Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays

MacPhail, Susan H.; Banáth, Judit P.; Takata, Yasuyuki; Chu, Eric; Lambur, H; Olive, Peggy L.

doi:10.1080/0955300032000093128

Cited by 299 publications

(248 citation statements)

References 11 publications

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“…Recently, γH2AX foci analysis has become a widely used tool to quantify DSBs on a single cell level after IR (2). γH2AX foci numbers of 30-35 per Gy per G1 cell have been reported (3)(4)(5)(6)(7)(8) suggesting that there is a 1:1 correlation between γH2AX foci and DSBs after IR of nonreplicating cells. However, the precise DSB induction yields following irradiation can depend on a number of factors (see, eg, (9)) and, we show here, are critically dependent upon the surface upon which the cells are grown and irradiated.…”

Section: Dna Double-strand Breaks (Dsbs) Represent Important Lesions mentioning

confidence: 99%

X-irradiation of cells on glass slides has a dose doubling impact

et al. 2007

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Immunofluorescence detection of γH2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks induced by ionising radiation. We observed that Xirradiation of mammalian cells exposed on glass slides induced 2-fold higher foci numbers compared to irradiation with γ-rays. Here, we show that the excess γH2AX foci after Xirradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120 kV X-rays and 137 Cs γ-rays induce ~20 γH2AX foci per Gy in cells growing on thin (~2 µm) plastic foils immersed in water. The same yield is obtained following γ-irradiation of cells growing on glass slides. However, 120 kV X-rays produce ~40 γH2AX foci per Gy in cells growing on glass, two-fold greater than obtained using cells irradiated on plastic surfaces. The same increase in γH2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess γH2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 kV relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X-or γ-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner.3

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Section: Dna Double-strand Breaks (Dsbs) Represent Important Lesions mentioning

confidence: 99%

X-irradiation of cells on glass slides has a dose doubling impact

et al. 2007

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“…Ectopic expression of TPD52 downregulated ATMmediated DNA repair signaling Since γH2AX foci are early markers of DSB sites undergoing DNA damage repair, 4,6 and the rate of γH2AX clearance represents an important factor associated with radiosensitivity, 27 we examined the effects of HA-TPD52 transfection on γH2AX focus formation. In vector-transfected SK-BR-3 cells, the intensity ( Fig.…”

Section: Overexpression Of Tpd52 Compromised Dna Repair Capacity In Smentioning

confidence: 99%

Tumor protein D52 represents a negative regulator of ATM protein levels

et al. 2013

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“…To test the suitability of our method for evaluation of DNA damage levels that are below the limits of reliable detection for both the physical methods and flow cytometry approaches, we first examined the effects of ionizing radiation (IR), taking advantage of the known linear response of mammalian cells to increasing radiation doses 9,17,36 and the possibility to expose the cells to precise doses of the clastogenic insult. In principle, however, virtually any type of DNA damaging agent can be used, as the readout we used, the induction of g-H2AX visualized by antibodies to ser139-phosphorylated H2AX, is a universal marker for DDR created by all three major apical DDR kinases.…”

Section: Resultsmentioning

confidence: 99%

“…[4][5][6][7][8]11,14 In practical terms, the so-called g-H2AX foci can be conveniently identified by immunostaining, and this has become a popular surrogate marker to highlight the presence of DSBs. [6][7][8][9][10][11]13,[15][16][17][18][19][20][21][22] Apart from g-H2AX, numerous additional proteins that participate in the multifaceted genome maintenance network form subnuclear 'foci' through recruitment to, and accumulation at, the DNA damage sites. [6][7][8]15,[23][24][25] Such foci, for example those formed by the MRN complex proteins, 53BP1, MDC1, 5,7,[23][24][25][26] and the components of the recently identified ubiquitin ligase cascade including RNF8, 27-29 RNF168, [30][31][32] and also BRCA1, 30 are often closely overlapping with the relatively large chromatin regions marked by g-H2AX.…”

Section: Introductionmentioning

confidence: 99%

Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

Mistrík

Oplustilova²,

Lukáš³

et al. 2009

Cell Cycle

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Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused by environmental or metabolic genotoxic insults is critical for contemporary biomedicine. The available physical, flow cytometry and sophisticated scanning approaches to DNA damage estimation each have some drawbacks such as insufficient sensitivity, limitation to analysis of cells in suspension, or high costs and demand for trained personnel. Here we present an option how to transform a regular fluorescence microscope and personal computer with common software into a functional alternative to high-throughput screening devices. In two detailed protocols we introduce a new semi-automatic procedure allowing for very sensitive, quantitative, rapid and simple fluorescence image analysis in thousands of adherent cells per day. Sensitive DNA breakage estimation through analysis of phosphorylated histone H2AX (g-H2AX), and homologous recombination (HR) assessed by a new RPA/Rad51 dual-marker approach illustrate the advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document the versatility of the method. We believe this affordable approach may facilitate mechanistic insights into the role of low-dose DNA damage in human diseases, and generally promote both basic and translational research in many areas of biomedicine where suitable fluorescence markers are available.

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Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays

Abstract: GammaH2AX can be detected with excellent sensitivity using both flow and image analysis. The rate of gammaH2AX loss may be an important factor in the response of cells to ionizing radiation, with more rapid loss and less retention associated with more radioresistant cell lines.

Cited by 299 publications

References 11 publications

X-irradiation of cells on glass slides has a dose doubling impact

X-irradiation of cells on glass slides has a dose doubling impact

Tumor protein D52 represents a negative regulator of ATM protein levels

Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

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