Defective Artemis causes mild telomere dysfunction

This article has been made available through the Brunel Open Access Publishing Fund.Background: Repair of DNA double strand breaks by non-homologous end joining (NHEJ) requires several proteins including Ku, DNA-PKcs, Artemis, XRCC4, Ligase IV and XLF. Two of these proteins, namely Ku and DNA-PKcs, are also involved in maintenance of telomeres, chromosome end-structures. In contrast, cells defective in Ligase IV and XRCC4 do not show changes in telomere length or function suggesting that these proteins are not involved in telomere maintenance. Since a mouse study indicated that defective Artemis may cause telomere dysfunction we investigated the effects of defective Artemis on telomere maintenance in human cells. Results: We observed significantly elevated frequencies of telomeric fusions in two primary fibroblast cell lines established from Artemis defective patients relative to the control cell line. The frequencies of telomeric fusions increased after exposure of Artemis defective cells to ionizing radiation. Furthermore, we observed increased incidence of DNA damage at telomeres in Artemis defective cells that underwent more than 32 population doublings using the TIF (Telomere dysfunction Induced Foci) assay. We have also inhibited the expression levels of DNA-PKcs in Artemis defective cell lines by either using synthetic inhibitor (IC86621) or RNAi and observed their greater sensitivity to telomere dysfunction relative to control cells. Conclusion: These results suggest that defective Artemis causes a mild telomere dysfunction phenotype in human cell lines.This article is available through the Brunel Open Access Publishing Fund. This study was supported by a grant from European Commission RISC-RAD contract FI6R-CT2003-50884

Analysis of telomere length and function in radiosensitive mouse and human cells in response to DNA-PKcs inhibition

© 2013 Yasaei et al.; licensee BioMed Central Ltd.This article has been made available through the Brunel Open Access Publishing Fund.Telomeres, the physical ends of chromosomes, play an important role in preserving genomic integrity. This protection is supported by telomere binding proteins collectively known as the shelterin complex. The shelterin complex protects chromosome ends by suppressing DNA damage response and acting as a regulator of telomere length maintenance by telomerase, an enzyme that elongates telomeres. Telomere dysfunction manifests in different forms including chromosomal end-to-end fusion, telomere shortening and p53-dependent apoptosis and/or senescence. An important shelterin-associated protein with critical role in telomere protection in human and mouse cells is the catalytic subunit of DNA-protein kinase (DNA-PKcs). DNA-PKcs deficiency in mouse cells results in elevated levels of spontaneous telomeric fusion, a marker of telomere dysfunction, but does not cause telomere length shortening. Similarly, inhibition of DNA-PKcs with chemical inhibitor, IC86621, prevents chromosomal end protection through mechanism reminiscent of dominant-negative reduction in DNA-PKcs activity.This study was supported by a grant from European Commission RISC-RAD contract FI6R-CT2003-508842 to P

Genome Integrity: A new open access journal

The full and final version of this article can be found at the link belowThis article has been made available through the Brunel Open Access Publishing Fund.This article is available through the Brunel Open Access Publishing Fund

Hydrogen peroxide induced genomic instability in nucleotide excision repair-deficient lymphoblastoid cells

Copyright @ 2010 Gopalakrishnan et al; licensee BioMed Central Ltd.Background The Nucleotide Excision Repair (NER) pathway specialises in UV-induced DNA damage repair. Inherited defects in the NER can predispose individuals to Xeroderma Pigmentosum (XP). UV-induced DNA damage cannot account for the manifestation of XP in organ systems not directly exposed to sunlight. While the NER has recently been implicated in the repair of oxidative DNA lesions, it is not well characterised. Therefore we sought to investigate the role of NER factors Xeroderma Pigmentosum A (XPA), XPB and XPD in oxidative DNA damage-repair by subjecting lymphoblastoid cells from patients suffering from XP-A, XP-D and XP-B with Cockayne Syndrome to hydrogen peroxide (H2O2). Results Loss of functional XPB or XPD but not XPA led to enhanced sensitivity towards H2O2-induced cell death. XP-deficient lymphoblastoid cells exhibited increased susceptibility to H2O2-induced DNA damage with XPD showing the highest susceptibility and lowest repair capacity. Furthermore, XPB- and XPD-deficient lymphoblastoid cells displayed enhanced DNA damage at the telomeres. XPA- and XPB-deficient lymphoblastoid cells also showed differential regulation of XPD following H2O2 treatment. Conclusions Taken together, our data implicate a role for the NER in H2O2-induced oxidative stress management and further corroborates that oxidative stress is a significant contributing factor in XP symptoms. Resistance of XPA-deficient lymphoblastoid cells to H2O2-induced cell death while harbouring DNA damage poses a potential cancer risk factor for XPA patients. Our data implicate XPB and XPD in the protection against oxidative stress-induced DNA damage and telomere shortening, and thus premature senescence.This research is supported by the Defence Innovative Research Programme, Defence Science and Technology Agency, Singapore (POD: 0613592) and the Academic Research Fund, Ministry of Education, Singapore (T206B3108). Supported in part by a grant from British Council, PMI2 Connect (Grant Number: RC134)

The effect of chemotherapeutic agents on telomere length maintenance in breast cancer cell lines

Copyright @ 2014 the authors. This article is made available through the Brunel Open Access Publishing Fund. It is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. Short telomeres in breast cancer cells confer telomere dysfunction and this can be related to Shelterin proteins and their level of expression in breast cancer cell lines. This study investigates whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA) were used in an attempt to reactivate the expression of silenced genes. Our studies have shown that Shelterin and Shelterin-associated genes were down-regulated in breast cancer cell lines; this may be due to epigenetic modification of DNA as the promoter region of POT1 was found to be partially methylated. Shelterin genes expression was up-regulated upon treatment of 21NT breast cancer cells with 5-aza-CdR and TSA. The telomere length of treated 21NT cells was measured by q-PCR showed an increase in telomere length at different time points. Our studies have shown that down-regulation of Shelterin genes is partially due to methylation in some epithelial breast cancer cell lines. Removal of epigenetic silencing results in up-regulation of Shelterin and Shelterin-associated genes which can then lead to telomere length elongation and stability

Serial Endosymbiosis Theory: From biology to astronomy and back to the origin of life

Serial Endosymbiosis Theory, or SET, was conceived and developed by Lynn Margulis, to explain the greatest discontinuity in the history of life, the origin of eukaryotic cells. Some predictions of SET, namely the origin of mitochondria and chloroplasts, withstood the test of the most recent evidence from a variety of disciplines including phylogenetics, biochemistry, and cell biology. Even though some other predictions fared less well, SET remains a seminal theory in biology. In this paper, I focus on two aspects of SET. First, using the concept of “universal symbiogenesis”, developed by Freeman Dyson to search for commonalities in astronomy and biology, I propose that SET can be extended beyond eukaryogenesis. The extension refers to the possibility that even prokaryotic organisms, themselves subject to the process of symbiogenesis in SET, could have emerged symbiotically. Second, I contrast a recent “viral eukaryogenesis” hypothesis, according to which the nucleus evolved from a complex DNA virus, with a view closer to SET, according to which the nucleus evolved through the interplay of the archaeal host, the eubacterial symbiont, and a non-LTR transposon, or telomerase. Viruses joined in later, through the process of viral endogenization, to shape eukaryotic chromosomes in the process of karyotype evolution. These two proposals based on SET are a testament to its longevity as a scientific theory.Brunel University Londo

Effects of BRCA2 deficiency on telomere recombination in non-ALT and ALT cells

This article has been made available through the Brunel Open Access Publishing Fund - Copyright @ 2011 Sapir et al.Background: Recent studies suggest that BRCA2 affects telomere maintenance. Interestingly, anti cancer treatments that involve BRCA2 and telomerase individually are currently being explored. In the light of the above recent studies their combinatorial targeting may be justified in the development of future treatments. In order to investigate effects of BRCA2 that can be explored for this combinatorial targeting we focused on the analysis of recombination rates at telomeres by monitoring T-SCEs (Telomere Sister Chromatid Exchanges). Results: We observed a significant increase in T-SCE frequencies in four BRCA2 defective human cell lines thus suggesting that BRCA2 suppresses recombination at telomeres. To test this hypothesis further we analyzed T-SCE frequencies in a set of Chinese hamster cell lines with or without functional BRCA2. Our results indicate that introduction of functional BRCA2 normalizes frequencies of T-SCEs thus supporting the notion that BRCA2 suppresses recombination at telomeres. Given that ALT (Alternative Lengthening of Telomeres) positive cells maintain telomeres by recombination we investigated the effect of BRCA2 depletion in these cells. Our results show that this depletion causes a dramatic reduction in T-SCE frequencies in ALT positive cells, but not in non-ALT cells. Conclusion: BRCA2 suppresses recombination at telomeres in cells that maintain them by conventional mechanisms. Furthermore, BRCA2 depletion in ALT positive cells reduces high levels of T-SCEs normally found in these cells. Our results could be potentially important for refining telomerase-based anti-cancer therapies.This work is supported in part by grants from European Commission RISC-RAD contract FI6RCT2003-508842 and British Counci

Reconfiguring SETI in the microbial context: Panspermia as a solution to Fermi's paradox

All SETI (Search for Extraterrestrial Intelligence) programmes that were conceived and put into practice since the 1960s have been based on anthropocentric ideas concerning the definition of intelligence on a cosmic-wide scale. Brain-based neuronal intelligence, augmented by AI, are currently thought of as being the only form of intelligence that can engage in SETI-type interactions, and this assumption is likely to be connected with the dilemma of the famous Fermi paradox. We argue that high levels of intelligence and cognition inherent in ensembles of bacteria are much more likely to be the dominant form of cosmic intelligence, and the transfer of such intelligence is enabled by the processes of panspermia. We outline the main principles of bacterial intelligence, and how this intelligence may be used by the planetary-scale bacterial system, or the bacteriosphere, through processes of biological tropism, to connect to any extra-terrestrial microbial forms, independently of human interference

Telomere elongation in the breast cancer cell line 21NT after treatment with an epigenetic modifying drug

Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.We thank Professor Robert Newbold for his support and for providing the opportunity to carry out this work within the Institute of Cancer Genetics and Pharmacogenomics, Brunel University London. HY was supported by a triennial project grant (Strategic Award) from the National Centre for Replacement, Refinement, and Reduction (NC3Rs) of animals in research (NC. K500045.1 and G0800697)

Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumours

Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.EThOS - Electronic Theses Online ServiceGrant to Dr. Predrag Slijepcevic from the Department of Health (RRX97)EC Euratom (Fig h-CT-2002-0021 7)my, Embryology, Histology and Medial Physics, Faculty of Medicine, University of Ghent, BelgiumGBUnited Kingdo