Kato, T. A., Nagasawa, H., Weil, M. M., Little, J. B. and Bedford, J. S. Levels of γ-H2AX Foci after Low-Dose-Rate Irradiation Reveal a DNA DSB Rejoining Defect in Cells from Human ATM Heterozygotes in Two AT Families and in Another Apparently Normal Individual. Radiat. Res. 166, 443– 453 (2006).

We have investigated the use of the γ-H2AX assay, reflecting the presence of DNA double-strand breaks, as a possible means for identifying individuals who are mildly hypersensitive to ionizing radiation, such as some ATM heterozygotes. We compared levels of γ-H2AX foci after irradiation in cells from six apparently normal individuals as well as from individuals from two separate AT families including the proband, mother, father and three unaffected siblings in each family. After a 1-Gy single acute (high-dose-rate) γ-ray dose delivered to noncycling contact-inhibited monolayers of cells, clear differences were seen between samples from normal individuals (ATM ^/ ) and probands (ATM^−/−) at nearly all sampling times after irradiation, but no clear distinctions were seen for cells from normal compared to obligate heterozygotes (ATM ^/−). In contrast, after 24 h of continuous irradiation at a dose rate of 10 cGy/h, appreciable differences in numbers of foci per cell were observed for cells from individuals for all the known ATM genotypes compared with controls. Four unaffected siblings had mean numbers of foci per cell similar to that for the obligate heterozygotes, whereas the other two had mean values similar to that for normal controls. We determined independently that those siblings with mean numbers of foci per cell in the range of ATM heterozygotes carried the mutant allele, while both siblings with a normal number of foci per cell after irradiation had normal alleles. A more limited set of experiments using lymphoblastoid cell strains in the low-dose-rate assay also revealed distinct differences for normal compared to ATM heterozygotes from the same families and opens the possibility of using peripheral blood lymphocytes as a more suitable material for an assay to detect mild hypersensitivities to radiation among individuals.

Ohnishi, K., Scuric, Z., Schiestl, R. H., Okamoto, N., Takahashi, A. and Ohnishi, T. siRNA Targeting NBS1 or XIAP Increases Radiation Sensitivity of Human Cancer Cells Independent of TP53 Status. Radiat. Res. 166, 454–462 (2006).

NBS1 is essential for the repair of radiation-induced DNA double-strand breaks (DSBs) in yeast and higher vertebrate cells. In this study, we examined whether suppressed NBS1 expression by small interference RNA (siRNA) could enhance radiation sensitivity in cancer cells with different TP53 status. We used human non-small cell lung cancer cells differing in TP53 gene status (H1299/wtp53 cells bearing wild-type TP53 or H1299/mp53 cells bearing mutant TP53). A DNA cassette expressing siRNA targeted for the NBS1 gene was transfected into those cell lines, and radiation sensitivity was examined with a colony-forming assay. Cellular levels of NBS1 and other proteins were analyzed using Western blotting. We found that the radiation sensitivity of H1299/wtp53 and H1299/mp53 cells was enhanced by transfection of the DNA cassette. In the NBS1-siRNA-transfected cells, we observed decreased constitutive expression of NBS1 protein and decreased radiation-induced accumulation of phosphorylated NBS1 protein. In addition, radiation-induced expression of the transcription factor NF-κB (NFKB) and XIAP (X-chromosome-linked inhibitor of apoptosis protein) was suppressed by NBS1-siRNA. Enhanced X-ray sensitivity after NBS1-siRNA transfection was achieved in TP53 wild-type cells and sensitivity was even more pronounced in TP53 mutant cells. The transfection of siRNA targeted for XIAP also enhanced X-ray sensitivity even more for TP53 mutant cells compared to TP53 wild-type cells. Our data suggest that the sensitization to radiation results from NBS1-siRNA-mediated suppression of DNA repair and/ or X-ray-induced cell survival signaling pathways through NFKB and XIAP. siRNA targeting appears to be a novel radiation-sensitizing agent, particularly in human TP53 mutant cancer cells.

Maier, P., Fleckenstein, K., Li, L., Laufs, S., Zeller, W. J., Baum, C., Fruehauf, S., Herskind, C. and Wenz, F. Overexpression of MDR1 Using a Retroviral Vector Differentially Regulates Genes Involved in Detoxification and Apoptosis and Confers Radioprotection. Radiat. Res. 166, 463–473 (2006).

Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). Sixty-one annotated genes showed a significant change in expression (P < 10⁻⁴) in MDR1-overexpressing cells compared to untransduced cells and cells transduced with a control virus expressing the neomycin phosphotransferase gene. Several genes coding for proteins involved in detoxification and exocytosis showed ∼1.4– 4-fold increases in transcript levels (e.g. ALDH1A, UNC13). Additionally, pro-apoptosis genes were down-regulated (e.g. twofold for CASP1, 2.5-fold for NALP7) with concomitant increased expression of the potential anti-apoptosis gene AKT3. In functional assays the influence of MDR1 overexpression on apoptosis signaling was further corroborated by showing reduced rates of apoptosis in response to irradiation in TK6 cells transduced with MDR1. In conclusion, the resistant phenotype of MDR1-mediated P-gp-overexpressing cells is associated with differential expression of genes coding for metabolic and apoptosis-related proteins. These results have important implications for understanding the mechanisms by which MDR1 gene therapy can protect normal tissues from radiation- or chemotherapy-induced damage during tumor treatment.

Otsuka, K., Koana, T., Tauchi, H. and Sakai, K. Activation of Antioxidative Enzymes Induced by Low-Dose-Rate Whole-Body γ Irradiation: Adaptive Response in Terms of Initial DNA Damage. Radiat. Res. 166, 474–478 (2006).

An adaptive response induced by long-term low-dose-rate irradiation in mice was evaluated in terms of the amount of DNA damage in the spleen analyzed by a comet assay. C57BL/ 6N female mice were irradiated with 0.5 Gy of ¹³⁷Cs γ rays at 1.2 mGy/h; thereafter, a challenge dose (0.4, 0.8 or 1.6 Gy) at a high dose rate was given. Less DNA damage was observed in the spleen cells of preirradiated mice than in those of mice that received the challenge dose only; an adaptive response in terms of DNA damage was induced by long-term low-dose-rate irradiation in mice. The gene expression of catalase and Mn-SOD was significantly increased in the spleen after 23 days of the low-dose-rate radiation (0.5 Gy). In addition, the enzymatic activity of catalase corresponded to the gene expression level; the increase in the activity was observed at day 23 (0.5 Gy). These results suggested that an enhancement of the antioxidative capacities played an important role in the reduction of initial DNA damage by low-dose-rate radiation.

Shao, C., Lyng, F. M., Folkard, M. and Prise, K. M. Calcium Fluxes Modulate the Radiation-Induced Bystander Responses in Targeted Glioma and Fibroblast Cells. Radiat. Res. 166, 479–487 (2006).

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca² channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage.

Zhou, G., Bennett, P. V., Cutter, N. C. and Sutherland, B. M. Proton-HZE-Particle Sequential Dual-Beam Exposures Increase Anchorage-Independent Growth Frequencies in Primary Human Fibroblasts. Radiat. Res. 166, 488–494 (2006).

The radiation field in deep space contains high levels of high-energy protons and substantially lower levels of high-atomic-number, high-energy (HZE) particles. Calculations indicate that cellular nuclei of human space travelers will be hit during a 3-year Mars mission by ∼400 protons and ∼0.4 HZE particles. Thus most cells in astronauts will be hit by a proton(s) before being hit by an HZE particle. To investigate effects of dual ion irradiations on human cells, we irradiated primary human neonatal fibroblasts with protons (1 GeV/nucleon, 20 cGy) followed from 2.5 min to 48 h later by iron or titanium ions (1 GeV/nucleon, 20 cGy) and then measured clonogenic survival and frequency of anchorage-independent growth. This frequency depends on the interval between hydrogen- and iron-ion irradiation, with a critical window between 2.5 min and 1 h producing about three times more anchorage-independent colonies per survivor than expected from simple addition of the two ions separately. The hydrogen-titanium-ion dual-beam irradiation produced similar increases that persisted to ∼6 h. At longer intervals, anchorage-independent growth frequencies were similar to those expected for additivity. However, irradiation of cells with either an iron or a titanium particle first followed by protons produced only additive levels.

Coderre, J. A., Morris, G. M., Micca, P. L., Hopewell, J. W., Verhagen, I., Kleiboer, B. J. and van der Kogel, A. J. Late Effects of Radiation on the Central Nervous System: Role of Vascular Endothelial Damage and Glial Stem Cell Survival. Radiat. Res. 166, 495–503 (2006).

Selective irradiation of the vasculature of the rat spinal cord was used in this study, which was designed specifically to address the question as to whether it is the endothelial cell or the glial progenitor cell that is the target responsible for late white matter necrosis in the CNS. Selective irradiation of the vascular endothelium was achieved by the intraperitoneal (ip) administration of a boron compound known as BSH (Na₂B₁₂H₁₁SH), followed by local irradiation with thermal neutrons. The blood-brain barrier is known to exclude BSH from the CNS parenchyma. Thirty minutes after the ip injection of BSH, the boron concentration in blood was 100 μg ¹⁰B/ g, while that in the CNS parenchyma was below the detection limit of the boron analysis system, <1 μg ¹⁰B/g. An ex vivo clonogenic assay of the O2A (oligodendrocyte-type 2 astrocyte) glial progenitor cell survival was performed 1 week after irradiation and at various times during the latent period before white matter necrosis in the spinal cord resulted in myelopathy. One week after 4.5 Gy of thermal neutron irradiation alone (approximately one-third of the dose required to produce a 50% incidence of radiation myelopathy), the average glial progenitor cell surviving fraction was 0.03. The surviving fraction of glial progenitor cells after a thermal neutron irradiation with BSH for a comparable effect was 0.46. The high level of glial progenitor cell survival after irradiation in the presence of BSH clearly reflects the lower dose delivered to the parenchyma due to the complete exclusion of BSH by the blood-brain barrier. The intermediate response of glial progenitor cells after irradiation with thermal neutrons in the presence of a boron compound known as BPA (p-dihydroxyboryl-phenylalanine), again for a dose that represents one-third the ED₅₀ for radiation-induced myelopathy, reflects the differential partition of boron-10 between blood and CNS parenchyma for this compound, which crosses the blood-brain barrier, at the time of irradiation. The large differences in glial progenitor survival seen 1 week after irradiation were also maintained during the 4–5-month latent period before the development of radiation myelopathy, due to selective white matter necrosis, after irradiation with doses that would produce a high incidence of radiation myelopathy. Glial progenitor survival was similar to control values at 100 days after irradiation with a dose of thermal neutrons in the presence of BSH, significantly greater than the ED₁₀₀, shortly before the normal time of onset of myelopathy. In contrast, glial progenitor survival was less than 1% of control levels after irradiation with 15 Gy of thermal neutrons alone. This dose of thermal neutrons represents the approximate ED_90–100 for myelopathy. The response to irradiation with an equivalent dose of X rays (ED₉₀: 23 Gy) was intermediate between these extremes as it was to thermal neutrons in the presence of BPA at a slightly lower dose equivalent to the approximate ED₆₀ for radiation myelopathy. The conclusions from these studies, performed at dose levels approximately iso-effective for radiation-induced myelopathy as a consequence of white matter necrosis, were that the large differences observed in glial progenitor survival were directly related to the dose distribution in the parenchyma. These observations clearly indicate the relative importance of the dose to the vascular endothelium as the primary event leading to white matter necrosis.

Prat, M., Demarquay, C., Frick, J., Dudoignon, N., Thierry, D. and Bertho, J. M. Use of Flt3 Ligand to Evaluate Residual Hematopoiesis after Heterogeneous Irradiation in Mice. Radiat. Res. 166, 504–511 (2006).

We evaluated the possibility of using plasma Flt3 ligand (FL) concentration as a biological indicator of bone marrow function after heterogeneous irradiation. Mice were irradiated with 4, 7.5 or 11 Gy with 25, 50, 75 or 100% of the bone marrow in the field of irradiation. This model of irradiation resulted in graded and controlled damage to the bone marrow. Mice exhibited a pancytopenia correlated with both the radiation dose and the percentage of bone marrow irradiated. The FL concentration in the blood increased with the severity of bone marrow aplasia. Nonlinear regression analysis showed that the FL concentration was strongly correlated with the total number of residual colony-forming cells 3 days after irradiation, allowing a precise estimate of residual hematopoiesis. Moreover, the FL concentration on day 3 postirradiation was correlated with the duration and severity of subsequent pancytopenia, suggesting that variations in FL concentrations might be used as a predictive indicator of bone marrow aplasia, especially by the use of linear regression equations describing these correlations. Our results provide a rationale for the use of FL concentration as a biological indicator of residual hematopoiesis after heterogeneous irradiation.

Wen, B., Urano, M., O'Donoghue, J. A. and Ling, C. C. Measurements of Partial Oxygen Pressure (pO₂) using the OxyLite System in R3327-AT Tumors under Isoflurane Anesthesia. Radiat. Res. 166, 512–518 (2006).

The presence of oxygen-deficient tumor cells is a critical issue in cancer therapy. To identify tumor hypoxia, tissue partial oxygen pressure (pO₂) can be measured directly. The OxyLite system allows determination of pO₂ in tumors and permits continuous measurements of pO₂ at a fixed point. In this study, this system was used to continuously measure pO₂ in R3327-AT tumors in animals anesthetized with isoflurane. In addition, continuous pO₂ measurement was performed in the muscle in non-tumor-bearing animals. In animals breathing isoflurane balanced by air, tumor pO₂ at fixed positions decreased rapidly within 1–2 min of probe positioning but remained stable thereafter. In animals breathing isoflurane balanced by pure oxygen, tumor pO₂ was higher and remained high. We also measured pO₂ values at multiple positions in R3327-AT tumors of various sizes, with anesthetized animals breathing either air or pure oxygen. Our data showed that the frequency of pO₂ measurements below 2.5 or 5.0 mmHg was significantly higher in animals breathing air than in animals breathing pure oxygen. Measurements in different-sized tumors showed that the mean pO₂ value decreased as tumor volume increased, with the largest change occurring between tumor volumes of 100 and 200 mm³. Our data demonstrate that the OxyLite system, when used with isoflurane anesthesia, is a valuable tool in the study of tumor hypoxia.

Kimmel, R. R., Zhao, L. P., Nguyen, D., Lee, S., Aronszajn, M., Cheng, C., Troshin, V. P., Abrosimov, A., Delrow, J., Tuttle, R. M., Tsyb, A. F., Kopecky, K. J., Davis, S. and Neiman, P. E. Microarray Comparative Genomic Hybridization Reveals Genome-Wide Patterns of DNA Gains and Losses in Post-Chernobyl Thyroid Cancer. Radiat. Res. 166, 519–531 (2006).

Genetic gains and losses resulting from DNA strand breakage by ionizing radiation have been demonstrated in vitro and suspected in radiation-associated thyroid cancer. We hypothesized that copy number deviations might be more prevalent, and/or occur in genomic patterns, in tumors associated with presumptive DNA strand breakage from radiation exposure than in their spontaneous counterparts. We used cDNA microarray-based comparative genome hybridization to obtain genome-wide, high-resolution copy number profiles at 14,573 genomic loci in 23 post-Chernobyl and 20 spontaneous thyroid cancers. The prevalence of DNA gains in tumors from cases in exposed individuals was two- to fourfold higher than for cases in unexposed individuals and up to 10-fold higher for the subset of recurrent gains. DNA losses for all cases were low and more prevalent in spontaneous cases. We identified unique patterns of copy variation (mostly gains) that depended on a history of radiation exposure. Exposed cases, especially the young, harbored more recurrent gains that covered more of the genome. The largest regions, spanning 1.2 to 4.9 Mbp, were located at 1p36.32-.33, 2p23.2-.3, 3p21.1-.31, 6p22.1-.2, 7q36.1, 8q24.3, 9q34.11, 9q34.3, 11p15.5, 11q13.2-12.3, 14q32.33, 16p13.3, 16p11.2, 16q21-q12.2, 17q25.1, 19p13.31-qter, 22q11.21 and 22q13.2. Copy number changes, particularly gains, in post-Chernobyl thyroid cancer are influenced by radiation exposure and age at exposure, in addition to the neoplastic process.

Vijayalaxmi, Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to 2.45 GHz or 8.2 GHz Radiofrequency Radiation. Radiat. Res. 166, 532–538 (2006).

Peripheral blood samples collected from healthy human volunteers were exposed in vitro to 2.45 GHz or 8.2 GHz pulsed-wave radiofrequency (RF) radiation. The net forward power, average power density, mean specific absorption rate, and the temperature maintained during the 2-h exposure of the cells to 2.45 GHz or 8.2 GHz were, respectively, 21 W or 60 W, 5 mW/cm² or 10 mW/cm², 2.13 W/kg or 20.71 W/kg, and 36.9 ± 0.1°C or 37.5 ± 0.2°C. Aliquots of the same blood samples that were either sham-exposed or exposed in vitro to an acute dose of 1.5 Gy γ radiation were used as unexposed and positive controls, respectively. Cultured lymphocytes were examined to determine the extent of cytogenetic damage assessed from the incidence of chromosomal aberrations and micronuclei. Under the conditions used to perform the experiments, the levels of damage in RF-radiation-exposed and sham-exposed lymphocytes were not significantly different. Also, there were no significant differences in the response of unstimulated lymphocytes and lymphocytes stimulated with phytohemagglutinin when exposed to 8.2 GHz RF radiation. In contrast, the positive control cells that had been subjected to γ irradiation exhibited significantly more damage than RF-radiation- and sham-exposed lymphocytes.

Lantow, M., Viergutz, T., Weiss, D. G. and Simkó, M. Comparative Study of Cell Cycle Kinetics and Induction of Apoptosis or Necrosis after Exposure of Human Mono Mac 6 Cells to Radiofrequency Radiation. Radiat. Res. 166, 539–543 (2006).

The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G₀/G₁-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used.

Sato, T. and Niita, K. Analytical Functions to Predict Cosmic-Ray Neutron Spectra in the Atmosphere. Radiat. Res. 166, 544–555 (2006).

Estimation of cosmic-ray neutron spectra in the atmosphere has been an essential issue in the evaluation of the aircrew doses and the soft-error rates of semiconductor devices. We therefore performed Monte Carlo simulations for estimating neutron spectra using the PHITS code in adopting the nuclear data library JENDL-High-Energy file. Excellent agreements were observed between the calculated and measured spectra for a wide altitude range even at the ground level. Based on a comprehensive analysis of the simulation results, we propose analytical functions that can predict the cosmic-ray neutron spectra for any location in the atmosphere at altitudes below 20 km, considering the influences of local geometries such as ground and aircraft on the spectra. The accuracy of the analytical functions was well verified by various experimental data.

Hofer, M., Pospíšil, M., Znojil, V., Holá, J., Vacek, A., Weiterová, L., Štreitová, D. and Kozub;aaik, A. Meloxicam, a Cyclooxygenase 2 Inhibitor, Supports Hematopoietic Recovery in Gamma-Irradiated Mice. Radiat. Res. 166, 556–560 (2006).

Meloxicam, a selective inhibitor of cyclooxygenase 2, a nonsteroidal anti-inflammatory drug with an improved side-effects profile in terms of gastrointestinal toxicity, has been found to stimulate hematopoiesis in whole-body γ-irradiated mice. A distinct corroboration of this positive action of meloxicam is an enhancement of the recovery of hematopoietic progenitor cells committed to granulocyte-macrophage and erythroid development, which has been demonstrated in sublethally irradiated animals treated with meloxicam at a dose of 20 mg/kg administered intraperitoneally either singly 1 h before irradiation or repeatedly after radiation exposure. The results suggest that meloxicam can be added to the list of biological response modifiers that can be used in the treatment of hematopoietic damage induced by ionizing radiation.