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Noon A.T.,University of Sussex | Noon A.T.,Research Center for Charged Particle Therapy | Shibata A.,University of Sussex | Rief N.,TU Darmstadt | And 4 more authors.
Nature Cell Biology | Year: 2010

DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, 15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling. 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1Β, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with γH2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11-NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair. © 2010 Macmillan Publishers Limited. All rights reserved. Source

Maeda J.,Colorado State University | Cartwright I.M.,Colorado State University | Haskins J.S.,Colorado State University | Fujii Y.,Ibaraki Prefectural University of Health Sciences | And 7 more authors.
Oncology Letters | Year: 2016

Heavy ions, characterized by high linear energy transfer (LET) radiation, have advantages compared with low LET protons and photons in their biological effects. The application of heavy ions within veterinary clinics requires additional background information to determine heavy ion efficacy. In the present study, comparison of the cell-killing effects of photons, protons and heavy ions was investigated in canine osteosarcoma (OSA) cells in vitro. A total of four canine OSA cell lines with various radiosensitivities were irradiated with 137Cs gamma-rays, monoenergetic proton beams, 50 keV/μm carbon ion spread out Bragg peak beams and 200 keV/μm iron ion monoenergetic beams. Clonogenic survival was examined using colony-forming assays, and relative biological effectiveness (RBE) values were calculated relative to gamma-rays using the D10 value, which is determined as the dose (Gy) resulting in 10% survival. For proton irradiation, the RBE values for all four cell lines were 1.0-1.1. For all four cell lines, exposure to carbon ions yielded a decreased cell survival compared with gamma-rays, with the RBE values ranging from 1.56-2.10. Iron ions yielded the lowest cell survival among tested radiation types, with RBE values ranging from 3.51-3.69 observed in the three radioresistant cell lines. The radiosensitive cell line investigated demonstrated similar cell survival for carbon and iron ion irradiation. The results of the present study suggest that heavy ions are more effective for killing radioresistant canine OSA cells when compared with gamma-rays and protons. This markedly increased efficiency of cell killing is an attractive reason for utilizing heavy ions for radioresistant canine OSA. © 2016, Spandidos Publications. All rights reserved. Source

Maeda J.,Colorado State University | Fujii Y.,Ibaraki Prefectural University of Health Sciences | Fujisawa H.,University of Tokyo | Hirakawa H.,Research Center for Charged Particle Therapy | And 5 more authors.
Oncology Letters | Year: 2015

The DNA repair mechanisms involved in hyperthermia-induced radiosensitization with proton and carbon ion radiation exposure were investigated in the present study. In a previous study, Chinese hamster ovary (CHO) cells were exposed to low linear energy transfer (LET) photon radiation. These cells can be sensitized by hyperthermia as a result of inhibition of homologous recombination (HR) repair. The present study used wild-type, non-homologous end joining (NHEJ) and HR repair-deficient CHO cells to define the contributions of each repair pathway to cellular lethality following hyperthermia-induced hadron radiation sensitization. The cells were exposed to ionizing radiation, followed by hyperthermia treatment (42.5˚C for 1 h). Hyperthermia-induced radiosensitization was determined by the colony formation assay and thermal enhancement ratio. HR repair-deficient cells exhibited no hyper-sensitization to X-rays, protons, or low and high LET carbon ions when combined with hyperthermia. Wild-type and NHEJ repair-deficient cells exhibited significant hyperthermia-induced sensitization to low LET photon and hadron radiation. Hyperthermia-induced sensitization to high LET carbon-ion radiation was less than at low LET radiation. Relative biological effectiveness (RBE) between radiation alone and radiation combined with hyperthermia cell groups was not significantly different in any of the cell lines, with the exception of wild-type cells exposed to high LET radiation, which exhibited a lower RBE in the combined group. The present study investigated additional cell lines to confirm the lower RBE observed in DNA repair-deficient cell lines. These findings suggested that hyperthermia-induced hyper-sensitization to hadron radiation is also dependent on inhibition of HR repair, as was observed with photon radiation in a previous study. © 2015, Spandidos Publications. All rights reserved. Source

Cartwright I.M.,Colorado State University | Bell J.J.,Colorado State University | Maeda J.,Colorado State University | Genet M.D.,Colorado State University | And 7 more authors.
Oncology Letters | Year: 2015

The present study investigated the effect of targeted mutations in the DNA-dependent protein kinase catalytic subunit and phosphorylation domains on the survival of cells in response to different qualities of ionizing radiation. Mutated Chinese hamster ovary V3 cells were exposed to 500 MeV/nucleon initial energy and 200 keV/μm monoenergetic Fe ions; 290 MeV/nucleon initial energy and average 50 keV/μm spread-out Bragg peak C ions; 70 MeV/nucleon initial energy and 1 keV/μm monoenergetic protons; and 0.663 MeV initial energy and 0.3 keV/μm Cs137 γ radiation. The results demonstrated that sensitivity to high linear energy transfer radiation is increased when both S2056 and T2609 clusters each contain a point mutation or multiple mutations are present in either cluster, whereas the phosphoinositide 3 kinase cluster only requires a single mutation to induce the sensitized phenotype of V3 cells. Additionally, the present study demonstrated that sensitivity to DNA cross-linking damage by cisplatin only requires a single mutation in one of the three clusters and that additional point mutations do not increase cell sensitivity. © 2014, Spandidos Publications. All rights reserved. Source

Schmutz V.,University of Strasbourg | Janel-Bintz R.,University of Strasbourg | Wagner J.,University of Strasbourg | Biard D.,French Institute of Health and Medical Research | And 3 more authors.
Nucleic Acids Research | Year: 2010

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but never-theless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA. © The Author(s) 2010. Published by Oxford University Press. Source

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