Measurement of Radiation-Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis

Ager, D.D.; Dewey, William C.; Gardiner, Katheleen; Harvey, W. F.; Johnson, Robert T.; Waldren, C

doi:10.2307/3577604

Cited by 104 publications

(20 citation statements)

References 20 publications

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“…Several sensitive methods have been used to measure DSBs in mammalian cells, including low-speed sucrose gradient centrifugation (41), neutral elution (42) and pulsedfield gel electrophoresis (34,37,43). These methods generally involve lysis of the cells and isolation of DNA under conditions that will melt out short stretches of DNA between staggered single-strand breaks.…”

Section: Discussionmentioning

confidence: 99%

“…The slices were transferred to individual liquid scintillation bottles containing 100 ml 1 M HC1. The sealed bottles were then immersed in a boiling water bath for 10 min to melt and hydrolyze the agarose (34). Five millititers Opti-Fluor (Packard) was added and the 14C radioactivity was counted in a Tri-Carb (Packard) liquid scintillation analyzer.…”

Section: Methodsmentioning

confidence: 99%

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DNA Double-Strand Breaks Induced by High-Energy Neon and Iron Ions in Human Fibroblasts. I. Pulsed-Field Gel Electrophoresis Method

Cooper¹,

Rydberg²,

Löbrich³

1994

Radiation Research

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The relative effectiveness of high-energy neon and iron ions for the production of DNA double-strand breaks was measured in one transformed and one nontransformed human fibroblast cell line using pulsed-field gel electrophoresis. The DNA released from the gel plug (fraction of activity released: FAR) as well as the size distribution of the DNA entering the gel were used to compare the effects of the heavy-ion exposure with X-ray exposure. Both methods gave similar results, indicating similar distributions of breaks over megabase-pair distances for the heavy ions and the X rays. The relative biological effectiveness (RBE) compared to 225 kVp X rays of initially induced DNA double-strand breaks was found to be 0.85 for 425 MeV/u neon ions (LET 32 keV/microns) and 0.42-0.55 for 250-600 MeV/u iron ions (LET 190-350 keV/microns). Postirradiation incubation showed less efficient repair of breaks induced by the neon ions and the 600 MeV/u iron ions compared to X rays. Survival experiments demonstrated RBE values larger than one for cell killing by the heavy ions in parallel experiments (neon: RBE = 1.2, iron: RBE = 2.3-3.0, based on D10 values). It is concluded that either the initial yield of DNA double-strand breaks induced by the high-energy particles is lower than the yield for X rays, or the breaks induced by heavy ions are present in clusters that cannot be resolved with the technique used. These results are confirmed in the accompanying paper (M. Löbrich, B. Rydberg and P. Cooper, Radiat. Res. 139, 142-151, 1994).

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

DNA Double-Strand Breaks Induced by High-Energy Neon and Iron Ions in Human Fibroblasts. I. Pulsed-Field Gel Electrophoresis Method

Cooper¹,

Rydberg²,

Löbrich³

1994

Radiation Research

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“… a Indicates the nominal number of DSBs recommended in the reference [13, 44]. The error of k value and α 0 value were estimated by propagation of error from β value and α value.…”

Section: Resultsmentioning

confidence: 99%

“…In general, the k estimated by the MK model fits well the number of γ-H2AX foci ( k exp ). The nominal number of DSBs for Chinese hamster ovary cell lines was taken from the reference [12, 44], and we adopted 30 as the expected k exp with 60 Co γ-rays for CHO-K1 cells. As is shown in Table 2, the number of DSBs ( k ) increases with the dose mean lineal energy y D [keV/μm], which suggests that k depends on the energy of photon beams.…”

Section: Resultsmentioning

confidence: 99%

Quantitative estimation of DNA damage by photon irradiation based on the microdosimetric-kinetic model

Matsuya

Ohtsubo

Tsutsumi

et al. 2014

Journal of Radiation Research

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The microdosimetric-kinetic (MK) model is one of the models that can describe the fraction of cells surviving after exposure to ionizing radiation. In the MK model, there are specific parameters, k and yD, where k is an inherent parameter to represent the number of potentially lethal lesions (PLLs) and yD indicates the dose-mean lineal energy in keV/μm. Assuming the PLLs to be DNA double-strand breaks (DSBs), the rate equations are derived for evaluating the DSB number in the cell nucleus. In this study, we estimated the ratio of DSBs for two types of photon irradiation (6 MV and 200 kVp X-rays) in Chinese hamster ovary (CHO-K1) cells and human non-small cell lung cancer (H1299) cells by observing the surviving fraction. The estimated ratio was then compared with the ratio of γ-H2AX foci using immunofluorescent staining. For making a comparison of the number of DSBs among a variety of radiation energy cases, we next utilized the survival data in the literature for both cells exposed to other photon types, such as 60Co γ-rays, 137Cs γ-rays and 100 kVp X-rays. The ratio of DSBs based on the MK model with conventional data was consistent with the ratio of γ-H2AX foci numbers, confirming that the γ-H2AX focus is indicative of DSBs. It was also shown that the larger yD is, the larger the DSB number is. These results suggest that k and yD represent the characteristics of the surviving fraction and the biological effects for photon irradiation.

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“…It may be indicative that differences of this magnitude are not present in pulsed-field gel electrophoresis, where exposure to 50 Gy results in a release from the well of approximately 50% of the loaded DNA in experiments carried out by different investigators using widely different pulsing schedules and electrode arrangements as well as different cell lines (e.g. Stamato and Denko 1990, Ager et al . 1990, Waters and Lyons 1990, Iliakis et al 1991 .…”

Section: Discussionmentioning

confidence: 95%

The Shape of DNA Elution Dose-response Curves Under Non-denaturing Conditions: The Contribution of the Degree of Chromatin Condensation

Okayasu

Iliakis

1992

International Journal of Radiation Biology

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We have re-examined the effect of detergent type, pH and temperature of lysis on the shape of the DNA elution dose response under non-denaturing conditions using plateau-phase CHO cells. Results practically identical to those previously published (Okayasu and Iliakis, 1989) were obtained, with a 1 h incubation at 60 degrees C during lysis with sodium-N-laurylsarcosine (NLS) resulting in almost linear dose-response curves. We also examined chromatin decondensation as a contributing factor in the linearization observed in the elution dose-response curve under the above conditions. When nuclei with condensed chromatin were prepared from irradiated cells, applied on the filter and lysed with NLS at room temperature, a shoulder-type elution dose-response curve was obtained only slightly higher than that of cells lysed under the same conditions. However, when nuclei prepared from irradiated cells were applied on the filter after relaxation of chromatin by incubation in low ionic strength buffer and lysed with NLS at room temperature, an almost linear dose-response curve was obtained similar to that of cells lysed with NLS at 60 degrees C. Lysis with NLS at 60 degrees C of nuclei with relaxed chromatin did not further modify the DNA elution dose-response curve. Based on these results we propose that the linearization of the DNA elution dose-response curve observed after chromatin decondensation reflects a reduction in the degree of chromatin compactness in the nuclear complexes that leads to a relatively uniform distribution of the DNA on the filter and reduces trapping of elutable material in the compact nuclear structures otherwise present. Since high radiation doses dissolve compact nuclear structures, trapping of elutable material is expected to be highest at low doses of radiation, leading to the observed reduction in the fraction of DNA eluted per Gy at low versus high radiation doses and thus to the observed shoulder. Furthermore, we propose that the linearization of the DNA elution dose-response curves observed in cells lysed in NLS at 60 degrees C may also be due to a decondensation of the nuclear complexes on the filter as a result of the combined action of detergent and high temperature. The notion of a correlation between DNA elution dose response and cell radiosensitivity was examined in two human (SQ20B, SCC61) and two Chinese hamster (V-79, irs-2) cell lines with widely different radiosensitivities.(ABSTRACT TRUNCATED AT 400 WORDS)

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Measurement of Radiation-Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis

Cited by 104 publications

References 20 publications

DNA Double-Strand Breaks Induced by High-Energy Neon and Iron Ions in Human Fibroblasts. I. Pulsed-Field Gel Electrophoresis Method

DNA Double-Strand Breaks Induced by High-Energy Neon and Iron Ions in Human Fibroblasts. I. Pulsed-Field Gel Electrophoresis Method

Quantitative estimation of DNA damage by photon irradiation based on the microdosimetric-kinetic model

The Shape of DNA Elution Dose-response Curves Under Non-denaturing Conditions: The Contribution of the Degree of Chromatin Condensation

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