Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

Wilson, Paul F.; Nham, Peter B.; Urbin, Salustra S.; Hinz, John M.; Jones, Irene M.; Thompson, Larry H.

doi:10.1016/j.mrfmmm.2009.10.013

Cited by 39 publications

(34 citation statements)

References 50 publications

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“…For example, in normal human fibroblasts (1) and in hamster V79 cells (10), we observed a maximum of 18-24 γH2AX RIF∕Gy after exposure to less than 1 Gy of X-rays, compared to 13-15 γH2AX RIF∕Gy for 1-4 Gy. Similarly, human fibroblasts showed a slight decrease with averages ranging from 21 to 17 RIF∕Gy between 0.05 and 0.25 Gy, which was consistent across 18 independent lines (11).…”

supporting

confidence: 67%

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells

Neumaier

Swenson

Pham

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

246

195

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The concept of DNA "repair centers" and the meaning of radiationinduced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 μm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF∕Gy after 2 Gy exposure compared to approximately 64 RIF∕Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation. DNA damage-sensing proteins localize at sites of DNA double-strand breaks (DSBs) within seconds to minutes following ionizing radiation (IR) exposure, resulting in the formation of immunofluorescently stainable nuclear domains referred to as radiation-induced foci (RIF) (1-3). RIF numbers are routinely used to assess the amount of DNA damage and repair kinetics after different treatments (4). However, there is a controversy surrounding the question of whether there is a 1∶1 correspondence between RIF and DSBs. For example, pulse field gel electrophoresis (PFGE) analysis suggests that DSBs decay exponentially with time immediately after exposure (5). In contrast, DNA damage-sensing proteins do not instantaneously detect DSBs, leading to delayed kinetics for both detection and resolution. More specifically, the maximum number of 53BP1 or γH2AX RIF is not reached until 15 to 30 min after exposure, and the yield of DSBs predicted by RIF is typically lower than the expected 25-40 DSB∕Gy measured by PFGE (4).Dose response provides another assay for assessing the relationship between DSBs and RIF. Based on theoretical Monte Carlo simulations and PFGE measurements (6, 7), the frequency of DSBs should be highly correlated with radiation dose. Confirming this prediction, two research groups reported that RIF number is proportional to radiation dosage from 1 mGy to 2 Gy (8, 9). In both studies, methods were applied to identify "real" RIF at low doses, where frequencies may be close to background levels before IR (e.g., 10 mGy would lead to about 0.3 DSB∕cell). They either used cells with very low γH2AX background foci (i.e., 0.05 background foci∕cell in primary human l...

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supporting

confidence: 67%

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells

Neumaier

Swenson

Pham

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

246

195

View full text Add to dashboard Cite

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“…Second, differences in the efficiency of DSB repair for low vs. high doses were not observed in cells analyzed by live cell imaging (23). Third, most cell lines in culture exhibit high background foci levels, which make it difficult to analyze DSB repair after low doses in vitro (26). Thus, we refined our previously published methodological approach to investigate DSB repair after low radiation doses in various tissues of irradiated mice (34).…”

Section: Discussionmentioning

confidence: 99%

“…We and others have shown that foci arise in a 1:1 relationship to DSBs and that the kinetics of foci loss reflect repair of DSBs (16,(20)(21)(22). This technology has the sensitivity that is necessary to investigate milligray doses and can be applied to various cell types and tissue samples (4,5,(23)(24)(25)(26)(27).…”

mentioning

confidence: 99%

Inducible response required for repair of low-dose radiation damage in human fibroblasts

Grudzenski

Raths

Conrad

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

130

115

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Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double-strand breaks (DSBs) are the biologically most significant. It is currently unclear if DSB repair is equally efficient after low and high doses. Here, we use γ-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of γ-H2AX and pATM foci loss in confluent primary human fibroblasts are substantially compromised after doses of 10 mGy and lower. Following 2.5 mGy, cells fail to show any foci loss. Strikingly, cells pretreated with 10 μM H 2 O 2 efficiently remove all γ-H2AX foci induced by 10 mGy. At the concentration used, H 2 O 2 produces single-strand breaks and base damages via the generation of oxygen radicals but no DSBs. Moreover, 10 μM H 2 O 2 up-regulates a set of genes that is also up-regulated after high (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low radical levels induce a response that is required for the repair of radiation-induced DSBs when the radiation damage is too low to cause the induction itself. To address the in vivo significance of this finding, we established γ-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient γ-H2AX and 53BP1 foci removal during 24 h post-IR, barely any foci loss was observed after 10 mGy. Our data suggest that the cellular response to DSBs is substantially different for low vs. high radiation doses.

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“…Instead, while the intermediates responsible for ATM localization at DSBs are not yet well described, ATM has been shown to be activated through transautophosphorylationinduced dissociation of ATM-inactive dimers. 17,23 Active ATM is shown to be prominently involved in the phosphorylation of H2AX and spreading of DSB signaling at the distance of IRIF. 9,24,25 In turn, γH2AX, together with a factor called mediator of DNA checkpoint 1 (MDC1), was shown to be necessary for the recruitment of key elements involved in DSB signaling such as 53BP1, BRCA1, and p53 tumor suppressor protein.…”

Section: Dna Damage Forward Signalingmentioning

confidence: 99%

Linking JNK Activity to the DNA Damage Response

Picco

Pagès

2013

Genes & Cancer

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The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet-and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents.

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Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

Cited by 39 publications

References 50 publications

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells

Inducible response required for repair of low-dose radiation damage in human fibroblasts

Linking JNK Activity to the DNA Damage Response

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