T-cells. Telomerase levels even correlated inversely with telomere length in clonal T-cells (r=−0.91, n=6). In addition, we could induce a dose-dependent cytotoxicity of T-PLL cells with the telomerase inhibitor BIBR1532 (viable cells as percentage of untreated controls (viability index) in % after 10 days of cell culture (mean ± std) with 0, 10, 40, 100 μM BIBR1532: 100 ± 0, 54 ± 14.3, 27.5 ± 11.7, 2.6 ± 1.6, n=6) whereas no effect was observed in normal, unstimulated T-cells (viability index in % (mean ± std) after 10 days with 0, 10, 40, 100 μM BIBR1532: 100 ± 0, 100.8 ± 8.6, 96.9 ± 7.9, 103.1 ± 22.4, n=3).In summary, clonal T-cells in T-PLL exhibit extremely short telomeres which could explain the genomic instability with cytogenetic aberrations. The high levels of telomerase found in T-PLL cells are sufficient to stably maintain the critically short telomeres and allow clonal expansion. In addition, we can demonstrate that inhibition of telomerase in vitro in the situation of T-PLL cells with already very short telomeres and high telomerase levels leads to rapid cytotoxicity of the T-cell clone without a time delay before telomeres get critically short as observed in the situation of longer telomeres. Targeting telomerase and telomeres seems therefore an attractive strategy for the future treatment of this devastating disease.

A role for Brca1 in chromosome end maintenance

The role of BRCA1 in breast and ovarian tumor suppression has been primarily ascribed to the maintenance of genome integrity. BRCA1 interacts with components of the non-homologous end-joining pathway previously shown to play a role in telomere maintenance in yeast. Here, we provide evidence that links Brca1 with telomere integrity. Brca1(-/-) T-cells display telomere dysfunction in both loss of telomere repeats as well as defective telomere capping. Loss of Brca1 synergizes with p53 deficiency in the onset and frequency of tumorigenesis. Karyotyping of tBrca1(-/-)p53(-/-) thymic lymphomas revealed the presence of telomere dysfunction accompanied by clonal chromosomal translocations. The telomere dysfunction phenotype in Brca1-deficient cells suggests that loss of telomere integrity might contribute to chromosome end dysfunction and permit the formation of potentially oncogenic translocations.

Does telomere elongation lead to a longer lifespan if cancer is considered?

As cell proliferation is limited due to the loss of telomere repeats in DNA of normal somatic cells during division, telomere attrition can possibly play an important role in determining the maximum life span of organisms as well as contribute to the process of biological ageing. With computer simulations of cell culture development in organisms, which consist of tissues of normal somatic cells with finite growth, we obtain an increase of life span and life expectancy for longer telomeric DNA in the zygote. By additionally considering a two-mutation model for carcinogenesis and indefinite proliferation by the activation of telomerase, we demonstrate that the risk of dying due to cancer can outweigh the positive effect of longer telomeres on the longevity.