Radiation-induced chromosome breaks in ataxia-telangiectasia cells remain open

Martı́n, Marga; Genescà, Anna; Latre, Laura; Ribas, M.; Miró, Rosa; Egozcue, J.; Tusell, Laura

doi:10.1080/0955300031000089601

Cited by 18 publications

(12 citation statements)

References 32 publications

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“…It is noteworthy that our experiments involve plateau-phase cells and therefore cannot be attributed to any impact of cell cycle checkpoints. Overall, our findings are consistent with cytogenetic studies involving premature chromosome condensation of G 1 -arrested primary human AT fibroblasts (36,37) or fluorescence in situ hybridization of mitotic cells (61,62). In these studies, AT cells exhibit a substantially increased level of unrepaired chromosome breaks after irradiation compared with control cells and a smaller increase of exchange-type chromosome aberrations.…”

Section: Discussionsupporting

confidence: 80%

A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity

et al. 2004

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The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of ␥-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.

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Section: Discussionsupporting

confidence: 80%

A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity

et al. 2004

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“…The karyotype stability, repair efficiency and normality of the DNA-repair kinetics of this cell line have been evaluated in previous studies. 27,28 After immunostaining of phosphorylated H2AX, mitotic chromosomes displayed some gH2AX foci of big size, and these were accompanied by a population of much smaller foci, but still clearly different from the background. Previous studies have reported the presence of gH2AX foci of variable sizes in mitotic chromosomes.…”

Section: Resultsmentioning

confidence: 99%

“…Immediately after irradiation, BrdU (Sigma; Sigma Aldrich, Buchs SG, Switzerland) was added to the culture at a final concentration of 12 mg/ml and the protocol was followed as previously described. 27 Metaphase slides were obtained and UV irradiated at different post-irradiation times. Slides showing a majority of metaphases in first division after irradiation (78.5% of first divisions and 21.5% of second divisions at 48 h after irradiation) were chosen to avoid the loss of acentric fragments and other unrepaired events in further cell divisions.…”

Section: Methodsmentioning

confidence: 99%

γH2AX foci on apparently intact mitotic chromosomes: Not signatures of misrejoining events but signals of unresolved DNA damage

et al. 2014

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Abbreviations: AU, arbitrary units; DSB, DNA double-strand break; FISH, fluorescence in situ hybridization; IRIF, ionizing radiation induced foci; FI, fluorescence intensity; mFISH, multicolor fluorescence in situ hybridization; MRN complex, MRE11-Rad50-Nbs1 complex; SD, standard deviation; TIF, telomere-dysfunction induced foci.The presence of gH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between gH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of gH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the gH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal gH2AX foci, but very rarely are they signaled with Small gH2AX foci. To evaluate if Interstitial gH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial gH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a gH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all gH2AX foci-regardless their coincidence with a visible break-always colocalized with MRE11, but not with 53BP1. This pattern suggests that these gH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.

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“…After solid-staining analysis, slides were washed in PBS, treated with pepsin, and fixed with 4% formaldehyde. Telomere labeling was applied as described before (32). Telomere-labeled binucleated MEFs were recaptured and analyzed.…”

Section: Methodsmentioning

confidence: 99%

Postreplicative Joining of DNA Double-Strand Breaks Causes Genomic Instability in DNA-PKcs–Deficient Mouse Embryonic Fibroblasts

Martı́n

Genescà²,

Latre³

et al. 2005

Cancer Research

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Combined cytogenetic and biochemical approaches were used to investigate the contributions of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the maintenance of genomic stability in nonirradiated and irradiated primary mouse embryo fibroblasts (MEF). We show that telomere dysfunction contributes only marginally to genomic instability associated with DNA-PKcs deficiency in the absence of radiation. Following exposure to ionizing radiation, DNAPKcs À/À MEFs are radiosensitized mainly as a result of the associated DNA double-strand break (DSB) repair defect. This defect manifests as an increase in the fraction of DSB rejoining with slow kinetics although nearly complete rejoining is achieved within 48 hours. Fifty-four hours after ionizing radiation, DNA-PKcs À/À cells present with a high number of simple and complex chromosome rearrangements as well as with unrepaired chromosome breaks. Overall, induction of chromosome aberrations is 6-fold higher in DNA-PKcs À/À MEFs than in their wild-type counterparts. Spectral karyotypingfluorescence in situ hybridization technology distinguishes between rearrangements formed by prereplicative and postreplicative DSB rejoining and identifies sister chromatid fusion as a significant source of genomic instability and radiation sensitivity in DNA-PKcs À/À MEFs. Because DNA-PKcsMEFs show a strong G 1 checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs À/À MEFs prolongs the mean life of broken chromosome ends and increases the probability of incorrect joining. The preponderance of sister chromatid fusion as a product of incorrect joining points to a possible defect in S-phase arrest and emphasizes proximity in these misrepair events. (Cancer Res 2005; 65(22): 10223-32)

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Radiation-induced chromosome breaks in ataxia-telangiectasia cells remain open

Abstract: The disability of cells to rejoin broken chromosome ends does not lead to the healing of DSBs by the acquisition of new telomeric sequences.

Cited by 18 publications

References 32 publications

A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity

A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity

γH2AX foci on apparently intact mitotic chromosomes: Not signatures of misrejoining events but signals of unresolved DNA damage

Postreplicative Joining of DNA Double-Strand Breaks Causes Genomic Instability in DNA-PKcs–Deficient Mouse Embryonic Fibroblasts

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