Okazaki, R., Moon, Y., Norimura, T. and Eling, T. Ionizing Radiation Enhances the Expression of the Nonsteroidal Anti-inflammatory Drug-Activated Gene (NAG1) by Increasing the Expression of TP53 in Human Colon Cancer Cells. Radiat. Res. 165, 125–130 (2006).

The induction of apoptosis in cells of human colon cancer cell lines after γ irradiation was investigated to determine whether apoptosis was mediated by TP53 and the subsequent expression of its downstream target, the NSAID-activated gene (NAG1). HCT116 (TP53 ^/ ), HCT15 (TP53 mutant) and TP53 null HCT116 (TP53^−/−) cells were irradiated with γ rays, and apoptosis was measured at various times after irradiation. In HCT116 TP53 ^/ cells, apoptosis was increased after irradiation; the increase was dependent on the time after treatment and the dose of γ rays. However, in HCT15 TP53 mutant cells and HCT116 TP53^−/− cells, there were no remarkable changes in apoptosis. The expression of TP53 protein in HCT116 cells was increased after irradiation and was followed by an increase in the expression of NAG1 protein. In contrast, the expression of NAG1 protein in TP53 mutant cells and TP53^−/− cells was not increased by the radiation treatment, suggesting that NAG1 was required for apoptosis. The expression of NAG1 increased apoptosis in HCT116 cells, but radiation treatment did not further increase apoptosis. The transfection of a NAG1 siRNA into HCT116 cells suppressed radiation-induced apoptosis and inhibited the induction of NAG1 protein without altering the expression of TP53. a NAG1 luciferase promoter construct that included both of the TP53 binding sites, was activated by radiation in dose-dependent manner, while the promoters lacking one or both of the TP53 binding sites in the NAG1 promoter activity either was less responsive or did not respond. The findings reported here indicate that γ radiation activates the TP53 tumor suppressor, which then increases the expression of NAG1. NAG1 mediates the induction of apoptosis in human colorectal cells.

Santini, M. T., Romano, R., Rainaldi, G., Ferrante, A., Motta, A. and Indovina, P. L. Increases in ¹H-NMR Mobile Lipids are not Always Associated with Overt Apoptosis: Evidence from MG-63 Human Osteosarcoma Three-Dimensional Spheroids Exposed to a Low Dose (2 Gy) of Ionizing Radiation. Radiat. Res. 165, 131–141 (2006).

The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Specifically, the ¹H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50–80 μm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH₂ and CH₃ mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that ¹H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.

Mahmoud-Ahmed, A. S., Atkinson, S. and Wong, C. S. Early Gene Expression Profile in Mouse Brain after Exposure to Ionizing Radiation. Radiat. Res. 165, 142–154 (2006).

Acute changes in the gene expression profile in mouse brain after exposure to ionizing radiation were studied using microarray analysis. RNA was isolated at 0.25, 1, 5 and 24 h after exposure to 20 Gy and at 5 h after exposure of the whole brain of adult mice to 2 or 10 Gy. RNA was hybridized onto 15K cDNA microarrays, and data were analyzed using GeneSpring and Significant Analysis of Microarray. Radiation modulated the expression of 128, 334, 325 and 155 genes and ESTs at 0.25, 1, 5 and 24 h after 20 Gy and 60 and 168 at 5 h after 2 and 10 Gy, respectively. The expression profiles showed dose- and time-dependent changes in both expression levels and numbers of differentially modulated genes and ESTs. Seventy-eight genes were modulated at two or more times. Differentially modulated genes were associated with 12 different classes of molecular function and 24 different biological pathways and showed time- and dose-dependent changes. The change in expression of four genes (Jak3, Dffb, Nsep1 and Terf1) after irradiation was validated using quantitative real-time PCR. Up-regulation of Jak3 was observed in another mouse strain. In mouse brain, there was an increase of Jak3 immunoreactivity after irradiation. In conclusion, changes in the gene profile in the brain after irradiation are complex and are dependent on time and dose, and genes with diverse functions and pathways are modulated.

Nowak, E., Etienne, O., Millet, P., Silva Lages, C., Mathieu, C., Mouthon, M. A. and Boussin, F. D. Radiation-Induced H2AX Phosphorylation and Neural Precursor Apoptosis in the Developing Brain of Mice. Radiat. Res. 165, 155–164 (2006).

We showed that γ irradiation of the developing mouse brain with 2 Gy induced a massive apoptosis of neural precursors but not of neurons within 24 h. Successive phosphorylation and dephosphorylation of histone H2AX have been linked to DNA breaks and repair. Similar numbers of nuclear foci of phosphorylated H2AX (γ-H2AX) were found 1 h postirradiation in neural precursors and in neurons, suggesting that differences in radiosensitivity were not related to variations in the numbers of DNA double-strand breaks induced by radiation. Surviving neural precursors like neurons totally lost γ-H2AX within 24 h after irradiation, but they had a slower kinetics of loss of γ-H2AX foci. This suggests that the DNA repair machinery processed damage more slowly in these neural precursors in relation to their greater radiosensitivity. We also found a bright and diffuse γ-H2AX staining of nuclei of cells at an early stage of apoptosis, whereas cells at later stages of apoptosis were unstained. This was probably related to phosphorylation and subsequent degradation of H2AX in the course of DNA fragmentation during apoptosis. Detection of γ-H2AX-bright nuclei may thus be a useful marker of neural cells at an early stage of apoptosis.

Imaoka, T., Okamoto, M., Nishimura, M., Nishimura, Y., Ootawara, M., Kakinuma, S., Tokairin, Y. and Shimada, Y. Mammary Tumorigenesis in Apc^Min/ Mice is Enhanced by X Irradiation with a Characteristic Age Dependence. Radiat. Res. 165, 165–173 (2006).

The Apc^Min/ (Min) mouse is genetically predisposed to both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X rays at 2, 5, 7 and 10 weeks and killed humanely at 18 weeks of age. Min mice irradiated at 7–10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type littermates did not. Interestingly, irradiation of Min mice at 2–5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear β-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by β-catenin signaling.

Yu, D., Shen, Y. H., Kuster, N., Fu, Y. T. and Chiang, H. Effects of 900 MHz GSM Wireless Communication Signals on DMBA-Induced Mammary Tumors in Rats. Radiat. Res. 165, 174–180 (2006).

The purpose of the study was to investigate whether exposure to 900 MHz GSM wireless communication signals enhances mammary tumor development and growth induced by low-dose DMBA. Five hundred female Sprague-Dawley rats were treated with a single dose of 35 mg/kg DMBA and then divided into five groups in a blinded fashion: one cage control group and four exposure groups, including three microwave exposure groups and one sham exposure with specific absorption rates (SARs) of 4.0, 1.33, 0.44 and 0 W/kg, respectively. Exposure started on the day after DMBA administration and lasted 4 h/day, 5 days/week for 26 weeks. Rats were weighed and palpated weekly for the presence of tumors and were killed humanely at the end of the 26-week exposure period. All mammary glands were examined histologically. There were no statistically significant differences in body weight between sham- and GSM microwave-exposed groups. No significant differences in overall mammary tumor incidence, latency to tumor onset, tumor multiplicity, or tumor size were observed between microwave- and sham-exposed groups. There was a tendency for reduction of mammary adenocarcinoma incidence in the lowest microwave exposure group (0.44 W/ kg) compared with the sham-exposed group (P = 0.058). Additionally, a higher incidence of adenocarcinoma was noticed in the 4.0 W/kg group from the 15th to 26th weeks, especially in the 19th week (P = 0.358 compared to sham). However, neither tendency was statistically significant; thus this study does not provide evidence that GSM microwave exposure promotes mammary tumor development in rats. In the present study there were significant differences between the cage controls and the experimental groups (sham and exposure). Body weight and mammary tumor (malignant plus benign) incidence in the cage control group were significantly higher than in the sham- and GSM microwave-exposed groups. The latency to the mammary tumor onset was significantly shorter in the cage control group than in the other groups.

Liu, W., Ding, I., Chen, K., Olschowka, J., Xu, J., Hu, D., Morrow, G. and Okunieff, P. Interleukin 1β (IL1B) Signaling is a Critical Component of Radiation-Induced Skin Fibrosis. Radiat. Res. 165, 181–191 (2006).

Interleukin 1 beta (IL1B), a potent pro-inflammatory cytokine, is directly up-regulated by radiation and is known to regulate other inflammation-related molecules, such as the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). However, the nature of the interaction of IL1B with MMPs and TIMPs in radiation-induced skin fibrosis is unknown. We examined the response of primary dermal keratinocytes, fibroblasts and endothelial cells to single-fraction radiation (10 Gy) and compared the results to a temporal sequence of histology from irradiated C57BL/6 and IL1R1 knockout mice. These studies showed that keratinocytes are the major IL1-producing cells in vitro and that radiation induces an immediate and chronic elevation in the expression of IL1B mRNA in the skin of C57BL/6 mice. This elevation was principally early and was less pronounced in the IL1R1 knockout strain, which also demonstrated reduced late radiation fibrosis. Radiation also increased expression of MMP mRNA in C57BL/6 mice. Finally, exogenous IL1B protein induced robust endogenous IL1B mRNA expression, along with a brisk increase in MMPs and collagen III, but only in the C57BL/6 mice. In conclusion, these data suggest that IL1B plays a critical role in radiation-induced fibrosis and that the increased MMPs fail to block the IL1-related collagen accumulation.

Kirkpatrick, J. P., Hardee, M. E., Snyder, S. A., Peltz, C. M., Zhao, Y., Brizel, D. M., Dewhirst, M. W. and Blackwell, K. L. The Effect of Darbepoetin Alfa on Growth, Oxygenation and Radioresponsiveness of a Breast Adenocarcinoma. Radiat. Res. 165, 192–201 (2006).

Tumor hypoxia is associated with poor clinical outcome in a variety of tumors, including cervical, head/neck and breast cancer. Administration of erythropoietic factors has been suggested as a means of improving tumor oxygenation (pO₂). This study randomized rats to treatment with low-dose or high-dose darbepoetin alfa or a placebo to determine the effect of darbepoetin alfa on the pO₂, growth and response to radiation therapy of R3230 mammary adenocarcinoma. Rats received 3 μg/kg (high dose) or 0.2 μg/kg (low dose) darbepoetin alfa or placebo for eight doses prior to either (1) pO₂ measurement and pimonidazole staining or (2) irradiation or sham irradiation on post-transplant day 20. In the animals randomized to radiation treatment, placebo or darbepoetin alfa administration at a reduced dose was continued for 9 weeks or until the tumor diameter exceeded 15 mm (defined as failure for survival analysis). Treatment with high-dose and low-dose darbepoetin alfa produced hematocrits of 68 and 56% compared to 44 and 45% in their respective control groups (both P < 10⁻⁵). At 18 days post-transplant, tumor volume was not different for either darbepoetin alfa group compared to the placebo group. Tumor oxygenation, as measured by the fraction of pO₂ measurement <10 mmHg and the intensity of pimonidazole staining, was significantly improved in the high-dose group (P = 0.046 and 0.03, respectively, compared with controls) but not in the low-dose group. Growth delay curves after irradiation did not differ significantly for high- or low-dose darbepoetin alfa compared to placebo. In this nonanemic animal model of mammary adenocarcinoma, darbepoetin alfa does not significantly alter tumor growth or radioresponsiveness, even though it improves oxygenation when administered at high doses.

Wilding, C. S., Cadwell, K., Tawn, E. J., Relton, C. L., Taylor, G. A., Chinnery, P. F. and Turnbull, D. M. Mitochondrial DNA Mutations in Individuals Occupationally Exposed to Ionizing Radiation. Radiat. Res. 165, 202–207 (2006).

Mutations in a 443-bp amplicon of the hypervariable region HVR1 of the D-loop of mitochondrial DNA (mtDNA) were quantified in DNA extracted from peripheral blood samples of 10 retired radiation workers who had accumulated external radiation doses of >0.9 Sv over the course of their working life and were compared to the levels of mutations in 10 control individuals matched for age and smoking status. The mutation rate in the 10 exposed individuals was 9.92 × 10⁻⁵ mutations/ nucleotide, and for the controls it was 8.65 × 10⁻⁵ mutations/ nucleotide, with a procedural error rate of 2.65 × 10⁻⁵ mutations/nucleotide. No increase in mtDNA mutations due to radiation exposure was detectable (P = 0.640). In contrast, chromosomal translocation frequencies, a validated radiobiological technique for retrospective dosimetric purposes, were significantly elevated in the exposed individuals. This suggests that mutations identified through sequencing of mtDNA in peripheral blood lymphocytes do not represent a promising genetic marker of DNA damage after low-dose or low-dose-rate exposures to ionizing radiation. There was an increase in singleton mutations above that attributable to procedural error in both exposed and control groups that is likely to reflect age-related somatic mutation.

Simon, S. L., Anspaugh, L. R., Hoffman, F. O., Scholl, A. E., Stone, M. B., Thomas, B. A. and Lyon, J. L. 2004 Update of Dosimetry for the Utah Thyroid Cohort Study. Radiat. Res. 165, 208–222 (2006).

In the 1980s, individual thyroid doses and uncertainties were estimated for members of a cohort of children identified in 1965 in Utah and Nevada who had potentially been exposed to fallout from the Nevada Test Site. That reconstruction represented the first comprehensive assessment of doses received by the cohort and was the first large effort to assess the uncertainty of dose on an individual person basis. The data on dose and thyroid disease prevalence during different periods were subsequently used in an analysis to determine risks of radiogenic thyroid disease. This cohort has received periodic medical follow-up to observe changes in disease frequency and to reassess the previously reported radiation-related risks, most recently after a Congressional mandate in 1998. In a recent effort to restore the databases and computer codes used to estimate doses in the 1980s, various deficiencies were found in the estimated doses due to improperly operating computer codes, corruption of secondary data files, and lack of quality control procedures. From 2001 through 2004, the dosimetry system was restored and corrected and all doses were recalculated. In addition, two parameter values were updated. While the mean of all doses has not changed significantly, many individual doses have changed by more than an order of magnitude.

Hada, M. and Sutherland, B. M. Spectrum of Complex DNA Damages Depends on the Incident Radiation. Radiat. Res. 165, 223–230 (2006).

Ionizing radiation induces bistranded clustered damages— two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy⁻¹) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles—including the high-energy, low-LET protons—than for the ionizing photons.

Heiß, M., Fischer, B. E., Jakob, B., Fournier, C., Becker, G. and Taucher-Scholz, G. Targeted Irradiation of Mammalian Cells Using a Heavy-Ion Microprobe. Radiat. Res. 165, 231–239 (2006).

The existing focusing heavy-ion microprobe at the Gesellschaft für Schwerionenforschung in Darmstadt (Germany) has been modified to enable the targeted irradiation of single, selected cells with a defined number of ions. With this setup, ions in the range from helium to uranium with linear energy transfers (LETs) up to ∼15,000 keV/μm can be positioned with a precision of a few micrometers in the nuclei of single cells that are growing in culture on a thin polypropylene film. To achieve this accuracy, the microbeam traverses a thin vacuum window with minimal scattering. Electron emission from that window is used for particle detection. The cells are kept in a specially designed dish that is mounted directly behind the vacuum window in a setup allowing the precise movement and the imaging of the sample with microscopic methods. The cells are located by an integrated software program that also controls the rapid deflection and switching of the beam. In this paper, the setup is described in detail together with the first experiments showing its performance. We describe the ability of the microprobe to reliably hit randomly positioned etched nuclear tracks in CR-39 with single ions as well as the ability to visualize the ion hits using immunofluorescence staining for 53BP1 as a marker of DNA damage in the targeted cell nuclei.

Lehmann, J., Stern, R. L., Daly, T. P., Rocke, D. M., Schwietert, C. W., Jones, G. E., Arnold, M. L., Hartmann Siantar, C. L. and Goldberg, Z. Dosimetry for Quantitative Analysis of the Effects of Low-Dose Ionizing Radiation in Radiation Therapy Patients. Radiat. Res. 165, 240–247 (2006).

We have developed and validated a practical approach to identifying the location on the skin surface that will receive a prespecified biopsy dose (ranging down to 1 cGy) in support of in vivo biological dosimetry in humans. This represents a significant technical challenge since the sites lie on the patient's surface outside the radiation fields. The PEREGRINE Monte Carlo simulation system was used to model radiation dose delivery, and TLDs were used for validation on phantoms and for confirmation during patient treatment. In the developmental studies, the Monte Carlo simulations consistently underestimated the dose at the biopsy site by approximately 15% (of the local dose) for a realistic treatment configuration, most likely due to lack of detail in the simulation of the linear accelerator outside the main beam line. Using a single, thickness-independent correction factor for the clinical calculations, the average of 36 measurements for the predicted 1-cGy point was 0.985 cGy (standard deviation: 0.110 cGy) despite patient breathing motion and other real-world challenges. Since the 10-cGy point is situated in the region of high-dose gradient at the edge of the field, patient motion had a greater effect, and the six measured points averaged 5.90 cGy (standard deviation: 1.01 cGy), a difference that is equivalent to approximately a 6-mm shift on the patient's surface.