skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: DNA Double-Strand Break Rejoining in Complex Normal Tissues

Abstract

Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive {gamma}H2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating {gamma}H2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that {gamma}H2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for {gamma}H2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measuredmore » in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis.« less

Authors:
 [1];  [2];  [3]; ;  [2]; ;  [4];  [2]
  1. Department of Radiation Oncology, Saarland University, Homburg/Saar, Saarland (Germany), E-mail: claudia.ruebe@uks.eu
  2. Department of Radiation Oncology, Saarland University, Homburg/Saar, Saarland (Germany)
  3. (China)
  4. Institute for Molecular Cell Biology, Saarland University, Homburg/Saar, Saarland (Germany)
Publication Date:
OSTI Identifier:
21172473
Resource Type:
Journal Article
Resource Relation:
Journal Name: International Journal of Radiation Oncology, Biology and Physics; Journal Volume: 72; Journal Issue: 4; Other Information: DOI: 10.1016/j.ijrobp.2008.07.017; PII: S0360-3016(08)03042-3; Copyright (c) 2008 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; BRAIN; DNA; DNA REPAIR; HEART; HOMEOSTASIS; IN VIVO; IONIZING RADIATIONS; KIDNEYS; LUNGS; MICE; RADIATION DOSES; RADIONUCLIDE KINETICS; RADIOSENSITIVITY; SMALL INTESTINE; STRAND BREAKS; WHOLE-BODY IRRADIATION

Citation Formats

Ruebe, Claudia E., Dong, Xiaorong, Cancer Center, Union Hospital Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Kuehne, Martin, Fricke, Andreas, Kaestner, Lars, Lipp, Peter, and Ruebe, Christian. DNA Double-Strand Break Rejoining in Complex Normal Tissues. United States: N. p., 2008. Web. doi:10.1016/j.ijrobp.2008.07.017.
Ruebe, Claudia E., Dong, Xiaorong, Cancer Center, Union Hospital Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Kuehne, Martin, Fricke, Andreas, Kaestner, Lars, Lipp, Peter, & Ruebe, Christian. DNA Double-Strand Break Rejoining in Complex Normal Tissues. United States. doi:10.1016/j.ijrobp.2008.07.017.
Ruebe, Claudia E., Dong, Xiaorong, Cancer Center, Union Hospital Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Kuehne, Martin, Fricke, Andreas, Kaestner, Lars, Lipp, Peter, and Ruebe, Christian. 2008. "DNA Double-Strand Break Rejoining in Complex Normal Tissues". United States. doi:10.1016/j.ijrobp.2008.07.017.
@article{osti_21172473,
title = {DNA Double-Strand Break Rejoining in Complex Normal Tissues},
author = {Ruebe, Claudia E. and Dong, Xiaorong and Cancer Center, Union Hospital Tongji Medical College, Huazhong University of Science and Technology, Wuhan and Kuehne, Martin and Fricke, Andreas and Kaestner, Lars and Lipp, Peter and Ruebe, Christian},
abstractNote = {Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive {gamma}H2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating {gamma}H2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that {gamma}H2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for {gamma}H2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis.},
doi = {10.1016/j.ijrobp.2008.07.017},
journal = {International Journal of Radiation Oncology, Biology and Physics},
number = 4,
volume = 72,
place = {United States},
year = 2008,
month =
}
  • The study was aimed at the measurement of effect-depth distributions of intracellularly induced DNA damage in water as tissue equivalent after heavy ion irradiation with therapy particle beams. An assay involving embedding of Chinese hamster ovary (CHO-K1) cells in large agarose plugs and electrophoretic elution of radiation induced DNA fragments by constant field gel electrophoresis was developed. Double-strand break production was quantified by densitometric analysis of DNA-fluorescence after staining with ethidium-bromide and determination of the fraction of DNA eluted out of the agarose plugs. Intracellular double-strand break induction and the effect of a 3 h rejoining incubation were investigated followingmore » irradiation with 250 kV x-rays and 190 MeV/u carbon- and 295 MeV/u neon-ions. While the DNA damage induced by x-irradiation decreased continuously with penetration depth, a steady increase in the yield of double-strand breaks was observed for particle radiation, reaching distinct maxima at the position of the physical Bragg peaks. Beyond this, the extent of radiation damage dropped drastically. From comparison of DNA damage and calculated dose profiles, relative biological efficiencies (RBEs) for both double-strand break induction and unrejoined strand breaks after 3 h were determined. While RBE for the induction of DNA double-strand breaks decreased continuously with penetration depth, RBE maxima greater than unity were found with carbon- and neon-ions for double-strand break rejoining near the maximum range of the particles. The method presented here allows for a fast and accurate determination of depth profiles of relevant radiobiological effects for mixed particle fields in tissue equivalent. DNA DSB-induction, Strand break rejoining, CHO-K1 cells, Heavy ion therapy beams, Effect-depth distribution. 35 refs., 8 figs.« less
  • No abstract prepared.
  • To better link biochemical processing of the DSB to cell killing, a two-lesion kinetic (TLK) model is proposed. In the TLK model, the family of all possible DSBs is sub-divided into simple and complex DSBs, and each kind of DSB may have its own repair characteristics. A unique aspect of the TLK model is that break-ends associated with both kinds of DSB are allowed to interact in pairwise fashion to form irreversible lethal and non-lethal damages. To test the performance of the TLK model, non-linear optimization methods are used to calibrate the model based on CHO cell survival data formore » an extensive set of single-dose and split-dose exposure conditions. Then, some of the postulated mechanisms of action are tested by comparing measured and predicted estimates of the initial DSB yield and the rate of DSB rejoining. TLK model predictions of CHO survival and the initial DSB yield and rejoining rate are all shown to be in good agreement with the measured data. Studies suggest a yield of about 25 DSB Gy-1 cell-1. About 20 DSB Gy-1 cell-1 are rejoined quickly (15-minute repair half-time), and 4 to 6 DSB Gy-1 cell-1 are rejoined very slowly (10 to 15 hour repair half-time). Both the slow- and fast-rejoining DSBs make a substantial contribution to the radiation killing of CHO cells. Although the TLK model provides a much more satisfactory formalism to relate biochemical processing of the DSB to cell killing than earlier kinetic models, some small differences among the measured and predicted CHO survival and DSB rejoining data suggest that additional factors and processes not considered in the present work may affect biochemical processing of the DSB and, hence, cell killing.« less
  • Purpose: To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. Methods and Materials: We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Results: Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early ({<=}4 h) after exposure to 8 Gy of {gamma}-radiation and also exhibitedmore » high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX ({gamma}-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Conclusion: Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and {gamma}-H2AX assays. Our data suggest that the {gamma}-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.« less
  • The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killings. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), andmore » 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy{sup -1} of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval=0-1.4%), but are 12.5% ({plus_minus}2.3%) and 43.8% ({plus_minus}1.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosomes breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand. 74 refs., 6 figs., 4 tabs.« less