Watson, C. N., Miller, D. A., Chin-Sinex, H., Losch, A. C., Hughes, W., Sweeney, C. and Mendonca, M. S. Suppression of NF-κB Activity by Parthenolide Induces X-Ray Sensitivity through Inhibition of Split-Dose Repair in TP53 Null Prostate Cancer Cells. Radiat. Res. 171, 389–396 (2009).

We have shown that parthenolide, a sesquiterpene lactone, is a radiation sensitizer for human CGL1 hybrid cells that have constitutively activated NF-κB and wild-type p53. Since many malignant cells have nonfunctional p53, we investigated whether parthenolide could alter the X-ray sensitivity of PC-3 prostate cancer cells, a p53 null cell line with constitutively activated NF-κB. The addition of 5 μM parthenolide induced non-apoptotic cell death, inhibited PC-3 proliferation, and increased the population doubling time from 23 ± 1 h to 49 ± 4 h. Parthenolide also inhibited constitutive and radiation-induced NF-κB binding activity and enhanced the X-ray sensitivity of these p53 null PC-3 cells by a dose modification factor of 1.7. Cell cycle analysis of PC-3 cells treated with parthenolide showed only small alterations in G₁ and G₂/M cells, and these appeared to be insufficient to explain the observed radiosensitization. Split-dose studies using clinically relevant 2- and 4-Gy fractions demonstrated that parthenolide completely inhibited split-dose repair in PC-3 cells. We hypothesized that inhibition of NF-κB activity by parthenolide was responsible for the observed X-ray sensitization and inhibition of split-dose repair. To test this hypothesis, we knocked down the expression of NF-κB p65 protein, an active component of NF-κB in both PC-3 and CGL1 cells, by siRNA. Inhibition of NF-κB activity by knockdown of p65 increased radiation sensitivity and completely inhibited split-dose repair in both cell lines in a nearly identical manner as parthenolide treatment alone. Treating p65-depleted PC-3 cells with 5 μM parthenolide did not further increase their radiation sensitivity or the inhibition of split-dose repair. We propose that the suppression of radiation-induced NF-κB activity by parthenolide leads to X-ray sensitization through inhibition of split-dose repair in p53 null PC-3 prostate cancer cells.

Mladenov, E., Kalev, P. and Anachkova, B. The Complexity of Double-Strand Break Ends is a Factor in the Repair Pathway Choice. Radiat. Res. 171, 397–404 (2009).

The repair of double-strand breaks in mammalian cells is carried out by two pathways: homologous recombination and nonhomologous end joining. The factors that regulate the mechanism through which a specific repair pathway is activated are still not clearly defined. To study whether the complexity of the double-strand break ends is a factor that determines the choice of the repair pathway, we examined the involvement of homologous recombination by the formation of Rad51 foci in human HeLa cells treated with bleomycin and ionizing radiation. The quantity of double-strand breaks was determined by gel electrophoresis and the formation of γ-H2AX foci. Two hours after treatment with low doses of the agents that induced similar quantities of double-strand breaks that could be repaired effectively by the cells, Rad51 foci were observed only in the irradiated cells. Rad51 foci appeared in bleomycin-treated cells after prolonged exposure to the drug when the cells were arrested in the G₂ phase of the cell cycle. Since bleomycin produces double-strand breaks that are less complex than the breaks induced by ionizing radiation, these results indicate that the complexity of the break ends is a factor in the choice of repair pathway and that homologous recombination is recruited in the repair of breaks with more complex multiply damaged ends during the late S and G₂ phases of the cell cycle.

Jakob, B., Splinter, J. and Taucher-Scholz, G. Positional Stability of Damaged Chromatin Domains along Radiation Tracks in Mammalian Cells. Radiat. Res. 171, 405–418 (2009).

Irradiation of cell nuclei with charged particles leads to the spatially defined production of DNA damage along the particle trajectories, thus facilitating studies on the dynamics of radiation-induced protein foci associated with lesion processing. Here we used visual inspection and computational analysis of the track morphology after immunodetection to describe the patterns of formation of γ-H2AX foci and the repair-related proteins 53BP1 and RPA. We addressed the influence of lesion density on γ-H2AX formation and the mobility of damaged chromatin sites by using low-angle irradiation of cell monolayers with low-energy carbon or uranium ions. We show the discrete formation of γ-H2AX foci and the recruitment of repair-related proteins along ion trajectories over an LET range from 200 to 14300 keV/μm in human fibroblasts and in HeLa cells. The marked DSBs exhibited a limited mobility that was independent of the LET. The moderate extent of mobility in human fibroblasts pointed to a relatively stable positioning of the damaged chromatin domains during repair, in contrast to HeLa cells, which showed significant changes in the streak patterns in a fraction of cells, suggesting greater mobility in the local processing of DSBs. Our data indicate that the presence of single or multiple DSBs is not associated with an altered potential for movement of damaged chromatin. We infer that the repair of high-LET radiation-induced DSBs in mammalian cells is not coupled to an increased motional activity of lesions enhancing the probability of translocations.

Solovjeva, L. V., Pleskach, N. M., Firsanov, D. V., Svetlova, M. P., Serikov, V. B. and Tomilin, N. V. Forskolin Decreases Phosphorylation of Histone H2AX in Human Cells Induced by Ionizing Radiation. Radiat. Res. 171, 419–424 (2009).

Forskolin is a natural compound found in the coleus herb that activates the enzyme adenylate cyclase and increases the concentration of intracellular cyclic AMP (cAMP). This chemical is widely used as a stimulating food additive. It is unknown whether forskolin can effect cellular responses to ionizing radiation, such as induction of phosphorylation of histone H2AX (γ-H2AX) in megabase chromatin domains near DNA double-strand breaks (DSBs). Here we report that treatment with forskolin decreases H2AX phosphorylation after irradiation detected by immunoblotting or by analysis of the overall γ-H2AX-associated fluorescence in the nuclei. However, this chemical does not affect the number of γ-H2AX foci, the frequency of radiation-induced chromosome aberrations, or cell survival after X irradiation, which is consistent with the view that it does not change the induction of repair of DSBs. We suggest that the overall decrease of H2AX phosphorylation after treatment with forskolin in irradiated cells reflects a lesser extent of apparent H2AX modification at individual DSBs that may be caused by inhibition of the initial spread of γ-H2AX and/or by stimulation of elimination of γ-H2AX from chromatin after DSB rejoining.

Duran, A., Barquinero, J. F., Caballín, M. R., Ribas, M. and Barrios, L. Persistence of Radiation-Induced Chromosome Aberrations in a Long-Term Cell Culture. Radiat. Res. 171, 425–437 (2009).

The aim of the present study was to evaluate the persistence of chromosome aberrations induced by X rays. FISH painting and mFISH techniques were applied to long-term cultures of irradiated cells. With painting, at 2 Gy the frequency of apparently simple translocations remained almost invariable during all the culture, whereas at 4 Gy a rapid decline was observed between the first and the second samples, followed by a slight decrease until the end of the culture. Apparently simple dicentrics and complex aberrations disappeared after the first sample at 2 and 4 Gy. By mFISH, at 2 Gy the frequency of complete plus one-way translocations remained invariable between the first and last sample, but at 4 Gy a 60% decline was observed. True incomplete simple translocations disappeared at 2 and 4 Gy, indicating that incompleteness could be a factor to consider when the persistence of translocations is analyzed. The analysis by mFISH showed that the frequency of complex aberrations and their complexity increased with dose and tended to disappear in the last sample. Our results indicate that the influence of dose on the decrease in the frequency of simple translocations with time postirradiation cannot be fully explained by the disappearance of true incomplete translocations and complex aberrations. The chromosome involvement was random for radiation-induced exchange aberrations and non-random for total aberrations. Chromosome 7 showed the highest deviations from expected, being less and more involved than expected in the first and last samples, respectively. Some preferential chromosome-chromosome associations were observed, including a coincidence with a cluster from radiogenic chromosome aberrations described in other studies.

Campa, A., Alloni, D., Antonelli, F., Ballarini, F., Belli, M., Dini, V., Esposito, G., Facoetti, A., Friedland, W., Furusawa, Y., Liotta, M., Ottolenghi, A., Paretzke, H. G., Simone, G., Sorrentino, E. and Tabocchini, M. A. DNA Fragmentation Induced in Human Fibroblasts by ⁵⁶Fe Ions: Experimental Data and Monte Carlo Simulations. Radiat. Res. 171, 438–445 (2009).

We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1–5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1–23 kbp and 23–5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1–5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since γ rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

Pan, Y., Yuan, D., Zhang, J., Xu, P., Chen, H. and Shao, C. Cadmium-Induced Adaptive Response in Cells of Chinese Hamster Ovary Cell Lines with Varying DNA Repair Capacity. Radiat. Res. 171, 446–453 (2009).

The combined exposure to environmental toxicants such as heavy metals and radiation is an important research area in health protection. The aim of this study was to investigate the role of DNA repair and the phosphatidylinositol 3-kinase (PI3K) family in the cadmium-induced adaptive response to toxicants or radiation. Using cells of three Chinese hamster ovary (CHO) cell lines with different capacities to repair DNA damage, we found that pre-exposure to cadmium at a nonlethal concentration could induce an adaptive response to a subsequent challenge with cadmium or radiation in all the cell lines. The magnitude of the adaptive response in adapted cells was dependent on several factors, including DNA repair capacity, the priming dose of cadmium, and the challenging dose of cadmium or radiation. When the cells were challenged with 50 μM CdCl₂, the adaptive response was less evident in XRCC1-defective EM-C11 cells than in cells of the other two cell lines. Moreover, treatment of cells with wortmannin or KU-55933 eliminated the adaptive response in all the cell lines. Our data suggest that the triggering of a cadmium-induced adaptive response was independent of DNA repair capacity. Single-strand break (SSB) repair or base excision repair (BER) rather than double-strand break (DSB) repair was mainly involved in the adaptive response. This response of cells to a further challenge with cadmium or radiation may be mediated through the ataxia telangiectasia mutated (ATM) pathway.

Scuric, Z., Chan, C. Y., Hafer, K. and Schiestl, R. H. Ionizing Radiation Induces Microhomology-Mediated End Joining in trans in Yeast and Mammalian Cells. Radiat. Res. 171, 454–463 (2009).

DNA double-strand breaks repaired through nonhomologous end joining require no extended sequence homology as a template for the repair. A subset of end-joining events, termed microhomology-mediated end joining, occur between a few base pairs of homology, and such pathways have been implicated in different human cancers and genetic diseases. Here we investigated the effect of exposure of yeast and mammalian cells to ionizing radiation on the frequency and mechanism of rejoining of transfected unirradiated linear plasmid DNA. Cells were exposed to γ radiation prior to plasmid transfection; subsequently the rejoined plasmids were recovered and the junction sequences were analyzed. In irradiated yeast cells, 68% of recovered plasmids contained microhomologies, compared to only 30% from unirradiated cells. Among them 57% of events used ≥4 bp of microhomology compared to only 11% from unirradiated cells. In irradiated mammalian cells, 54% of plasmids used ≥4 bp of microhomology compared to none from unirradiated cells. We conclude that exposure of yeast and mammalian cells to radiation prior to plasmid transfection enhances the frequency of microhomology-mediated end-joining events in trans. If such events occur within genomic locations, they may be involved in the generation of large deletions and other chromosomal aberrations that occur in cancer cells.

O'Dowd, C., Mothersill, C. E., Cairns, M. T., Austin, B., Lyng, F. M., McClean, B., Talbot, A. and Murphy, J. E. J. Gene Expression and Enzyme Activity of Mitochondrial Proteins in Irradiated Rainbow Trout (Oncorhynchus Mykiss, Walbaum) Tissues In Vitro. Radiat. Res. 171, 464–473 (2009).

In recent years ethical, legislative and economic pressures have created a renewed interest in the development of alternatives to in vivo animal experiments. In vitro studies, particularly those using cell cultures, have been used increasingly as tools to assess the degree of toxicity associated with or present in particular environments. While cell cultures are useful to give relative toxicity values, genotypic and phenotypic integrity may be compromised in the continuous artificial environment they experience. In addition, cell cultures lack the complexity of functional organs and thus do not truly represent the effects that toxins exert on organ and organism functionality. In this study, ex vivo tissue cultures of rainbow trout gill, skin and spleen samples were analyzed for variation of expression in genes associated with oxidative phosphorylation after exposure to ionizing radiation. Significant radiation-induced changes in gene expression and enzyme activity associated with the mitochondrial oxidative phosphorylation process were identified. The tissues examined in this study demonstrated an exposure threshold at which radiation dose stimulates an alteration in the regulatory activity of mitochondrial-associated genes. Spleen tissues exposed to low levels of radiation (0.1 Gy) appeared most sensitive whereas skin tissues proved least sensitive, reacting only to higher doses (>1 Gy). We propose this investigative approach as an innovative alternative to in vivo studies because it identifies toxic exposure in vitro and could significantly reduce the number of live-animal toxicity tests required.

Peng, Y., Brown, N., Finnon, R., Warner, C. L., Liu, X., Genik, P. C., Callan, M. A., Ray, F. A., Borak, T. B., Badie, C., Bouffler, S. D., Ullrich, R. L., Bedford, J. S. and Weil, M. M. Radiation Leukemogenesis in Mice: Loss of PU.1 on Chromosome 2 in CBA and C57BL/6 Mice after Irradiation with 1 GeV/nucleon ⁵⁶Fe Ions, X Rays or γ Rays. Part I. Experimental Observations. Radiat. Res. 171, 474–483 (2009).

Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of γ-, X- and ⁵⁶Fe-ion-irradiated mice at various times postirradiation. Although ⁵⁶Fe ions were initially some two to three times more effective than X or γ rays in inducing PU.1 deletions, by 1 month postirradiation, the proportions of cells with PU.1 deletions were similar for the HZE particles and the sparsely ionizing radiations. These results indicate that while ⁵⁶Fe ions are more effective in inducing PU.1 deletions, they are also more effective in causing collateral damage that removes hit cells from the bone marrow. After X, γ or ⁵⁶Fe-ion irradiation, AML-resistant C57BL/6 mice have fewer cells with PU.1 deletions than CBA mice, and those cells do not persist in the bone marrow of the C57B6/6 mice. Our findings suggest that quantification of PU.1 deleted bone marrow cells 1 month postirradiation can be used as surrogate for the incidence of radiation-induced AML measured in large-scale mouse studies. If so, PU.1 loss could be used to systematically assess the potential leukemogenic effects of other ions and energies in the space radiation environment.

Peng, Y., Borak, T. B., Bouffler, S. D., Ullrich, R. L., Weil, M. M. and Bedford, J. S. Radiation Leukemogenesis in Mice: Loss of PU.1 on Chromosome 2 in CBA and C57BL/6 Mice after Irradiation with 1 GeV/nucleon ⁵⁶Fe Ions, X Rays or γ Rays. Part II. Theoretical Considerations Based on Microdosimetry and the Initial Induction of Chromosome Aberrations. Radiat. Res. 171, 484–493 (2009).

Chromosome aberrations in mitotic bone marrow cells of CBA/Ca and C57BL/6 mice were measured 1 day after exposure to 1 Gy of 1 GeV/nucleon ⁵⁶Fe ions or 3 Gy of γ rays. The proportion that have lost a region of chromosome 2 containing the PU.1 gene could be explained by a model based on these measurements. The distribution of aberrations among cells was close to the expected Poisson for the γ-irradiated cells, but for the HZE ⁵⁶Fe ions the distribution was highly dispersed. The observations were consistent with the results of an analysis similar to that of Edwards and co-workers in 1980 after ex vivo irradiation of human blood with α particles. The analysis used to fit the current data was based on a compound Poisson process, also used previously by others, but in addition included the random nature of parameters involved such as cell nuclear diameter, particle traversal lengths through cell nuclei, production of aberrations, and cell cycle arrest per traversal. From the measured numbers of acentric fragments produced, the relative size of chromosome 2 and the region associated with PU.1 deletions, an independent prediction of PU.1 loss agreed well with measurements described in the accompanying paper.

Yamamoto, Y., Usuda, N., Takatsuji, T., Kuwahara, Y. and Fukumoto, M. Long Incubation Period for the Induction of Cancer by Thorotrast is Attributed to the Uneven Irradiation of Liver Cells at the Microscopic Level. Radiat. Res. 171, 494– 503 (2009).

Irradiation from internally deposited radionuclides induces malignant tumors. Ingested long-lived radionuclides accumulate in specific organs that are irradiated throughout life. To elucidate why the development of malignant tumors requires long-term internal exposure, of the order of decades, despite the fact that irradiation is continuous over this period, we analyzed intrahepatic cholangiocarcinoma in Thorotrast patients (Th-ICC). Autoradiography showed that the density of α-particle tracks was 50 times more concentrated than would be expected if Thorotrast were evenly distributed throughout the liver. The age–incidence curves revealed that while the incidence of hepatobiliary cancer in Japan increased in proportion to the 7th power of age, that of Th-ICC increased to the 6th power. Internal radiation significantly increased the randomness of hepatocyte distribution but not the density. Three major factors are considered to be responsible for the long incubation time: the uneven distribution of radionuclides, the limited range of radiation, and the movement of tumor precursor cells. Target cells susceptible to malignant transformation may undergo one event and may then migrate outside of the range of α particles, thereby avoiding immediate induction of successive additional events that would lead to cell death or neoplastic changes.

Hammer, G. P., Seidenbusch, M. C., Schneider, K., Regulla, D. F., Zeeb, H., Spix, C. and Blettner, M. A Cohort Study of Childhood Cancer Incidence after Postnatal Diagnostic X-Ray Exposure. Radiat. Res. 171, 504–512 (2009).

Ionizing radiation is an established cause of cancer, yet little is known about the health effects of doses from diagnostic examinations in children. The risk of childhood cancer was studied in a cohort of 92.957 children who had been examined with diagnostic X rays in a large German hospital during 1976–2003. Radiation doses were reconstructed using the individual dose area product and other exposure parameters, together with conversion coefficients developed specifically for the medical devices and standards used at the radiology department. Newly diagnosed cancers occurring between 1980 and 2006 were determined through record linkage to the German Childhood Cancer Registry. The median radiation dose was 7 μSv. Eight-seven incident cases were found in the cohort: 33 leukemia, 13 lymphoma, 10 central nervous system tumors, and 31 other tumors. The standardized incidence ratio (SIR) for all cancers was 0.99 (95% CI: 0.79–1.22). No trend in the incidence of total cancer, leukemia or solid tumors with increasing radiation dose was observed in the SIR analysis or in the multivariate Poisson regression. Risk did not differ significantly in girls and boys. Overall, while no increase in cancer risk with diagnostic radiation was observed, the results are compatible with a broad range of risk estimates.