Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis

Copyright @ 2012 Nature Publishing GroupThis article has been made available through the Brunel Open Access Publishing Fund.Background: The objective of this study was to determine the molecular mechanism(s) responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Methods: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double strand break (DSB) repair in cells. Quantitative-PCR (Q-PCR) established the expression levels of key DNA DSB repair proteins. Imaging flow cytometry using Annexin V-FITC was used to compare artemis expression and apoptosis in cells. Results: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Over-expression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. Conclusion: We conclude elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.This work was supported in part by The Vidal Sassoon Foundation USA. This article is made available through the Brunel Open Access Publishing Fund

Radiation-hypersensitive cancer patients do not manifest protein expression abnormalities in components of the nonhomologous end-joining (NHEJ) pathway

Radiation therapy (RT) is utilised for the treatment of around half of all oncology patients during the course of their illness. Despite great clinical progress in the rational deployment of RT, the underlying molecular basis for its efficacy and toxicity are currently imperfectly understood. In this study, we took a biochemical approach to evaluate the potential role of key ionising radiation repair proteins in the treatment outcomes of patients with severe acute or late RT side effects. Lymphoblastoid cell lines were established from blood samples from 36 radiosensitive cases and a number of controls (the latter had had RT but did not develop significant toxicity). The expression level and migration of key proteins from the nonhomologous end-joining (NHEJ) pathway was evaluated by Western blot analysis on cases and controls. We did not observe any abnormalities in expression level or migration pattern of the following NHEJ proteins in radiosensitive cancer cases: Ku70, Ku80, XRCC4, DNA Ligase IV. These important negative results provide evidence that mutations that affect protein expression of these NHEJ components are unlikely to underlie clinical radiation sensitivity

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

CsA can induce DNA double-strand breaks: implications for BMT regimens particularly for individuals with defective DNA repair

Several human disorders mutated in core components of the major DNA double-strand break (DSB) repair pathway, non-homologous end joining (NHEJ), have been described. Cell lines from these patients are characterized by sensitivity to DSB-inducing agents. DNA ligase IV syndrome (LIG4) patients specifically, for unknown reasons, respond particularly badly following treatment for malignancy or BMT. We report the first systematic evaluation of the response of LIG4 syndrome to compounds routinely employed for BMT conditioning. We found human pre-B lymphocytes, a key target population for BMT conditioning, when deficient for DNA ligase IV, unexpectedly exhibit significant sensitivity to CsA the principal prophylaxis for GVHD. Furthermore, we found that CsA treatment alone or in combination with BU and fludarabine resulted in increased levels of DSBs specifically in LIG4 syndrome cells compared to wild-type or Artemis-deficient cells. Our study shows that CsA can induce DSBs and that LIG4 syndrome patient's fail to adequately repair this damage. These DSBs likely arise as a consequence of DNA replication in the presence of CsA. This work has implications for BMT and GVHD management in general and specifically for LIG4 syndrome

ATM Limits Incorrect End Utilization during Non-Homologous End Joining of Multiple Chromosome Breaks

Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI–resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ

Ribosomal S6 Kinase 2 (RSK2) Maintains Genomic Stability by Activating the Atm/p53-Dependent DNA Damage Pathway

10.1371/journal.pone.0074334PLoS ONE89-POLN

Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron–sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.Ataxia UK, FARA Australasia and FARA US

Heavy Ion Carcinogenesis and Human Space Exploration

Prior to the human exploration of Mars or long duration stays on the Earth s moon, the risk of cancer and other diseases from space radiation must be accurately estimated and mitigated. Space radiation, comprised of energetic protons and heavy nuclei, has been show to produce distinct biological damage compared to radiation on Earth, leading to large uncertainties in the projection of cancer and other health risks, while obscuring evaluation of the effectiveness of possible countermeasures. Here, we describe how research in cancer radiobiology can support human missions to Mars and other planets

HIV-1 Tat and AIDS-associated cancer: targeting the cellular anti-cancer barrier?

The acquired immunodeficiency syndrome (AIDS) is accompanied by a significant increase in the incidence of neoplasms. Several causative agents have been proposed for this phenomenon. These include immunodeficiency and oncogenic DNA viruses and the HIV-1 protein Tat. Cancer in general is closely linked to genomic instability and DNA repair mechanisms. The latter maintains genomic stability and serves as a cellular anti-cancer barrier. Defects in DNA repair pathway are associated with carcinogenesis