Piening, B. D., Wang, P., Subramanian, A. and Paulovich, A. G. A Radiation-Derived Gene Expression Signature Predicts Clinical Outcome for Breast Cancer Patients. Radiat. Res. 171, 141–154 (2009).

Activation of the DNA damage response pathway is a hallmark for early tumorigenesis, while loss of pathway activity is associated with disease progression. Thus we hypothesized that a gene expression signature associated with the DNA damage response may serve as a prognostic signature for outcome in cancer patients. We identified ionizing radiation-responsive transcripts in human lymphoblast cells derived from 12 individuals and used this signature to screen a panel of cancer data sets for the ability to predict long-term survival of cancer patients. We demonstrate that gene sets induced or repressed by ionizing radiation can predict clinical outcome in two independent breast cancer data sets, and we compare the radiation signature to previously described gene expression-based outcome predictors. While genes repressed in response to radiation likely represent the well-characterized proliferation signature predictive of breast cancer outcome, genes induced by radiation likely encode additional information representing other deregulated biological properties of tumors such as checkpoint or apoptotic responses.

Schwartz, J. L., Kopecky, K. J., Mathes, R. W., Leisenring, W. M., Friedman, D. L. and Deeg, H. J. Basal Cell Skin Cancer after Total-Body Irradiation and Hematopoietic Cell Transplantation. Radiat. Res. 171, 155–163 (2009).

Previous studies identified radiation therapy as a key modifier of basal cell carcinoma (BCC) risk in survivors of hematopoietic cell transplantation (HCT). In the present analysis, risk of BCC was analyzed in relation to age at transplant, attained age, race, total-body irradiation (TBI), and radiation fractionation in 6,306 patients who received HCT at ages 0–65 years after conditioning regimens with (n = 3870) or without (n = 2436) TBI, and who were followed from 100 days to 36.2 years after HCT. While age-specific BCC rates in the unirradiated patient population were higher than those reported for two non-patient populations, the general characteristics were similar; rates increased with attained age, were eightfold lower for non-white patients, and were higher in more recent birth cohorts. After adjusting for these effects, risk in unirradiated patients did not vary significantly with age at HCT. The additional BCC risk associated with radiation exposure was largest for the youngest ages at exposure to radiation, with relative risks exceeding 20 for those transplanted at ages less than 10 years, and decreased with increasing age at exposure until age 40 years, above which no excess risk was identified. Relative risk in the irradiated population did not vary significantly with attained age, dose fractionation or race. Risks per unit dose in HCT patients were similar to other populations exposed under clinical settings to similar radiation doses and were more than 10-fold lower than seen in the atomic bomb survivors, 97% of whom were exposed to doses <1 Sv.

Lenarczyk, M., Cohen, E. P., Fish, B. L., Irving, A. A., Sharma, M., Driscoll, C. D. and Moulder, J. E. Chronic Oxidative Stress as a Mechanism for Radiation Nephropathy. Radiat. Res. 171, 164–172 (2009).

Suppression of the renin-angiotensin system has proven efficacy for mitigation and treatment of radiation nephropathy, and it has been hypothesized that this efficacy is due to suppression of radiation-induced chronic oxidative stress. It is known that radiation exposure leads to acute oxidative stress, but direct evidence for radiation-induced chronic renal oxidative stress is sparse. We looked for evidence of oxidative stress after total-body irradiation in a rat model, focusing on the period before there is physiologically significant renal damage. No statistically significant increase in urinary 8-isoprostane (a marker of lipid peroxidation) or carbonylated proteins (a marker of protein oxidation) was found over the first 42 days after irradiation, while a small but statistically significant increase in urinary 8-hydroxydeoxy-guanosine (a marker of DNA oxidation) was detected at 35–55 days. When we examined renal tissue from these animals, we found no significant increase in either DNA or protein oxidation products over the first 89 days after irradiation. Using five different standard methods for detecting oxidative stress in vivo, we found no definitive evidence for radiation-induced renal chronic oxidative stress. If chronic oxidative stress is part of the pathogenesis of radiation nephropathy, it does not leave widespread or easily detectable evidence behind.

Ghosh, S. P., Perkins, M. W., Hieber, K., Kulkarni, S., Kao, T-C., Reddy, E. P., Reddy, M. V. R., Maniar, M., Seed, T. and Kumar, K. S. Radiation Protection by a New Chemical Entity, Ex-Rad^TM : Efficacy and Mechanisms. Radiat. Res. 171, 173–179 (2009).

Ex-Rad™ is among a series of small molecule kinase inhibitors developed for modifying cell cycle distribution patterns in cancer cells subjected to radiation therapy, and it has been identified as a potential candidate for radiation protection studies. We have investigated its radioprotective efficacy using mouse and in vitro models. Thirty-day survival studies with C3H/HeN male mice revealed 88% survival when 500 mg/kg of Ex-Rad was injected subcutaneously 24 h and 15 min before γ irradiation with 8.0 Gy. To understand Ex-Rad's mechanism of action, we also studied its radioprotective efficacy in lung fibroblast (HFL-1), skin fibroblast (AG1522) and human umbilical vein endothelial cells (HUVECs). Colony-forming assays indicated that Ex-Rad protected cells from radiation damage after exposure to ⁶⁰Co γ radiation. A study using single-cell gel electrophoresis (SCGE; also known as the alkaline comet assay) showed that Ex-Rad protected cells from radiation-induced DNA damage. Western blot analyses indicated that the radiation protection provided by Ex-Rad resulted in reduced levels of pro-apoptosis proteins such as p53 as well as its downstream regulators p21, Bax, c-Abl and p73, indicating that Ex-Rad could rescue cells from ionizing radiation-induced p53-dependent apoptosis. In conclusion, it appears that Ex-Rad's radioprotective mechanisms involve prevention of p53-dependent and independent radiation-induced apoptosis.

Shukla, J., Chatterjee, S., Thakur, V. S., Premachandran, S., Checker, R. and Poduval, T. B. l-Arginine Reverses Radiation-Induced Immune Dysfunction: The Need for Optimum Treatment Window. Radiat. Res. 171, 180–187 (2009).

The aim of the present study was to investigate the protective efficacy of l-arginine in mitigating the injury induced by 2 Gy of total-body γ radiation (TBI). Mice exposed to radiation (TBI group) had significantly decreased spleen weight, splenocyte numbers and bone marrow cellularity. Administration of l-arginine 2 h after TBI (TBI l-arginine group) was effective in reducing the radiation-induced depletion of spleen and bone marrow cellularity but was not effective when administered before TBI (l-arginine TBI group). The radiation-induced decrease in Con A-induced spleen cell proliferation, specific antibody response of spleen B cells to sheep red blood cells, and spleen RNA content was reversed in mice in the TBI l-arginine group. The radiation-induced increase in serum TNF-α levels, serum nitrate/nitrite (NOx) levels, spleen DNA fragmentation, spleen nitric oxide synthase (NOS) activity, spleen inducible NOS (iNOS) activity, and hepatic iNOS activity was reversed in mice in the TBI l-arginine group. l-Arginine administered before TBI could not reverse these changes. Mice in the TBI l-arginine group had significantly increased spleen arginase activity compared to mice from either the TBI or l-arginine TBI group. The results suggest the importance of the time of administration of l-arginine and the l-arginine pathway in mitigating the radiation-induced host immune dysfunction.

Kruse, J. J. C. M., Floot, B. G. J., Te Poele, J. A. M., Russell, N. S. and Stewart, F. A. Radiation-Induced Activation of TGF-β Signaling Pathways in Relation to Vascular Damage in Mouse Kidneys. Radiat. Res. 171, 188–197 (2009).

The purpose of this study was to investigate the long-term effects of radiation-induced alterations in TGF-β signaling pathways with respect to the development of vascular damage in the irradiated kidney. Total RNA was isolated from mouse kidneys at 1–30 weeks after irradiation, and quantitative real-time PCR analyses were performed for TGF-β receptors (ALK1, ALK5, endoglin), downstream mediators (Smad7, CTGF), and downstream targets (PAI-1 and Id-1). Expression of endoglin and Smad7 protein as well as nucleo-cytoplasmic distribution of phospho Smad 2/3 and phospho Smad 1/5 was analyzed by immunohistochemistry. Radiation caused a rapid and persistent increase in expression of TGF-β receptors and mediators from 1–30 weeks after treatment. Expression of Id-1, a downstream target of endothelial cell specific receptor ALK1, was transiently increased (1–10 weeks after irradiation) but returned to control levels at later times. Expression of PAI-1, a downstream target of ALK5, increased progressively from 10–30 weeks after irradiation. These results show that radiation activated TGF-β signaling pathways in the kidney and shifted the balance in favor of ALK5 signaling, which generally inhibits endothelial cell proliferation and migration. We hypothesize that prolonged activation of ALK5 signaling and relative suppression of ALK1 signaling may provide an explanation for the telangiectatic phenotype observed in irradiated kidneys.

Toyokuni, H., Maruo, A., Suzuki, K. and Watanabe, M. The Contribution of Radiation-Induced Large Deletion of the Genome to Chromosomal Instability. Radiat. Res. 171, 198–203 (2009).

Ionizing radiation is known to induce genomic instability that is transmitted across many generations of the progeny of surviving cells. However, the mechanism underlying the initiation, perpetuation and manifestation of radiation-induced genomic instability remains unclear. We expect that large radiation-induced deletions destabilize the structure of chromatin and that this destabilization is transmitted across many generations and plays a role in the perpetuation of genomic instability. Therefore, in this study, we examined the relationship between deletion size and the frequency of delayed chromosomal aberrations in SV40-immortalized normal human fibroblast (GM638) cells. GM638 cells were irradiated with 3 Gy of X rays, and chromosomal aberrations were analyzed in clones derived after irradiation. To determine the size of each deletion, we isolated mutants of the HPRT gene from the X-irradiated cell population and examined the genes around the HPRT locus, which is located in the q-arm of chromosome X. The results indicated that X chromosomes with large (>0.5 Mb) deletions have a higher probability of exhibiting delayed chromosomal aberrations and that these aberrations were induced more frequently in q-arms than in p-arms. Because no induction of X-chromosomal instability was observed in clones that lacked such large deletions, the present findings suggest that chromosomes with large radiation-induced deletions can be genomically unstable.

Whiteside, J. R. and McMillan, T. J. A Bystander Effect is Induced in Human Cells Treated with UVA Radiation but Not UVB Radiation. Radiat. Res. 171, 204–211 (2009).

The bystander effect is defined as the induction of damage in nonirradiated cells by irradiated cells. The significance on health is unknown, but the effective amplification of deleterious effects of cytotoxic agents is a concern. In this study the ability of UVA or UVB radiation to induce the bystander effect in human keratinocytes (HaCaT) and fibroblasts (MRC5) was examined using a method that involved the co-incubation of two differentially treated cell populations separated by a medium-permeable insert. This allowed the study of interactions between cells in the absence of direct cell-to-cell contact. Irradiation of one population with 100 kJ/m² UVA radiation induced a bystander effect in a second population of unirradiated cells that was manifested as reduced clonogenic survival. This effect was induced within and between the two cell lines but was not seen after treatment with 400 J/m² UVB radiation. An additional reduction in survival above that expected to occur as a result of direct exposure was observed when the two UVA-irradiated populations were co-incubated. As well as providing some potentially important information regarding the biological effects of UV light, the spectral variation in the induction of the effect provides a useful approach to dissecting the mechanisms underlying such effects.

Hirayama, R., Ito, A., Tomita, M., Tsukada, T., Yatagai, F., Noguchi, M., Matsumoto, Y., Kase, Y., Ando, K., Okayasu, R., and Furusawa, F. Contributions of Direct and Indirect Actions in Cell Killing by High-LET Radiations. Radiat. Res. 171, 212–218 (2009).

The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/μm in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/μm. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/μm, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/μm, and decreased thereafter. When the RBE was estimated separately for direct action (RBE_D) and indirect action (RBE_I); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/μm. However, the peak value was much higher for RBE_D (5.99) than RBE_I (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.

Tsukimoto, M., Homma, T., Mutou, Y. and Kojima, S. 0.5 Gy Gamma Radiation Suppresses Production of TNF-α through Up-regulation of MKP-1 in Mouse Macrophage RAW264.7 Cells. Radiat. Res. 171, 219–224 (2009).

Low- or intermediate-dose γ radiation appears to have the capacity to ameliorate certain types of diseases, including allergic conditions, when examined under specific exposure conditions and with specific animal models, though the molecular mechanisms involved remain to be fully clarified. We investigated the anti-inflammatory effects of intermediate-dose γ radiation by examining its effects on the activation state of p38 MAPK and the production of cytokines in mouse macrophage RAW264.7 cells. Dephosphorylation of both ERK1/2 and p38 MAPK was observed at 15 min after irradiation (0.5–1 Gy from a ¹³⁷Cs source) concomitant with a significant increase in the expression of MKP-1, which dephosphorylates ERK1/2 and p38 MAPK. Since activated p38 MAPK mediates TNF-α production, we examined the effect of radiation on LPS-induced activation of p38 MAPK and TNF-α production. The activation of p38 MAPK and production of TNF-α induced by LPS treatment were both suppressed in preirradiated cells. LPS-induced production of TNF-α was enhanced by knockdown of MKP-1. These results indicate that 0.5 Gy γ radiation would cause up-regulation of MKP-1, leading to inactivation of p38 MAPK and suppression of TNF-α production, in cells of mouse macrophages cell line.

Reitz, G., Berger, T., Bilski, P., Facius, R., Hajek, M., Petrov, V., Puchalska, M., Zhou, D., Bossler, J., Akatov, Y., Shurshakov, V., Olko, P., Ptaszkiewicz, M., Bergmann, R., Fugger, M., Vana, N., Beaujean, R., Burmeister, S., Bartlett, D., Hager, L., Pálfalvi, J., Szabó, J., O'Sullivan, D., Kitamura, H., Uchihori, Y., Yasuda, N., Nagamatsu, A., Tawara, H., Benton, E., Gaza, R., McKeever, S., Sawakuchi, G., Yukihara, E., Cucinotta, F., Semones, E., Zapp, N., Miller, J. and Dettmann, J. Astronaut's Organ Doses Inferred from Measurements in a Human Phantom Outside the International Space Station. Radiat. Res. 171, 225–235 (2009).

Space radiation hazards are recognized as a key concern for human space flight. For long-term interplanetary missions, they constitute a potentially limiting factor since current protection limits for low-Earth orbit missions may be approached or even exceeded. In such a situation, an accurate risk assessment requires knowledge of equivalent doses in critical radiosensitive organs rather than only skin doses or ambient doses from area monitoring. To achieve this, the MATROSHKA experiment uses a human phantom torso equipped with dedicated detector systems. We measured for the first time the doses from the diverse components of ionizing space radiation at the surface and at different locations inside the phantom positioned outside the International Space Station, thereby simulating an extravehicular activity of an astronaut. The relationships between the skin and organ absorbed doses obtained in such an exposure show a steep gradient between the doses in the uppermost layer of the skin and the deep organs with a ratio close to 20. This decrease due to the body self-shielding and a concomitant increase of the radiation quality factor by 1.7 highlight the complexities of an adequate dosimetry of space radiation. The depth-dose distributions established by MATROSHKA serve as benchmarks for space radiation models and radiation transport calculations that are needed for mission planning.

Morgan, N. Y., Kramer-Marek, G., Smith, P. D., Camphausen, K. and Capala, J. Nanoscintillator Conjugates as Photodynamic Therapy-Based Radiosensitizers: Calculation of Required Physical Parameters. Radiat. Res. 171, 236–244 (2009).

The recent demonstration of nanoscale scintillators has led to interest in the combination of radiation and photodynamic therapy. In this model, scintillating nanoparticles conjugated to photosensitizers and molecular targeting agents would enhance the targeting and improve the efficacy of radiotherapy and extend the application of photodynamic therapy to deeply seated tumors. In this study, we calculated the physical parameters required for these nanoparticle conjugates to deliver cytotoxic levels of singlet oxygen at therapeutic radiation doses, drawing on the published literature from several disparate fields. Although uncertainties remain, it appears that the light yield of the nanoscintillators, the efficiency of energy transfer to the photosensitizers, and the cellular uptake of the nanoparticles all need to be fairly well optimized to observe a cytotoxic effect. Even so, the efficacy of the combination therapy will likely be restricted to X-ray energies below 300 keV, which limits the application to brachytherapy.

Chun, K. J., Park, Y., Choi, Y. and Hyun, S. A Numerical Approach to Dose Optimization for Moving Targets Using Monte Carlo Simulations. Radiat. Res. 171, 245–253 (2009).

A novel simulation model for the dose distribution of moving targets for high-energy photons was analyzed using the EGSnrc Monte Carlo simulation. We provide here a fundamental numerical framework for the calculation of doses delivered to moving tissues in respiratory systems with improved accuracy. A spherical object with periodic motions inside a water phantom irradiated with incident photons was taken into consideration. The dose distributions of the target and its surrounding region were calculated for a variety of radiation conditions such as photon energy, beam numbers, and beam orientations as well as the target motions determined by realistic respiratory patterns. To determine the optimal dose, two parameters, the average absorbed dose ratio and dose deviation, were newly defined for the moving targets in the phantom. Optimal conditions were examined for treatment planning on tumors in motions based on the defined parameters. We found that the actual doses delivered to the tumor generally were not correlated to the respiratory patterns. Our quantitative assessment suggests useful guidelines for improved clinical radiotherapy to escalate dose concentration in the tumors by using multiple photon beams.

Taguchi, M., Kimura, A., Watanabe, R. and Hirota, K. Estimation of Yields of Hydroxyl Radicals in Water under Various Energy Heavy Ions. Radiat. Res. 171, 254–263 (2009).

This article reports the determination of yields of OH (hydroxyl) radicals in water irradiated with helium, carbon, neon and argon ions ranging from 2 to 18 MeV/nucleon. The yields of the OH radicals depend on the atomic number and energy of the incident ion and the reaction time just after the irradiation based on the track structure theory. The yields of the OH radicals estimated by analyzing the yields of the irradiation products from phenol were at almost 0 to 3.1 per 100 eV absorbed energy on a time scale from 0.75 to 300 ns and were lower than the corresponding ones after exposure to low-LET radiation. The yields of OH radicals decreased with decreasing specific energy for each ion, with increasing atomic number of each ion at a similar specific energy, and with the average reaction time after irradiation. In addition, Monte Carlo simulations were conducted and compared with the OH radical yields obtained experimentally.