Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue.

Charged particles are increasingly used in cancer radiotherapy and contribute significantly to the natural radiation risk. The difference in the biological effects of high-energy charged particles compared with X-rays or γ-rays is determined largely by the spatial distribution of their energy deposition events. Part of the energy is deposited in a densely ionizing manner in the inner part of the track, with the remainder spread out more sparsely over the outer track region. Our knowledge about the dose distribution is derived solely from modeling approaches and physical measurements in inorganic material. Here we exploited the exceptional sensitivity of γH2AX foci technology and quantified the spatial distribution of DNA lesions induced by charged particles in a mouse model tissue. We observed that charged particles damage tissue nonhomogenously, with single cells receiving high doses and many other cells exposed to isolated damage resulting from high-energy secondary electrons. Using calibration experiments, we transformed the 3D lesion distribution into a dose distribution and compared it with predictions from modeling approaches. We obtained a radial dose distribution with sub-micrometer resolution that decreased with increasing distance to the particle path following a 1/r2 dependency. The analysis further revealed the existence of a background dose at larger distances from the particle path arising from overlapping dose deposition events from independent particles. Our study provides, to our knowledge, the first quantification of the spatial dose distribution of charged particles in biologically relevant material, and will serve as a benchmark for biophysical models that predict the biological effects of these particles.

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Opposing roles for 53BP1 during homologous recombination.

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2.

Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of approximately 15% of X- or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin.

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ∼ 20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces.

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

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 gamma-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of gamma-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 microM H(2)O(2) efficiently remove all gamma-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 microM 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 gamma-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient gamma-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.

Differential effects of irradiation with carbon ions and x-rays on macrophage function.

Macrophages are potent elicitors of inflammatory reactions that can play both positive and negative roles in radiotherapy. While several studies have investigated the effects of X-rays or gamma-rays on macrophages, virtually no work has been done on the responses of these cells to irradiation with carbon ions. Investigations into the effects of carbon ion irradiation are of particular interest in light of the fact that this type of radiation is being used increasingly for cancer therapy. In the present investigation we compared the effects of 250 kV X-rays with those of 9.8 MeV/u carbon ions on RAW 264.7 macrophages over a wide range of radiation doses. Macrophage functions including vitality, phagocytic activity, production of the proinflammatory cytokines IL-1beta and TNFalpha and production of nitric oxide (NO) were measured. In comparison to lymphocytes and fibroblasts, macrophages showed only a small decrease in vitality after irradiation with either X-rays or carbon ions. Proinflammatory cytokines and NO were induced in macrophages by LPS but not by irradiation alone. X-rays or carbon ions had little modulating effect on LPS-induced TNFalpha production. However, LPS-induced NO increased in a dose dependent manner up to 6-fold after carbon ion irradiation, while X-ray irradiation did not have this effect. Carbon ion irradiation mediated a concomitant decrease in IL-1beta production. Carbon ions also had a greater effect than X-rays in enhancing the phagocytic activity of macrophages. These results underscore the greater potential of carbon ion irradiation with regard to radiobiological effectiveness.

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1.

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

Sister chromatid exchanges occur in G2-irradiated cells.

DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or mis-repaired DSBs can lead to chromosomal aberrations and cell death. The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. Following S-phase-dependent genotoxins, HR events give rise to sister chromatid exchanges (SCEs), which can be detected cytogenetically in mitosis. Here, we describe that HR occurring in G2-irradiated cells also generates SCEs in ~50% of HR events. Since HR of IR-induced DSBs in G2 is a slow process, SCE formation in G2-irradiated cells requires several hours. During this time, irradiated S-phase cells can also reach mitosis, which has contributed to the widely held belief that SCEs form only during S phase. We describe procedures to measure SCEs exclusively in G2-irradiated cells and provide evidence that following IR cells do not need to progress through S phase in order to form SCEs.