DNA Double-strand Break Measurement in Mammalian Cells by Pulsed-field Gel Electrophoresis: An Approach Using Restriction Enzymes and Gene Probing

Löbrich, Markus; Ikpeme, S.; Kiefer, Jürgen

doi:10.1080/09553009414550731

Cited by 39 publications

(15 citation statements)

References 20 publications

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“…DSBs are 20 times less frequent than SSBs and difficult to measure at "physiologic" radiation doses [5,14]. Even at high doses, an exclusively linear dose response of DNA fragmentation in different cell lines has been described [21,34], but also nonlinear results have been reported [29]. Our data are in good agreement with the linear-quadratic model [3], already well established for other biological endpoints, such as colony formation, population growth or the frequency of chromosome aberrations.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The Influence of Bromodeoxyuridine on the Induction and Repair of DNA Double-Strand Breaks in Glioblastoma Cells

Nusser

Bartkowiak

Röttinger

2002

Strahlentherapie und Onkologie

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The linear-quadratic model appropriately describes the X-ray-induced fragmentation of DNA. BrdU sensitizing acts predominantly by increasing DNA fragility, and not by impairing damage repair. The amount of DSBs persistent after 24 h of repair is minimal, even after highly cytotoxic doses. However, it appears to depend on the extent of initial damage, causing sensitized cells to retain more DSBs than unsensitized cells.

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Section: Discussionmentioning

confidence: 99%

“…Of these types of damage, DSBs are the major cause of cell death [21]. DSBs may be induced directly or by the combination of SSBs, which are located in close vicinity [3,35].…”

Section: Introductionmentioning

confidence: 99%

The Influence of Bromodeoxyuridine on the Induction and Repair of DNA Double-Strand Breaks in Glioblastoma Cells

Nusser

Bartkowiak

Röttinger

2002

Strahlentherapie und Onkologie

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“…The samples were collected at −20°C, again permeabilised in 0.25% Triton X-100, blocked by 5% Goat serum and labelled for their H2AX phosphorylation using the monoclonal Anti-phospho-Histone H2A.X (Ser139) antibody, clone JBW301 (UPSTATE, Lake Placid, USA). Fluorescence labelling took place by using an Alexa Fluor 488 conjugated goat anti-mouse antibody (Invitrogen, Karlsruhe, Germany) and fluorescence intensity was measured on a flow cytometer [16] (München: BD LSR II, Becton Dickinson Biosciences, Heidelberg, Germany; Jena: FACS Calibur, Becton Dickinson Biosciences, Heidelberg, Germany). For statistical analysis the median of fluorescence at 530 nm (530/30 BP) was used to calculate the relative H2AX phosphorylation.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Different Biomarkers to Predict Individual Radiosensitivity in an Inter-Laboratory Comparison–Lessons for Future Studies

Greve

Bölling

Amler³

et al. 2012

PLoS ONE

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Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.

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“…The use of rarely cutting restriction enzymes and probing of individual restriction fragments bypasses these complications and allows direct quantification of DSB yields in specific regions of the genome [32,33]. However, as both these PFGE approaches measure the electrophoretic migration of DNA fragments in the size range of hundreds to thousands of kilo base pairs (to achieve the highest assay sensitivity), they fail to detect clustered DSB which produce smaller fragments and therefore underestimate the total yield of DSB, especially for densely ionising radiations.…”

Section: Introductionmentioning

confidence: 99%

The shape of the radiation dose response for DNA double-strand break induction and repair

Barnard¹,

Bouffler²,

Rothkamm³

2013

Genome Integrity

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DNA double-strand breaks are among the most deleterious lesions induced by ionising radiation. A range of inter-connected cellular response mechanisms has evolved to enable their efficient repair and thus protect the cell from the harmful consequences of un- or mis-repaired breaks which may include early effects such as cell killing and associated acute toxicities and late effects such as cancer. A number of studies suggest that the induction and repair of double-strand breaks may not always occur linearly with ionising radiation dose. Here we have aimed to identify and discuss some of the biological and methodological factors that can potentially modify the shape of the dose response curve obtained for these endpoints using the most common assays for double-strand breaks, pulsed-field gel electrophoresis and microscopic scoring of radiation-induced foci.

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DNA Double-strand Break Measurement in Mammalian Cells by Pulsed-field Gel Electrophoresis: An Approach Using Restriction Enzymes and Gene Probing

Cited by 39 publications

References 20 publications

The Influence of Bromodeoxyuridine on the Induction and Repair of DNA Double-Strand Breaks in Glioblastoma Cells

The Influence of Bromodeoxyuridine on the Induction and Repair of DNA Double-Strand Breaks in Glioblastoma Cells

Evaluation of Different Biomarkers to Predict Individual Radiosensitivity in an Inter-Laboratory Comparison–Lessons for Future Studies

The shape of the radiation dose response for DNA double-strand break induction and repair

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