Willers, H., Husson, J., Lee, L. W., Hubbe, P., Gazemeirer, F., Powell, S. N. and Dahm-Daphi, J. Distinct Mechanisms of Nonhomologous End Joining in the Repair of Site-Directed Chromosomal Breaks with Noncomplementary and Complementary Ends. Radiat. Res. 166, 567–574 (2006).

DNA double-strand breaks (DSBs) are considered the most important type of DNA damage inflicted by ionizing radiation. The molecular mechanisms of DSB repair by nonhomologous end joining (NHEJ) have not been well studied in live mammalian cells, due in part to the lack of suitable chromosomal repair assays. We previously introduced a novel plasmid-based assay to monitor NHEJ of site-directed chromosomal I-SceI breaks. In the current study, we expanded the analysis of chromosomal NHEJ products in murine fibroblasts to focus on the error-prone rejoining of DSBs with noncomplementary ends, which may serve as a model for radiation damage repair. We found that noncomplementary ends were efficiently repaired using microhomologies of 1–2 nucleotides (nt) present in the single-stranded overhangs, thereby keeping repair-associated end degradation to a minimum (2–3 nt). Microhomology-mediated end joining was disrupted by Wortmannin, a known inhibitor of DNA-PKcs. However, Wortmannin did not significantly impair the proficiency of end joining. In contrast to noncomplementary ends, the rejoining of cohesive ends showed only a minor dependence on microhomologies but produced fivefold larger deletions than the repair of noncomplementary ends. Together, these data suggest the presence of several distinct NHEJ mechanisms in live cells, which are characterized by the degree of sequence deletion and microhomology use. Our NHEJ assay should prove a useful system to further elucidate the genetic determinants and molecular mechanisms of site-directed DSBs in living cells.

Someya, M., Sakata, K., Tauchi, H., Matsumoto, Y., Nakamura, A., Komatsu, K. and Hareyama, M. Association of Ionizing Radiation-Induced Foci of NBS1 with Chromosomal Instability and Breast Cancer Susceptibility. Radiat. Res. 166, 575–582 (2006).

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.

Wu, H., Hada, M., Meador, J., Hu, X., Rusek, A. and Cucinotta, F. A. Induction of Micronuclei in Human Fibroblasts across the Bragg Curve of Energetic Heavy Ions. Radiat. Res. 166, 583–589 (2006).

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LΕ;Τ; γ or X rays, the presence of shielding does not always reduce the radiation risks for energetic charged-particle exposure. The dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak. However, the Bragg curve does not necessarily represent the biological damage along the particle path since biological effects are influenced by the track structures of both primary and secondary particles. Therefore, the “biological Bragg curve” is dependent on the energy and the type of the primary particle and may vary for different biological end points. Here we report measurements of the biological response across the Bragg curve in human fibroblasts exposed to energetic silicon and iron ions in vitro at two different energies, 300 MeV/nucleon and 1 GeV/nucleon. A quantitative biological response curve generated for micronuclei per binucleated cell across the Bragg curve did not reveal an increased yield of micronuclei at the location of the Bragg peak. However, the ratio of mono- to binucleated cells, which indicates inhibition of cell progression, increased at the Bragg peak location. These results confirm the hypothesis that severely damaged cells at the Bragg peak are more likely to go through reproductive death and not be evaluated for micronuclei.

Qutob, S. S., Multani, A. S., Pathak, S., McNamee, J. P., Bellier, P. V., Liu, Q. Y. and Ng, C. E. Fractionated X-Radiation Treatment can Elicit an Inducible-Like Radioprotective Response that is not Dependent on the Intrinsic Cellular X-Radiation Resistance/Sensitivity. Radiat. Res. 166, 590–599 (2006).

Inducible responses are well documented to play a role in the radiation response of cells. However, it is not known whether clinically relevant fractionated X-radiation treatment could elicit an inducible-like radioprotective response and whether there is a direct correlation between the inducible radiation response phenomenon and the intrinsic radiation response of the cell. Therefore, the purpose of this study was to determine whether closely related human colorectal tumor (HCT116) clones treated with fractionated X rays could elicit an inducible-like radiation response to a subsequent acute (i.e. single) X-ray challenge, and whether the magnitude of the inducible-like response correlates with the intrinsic X-ray resistance of the responding clones. After fractionated X irradiation, only the radiosensitive clone showed enhanced clonogenic survival with a subsequent acute X-ray exposure. Cell cycle changes or the selection of subclones with increased intrinsic radiation resistance induced by the fractionated X rays were excluded as the basis of this enhanced tolerance, suggesting the presence of an inducible-like radioprotective response. Using the comet assay, we found similar amounts of intrinsic DNA damage among the clones after acute X irradiation. Our findings demonstrate that fractionated X-ray treatment can elicit an inducible-like radioprotective response and represent the first evidence that this response is independent of the intrinsic radiation resistance/sensitivity of the responding cells.

Amine, A., Vozenin-Brotons, M-C., Abdulkarim, B., Violot, D., Aubel, C. and Bourhis, J. Cidofovir Administered with Radiation Displays an Antiangiogenic Effect Mediated by E6 Inhibition and Subsequent TP53-Dependent VEGF Repression in HPV18 Cell Lines. Radiat. Res. 166, 600–610 (2006).

Therapeutic administration of the antiviral agent cidofovir with radiation markedly enhanced the antitumor effect of ionizing radiation in cells of two HPV18 human cervical carcinoma cell lines. Although this potent radiosensitizing effect was associated with repression of the viral oncoproteins E6/ E7 and restoration of TP53 as shown previously, additional mechanisms may be involved. In the present study, we investigated the antiangiogenic effect of the combination of cidofovir and radiation in cells of two HPV18 cervical cancer cell lines, HeLa and ME180, and assessed the molecular mechanisms associated with the antiangiogenic effect observed. Cells were exposed to cidofovir (10 μg/ml) and irradiated (1– 9 Gy). The angiogenic response was studied in vitro by a matrigel invasion assay. Modulations of E6, TP53 and VEGF mRNA and protein levels were studied by real-time RT-PCR, Western blot analysis and ELISA, respectively. Then a double RNA interference approach was used to analyze the connection between E6/TP53 and VEGF. The combination of cidofovir and radiation had a potent antiangiogenic effect. It induced E6 inhibition, restoration of TP53, and reduction of the proangiogenic phenotype of HPV18 cells associated with VEGF inhibition. A siRNA strategy showed an anti-VEGF action of the combination mediated directly by E6 inhibition and TP53 restoration, since E6 siRNA inhibited VEGF whereas co-transfection with E6 and TP53 siRNA abrogated the anti-VEGF effect. This study showed that the combination of cidofovir with ionizing radiation has an antiangiogenic effect associated with VEGF inhibition subsequent to E6 inhibition and TP53 restoration.

O'Dowd, C., Mothersill, C. E., Cairns, M. T., Austin, B., McClean, B., Lyng, F. M. and Murphy, J. E. J. The Release of Bystander Factor(s) from Tissue Explant Cultures of Rainbow Trout (Onchorhynchus mykiss) after Exposure to γ Radiation. Radiat. Res. 166, 611–617 (2006).

The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing γ radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy γ radiation from a ⁶⁰Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to γ-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

Ronckers, C. M., Sigurdson, A. J., Stovall, M., Smith, S. A., Mertens, A. C., Liu, Y., Hammond, S., Land, C. E., Neglia, J. P., Donaldson, S. S., Meadows, A. T., Sklar, C. A., Robison, L. L. and Inskip, P. D. Thyroid Cancer in Childhood Cancer Survivors: A Detailed Evaluation of Radiation Dose Response and its Modifiers. Radiat. Res. 166, 618–628 (2006).

Radiation exposure at a young age is a strong risk factor for thyroid cancer. We conducted a nested case-control study of 69 thyroid cancer cases and 265 controls from a cohort of 14,054 childhood cancer survivors to evaluate the shape of the radiation dose–response relationship, in particular at high doses, and to assess modification of the radiation effects by patient and treatment characteristics. We considered several types of statistical models to estimate the excess relative risk (ERR), mainly guided by radiobiological models. A two-parameter model with a term linear in dose and a negative exponential in dose squared provided the best parsimonious description with an ERR of 1.3 per gray (95% confidence interval 0.4–4.1) at doses below 6 Gy and a relative decrease in ERR of 0.2% per unit dose squared with increasing dose, that is, decreases in the ERR/Gy of 53% at 20 Gy and 95% at 40 Gy. Further analyses using spline models suggested that the significant nonlinearity at high doses was characterized most appropriately as a true downturn rather than a flattening of the dose–response curve. We found no statistically significant modification of the dose–response relationship by patient characteristics; however, the linear parameter (i.e., the ERR/ Gy at doses less than 6 Gy) did decrease consistently and linearly with increasing age at childhood cancer diagnosis, from 4.45 for 0–1-year-olds to 0.48 for 15–20-year-olds. In summary, we applied models derived from radiobiology to describe the radiation dose–response curve for thyroid cancer in an epidemiological study and found convincing evidence for a downturn in risk at high doses.

Kase, Y., Kanai, T., Matsumoto, Y., Furusawa, Y., Okamoto, H., Asaba, T., Sakama, M. and Shinoda, H. Microdosimetric Measurements and Estimation of Human Cell Survival for Heavy-Ion Beams. Radiat. Res. 166, 629–638 (2006).

The microdosimetric spectra for high-energy beams of photons and proton, helium, carbon, neon, silicon and iron ions (LET = 0.5–880 keV/μm) were measured with a spherical-walled tissue-equivalent proportional counter at various depths in a plastic phantom. Survival curves for human tumor cells were also obtained under the same conditions. Then the survival curves were compared with those estimated by a microdosimetric model based on the spectra and the biological parameters for each cell line. The estimated α terms of the liner-quadratic model with a fixed β value reproduced the experimental results for cell irradiation for ion beams with LETs of less than 450 keV/μm, except in the region near the distal peak.

Shi, W-Q., Dong, J-C., Zhao, Y-F. and Li, Y-M. Hydroxylation of 3-Nitrotyrosine and Its Derivatives by Gamma Irradiation. Radiat. Res. 166, 639–645 (2006).

Radiation-induced hydroxylation of 3-nitrotyrosine (3-NY) and its derivatives in aqueous solution were investigated as a function of γ-radiation dose. Irradiated 3-NY, 3-nitrotyrosine ethyl ester (3-NYE) and 3-NY containing peptide Gly-nitroTyr-Gly were separated and analyzed with reverse-phase HPLC and UV-Vis absorption spectroscopy. The structures of the hydroxylated products were confirmed by electrospray ionization mass spectrometry and ¹H-NMR spectrometry. The amounts of the hydroxylated products in irradiated 3-NY and Gly-nitroTyr-Gly solutions increased with increasing radiation dose. Tandem electrospray ionization mass spectrometry (ESI-Mass²) was performed to investigate the hydroxylation of peptide Gly-nitroTyr-Gly. These studies showed that the hydroxylation occurred at 3-NY residue. We also found that the identification of 3-NY hydroxylation in peptide could be identified by ion scanning for the specific immonium ion at m/z 197.0.

Gibson, S. L., Bindra, R. S. and Glazer, P. M. CHK2-Dependent Phosphorylation of BRCA1 in Hypoxia. Radiat. Res. 166, 646–651 (2006).

Hypoxia induces a diverse spectrum of changes in the expression and activity of numerous DNA repair factors within the tumor microenvironment. In particular, we and others have shown that hypoxia induces phosphorylation and activation of the checkpoint kinase, CHK2, in an ATM-dependent manner. One downstream target of CHK2, the BRCA1 protein, plays a critical role in both DNA repair and cell cycle checkpoint regulation in mammalian cells. Here we report that BRCA1 is specifically phosphorylated on Serine 988 in response to hypoxic stress, and phosphorylation at this site is dependent on CHK2 expression. These findings enhance our understanding of ATM-CHK2 pathway activation in hypoxia, and they identify a novel role for BRCA1 in the response to hypoxic stress.