When the caps fall off: Responses to telomere uncapping in yeast

Telomeres protect the ends of linear chromosomes from activities that cause sequence losses or challenge chromosome integrity. Furthermore, these ends must be hidden from detection by the DNA damage recognition and response pathways. In particular, they must not fuse with each other. These fundamental and very first functions attributed to telomeres are also summarized with the term ‘chromosome capping’. However, telomeres can become uncapped and the foremost cellular responses to such events aim to restore genome stability in the most conservative fashion possible. I will provide an outline of cellular responses to uncapping in budding yeast and briefly discuss the reverse, namely avoidance mechanisms that prevent telomere formation at inappropriate places.     

Race/ethnicity and telomere length in the Multi-Ethnic Study of Atherosclerosis

Telomere length has emerged as a marker of exposure to oxidative stress and aging. Race/ethnic differences in telomere length have been infrequently investigated. Leukocyte telomere length (LTL) was assessed 981 white, black and Hispanic men and women aged 45–84 years participating in the Multi-Ethnic Study of Atherosclerosis. Direct measurement and questionnaire were used to assess covariates. Linear regression was used to estimate associations of LTL with race/ethnicity and age after adjustment for sex, income, education, smoking, physical activity, diet and body mass index. On average blacks and Hispanics had shorter telomeres than whites [adjusted mean differences (standard error) in T/S ratio compared to whites: −0.041 (0.018) for blacks and −0.044 (0.018) for Hispanics]. Blacks and Hispanics showed greater differences in telomere length associated with age than whites (adjusted mean differences in T / S ratio per 1 year increase in age −0.0018, −0.0047 and −0.0055 in whites, blacks and Hispanics respectively). Differences in age associations were more pronounced and only statistically significant in women. Race/ethnic differences in LTL may reflect the cumulative burden of differential exposure to oxidative stress (and its predictors) over the lifecourse.     

Telomerase regulation and progressive telomere shortening of rat hepatic stem-like epithelial cells during in vitro aging

Rat hepatic stem-like epithelial cells, LE/2, LE/6, and WB-F344, share some phenotypic properties with oval cells, observed in the early stages of hepatocarcinogenesis. Here, we describe regulations of telomerase and telomere length during in vitro aging of LEs and WB-F344. These cells displayed no apparent aging phenotypes for over 140 passages. Telomerase activity and telomere length of these cells progressively decreased with the passages, and at the late passages, telomere shortening appeared to be reduced as telomerase activity increased. Regulation of TERT and TR, key components of telomerase, was similar to that of the telomerase activity. LEs possessed weak telomerase activity with a slow rate of proliferation compared to WB-F344, and were not tumorigenic, whereas WB-F344 was transformed in vitro from intermediate passage. In conclusion, LEs and WB-F344 have different biochemical properties, and telomerase activation and short telomeres are unlikely necessary for the transformation of WB-F344. TERT and TR seem to be the regulators of the telomerase activity. The relationship between telomere length and telomerase activity suggests that telomerase contributes to the regulation of telomere length in these cells. Our findings provide a better understanding of mechanisms in neoplastic transformation of rat hepatic stem-like epithelial cells.     

Increasing the accuracy and precision of relative telomere length estimates by RT qPCR

As attrition of telomeres, DNA caps that protect chromosome integrity, is accelerated by various forms of stress, telomere length (TL) has been proposed as an indicator of lifetime accumulated stress. In ecological studies, it has been used to provide insights into ageing, life history trade‐offs, the costs of reproduction and disease. qPCR is a high‐throughput and cost‐effective tool to measure relative TL (rTL) that can be applied to newly collected and archived ecological samples. However, qPCR is susceptible to error both from the method itself and pre‐analytical steps. Here, repeatability was assessed overall and separately across multiple levels (intra‐assay, inter‐assay and inter‐extraction) to elucidate the causes of measurement error, as a step towards improving precision. We also tested how accuracy, defined as the correlation between the “gold standard” for TL estimation (telomere restriction fragment length analysis with in‐gel hybridization), could be improved. We find qPCR repeatability (intra‐ and inter‐assay levels) to be at similar levels across three common storage media (ethanol, Longmire's and Queen's). However, inter‐extraction repeatability was 50% lower for samples stored in Queen's lysis buffer, indicating storage medium can influence precision. Precision as well as accuracy could be increased by estimating rTL from multiple qPCR reactions and from multiple extractions. Repetition increased statistical power equivalent to a 25% (single extraction analysed twice) and 17% (two extractions) increase in sample size. Overall, this study identifies novel sources of variability in high‐throughput telomere quantification and provides guidance on sampling strategy design and how to increase rTL precision and accuracy.     

A model with 'growth retardation' for the kinetic heterogeneity of tumour cell populations

In the present paper we propose a continuous cell population model based on Shackney's idea of growth retardation. Cells are characterized by two state variables: the cell maturity x, 0 < or = x < or = 1, and a state variable T that identifies the rate of maturation along cell cycle. During their life span, cells can change T at random by jump transitions to T values corresponding to slower maturation rates, while at each jump the maturity x is conserved. Both the time evolution of the population and the exponential stationary solution are numerically computed. The distribution of the cell cycle transit time in asynchronous exponential growth is investigated by Monte Carlo simulation. An approximated formula for the distribution of cell cycle time is also provided.     

An in silico structural insights into Plasmodium LytB protein and its inhibition

In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new anti-malarial compounds or inhibitors. Here a systematic in silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three-dimensional (3D) structure of Plasmodium LytB taking Escherichia coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand data-set containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, five were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any “drug like” molecule and the knowledge of Plasmodium LytB-inhibitory mechanism which can provide valuable support for the anti-malarial-inhibitor design in future.     

gamma-Tubulin in Leishmania: cell cycle-dependent changes in subcellular localization and heterogeneity of its isoforms

A panel of six anti-peptide antibodies recognizing epitopes in different regions of the gamma-tubulin molecule was used for the characterization and localization of gamma-tubulin during cell cycle in Leishmania promastigotes. Immunofluorescence microscopy revealed the presence of gamma-tubulin in the basal bodies, posterior pole of the cell, and in the flagellum. Furthermore, the antibodies showed punctuate staining in the subpellicular microtubule. This complex localization pattern was observed in both interphase and dividing cells, where staining of posterior poles and the subpellicular corset was more prominent. In posterior poles, gamma-tubulin co-distributed with the 210-kDa microtubule-interacting protein and the 57-kDa protein immunodetected with anti-vimentin antibody. Immunogold electron microscopy on thin sections of isolated flagella showed that gamma-tubulin was associated with the paraflagellar rod (PFR) that runs adjacent to the axonemal microtubules. Under different extraction conditions, gamma-tubulin in Leishmania was found only in insoluble cytoskeletal fractions, in contrast to tubulin dimers that were both in soluble and cytoskeletal pool. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin. Posttranslational modifications of Leishmania gamma-tubulin might therefore have an important role in the regulation of microtubule nucleation and interaction with other proteins. The complex pattern of gamma-tubulin localization and its properties indicate that gamma-tubulin in Leishmania might have other function(s) besides microtubule nucleation.     

Nature vs nurture: Interplay between the genetic control of telomere length and environmental factors

Telomeres are nucleoprotein structures that cap the ends of the linear eukaryotic chromosomes, thus protecting their stability and integrity. They play important roles in DNA replication and repair and are central to our understanding of aging and cancer development. In rapidly dividing cells, telomere length is maintained by the activity of telomerase. About 400 TLM (telomere length maintenance) genes have been identified in yeast, as participants of an intricate homeostasis network that keeps telomere length constant. Two papers have recently shown that despite this extremely complex control, telomere length can be manipulated by external stimuli. These results have profound implications for our understanding of cellular homeostatic systems in general and of telomere length maintenance in particular. In addition, they point to the possibility of developing aging and cancer therapies based on telomere length manipulation.     

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.     

Combined effects of leukocyte telomere length,p53 polymorphism and human papillomavirus infection on esophageal squamous cell carcinoma in a Han Chinese population

Telomere shortening has been suggested to be a genetic predictor for various cancers. However, evidences about this point with respect to esophageal squamous cell carcinoma (ESCC) in Han Chinese populations remain limited. Our previous study demonstrated that p53 Arg72Pro polymorphism was associated with the risk of human papillomavirus (HPV)-related ESCC. Telomeres and p53 play important roles in maintaining genomic stability and regulating the cell cycle. HPV impacts both telomere length stabilization and p53 degradation. Given the roles of the three factors, we evaluated leukocyte telomere length, p53 variants and HPV-16 serology to examine the potential associations between them and ESCC risk in a case–control study with 308 patients and 309 cancer-free controls matched by age and sex. Compared with long telomere length, short telomere length was significantly associated with an increased risk of ESCC (adjusted OR 2.01; 95% CI 1.41–2.80). Moreover, this association was enhanced when combined with HPV-16 seropositivity and p53 Arg/Arg or Arg/Pro genotypes. Notably, individuals with short telomere length, Arg/Pro or Arg/Arg genotypes and HPV-16 seropositivity had a 12.08-fold (95% CI 5.49–26.56) increased risk of ESCC compared to those with none of the three investigated risk factors. Taken together, these results indicate that short telomere length in peripheral blood leukocytes is a biomarker for ESCC risk, and has statistically additive effects with p53 variants and HPV seropositivity with regard to the risk of ESCC in a Han Chinese population.     

Mid-life leukocyte telomere length and dementia risk: An observational and mendelian randomization study of 435,046 UK Biobank participants

Telomere attrition is one of biological aging hallmarks and may be intervened to target multiple aging-related diseases, including Alzheimer's disease and Alzheimer's disease related dementias (AD/ADRD). The objective of this study was to assess associations of leukocyte telomere length (TL) with AD/ADRD and early markers of AD/ADRD, including cognitive performance and brain magnetic resonance imaging (MRI) phenotypes. Data from European-ancestry participants in the UK Biobank (n = 435,046) were used to evaluate whether mid-life leukocyte TL is associated with incident AD/ADRD over a mean follow-up of 12.2 years. In a subsample without AD/ADRD and with brain imaging data (n = 43,390), we associated TL with brain MRI phenotypes related to AD or vascular dementia pathology. Longer TL was associated with a lower risk of incident AD/ADRD (adjusted Hazard Ratio [aHR] per SD = 0.93, 95% CI 0.90–0.96, p = 3.37 × 10⁻⁷). Longer TL also was associated with better cognitive performance in specific cognitive domains, larger hippocampus volume, lower total volume of white matter hyperintensities, and higher fractional anisotropy and lower mean diffusivity in the fornix. In conclusion, longer TL is inversely associated with AD/ADRD, cognitive impairment, and brain structural lesions toward the development of AD/ADRD. However, the relationships between genetically determined TL and the outcomes above were not statistically significant based on the results from Mendelian randomization analysis results. Our findings add to the literature of prioritizing risk for AD/ADRD. The causality needs to be ascertained in mechanistic studies.     

An investigation of the plausibility of stochastic resonance in tubulin dimers

This paper considers the possibility of stochastic resonance (SR) in tubulin dimers. A formula for the signal-to-noise ratio (SNR) of tubulin as a function of temperature is derived. The effective potential experienced by a delocalized electron in such a dimer is postulated to be a symmetric bimodal well. Inter-well and intra-well motions are described by Kramers rate theory and the Langevin formalism respectively. The frequency-dependent expression for the SNR shows that the response of the electron-tubulin dimer system is enhanced by ambient dipolar oscillations in specific frequency regimes. This is a characteristic of SR. Biophysical implications of this property such as the relevance to 8.085 MHz microtubule resonance and the clocking mechanism are detailed.     

Effects of cisplatin on telomerase activity and telomere length in BEL—7404 human hepatoma cells

INTRODUCTIONPrimary hepatocellular carcinoma (PHCC), oneof the most common malignancies in the world, isan aggressive cancer. The mean survival time fromestablishment of diagnosi8 is only about 4 months(2 months if the diagnosis is made 1ate). It causesab…     

Evolutionary hypothesis of telomere length in primary breast cancer and brain tumour patients: a tracer for genomic-tumour heterogeneity and instability

It was previously reported that tumour samples had shorter telomeres than the surrounding normal tissue. Hereby, the initial sign of correlation between malignant tissue and telomere behaviour could be noticed. Bridging knowledge between germ and somatic cells could facilitate understanding cellular evolution. The aim of our investigation was to provide evidence for the evolutionary hypothesis of TL (telomere length) in primary BC (breast cancer) and BTs (brain tumours), which might be applied as a prognostic and/or predictive marker. DNA extraction from the frozen tissues was performed using high pure PCR template preparation kit. Standard protocol of Telo TTAGGG Telomere Length Assay kit, a non‐radioactive chemiluminescent assay, was used. The protein expression in extracted cells was analysed by immunofluorescence. We also detected telomerase activity. The G/T (genomic/tumour ratio) for TL in two groups of patients affected with primary BC and primary BT revealed significant differences in both BC patients (P=0.025) and in BTs (P=0.001). The pattern of telomere signals by Q‐FISH (quantitative fluorescent in situ hybridization) show that in all samples, except one, SI (signal intensity) has been significantly decreased in tissue related to blood, either in BC patients or in patients with BTs (0.041≥P≥0.001). However, the data achieved by Q‐FISH support the results of Southern blot. These data reflect a significant diversity either in BC or in BT patients, providing evidence for the evolutionary hypothesis of TL in cancer development and progression.     

Imbalance in dosage of the genes for the heterochromatin components Sir3p and histone H4 results in changes in the length and sequence organization of yeast telomeres

Telomeric heterochromatin plays an essential role in telomere function, including the regulation of telomere length. We observe that in Saccharomyces cerevisiae an imbalance in the dosage of genes for two protein components of heterochromatin (namely Sir3p and histone H4) causes modifications in telomere length and telomere sequence organization. The effects of Sir3p/H4 imbalance were analyzed in yeast strains in which the wild-type SIR3 gene (normally a single-copy gene) was either absent or present in 20–30 copies, and both histone H4 genes (HHF1 and HHF2) were present or HHF1 was deleted, thus covering a wide range of viable gene-dosage combinations. Modifications of telomeres and of subtelomeric regions were identified by analyzing both the overall telomere population and by focusing on two single telomeric regions: the left telomere of chromosome III (LIII) and the right telomere of chromosome XI (RXI). The modifications induced by alteration of the Sir3p/H4 ratio consist of a reduction in the length and an increase in the instability of the terminal block of (C_1–3A)_n repeats and in susceptibility to insertion of Y′ elements into this repeat element. Restoration of the wild-type gene ratio (by removal of the extra copies of SIR3 or by complementation with the missing second copy of HHF) restored the original telomere organization, both with respect to the length of the (C_1–3A)_n repeat stretch and the absence of Y′ elements. This behavior shows that the stability of the wild-type sequence organization requires maintenance of the normal structure of telomeric heterochromatin. Received: 23 March 1999 / Accepted: 10 June 1999