| 1. |
- Jucha, Anna, et al.
(författare)
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FociCounter : A freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci
- 2010
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Ingår i: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 696:1, s. 16-20
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Tidskriftsartikel (refereegranskat)abstract
- Gamma-H2AX foci are sensitive and specific indicator for the induction of DNA double-strand breaks (DSBs) and an immunocytochemical assay with antibodies recognizing gamma-H2AX has become the gold standard for the detection of this type of DNA lesion. Quantification of gamma-H2AX foci can be achieved by various methods such as Western blotting, flow cytometry, visual analysis and computational analysis with a fluorescence microscope. The best sensitivity is achieved by computer analysis. Since no freeware programme for the analysis of gamma-H2AX foci exists for a PC platform, the aim of our study was to develop a simple and user-friendly public-domain software. The algorithm applied in our programme allows determination of the number of foci in a single cell, a focus intensity per cell, as well as a cell intensity. Its graphical user interface is based on a GTK+ library and the whole application can be run under a variety of operating systems, including MS Windows and Linux. The programme called FociCounter is publicly available at http://focicounter.sourceforge.net. Application of the programme was tested by analysing gamma-H2AX foci in CHO and MO59K cells irradiated in vitro with X-rays and validated by comparing the results obtained with the outcome of automated image analysis and flow cytometry.
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| 2. |
- Lisowska, Halina, et al.
(författare)
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The dose-response relationship for dicentric chromosomes and gamma-H2AX foci in human peripheral blood lymphocytes : Influence of temperature during exposure and intra- and inter-individual variability of donors
- 2013
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 89:3, s. 191-199
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Tidskriftsartikel (refereegranskat)abstract
- Purpose : Hypothermia during in vitro irradiation of human peripheral blood lymphocytes (PBL) affects the level of chromosome aberrations. The molecular mechanisms of this phenomenon are not fully understood. The aim of our study was to examine the effect of hypothermia on the dose-response relationship for dicentric chromosomes and the level of gamma-H2AX (phosphorylated histone H2AX) foci. In addition, the inter- and intra-individual variability was assessed in relation to temperature. Materials and methods : PBL were kept at 0.8, 20 and 37 degrees C and then exposed to gamma-rays (from 0-3 Gy). Dicentric chromosomes were scored in first post-treatment mitoses. gamma H2AX foci were scored 15, 30, 60, 120 min and 24 h post irradiation. Results : Our results revealed that the frequency of dicentric chromosomes in cells exposed at 37 degrees C to gamma-rays was higher than after exposure at 0.8 and 20 degrees C. No effect of temperature was observed on the number of gamma-H2AX foci as well as on the intra-and inter-individual variations of the dicentric yield and the number of gamma-H2AX foci. Conclusions : Temperature at exposure to ionizing radiation has a pronounced effect on the level of cytogenetic damage but not gamma-H2AX foci.
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| 3. |
- Padjas, Anna, et al.
(författare)
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Comparative analysis of three functional predictive assays in lymphocytes of patients with breast and gynaecological cancer treated by radiotherapy
- 2012
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Ingår i: Journal of Contemporary Brachytherapy. - : Termedia Sp. z.o.o.. - 1689-832X .- 2081-2841. ; 4:4, s. 219-226
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: There is a need to develop predictive tests that would allow identifying cancer patients with a high risk of developing side effects to radiotherapy. We compared the predictive value of three functional assays: the G(0) aberration assay, the G(2) aberration assay and the alkaline comet assay in lymphocytes of breast cancer and gynaecological cancer patients.Material and methods: Peripheral blood was collected from 35 patients with breast cancer and 34 patients with gynaecological cancer before the onset of therapy. Chromosomal aberrations were scored in lymphocytes irradiated in the G(0) or G(2) phase of the cell cycle. DNA repair kinetics was performed with the alkaline comet assay following irradiation of unstimulated lymphocytes. The results were compared with the severity of early and late side effects to radiotherapy.Results: No correlation was observed between the results of the assays and the severity of side effects. Moreover, each assay identified different patients as radiosensitive.Conclusions: There is no simple correlation between the in vitro sensitivity of lymphocytes and the risk of developing early and late side effects.
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| 4. |
- Cheng, Lei, et al.
(författare)
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Modulation of radiation-induced cytogenetic damage in human peripheral blood lymphocytes by hypothermia
- 2015
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 793:SI, s. 96-100
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Recent studies have shown that low temperature (hypothermia) at exposure can act in a radioprotective manner at the level of cytogenetic damage. The mechanisms of this phenomenon are not understood, but it was suggested to be due to hypothermia-induced perturbations of the cell cycle. The purpose of the present study was to detect whether a reduced frequency of micronuclei is observed in peripheral blood lymphocytes (PBL) irradiated at low temperature and harvested sequentially at 3 time points. Additionally, the level of apoptosis was estimated by microscopic analysis of the MN slides. Materials and methods: Experiments were carried out with blood drawn from three donors at the Stockholm University and from three donors at the Jan Kochanowski University. Prior to irradiation, blood samples were incubated for 20 mm and irradiated at the respective temperature (0 degrees C and 37 degrees C) with gamma rays. Whole blood cultures were set up, cytochalasin B was added after 44h of irradiation and the samples were harvested after 72,96 and 120 h of incubation time. Results and conclusions: The frequency of micronuclei was markedly lower in PBL harvested at 72h, 96 h and 120 h following irradiation at 0 degrees C as compared to 37 degrees C. This indicates that the temperature effect observed in peripheral blood lymphocytes after irradiation is not related to a temporary perturbation of the cell cycle. Also, it is not due to selective elimination of damaged cells by apoptosis.
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| 5. |
- Czub, Joanna, et al.
(författare)
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Biological effects of mixed-ion beams. Part 1 : Effect of irradiation of the CHO-K1 cells with a mixed-ion beam containing the carbon and oxygen ions
- 2018
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Ingår i: Applied Radiation and Isotopes. - : Elsevier BV. - 0969-8043 .- 1872-9800. ; 139, s. 304-309
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Tidskriftsartikel (refereegranskat)abstract
- Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found.
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| 6. |
- Czub, Joanna, et al.
(författare)
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Biological effects of mixed-ion beams. Part 2 : The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams
- 2019
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Ingår i: Applied Radiation and Isotopes. - : Elsevier BV. - 0969-8043 .- 1872-9800. ; 150, s. 192-198
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Tidskriftsartikel (refereegranskat)abstract
- The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236-300 and 461-470 keV/mu m for C-12 and O-16 ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (C-12) and 470 keV/mu m (O-16).
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| 7. |
- Kacprzak, Justyna, et al.
(författare)
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Individual variations in the micronucleus assay for biological dosimetry after high dose exposure
- 2013
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 756:1-2, s. 196-200
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Tidskriftsartikel (refereegranskat)abstract
- The micronucleus assay is widely used as a biological dosimeter. Due to an inhibitory effect of radiation on cell proliferation the assay yields satisfactory results only when the absorbed dose is below about 5 Gy. In 2002 Muller and Rode suggested that a modified version of the test, based on the analysis of the ratio of trinucleated to tetranucleated cells and the frequency of micronuclei (Mn) in binucleated cells containing at least one Mn, can be applied to detect a dose reaching 15 Gy (Mutat. Res. 502 (2002) 47-51). Their conclusion was based on the results of experiments with lymphocytes from one donor and nothing is known about the possible influence of individual variability on the applicability of the Mn test to detect high doses of radiation. The aim of the present study was to validate the modified micronucleus assay with lymphocytes of 5 donors. Their blood was exposed to 0, 5, 10, 15 and 20 Gy of Co-60 gamma rays. The levels of Mn and of cell proliferation were assessed using various approaches. A strong inter-individual variability was observed for all endpoints. The results clearly show that the assessment of cell proliferation is essential for the interpretation of results. Unfortunately, it was not possible to identify one single proliferation marker that gives all necessary information.
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| 8. |
- Lisowska, Halina, et al.
(författare)
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Effect of hypothermia on radiation-induced micronuclei and delay of cell cycle progression in TK6 cells
- 2014
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 90:4, s. 318-324
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Low temperature (hypothermia) during irradiation leads to a reduced frequency of micronuclei in TK6 cells and it has been suggested that perturbation of cell cycle progression is responsible for this effect. The aim of the study was to test this hypothesis. Materials and methods: Human lymphoblastoid TK6 cells were treated by a combination of hypothermia (0.8 degrees C) and ionizing radiation in varying order (hypothermia before, during or after irradiation) and micronuclei were scored. Growth assay and two-dimensional flow cytometry was used to analyze cell cycle kinetics following irradiated of cells at 0.8 degrees C or 37.0 degrees C. Results: The temperature effect was observed at the level of micronuclei regardless of whether cells were cooled during or immediately before or after the radiation exposure. No indication of cell cycle perturbation by combined exposure to hypothermia and radiation could be detected. Conclusions: The protective effect of hypothermia observed at the level of cytogenetic damage was not due to a modulation of cell cycle progression. A possible alternative mechanism and experiments to test it are discussed.
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| 9. |
- Lisowska, Halina, et al.
(författare)
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Hypothermia modulates the DNA damage response to ionizing radiation in human peripheral blood lymphocytes
- 2018
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 94:6, s. 551-557
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8 degrees C and 37 degrees C.Materials and methods: To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A.Results: A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8 degrees C as compared to 37 degrees C already after 30minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes.Conclusions: Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.
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| 10. |
- Płódowska, Magdalena, et al.
(författare)
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Hypothermia differentially modulates the formation and decay of NBS1, γH2AX and 53BP1 foci in U2OS cells exposed to gamma radiation
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage.
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