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The accident that occurred at the Chornobyl nuclear power plant (Ukraine, 1986) contaminated a large extension of territory after the deposition of radioactive material. It is still under debate whether the chronic exposure to the radiation levels currently present in the area has long-term effects on organisms, such as decreases in longevity. Here, we investigate whether current levels of radiation in Chornobyl negatively impact the age of the Eastern tree frog Hyla orientalis . We also explore whether radiation induces changes in an ageing marker, telomere length or the stress hormone corticosterone. We found no effect of total individual absorbed radiation (including both external and internal exposure) on frog age ( n = 197 individuals sampled in 3 consecutive years). We also did not find any relationship between individual absorbed radiation and telomere length, nor between individual absorbed radiation and corticosterone levels. Our results suggest that radiation levels currently experienced by Chornobyl tree frogs may not be high enough to cause severe chronic damage to semi-aquatic vertebrates such as this species. This is the first study addressing age and stress hormones in Chornobyl wildlife, and thus future research will confirm if these results can be extended to other taxa.

Cancer cells have the ability to undergo an unlimited number of cell divisions, which gives them immortality. Thus, the cancer cell can extend the length of its telomeres, allowing these cells to divide unlimitedly and avoid entering the state of senescence or cellular apoptosis. One of the main effects of photobiomodulation (PBM) is the increase in the production of adenosine triphosphate (ATP) and free radicals, mainly reactive oxygen species (ROS). Existent data indicates that high levels of ROS can cause shortening and dysfunctional telomeres. Therefore, a better understanding of the effects induced by PBM on cancer cell telomere maintenance is needed. This work aimed to evaluate the effects of low-power red laser (658 nm) and blue LED (470 nm) on the TRF1 and TRF2 mRNA levels and telomere length in human breast cancer cells. MCF-7 and MDA-MB-231 cells were irradiated with a low-power red laser (69 J cm -2 , 0.77 W/cm -2 ) and blue LED (482 J cm -2 , 5.35 W/cm -2 ), alone or in combination, and the relative mRNA levels of the genes and telomere length were assessed by quantitative reverse transcription polymerase chain reaction. The results suggested that exposure to certain red laser and blue LED fluences decreased the TRF1 and TRF2 mRNA levels in both human breast cancer cells. Telomere length was increased in MCF-7 cells after exposure to red laser and blue LED. However, telomere length in MDA-MB-231 was shortened after exposure to red laser and blue LED at fluences evaluated. Our research suggests that photobiomodulation induced by red laser and low-power blue LED could alter telomere maintenance and length., (© 2024. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be investigated. In this study, telomere length (TL) as endpoint of genetic damage was analyzed in human blood cells (leukocytes) and K562 leukemic cells irradiated with laser-generated ultrashort electron beam. Metaphases and interphases were analyzed in quantitative fluorescence in situ hybridization (Q-FISH) to assess TL. TLs were shortened compared to non-irradiated controls in both settings (metaphase and interphase) after irradiation with 0.5, 1.5, and 3.0 Gy in blood leukocytes. Radiation also caused a significant TL shortening detectable in the interphase of K562 cells. Overall, a negative correlation between TL and radiation doses was observed in normal and leukemic cells in a dose-dependent manner. K562 cells were more sensitive than normal blood cells to increasing doses of ultrashort electron beam radiation. As telomere shortening leads to genome instability and cell death, the results obtained confirm the suitability of this biomarker for assessing genotoxic effects of accelerated electrons for their further use in radiation therapy. Observed differences in TL shortening between normal and K562 cells provide an opportunity for further development of optimal radiation parameters to reduce side effects in normal cells during radiotherapy.

Background: Telomere length is associated with cancer risk and cancer aggressiveness. Radioactive iodine (RAI) therapy for thyroid cancer has raised concerns for second primary malignancy (SPM) in patients with high cumulative doses. The association between RAI dose and peripheral blood leukocyte telomere length was examined., Methods: A total of 425 patients were included who underwent total thyroidectomy and were followed up for at least 1 year with or without RAI treatment. The relative telomere length (RTL) of the patients was assessed via a quantitative polymerase chain reaction amplification method. RAI doses were divided into five groups on the basis of cumulative dose, and a comparison was made among these groups., Results: The number of patients with RAI treatment was 287 (67.5%), and the cumulative RAI dose was 3.33 GBq (range, 1.11-131.35 GBq). The mean RTL was significantly shorter in the highest RAI group (>22.2 GBq) compared to both the no-RAI and lower dose groups. The association between RAI dose and RTL was positive in the lower RAI group (1.1-3.7 GBq) and negative in the highest RAI group in both univariate and multivariate analyses. We observed 59 (13.9%) SPMs and 20 (4.7%) mortalities, and RTL did not show a significant risk effect for all-cause, thyroid cancer-specific, or SPM-specific mortality., Conclusions: In patients with thyroid cancer who underwent total thyroidectomy, peripheral blood leukocyte telomere length exhibited a significant association with cumulative RAI dose higher than 22.2 GBq. These results suggest the possibility of telomere length shortening in patients who undergo high-dose RAI treatment., (© 2024 American Cancer Society.)

Purpose: DNA, the hereditary material of a human cell generally exists as Watson-Crick base paired double-stranded B-DNA. Studies suggest that DNA can also exist in non-B forms, such as four stranded G-quadruplexes (G4 DNA). Recently, our studies revealed that the regions of DNA that can fold into G-quadruplex structures are less sensitive to ionizing radiation (IR) compared to B-DNA. Importantly, we reported that the planar G-quartet of a G4 structure is shielded from radiation induced DNA breaks, while the single- and double-stranded DNA regions remained susceptible. Thus, in the present study, we investigate whether telomeric repeat DNA present at the end of telomere, known to fold into G4 DNA can protect from radiation induced damages including strand breaks, oxidation of purines and bulky adduct formation on DNA., Materials and Methods: For plasmid irradiation assay, plasmids containing human telomeric repeat DNA sequence TTAGGG (0.8 kb or 1.8 kb) were irradiated with increasing doses of IR along with appropriate control plasmids and products were resolved on 1% agarose gel. Radioprotection was evaluated based on extent of conversion of supercoiled to nicked or linear forms of the DNA following irradiation. Formation of G-quadruplex structure on supercoiled DNA was evaluated based on circular dichroism (CD) spectroscopy studies. Cleavage of radiation induced oxidative damage and extent of formation of nicks was further evaluated using base and nucleotide excision repair proteins., Results: Results from CD studies showed that the plasmid DNA harboring human telomeric repeats (TTAGGG) can fold into G-quadruplex DNA structures. Further, results showed that human telomeric repeat sequence when present on a plasmid can protect the plasmid DNA against IR induced DNA strand breaks, unlike control plasmids bearing random DNA sequence., Conclusions: Human telomeric repeat sequence when present on plasmids can fold into G-quadruplex DNA structures, and can protect the DNA against IR induced DNA strand breaks and oxidative damage. These results in conjunction with our previous studies suggest that telomeric repeat sequence imparts less sensitivity to IR and thus telomeres of chromosomes are protected from radiation.

Telomeres, repetitive terminal features of chromosomes essential for maintaining genome integrity, shorten with cell division, lifestyle factors and stresses, and environmental exposures, and so they provide a robust biomarker of health, aging, and age-related diseases. We assessed telomere length dynamics (changes over time) in three unrelated astronauts before, during, and after 1-year or 6-month missions aboard the International Space Station (ISS). Similar to our results for National Aeronautics and Space Administration's (NASA's) One-Year Mission twin astronaut (Garrett-Bakelman et al., 2019), significantly longer telomeres were observed during spaceflight for two 6-month mission astronauts. Furthermore, telomere length shortened rapidly after return to Earth for all three crewmembers and, overall, telomere length tended to be shorter after spaceflight than before spaceflight. Consistent with chronic exposure to the space radiation environment, signatures of persistent DNA damage responses were also detected, including mitochondrial and oxidative stress, inflammation, and telomeric and chromosomal aberrations, which together provide potential mechanistic insight into spaceflight-specific telomere elongation., Competing Interests: Declaration of Interests S.M.B. is a cofounder and Scientific Advisory Board member of KromaTiD. C.E.M. is a cofounder and board member for Biotia and Onegevity Health., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The premature chromosome condensation (PCC) technique is used to study exposure to external radiation through the determination of chromosome fragments observed in interphase cells. The presence of large telomeric signals in CHO cells interferes with the detection of PCC fragments and the identification of dicentric chromosomes. We present an improved method for the fusion of G0-lymphocytes with mitotic Akodon cells (few chromosomes and weakly-staining telomeric sequences) to induce PCC in combination with rapid quantification of dicentric chromosomes and centric rings as an alternative to the classical CHO cell fusion technique. Materials and methods: Whole blood from three healthy volunteers was γ-irradiated with 0, 2, or 4 Gy. Following a 24 h incubation post-exposure at 37 °C, chromosome spreads of isolated lymphocytes were prepared by standard PCC procedures using mitotic Akodon cells. Results: The percentage of scorable fusions, measured by telomere/centromere (T/C) staining, for Akodon -induced PCC was higher than that for CHO-induced PCC, irrespective of radiation exposure. Importantly, both techniques gave the same result for biodosimetry evaluation. Conclusion: The mitotic Akodon cell-induced PCC fusion assay, in combination with the scoring of dicentric chromosomes and rings by T/C staining of G0-lymphocytes is a suitable alternative for fast and reliable dose estimation after accidental radiation exposure.

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3-4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6-8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes., Competing Interests: The authors have declared that no competing interests exist.

The polymorphic nature of G-quadruplex (G4) DNA structures points to a range of potential applications in nanodevices and an opportunity to control G4 in biological settings. Light is an attractive means for the regulation of oligonucleotide structure as it can be delivered with high spatiotemporal precision. However, surprisingly little attention has been devoted towards the development of ligands for G4 that allow photoregulation of G4 folding. We report a novel G4-binding chemotype derived from stiff-stilbene. Surprisingly however, whilst the ligand induces high stabilization in the potassium form of human telomeric DNA, it causes the unfolding of the same G4 sequence in sodium buffer. This effect can be reversed on demand by irradiation with 400 nm light through deactivation of the ligand by photo-oxidation. By fuelling the system with the photolabile ligand, the conformation of G4 DNA was switched five times., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Telomere repeats at chromosomal ends are essential for genome stability and sustained cellular proliferation but are susceptible to DNA damage. Repair of damage at telomeres is influenced by numerous factors including telomeric binding proteins, sequence and structure. Ultraviolet (UV) light irradiation induces DNA photoproducts at telomeres that can interfere with telomere maintenance. Here we describe a highly sensitive method for quantifying the formation and removal of UV photoproducts in telomeres isolated from UV irradiated cultured human cells. Damage is detected by immunospot blotting of telomeres with highly specific antibodies against UV photoproducts. This method is adaptable for measuring other types of DNA damage at telomeres as well.

Exposure of cells to ionizing radiation induces DNA double-strand breaks. To repair double-strand breaks correctly, cells must distinguish between the ends of chromosomes (telomeres) and DNA double-strand breaks within chromosomes. Double-strand breaks in telomeric DNA may lead to telomere shortening and mutagenesis. Eukaryotic cells repair double-strand breaks primarily by two mechanisms: error-free homologous recombination and error-prone nonhomologous end joining, of which homologous recombination is used in early meiotic prophase I to create recombined haploid gametes by two meiotic cell divisions lacking an intervening S-phase. Genotoxic exposures put meiosis at risk to transmit mutations, and ionizing radiation is known to induce large double-strand break-marking phospho (gamma)-H2AX foci along the cores and ends of mouse meiotic chromosomes. However, it remained unclear through which repair pathway the ionizing radiation-induced telomeric double-strand breaks are repaired in late prophase I spermatocytes. Using male wild-type and nonhomologous end joining-deficient (severe combined immunodeficient) mice, this study investigated the kinetics of in vivo double-strand break formation and repair at telomeres of late prophase I chromosomes up to 12 h after 0.5 Gy of whole-body gamma irradiation. Late pachytene and diplotene spermatocytes revealed overlapping gamma-H2AX and telomere repeat signal foci, indicating telomeric DNA damage. The comparison of double-strand break repair rates at telomeres and internal prophase chromosome sites revealed a more rapid double-strand break repair at wild-type telomeres during the first hour after irradiation. Increased double-strand break foci numbers at nonhomologous end joining-deficient telomeres and chromosomes and a slowed repair rate in this DNA-dependent protein kinase catalytic subunit mutant suggest that the fast repair of double-strand breaks in telomeric DNA repeats during late prophase I is largely mediated by canonical nonhomologous end joining.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Although the vast majority of DNA damage induced by radiation exposure disappears rapidly, some lesions remain in the cell nucleus in very small quantities for days to months. These lesions may cause a considerable threat to an organism and include certain types of DNA double-strand breaks (DSBs) called 'unrepairable DSBs'. Unrepairable DSBs are thought to cause persistent malfunctioning of cells and tissues or cause late effects of radiation, especially the induction of delayed cell death, mutation, senescence, or carcinogenesis. Moreover, the measurement of unrepairable DSBs could potentially be used for retrospective biodosimetry or for identifying individuals at greater risk for developing the adverse effects associated with radiotherapy or chemotherapy. This review summarizes the concept of unrepairable DSBs in the context of persistent repair foci formed at DSBs.

Background: Large epidemiologic studies have reported the premature onset of age-related conditions, such as ischemic heart disease and diabetes mellitus, in childhood cancer survivors, decades earlier than in their peers. The authors investigated whether young adult survivors of childhood acute lymphoblastic leukemia (ALL) have a biologic phenotype of cellular ageing and chronic inflammation., Methods: Plasma inflammatory cytokines were measured using a cytometric bead array in 87 asymptomatic young adult survivors of childhood ALL (median age, 25 years; age range, 18-35 years) who attended annual follow-up clinic and compared with healthy, age-matched and sex-matched controls. Leukocyte telomere length (LTL) was measured using Southern blot analysis., Results: Survivors had significant elevation of plasma interleukin-2 (IL-2), IL-10, IL-17a, and high-sensitivity C-reactive protein levels (all P < .05). A raised high-sensitivity C-reactive protein level (>0.8 mg/dL) was related to increased odds of having metabolic syndrome (odds ratio, 7.256; 95% confidence interval, 1.501-35.074). Survivors also had significantly shorter LTL compared with controls (median, 9866 vs 10,392 base pairs; P = .021). Compared with published data, LTL in survivors was similar to that in healthy individuals aged 20 years older. Survivors who received cranial irradiation had shorter LTL compared with those who had not (P = .013)., Conclusions: Asymptomatic young adult survivors of childhood ALL demonstrate a biologic profile of chronic inflammation and telomere attrition, consistent with an early onset of cellular processes that drive accelerated aging. These processes may explain the premature development of age-related chronic conditions in childhood cancer survivors. Understanding their molecular basis may facilitate targeted interventions to disrupt the accelerated aging process and its long-term impact on overall health. Cancer 2017;123:4207-4214. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Purpose: Cohorts allowing joint epidemiological and biological analyses are essential for radiation risk assessment. The French Hemangioma Cohort (FHC), studied within the European project EpiRadBio, is one of the rare cohorts suitable for studying the effect of low dose radiation exposure (<100 mGy at organs), with a long-term follow-up. This highly homogeneous cohort consists of healthy individuals belonging to a normal population, except for the presence of skin hemangioma (age at exposure: between 6 months and 3 years of age). Published epidemiological studies have demonstrated that the risk of developing cancer is three times higher in the exposed individuals than in the general population. Here, we present the biobanking of samples (nucleated blood cells, cytogenetic slides of T and B lymphocytes) from the FHC and a primary feasibility study of biomarker analysis focusing on mean telomere length (MTL). Telomeres act as an internal clock, regulating the lifetime of the cell by their shortening during cell division. MTL is thus a biomarker of age. Many in vitro studies have linked MTL and radiosensitivity. The FHC will make it possible to discriminate between the effects of aging and radiation on this biomarker., Conclusion: The establishment of a biobank of essentially healthy individuals (369 in total), exposed 40-70 years before, during their early childhood, is a logistical challenge. Even among those who previously participated to a self-questionnaire based study, the response rate was only 30%. The first biomarker to be studied was the MTL to discriminate age effects from those of radiation exposure. MTL showed significant variation within age groups (4-11 kb) in both the exposed and non-exposed groups. MTL within the limited age window (i.e. 40-73 year) examined, showed age-dependent changes of 46 bp/year, consistent with the age-dependent decline of 41 bp/year previously reported. We observed no significant changes in MTL according to the average active bone marrow dose. However, we were able to demonstrate that exposure to radiation causes the loss of cells with, on average, shorter telomeres, by applying a model in which both the heterogeneity of the individual dose received at the bone marrow and the heterogeneity of the intercellular distribution of MTL were taken into account.

Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y'-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y'-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu-Sir4 interaction, but Sir2 and Sir3 independent., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeric repeat binding factor 1 (TRF1) is essential to the maintenance of telomere chromatin structure and integrity. However, how telomere integrity is maintained, especially in response to damage, remains poorly understood. Here, we identify Nek7, a member of the Never in Mitosis Gene A (NIMA) kinase family, as a regulator of telomere integrity. Nek7 is recruited to telomeres and stabilizes TRF1 at telomeres after damage in an ATM activation-dependent manner. Nek7 deficiency leads to telomere aberrations, long-lasting γH2AX and 53BP1 foci, and augmented cell death upon oxidative telomeric DNA damage. Mechanistically, Nek7 interacts with and phosphorylates TRF1 on Ser114, which prevents TRF1 from binding to Fbx4, an Skp1-Cul1-F box E3 ligase subunit, thereby alleviating proteasomal degradation of TRF1, leading to a stable association of TRF1 with Tin2 to form a shelterin complex. Our data reveal a mechanism of efficient protection of telomeres from damage through Nek7-dependent stabilization of TRF1., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: Accurate assessment and monitoring of the therapeutic efficacy of locally advanced prostate cancer remains a major clinical challenge. Contrary to prostate biopsies, circulating tumor cells (CTCs) are a cellular source repeatedly obtainable by blood sampling and could serve as a surrogate marker for treatment efficacy. In this study, we used size-based filtration to isolate and enumerate CTCs from the blood of 20 patients with high-risk (any one of cT3, Gleason 8-10, or prostate-specific antigen>20ng/ml), nonmetastatic, and treatment-naive prostate cancer before and after androgen deprivation therapy (ADT) and radiation therapy (RT)., Materials and Methods: We performed 3D telomere-specific quantitative fluorescence in situ hybridization on isolated CTCs to determine 3D telomere profiles for each patient before and throughout the course of both ADT and RT., Results: Based on the distinct 3D telomere signatures of CTC before treatment, patients were divided into 3 groups. ADT and RT resulted in distinct changes in 3D telomere signatures of CTCs, which were unique for each of the 3 patient groups., Conclusion: The ability of 3D telomere analysis of CTCs to identify disease heterogeneity among a clinically homogeneous group of patients, which reveals differences in therapeutic responses, provides a new opportunity for better treatment monitoring and management of patients with high-risk prostate cancer., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In directly irradiating cells, telomere metabolism is altered and similar effects have been observed in nontargeted cells. Exosomes and their cargo play dominant roles in communicating radiation-induced bystander effects with end points related to DNA damage. Here we report novel evidence that exosomes are also responsible for inducing telomere-related bystander effects. Breast epithelial cancer cells were exposed to either 2 Gy X rays, or exposed to irradiated cell conditioned media (ICCM), or exosomes purified from ICCM. Compared to control cells, telomerase activity decreased in the 2 Gy irradiated cells and both bystander samples after one population doubling. At the first population doubling, telomere length was shorter in the 2 Gy irradiated sample but not in the bystander samples. By 24 population doublings telomerase activity recovered to control levels in all samples; however, the 2 Gy irradiated sample continued to demonstrate short telomeres and both bystander samples acquired shorter telomeres. RNase treatment of exosomes prevented the bystander effects on telomerase and telomere length that were observed at 1 population doubling and 24 population doublings, respectively. Thermal denaturation by boiling eliminated the reduction of telomere length in bystander samples, suggesting that the protein fraction of exosomes also contributes to the telomeric effect. RNase treatment plus boiling abrogated all telomere-related effects in directly irradiated and bystander cell populations. These findings suggest that both proteins and RNAs of exosomes can induce alterations in telomeric metabolism, which can instigate genomic instability in epithelial cancer cells after X-ray irradiation.

In a series of studies of atomic bomb survivors, radiation-dose-dependent alterations in peripheral T-cell populations have been reported. For example, reduced size in naïve T-cell pools and impaired proliferation ability of T cells were observed. Because these alterations are also generally observed with human aging, we hypothesized that radiation exposure may accelerate the aging process of the T-cell immune system. To further test this hypothesis, we conducted cross-sectional analyses of telomere length, a hallmark of cellular aging, of naïve and memory CD4 T cells and total CD8 T cells in the peripheral blood of 620 atomic bomb survivors as it relates to age and radiation dose, using fluorescence in situ hybridization with flow cytometry. Since telomere shortening has been recently demonstrated in obesity-related metabolic abnormalities and diseases, the modifying effects of metabolic status were also examined. Our results indicated nonlinear relationships between T-cell telomere length and prior radiation exposure, i.e., longer telomeres with lower dose exposure and a decreasing trend of telomere length with individuals exposed to doses higher than 0.5 Gy. There were associations between shorter T-cell telomeres and higher hemoglobin Alc levels or fatty liver development. In naïve and memory CD4 T cells, radiation dose and high-density lipoprotein (HDL) cholesterol were found to positively interact with telomere length, suggesting that the decreasing trend of telomere length from a higher radiation dose was less conspicuous in individuals with a higher HDL cholesterol. It is therefore likely that radiation exposure perturbs T-cell homeostasis involving telomere length maintenance by multiple biological mechanisms, depending on dose, and that long-term-radiation-induced effects on the maintenance of T-cell telomeres may be modified by the subsequent metabolic conditions of individuals.

Ionizing radiation (IR) is a major source of cellular damage and the immediate cellular response to IR has been well characterized. But the long-term impact of IR on cell function and its relationship with aging are not known. Here, we examined the IR effects on telomere length and other biomarkers 50 to 68 years post-exposure (two time points per person) in survivors of the atomic bombing at Hiroshima during WWII. We found that telomere length of leukocytes was inversely correlated with the dose of IR (p=0.008), and this effect was primarily found in survivors who were exposed at younger ages; specifically those <12 years old (p=0.0004). Although a dose-related retardation of telomere shortening with age was observed in the cross-sectional data, longitudinal follow-up after 11 years did not show IR exposure-related alteration of the rate of telomere shortening with age. In addition, IR diminished the associations between telomere length and selected aging biomarkers that were observed in survivors with no dose. These included uric acid metabolism, cytokines, and blood T cell counts. These findings showed long-lasting detrimental effects of IR on telomere length of leukocytes in both dose- and age-at-exposure dependent manner, and on alterations of biomarkers with aging., Competing Interests: CONFLICTS OF INTREST The authors declare no conflict of interests.

Telomeres consist of GC-rich DNA repeats and the "shelterin" protein complex that together protect chromosome ends from fusion and degradation. Telomeres shorten with age due to incomplete end replication and upon exposure to environmental and intrinsic stressors. Exposure to ionizing radiation is known to modulate telomere length. However, the response of telomere length in humans chronically exposed to radiation is poorly understood. Here, we studied relative telomere length (RTL) by IQ-FISH to leukocyte nuclei in a group of 100 workers from the plutonium production facility at the Mayak Production Association (PA) who were chronically exposed to alpha-emitting ((239)Pu) radiation and/or gamma (photon) radiation, and 51 local residents serving as controls, with a similar mean age of about 80 years. We applied generalized linear statistical models adjusted for age at biosampling and the second exposure type on a linear scale and observed an age-dependent telomere length reduction. In those individuals with the lowest exposure, a significant reduction of about 20% RTL was observed, both for external gamma radiation (≤1 Gy) and internal alpha radiation (≤0.05-0.1 Gy to the red bone marrow). In highly exposed individuals (>0.1 Gy alpha, 1-1.5 Gy gamma), the RTL was similar to control. Stratification by gender revealed a significant (∼30%) telomere reduction in low-dose-exposed males, which was absent in females. While the gender differences in RTL may reflect different working conditions, lifestyle and/or telomere biology, absence of a dose response in the highly exposed individuals may reflect selection against cells with short telomeres or induction of telomere-protective effects. Our observations suggest that chronic systemic exposure to radiation leads to variable dose-dependent effects on telomere length.

Ultraviolet light induces cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts, which interfere with DNA replication and transcription. Nucleotide excision repair (NER) removes these photoproducts, but whether NER functions at telomeres is unresolved. Here we use immunospot blotting to examine the efficiency of photoproduct formation and removal at telomeres purified from UVC irradiated cells at various recovery times. Telomeres exhibit approximately twofold fewer photoproducts compared with the bulk genome in cells, and telomere-binding protein TRF1 significantly reduces photoproduct formation in telomeric fragments in vitro. CPD removal from telomeres occurs 1.5-fold faster than the bulk genome, and is completed by 48 h. 6-4PP removal is rapidly completed by 6 h in both telomeres and the overall genome. A requirement for XPA protein indicates the mechanism of telomeric photoproduct removal is NER. These data provide new evidence that telomeres are partially protected from ultraviolet irradiation and that NER preserves telomere integrity.

We report the application of a photoreaction method for probing two consecutive G-quadruplexes formed by human telomeric DNA. This method can discriminate the loop structure located between two consecutive G-quadruplexes formed by eight TTAGGG repeats in K(+) and Na(+) solutions.

Objectives: This study sought to assess the association between long-term radiation exposure in the catheterization laboratory (cath lab) and early signs of subclinical atherosclerosis., Background: There is growing evidence of an excess risk of cardiovascular disease at low-dose levels of ionizing radiation exposure., Methods: Left and right carotid intima-media thickness (CIMT) was measured in 223 cath lab personnel (141 male; age, 45 ± 8 years) and 222 unexposed subjects (113 male; age, 44±10 years). Leukocyte telomere length (LTL) was evaluated by quantitative reverse transcriptase polymerase chain reaction. The DNA repair gene XRCC3 Thr241Met polymorphism was also analyzed to explore the possible interaction with radiation exposure. The occupational radiological risk score (ORRS) was computed for each subject on the basis of the length of employment, individual caseload, and proximity to the radiation source. A complete lifetime effective dose (mSv) was recorded for 57 workers., Results: Left, right, and averaged CIMTs were significantly increased in high-exposure workers compared with both control subjects and low-exposure workers (all p values<0.04). On the left side, but not on the right, there was a significant correlation between CIMT and ORRS (p=0.001) as well as lifetime dose (p=0.006). LTL was significantly reduced in exposed workers compared with control subjects (p=0.008). There was a significant correlation between LTL and both ORRS (p=0.002) and lifetime dose (p=0.03). The XRCC3 Met241 allele presented a significant interaction with high exposure for right side (pinteraction=0.002), left side (pinteraction<0.0001), and averaged (pinteraction<0.0001) CIMTs., Conclusions: Long-term radiation exposure in a cath lab may be associated with increased subclinical CIMT and telomere length shortening, suggesting evidence of accelerated vascular aging and early atherosclerosis., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

SLX4, a coordinator of multiple DNA structure-specific endonucleases, is important for several DNA repair pathways. Noncovalent interactions of SLX4 with ubiquitin are required for localizing SLX4 to DNA interstrand crosslinks (ICLs), yet how SLX4 is targeted to other functional contexts remains unclear. Here, we show that SLX4 binds SUMO-2/3 chains via SUMO-interacting motifs (SIMs). The SIMs of SLX4 are dispensable for ICL repair but important for processing CPT-induced replication intermediates, suppressing fragile site instability, and localizing SLX4 to ALT telomeres. The localization of SLX4 to laser-induced DNA damage also requires the SIMs, as well as DNA end resection, UBC9, and MDC1. Furthermore, the SUMO binding of SLX4 enhances its interaction with specific DNA-damage sensors or telomere-binding proteins, including RPA, MRE11-RAD50-NBS1, and TRF2. Thus, the interactions of SLX4 with SUMO and ubiquitin increase its affinity for factors recognizing different DNA lesions or telomeres, helping to direct the SLX4 complex in distinct functional contexts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG-3' human telomere sequence can fold into G-quadruplexes that adopt the hybrid, basket, or propeller fold. The telomere sequence is hypersensitive to UV-induced thymine dimer (T=T) formation, yet it does not cause telomere shortening. In this work, the potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand why this damage is tolerated in telomeres. First, established methods, such as thermal melting measurements, electrophoretic mobility shift assays, and circular dichroism spectroscopy, were utilized to determine the effects of the damage on these structures. Second, a single-molecule ion channel recording technique using α-hemolysin (α-HL) was employed to examine further the structural differences between the damaged sequences. It was observed that the damage caused slightly lower thermal stabilities and subtle changes in the circular dichroism spectra for hybrid and basket folds. The α-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are tolerated in edgewise and diagonal loops. The largest change was observed for the T=T-containing natural telomere sequence when the propeller fold (all double-chain reversal loops) was studied. On the basis of the α-HL experiments, it was determined that a triplexlike structure exists under conditions that favor a propeller structure. The biological significance of these observations is discussed.

Purpose: The dicentric chromosome (dicentric) assay is the international gold-standard method for biological dosimetry and classification of genotoxic agents. The introduction of telomere and centromere (TC) staining offers the potential to render dicentric scoring more efficient and robust. In this study, we improved the detection of dicentrics and all unstable chromosomal aberrations (CA) leading to a significant reevaluation of the dose-effect curve and developed an automated approach following TC staining., Material and Methods: Blood samples from 16 healthy donors were exposed to (137)Cs at 8 doses from 0.1 to 6Gy. CA were manually and automatically scored following uniform (Giemsa) or TC staining. The detection of centromeric regions and telomeric sequences using PNA probes allowed the detection of all unstable CA: dicentrics, centric and acentric rings, and all acentric fragments (with 2, 4 or no telomeres) leading to the precise quantification of estimated double strand breaks (DSB)., Results: Manual scoring following TC staining revealed a significantly higher frequency of dicentrics (p<10(-3)) (up to 30%) and estimated DSB (p<10(-4)) compared to uniform staining due to improved detection of dicentrics with centromeres juxtaposed with other centromeres or telomeres. This improvement permitted the development of the software, TCScore, that detected 95% of manually scored dicentrics compared to 50% for the best currently available software (DCScore™)., Conclusion: The use of TC staining has permitted a reevaluation of the dose-response curve and the highly efficient automation of the scoring process, marking a new step in the management and follow-up of populations exposed to genotoxic agents including ionizing radiation., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη's ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ionizing radiation (IR) is known to be a cause of telomere dysfunction in tumor cells; however, very few studies have investigated X-ray-related changes in telomere length and the telomerase activity in normal human cells, such as umbilical vein endothelial cells (HUVECs). The loss of a few hundred base pairs from a shortened telomere has been shown to be important with respect to cellular senescence, although it may not be detected according to traditional mean telomere length [assessed as the terminal restriction fragment (TRF)] analyses. In the present study, a continuous time window from irradiation was selected to examine changes in the telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, apoptotic rate, and survival rate in HUVECs from the first day to the fourth day after the administration of a 0.5-Gy dose of irradiation. The mean TRF length in the irradiated HUVECs showed shorter telomere length in first 3 days, but they were not statistically significant. On the other hand, according to the percentage analysis of the telomere length, a decreasing tendency was noted in the longer telomere lengths (9.4-4.4 kb), with a significant increase in the shortest telomeres (4.4-2.3 kb) among the irradiated cells versus the controls from the first day to the third after irradiation; no significant differences were noted on the fourth day. These results suggest that the shortest telomeres are sensitive to the late stage of radiation damage. The proliferation of irradiated cells was suppressed after IR in contrast to the non-irradiated cells. The apoptotic rate was elevated initially both in IR- and non-IR-cells, but that of IR-cells was maintained at an elevated level thereafter in contrast to that of non-IR-cells decreasing promptly. Therefore, a 0.5-Gy dose of IR induces persistent apoptosis leading to an apparent growth arrest of the normal HUVECs.

The outcome of the Chernobyl nuclear power plant (CNPP) accident was that a huge number of people were exposed to ionizing radiation. Previous studies of CNPP clean-up workers from Latvia revealed a high occurrence of age-associated degenerative diseases and cancer in young adults, as well as a high mortality as a result of cardiovascular disorders at age 45-54 years. DNA tandem repeats that cap chromosome ends, known as telomeres, are sensitive to oxidative damage and exposure to ionizing radiation. Telomeres are important in aging processes and carcinogenesis. The aim of this study was to investigate the long-term effect of protracted ionizing radiation exposure on telomere length in CNPP clean-up workers. Relative telomere length (RTL) was measured in peripheral blood leukocytes of 595 CNPP clean-up workers and 236 gender- and age-matched controls using real-time quantitative polymerase chain reaction (q-PCR). Close attention was paid to participation year and tasks performed during the worker's stay in Chernobyl, health status, and RTL differences between subgroups. Telomere shortening was not found in CNPP clean-up workers; on the contrary, their RTL was slightly greater than in controls (P = 0.001). Longer telomeres were found in people who worked during 1986, in those undertaking 'dirty' tasks (digging and deactivation), and in people with cancer. Shorter telomeres appeared frequently in those with cataract, osteoporosis, atherosclerosis, or coronary heart disease. We conclude that the longer telomeres revealed in people more heavily exposed to ionizing radiation probably indicate activation of telomerase as a chromosome healing mechanism following damage, and reflect defects in telomerase regulation that could potentiate carcinogenesis., (© The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

The incidence of skin cancer is increasing worldwide and cutaneous squamous cell carcinomas (SCCs) are associated with considerable morbidity and mortality, particularly in immunosuppressed individuals ('carcinomatous catastrophy'). Yet, molecular mechanisms are still insufficiently understood. Besides ultraviolet (UV)-indicative mutations, chromosomal aberrations are prominent. As telomeres are essential in preserving chromosome integrity, and telomere erosion as well as aberrant spatial telomere distribution contribute to genomic instability, we first established telomere length profiles across the whole tissue and identified normal skin (10/30) harboring discrete epidermal sites (stem cell territories) of evenly short telomeres. Precancerous actinic keratoses (AKs) (17) and SCCs (27) expressed two telomere phenotypes: (i) tissue-wide evenly short to intermediate and (ii) longer and tissue-wide heterogeneous telomere lengths, suggesting two modes of initiation, with one likely to originate in the epidermal stem cells. Although tumor histotype, location, patient gender or age failed to distinguish the two SCC telomere phenotypes, as did telomerase activity, we found a trend for a higher degree of aberrant p53 and cyclin D1 expression with long/heterogeneous telomeres. In addition, we established an association for the short/homogeneous telomeres with a simpler and the heterogeneous telomeres with a more complex karyotype correlating also with distinct chromosomal changes. SCCs (13) from renal transplant recipients displayed the same telomere dichotomy, suggesting that both telomere subtypes contribute to 'carcinomatous catastrophy' under immunosuppression by selecting for a common set (3, 9p and 17q) and subtype-specific aberrations (e.g., 6p gain, 13q loss). As a second mechanism of telomere-dependent genomic instability, we investigated changes in telomere distribution with its most severe form of telomeric aggregates (TAs). We identified a telomere length-independent but progression-dependent increase in cells with small telomere associations in AKs (17/17) and additional TAs in SCCs (24/32), basal cell carcinomas (30/31) and malignant melanomas (15/15), and provide evidence for a reactive oxygen species-dependent mechanism in this UV-induced telomere organization-dependent genomic instability.

Exposure to total body irradiation (TBI) induces not only acute hematopoietic radiation syndrome but also long-term or residual bone marrow (BM) injury. This residual BM injury is mainly attributed to permanent damage to hematopoietic stem cells (HSCs), including impaired self-renewal, decreased long-term repopulating capacity, and myeloid skewing. These HSC defects were associated with significant increases in production of reactive oxygen species (ROS), expression of p16(Ink4a) (p16) and Arf mRNA, and senescence-associated β-galacotosidase (SA-β-gal) activity, but not with telomere shortening or increased apoptosis, suggesting that TBI induces residual BM injury via induction of HSC premature senescence. This suggestion is supported by the finding that SA-β-gal(+) HSC-enriched LSK cells showed more pronounced defects in clonogenic activity in vitro and long-term engraftment after transplantation than SA-β-gal(-) LSK cells isolated from irradiated mice. However, genetic deletion of p16 and/or Arf had no effect on TBI-induced residual BM suppression and HSC senescence, because HSCs from irradiated p16 and/or Arf knockout (KO) mice exhibited changes similar to those seen in HSCs from wild-type mice after exposure to TBI. These findings provide important new insights into the mechanism by which TBI causes long-term BM suppression (eg, via induction of premature senescence of HSCs in a p16-Arf-independent manner)., (© 2014 by The American Society of Hematology.)

Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Radiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear., Principal Findings: In this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation., Conclusions: We demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer.

Purpose: Biological effects produced by low doses of ionizing radiations, though relevant for the risk assessment, have not been fully elucidated. The aim of the present work was to evaluate cytogenetic endpoints, as telomere dysfunctions and chromosome instability in the low-dose range as a function of radiation quality. In particular, we analyzed whether the telomere length was modulated, as well as the involvement of telomeres in chromosomal alterations at anaphase, and the yield of stable simple and complex chromosome aberrations., Materials and Methods: AG01522 human primary fibroblasts were irradiated with 0.1-1 Gy of X-rays, protons (28.5 keV/μm), and 4He(2+) ions (62 keV/μm). Frequency of chromosome bridges carrying or not telomeric signals and telomere length were measured in irradiated samples up to 72 h. Moreover, chromosome instability was measured using multicolor fluorescence in situ hybridization (mFISH)., Results: The results evidenced a linear energy transfer (LET)- and dose-dependent response in the frequency of anaphase bridges induction and in their persistence as a function of time. However, neither variation in telomere length and telomere loss, nor in the proportion of bridges bearing telomeric signals, was detected, thus indicating a minor role of telomeres in the generation of the radiation-induced chromosome bridges. Chromosome instability followed a linear-dependence with dose and LET, showing a far higher extent of complex translocations in helium-ion-irradiated cells than in proton- or X-ray-irradiated samples., Conclusions: Altogether, the results indicated the lack of telomere involvement in cytogenetic effects induced by low-dose ionizing radiation. On the contrary, chromosome aberration yield and spectrum were LET- and dose-dependent.

Telomeres are at the ends of chromosomes. Previous evidence suggests that laser-induced deoxyribose nucleic acid (DNA) breaks at chromosome ends during anaphase results in delayed cytokinesis. A possible explanation for this delay is that the DNA damage response (DDR) mechanism has been activated. We describe a live imaging method to study the effects of DDR activation following focal point near-infrared femtosecond laser microirradiation either at a single chromosome end or at a chromosome arm in mitotic anaphase cells. Laser microirradiation is used in combination with dual fluorescent labeling to monitor the co-localization of double-strand break marker γH2AX along with the DDR factors in PtK2 (Potorous tridactylus) cells. Laser-induced DNA breaks in chromosome ends as well as in chromosome arms results in recruitment of the following: poly(ADP-ribose) polymerase 1, checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen. However, phosphorylated p53 at serine 15 is detected only at chromosome ends and not at chromosome arms. Full activation of DDR on damaged chromosome ends may explain previously published results that showed the delay of cytokinesis.

Telomere integrity is important for chromosome stability. The main objective of our study was to investigate the relationship between telomere length modulation and mitotic chromosome segregation induced by ionizing radiation in human primary fibroblasts. We used X-rays and low-energy protons because of their ability to induce different telomeric responses. Samples irradiated with 4 Gy were fixed at different times up to 6 days from exposure and telomere length, anaphase abnormalities, and chromosome aberrations were analyzed. We observed that X-rays induced telomere shortening in cells harvested at 96 hrs, whereas protons induced a significant increase in telomere length at short as well as at long harvesting times (24 and 96 hrs). Consistent with this, the analysis of anaphase bridges at 96 hrs showed a fourfold increase in X-ray- compared with proton-irradiated samples, suggesting a correlation between telomere length/dysfunction and chromosome missegregation. In line with these findings, the frequency of dicentrics and rings decreased with time for protons whereas it remained stable after X-rays irradiation. Telomeric FISH staining on anaphases revealed a higher percentage of bridges with telomere signals in X-ray-treated samples than that observed after proton irradiation, thus suggesting that the aberrations observed after X-ray irradiation originated from telomere attrition and consequent chromosome end-to-end fusion. This study shows that, beside an expected "early" chromosome instability induced shortly after irradiation, a delayed one occurs as a result of alterations in telomere metabolism and that this mechanism may play an important role in genomic stability., (Copyright © 2013 Wiley Periodicals, Inc.)

Telomerase maintains the ends of eukaryotic chromosomes, and its activity is an important parameter correlating with the proliferative capacity of cells. We have investigated cell cycle-specific changes in telomerase activity using cultures of Desmodesmus quadricauda, a model alga naturally synchronized by light/dark entrainment. A quantitative telomerase assay revealed high activity in algal cultures, with slight changes during the light period. Significantly increased telomerase activity was observed at the end of the dark phase, when cell division was complete. In contrast to other models, a natural separation between nuclear and cellular division typical for the cell cycle in D. quadricauda made this observation possible., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Purpose: In recent years, growing epidemiological evidence has linked ionizing radiation exposure to cardiovascular atherosclerotic disease. However, there are still major gaps in the knowledge of the molecular mechanisms of radiation-induced vascular disease, especially for low-dose levels. Telomeres, repetitive DNA sequences of (TTAGGG)(n) located at the ends of eukaryotic chromosomes, play a role in regulating vascular aging, and shorter leukocyte telomere length has been demonstrated to predict cardiovascular disease and mortality. There is also evidence supporting the crucial role of telomeres in the formation of chromosome and chromatid aberrations induced by ionizing radiation., Conclusions: The purpose of the present paper is to review the recent advances in the biological mechanisms determining telomere length erosion after ionizing radiation exposure as well as to examine the hypothesis that telomere shortening may be the crucial mediator leading to detrimental vascular effects after ionizing radiation exposure.

The number of long-term survivors of patients with various malignancies (>5 years) is increasing mainly owing to advances in cancer therapeutics, but long-term side effects of the cancer treatment in this population have emerged as an important health and socio-economical issue. Telomeres and telomerase are known to be essential for regulation of cellular life-span and maintenance of genomic stability, and earlier studies have demonstrated that cancer patients who receive chemotherapy have shorter telomeres in their blood cells, indicating accelerated telomere erosion and a potential contribution of telomere loss to late side-effects. Little is currently known about the effect of chemotherapeutic agents and radiation on telomere dynamics including potential effects on telomere length, structure, function, telomerase activity, and telomere shelterin proteins in normal human cells. In the present study, we had addressed this issue experimentally. The treatment of normal human T lymphocytes and fibroblasts with chemotherapeutic agents doxorubicin (DOX) or etoposide (VP16) led to significant shortening of telomeres, down-regulation of telomerase activity, and diminished expression of telomerase reverse transcriptase (hTERT) and the telomere binding proteins TPP1 and POT1. More importantly, telomere dysfunction was observed in cells treated with DOX or VP16. Furthermore, all the above alterations were similarly found in the cells receiving γ-irradiation. Taken together, both chemotherapy and radiotherapy significantly impair telomere maintenance and function in normal human cells. Conceivably telomere dysfunction causes shortened life-span and genomic instability of normal human cells, and thereby contributes to tissue/organ damage and secondary malignancies in long-term survivors of cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: The tandemly repeated hexamers (TTAGGG)n present at the telomeric ends protect the human genome from a variety of environmental exposures including ionizing radiation. In order to find out the effect of chronic low dose radiation exposure, we have determined telomere length among newborns from high level natural radiation areas (HLNRA) of the Kerala coast in South west India., Methodology: Umbilical cord blood samples were collected from 128 newborns from HLNRA and 43 newborns from normal level natural radiation areas (NLNRA) and genomic DNA was isolated using a salt precipitation method. The mean telomere length was determined using SYBR green-based real time quantitative Polymerase chain reaction (qPCR), where telomere gene specific (T) and single copy gene specific (S) primers were used. The average of telomere versus single copy gene (T/S) ratio was calculated which was proportional to telomere length of each individual., Results: The mean relative telomere length was found to be 1.03 ± 0.01 (95% CI, 0.99-1.05) and 1.10 ± 0.03 (95% CI, 1.04-1.17) in HLNRA and NLNRA newborns, respectively (P > 0.05). Based on the level of background radiation dose, samples were divided into four groups, i.e., NLNRA: < 1.50 mGy/year and three HNLRA groups: 1.51-3.00 mGy/year, 3.01-5.00 mGy/year, and > 5.00 mGy/year. The mean relative telomere length in these groups were found to be 1.10 ± 0.03 (95% CI, 1.03-1.17), 0.98 ± 0.01 (95% CI, 0.95-1.01), 1.05 ± 0.02 (95% CI, 1.01-1.10) and 1.07 ± 0.03 (95% CI, 1.04-1.10), respectively. No significant difference was observed between the mean telomere length of male and female newborns., Conclusions: The elevated level of natural chronic background radiation prevailing in Kerala coast did not show any significant effect on telomere length of newborns. To our knowledge, this is the first report on newborn telomere length from a high level natural radiation area.

Objective: Telomere length was used as a biomarker of cell senescence to explore the role of telomere shortening in photoageing induced by ultraviolet A (UVA) light., Methods: Real-time polymerase chain reaction was used to determine telomere length in cultured human fibroblasts of different generations and after exposure to UVA at doses up to 10 000 mJ/cm(2). Twoway analysis of variance was used to determine whether passaging or UVA was the main factor contributing to telomere shortening., Results: In nonirradiated cells, telomere length was inversely related to cell generation number. In fibroblasts exposed to UVA at a dose of 1000 or 10 000 mJ/cm(2), telomere length was significantly shorter than that of nonirradiated controls and was negatively related to UVA dose., Conclusions: Telomere length and subsequent cell viability may be affected by UVA irradiation. DNA damage caused by UVA irradiation may initiate the photoageing process and telomeres may be a useful new target for attempts to prevent photoageing.

Background: Ionizing radiation could modify lymphocyte function via oxidative damage, DNA breaks, and resulting changes of proliferation, apoptosis and cellular senescence, where telomeres may play a critical role., Aim: To study the effect of low-dose irradiation on the telomere length and apoptosis rates in peripheral blood lymphocytes of irradiated persons., Patients and Methods: A study was performed on 83 peripheral blood samples from the Chornobyl clean-up workers, radiation workers exposed under the professional limits at construction works at the "Shelter" object and healthy controls. Bone marrow leukocyte telomere length was estimated in 15 patients with myelodysplastic syndrome secondary to low-dose radiation exposure and 12 age-standardized healthy donors. Relative telomere length was studied by the combination of a fluorescence hybridization in situ with PNA probe and flow cytometry, apoptosis - by Annexin-V test., Results: A significant relative telomere length decrease has been demonstrated in Chornobyl clean-up workers compared to healthy donors (13.2 ± 0.69 and 18.6 ± 0.73 respectively, p < 0.05), and a tendency (p < 0.1) in radiation workers. At doses over professional limits an inverse dependency is demonstrated between the relative telomere length and a number of lymphocytes in early stage of apoptosis. In MDS group a tendency of telomere elongation was demonstrated in bone marrow granulocytes in RAEB-t and RAEB as comparing with RA., Conclusion: This study shows telomere shortening after low-dose irradiation and preservation of these changes even 20 years after exposure. Apoptosis induction is possible by the telomere region changes at least in individuals with shorter telomeres. Apoptosis decrease in MDS clonal transformation is associated with a substantially longer telomeres.

Background: Because the long-term toxicity of myeloablative radioimmunotherapy remains a matter of concern, the authors evaluated the hematopoietic damage and incidence of secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/AML) in patients who received myeloablative doses of the radiolabeled antibody yttrium-90 (⁹⁰Y)-ibritumomab tiuxetan., Methods: The occurrence of sMDS/AML was investigated prospectively in 53 elderly patients with non-Hodgkin lymphoma (NHL) who underwent an autograft after high-dose radioimmunotherapy (HD-RIT) myeloablative conditioning with ⁹⁰Y-ibritumomab tiuxetan. Bone marrow (BM) hematopoietic progenitors and telomere length (TL) also were investigated., Results: At a median follow-up of 49 months, 4 patients developed sMDS/AML at 6 months, 12 months, 27 months, and 36 months after HD-RIT, and the 5-year cumulative incidence of sMDS/AML was 8.29%. A significant but transient decrease in BM granulocyte-macrophage progenitors was observed; whereas multilineage, erythroid, and fibroblast progenitors were unaffected. A significant and persistent shortening of BM TL also was detected. A matched-pair analysis comparing the study patients with 55 NHL patients who underwent autografts after chemotherapy-based myeloablative conditioning demonstrated a 8.05% 5-year cumulative incidence of sMDS/AML., Conclusions: HD-RIT for patients with NHL was associated with 1) limited toxicity on hematopoietic progenitors, 2) accelerated TL shortening, and 3) non-negligible incidence of sMDS/AML, which nevertheless was comparable to the incidence observed in a matched group of patients who received chemotherapy-based conditioning. Thus, in the current series of elderly patients with NHL, the development of sMDS/AML was not influenced substantially by HD-RIT., (Copyright © 2011 American Cancer Society.)

The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, as a potential radiosensitizing agent for glioblastoma multiforme (GBM). Two human radioresistant telomerase-positive GBM cell lines (SF763 and SF767) were analyzed, with and without TAC treatment, for telomere length, cell proliferation, apoptosis, cell-cycle distribution, gene expression, cytogenetic aberrations, clonogenic survival assay, 53BP1 immunofluorescence staining, and γH2AX phosphorylation. We found that low concentrations of TAC (0.5 and 1 μmol/L) inhibited the proliferation of GBM cells in a concentration-dependent manner after only 1 week of treatment, with minimal effects on cell cycle and apoptosis. TAC treatment had no visible effect on average telomere length but modified expression levels of telomere-related genes (hTERT, TRF1, and TRF2) and induced concentration-dependent DNA damage response and dicentric chromosomes. Survival curves analysis showed that exposure to nontoxic, subapoptotic concentrations of TAC enhanced radiation-induced killing of GBM cells. Analysis of DNA repair after irradiation revealed delayed repair kinetics in GBM cells treated with TAC. Furthermore, the combined treatment (TAC and radiation) significantly increased the frequency of chromosomal aberrations as compared with radiation alone. These findings provide the first evidence that exposure to a G4 ligand radiosensitizes human glioblastoma cells and suggest the prospect of future therapeutic applications.

To study the bystander effects, G(0) human peripheral blood lymphocytes were X-irradiated with 0.1, 0.5 and 3 Gy. After 24h, cell-free conditioned media from irradiated cultures were transferred to unexposed lymphocytes. Following 48 h of medium transfer, viability, induction of apoptosis, telomere shortening, reactive oxygen species (ROS) levels and micronuclei (after stimulation) were analyzed. A statistically significant decrement in cell viability, concomitant with the loss of mitochondrial membrane potential, telomere shortening, increases in hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) with depletion of intracellular glutathione (GSH) level, and higher frequencies of micronuclei, were observed in bystander lymphocytes incubated with medium from 0.5 and 3 Gy irradiated samples, compared to lymphocytes unexposed. Furthermore, no statistically significant difference between the response to 0.5 and 3 Gy of irradiation in bystander lymphocytes, was found. However, when lymphocytes were irradiated with 0.1 Gy, no bystander effect with regard to viability, apoptosis, telomere length, and micronuclei was observed, although a high production of ROS level persisted. Radiation in the presence of the radical scavenger dimethyl sulfoxide (DMSO) suppressed oxidative stress induced by 3 Gy of X-rays with the effective elimination of bystander effects, suggesting a correlation between ROS and bystander signal formation in irradiated cells. The data propose that bystander effect might be mostly due to the reactions of radiation induced free radicals on DNA, with the existence of a threshold at which the bystander signal is not operative (0.1 Gy dose of X-rays). Our results may have clinical implications for health risk associated with radiation exposure., (2011 Elsevier B.V. All rights reserved.)

Genome instability continuously presents perils of cancer, genetic disease and death of a cell or an organism. At the same time, it provides for genome plasticity that is essential for development and evolution. We address here the genome instability confined to a small fraction of DNA adjacent to free DNA ends at uncapped telomeres and double-strand breaks. We found that budding yeast cells can tolerate nearly 20 kilobase regions of subtelomeric single-strand DNA that contain multiple UV-damaged nucleotides. During restoration to the double-strand state, multiple mutations are generated by error-prone translesion synthesis. Genome-wide sequencing demonstrated that multiple regions of damage-induced localized hypermutability can be tolerated, which leads to the simultaneous appearance of multiple mutation clusters in the genomes of UV- irradiated cells. High multiplicity and density of mutations suggest that this novel form of genome instability may play significant roles in generating new alleles for evolutionary selection as well as in the incidence of cancer and genetic disease.

We examined phosphorylation of H2AX, a marker for DNA double-strand breaks over the life of a human fibroblast cell line. This marker was compared with a number of other cellular senescence and DNA repair endpoints. An increase in γH2AX foci number was observed after 24 hours of repair time following DNA damage over the course of fibroblast passaging. Progressive and relatively constant changes in growth retardation, doubling time, and telomere length were also observed. The fraction of cells expressing β-gal, a marker of cellular senescence, increased considerably around the 40th passage as did some other cell morphology endpoints. The detectable γH2AX foci at 24 hours after ionizing radiation were far fewer than the number detected at 1 hour across all passage numbers. We conclude that although residual DNA damage level increases with passage number, it is unlikely to be the result of less efficient DNA repair in the aged fibroblast since most DNA damage is repaired, even at late passages.