Spicakova, T., McCann, K. and Brown, J. M. A Role for Lsm1p in Response to Ultraviolet-Radiation Damage in Saccharomyces cerevisiae. Radiat. Res. 170, 411–421 (2008).

A genome-wide screen in Saccharomyces cerevisiae identified LSM1 as a new gene affecting sensitivity to ultraviolet (UV) radiation. Lsm1p is a member of a cytoplasmic complex composed of Lsm1p–7p that interacts with the yeast mRNA degradation machinery. To investigate the potential role of Lsm1p in response to UV radiation, we constructed double mutant strains in which LSM1 was deleted in combination with a representative gene from each of three known yeast DNA repair pathways. Our results show that lsm1Δ increases the UV-radiation sensitivity of the rad1Δ and rad51Δ mutants, but not the rad18Δ mutant, placing LSM1 within the post-replication repair/damage tolerance pathway (PRR). When combined with other deletions affecting PRR, lsm1Δ increases the UV-radiation sensitivity of the rev3Δ, rad30Δ and pol30-K164R mutants but not rad5Δ. Furthermore, the UV-radiation sensitivity phenotype of lsm1Δ is partially rescued by mutations in genes involved in 3′ to 5′ mRNA degradation, and mutations predicted to function in RNA interactions confer the most UV-radiation sensitivity. Together, these results suggest that Lsm1p may confer protection against UV-radiation damage by protecting the 3′ ends of mRNAs from exosome-dependent 3′ to 5′ degradation as part of a novel RAD5-mediated, PCNA-K164 ubiquitylation-independent subpathway of PRR.

Acevedo, S. F., McGinnis, G. and Raber, J. Effects of ¹³⁷Cs γ Irradiation on Cognitive Performance and Measures of Anxiety of Apoe^−/− and Wild-Type Female Mice. Radiat. Res. 170, 422–428 (2008).

Mice deficient in apoE (Apoe^−/−) can be used to assess the potential role of apoE in the effects of cranial irradiation on hippocampal function. Radiation-induced impairments in hippocampal function may be more pronounced in female Apoe^−/− mice and more pronounced in mice irradiated and tested cognitively later in life. To assess this possibility, female wild-type and Apoe^−/− mice were irradiated at 6 months of age with 10 Gy ¹³⁷Cs γ rays and tested cognitively 3 months later. Sham-irradiated wild-type female mice showed enhanced hippocampal-dependent novel location recognition compared to sham-irradiated Apoe^−/− female mice. However, cranial irradiation impaired novel location recognition similarly in both genotypes. Cranial irradiation also impaired hippocampal-dependent spatial memory retention similarly in wild-type and Apoe^−/− female mice in the water maze. Because novel location recognition was not affected after ¹³⁷Cs γ irradiation in younger mice, these data support the possibility that older mice are more susceptible to the effects of γ radiation on novel location recognition. Together with the impairments in spatial memory retention in the water maze after irradiation, these data support the existence of detrimental effects of cranial irradiation on hippocampal function. In addition, compared to wild-type female mice, Apoe^−/− female mice showed enhanced levels of anxiety, and in Apoe^−/−, but not in wild-type, female mice, radiation decreased levels of anxiety. Because levels of anxiety during the hidden session of the water maze were associated with ability to locate the hidden platform, assessments of anxiety need to be considered in evaluating the effects of cranial irradiation on cognitive performance after cranial irradiation.

Tsukimoto, M., Nakatsukasa, H., Sugawara, K., Yamashita, K. and Kojima, S. Repeated 0.5-Gy γ Irradiation Attenuates Experimental Autoimmune Encephalomyelitis with Up-regulation of Regulatory T Cells and Suppression of IL17 Production. Radiat. Res. 170, 429–436 (2008).

We recently reported attenuation of autoimmune disease by low-dose γ irradiation (repeated doses of 0.5 Gy) in MRL-lpr/ lpr mice. Here we studied the effect of low-dose γ irradiation on experimental autoimmune encephalomyelitis (EAE), which is a rodent model of multiple sclerosis. SJL/J mice were immunized with myelin basic protein for the induction of EAE and were exposed to 0.5 Gy γ rays once a week for 4 weeks. Radiation suppressed incidence and disease severity scores and delayed the onset of pathological changes. Pro-inflammatory cytokines (IFN-γ and IL6), autoantibody and CD8 cytotoxic T cells are involved in the appearance of EAE. Radiation suppressed the increases in these cytokines and the autoantibody production and blocked the increase in the population of CD8 cytotoxic T cells. Production of IL17 by Th17 cells and/or γδ T cells, which plays a crucial role in EAE, was also significantly suppressed by radiation. Furthermore, a significant increase in regulatory T cells, which are involved in suppression of autoimmune disease, was found in irradiated EAE mice. These data suggest that low-dose γ irradiation attenuates EAE through suppression of pro-inflammatory cytokines, reduction of cytotoxic T cells and induction of regulatory T cells.

Epperly, M. W., Dixon, T., Wang, H., Schlesselman, J., Franicola, D. and Greenberger, J. S. Modulation of Radiation-Induced Life Shortening by Systemic Intravenous MnSOD-Plasmid Liposome Gene Therapy. Radiat. Res. 170, 437–443 (2008).

To determine whether systemic administration of MnSOD-PL protected mice from the acute hematopoietic syndrome and delayed death after total-body irradiation (TBI), C57BL/ 6J mice were injected intravenously with 100 μl liposomes containing 100 μg of human MnSOD-transgene plasmid 24 h prior to irradiation with 9.5 Gy or 1.0 Gy. The dose of 9.5 Gy was lethal to 42% of irradiated control female mice and 74% of irradiated control male mice at 30 days, with bone marrow hypocellularity consistent with the hematopoietic syndrome. A statistically significant increase in survival was observed in MnSOD-PL-treated female mice out to 400 days and in male mice out to 340 days. The incidence of tumors was similar between surviving groups. Between 350 and 600 days, the outcome was similar for both MnSOD-PL-treated and control irradiated groups, consistent with aging, with no difference in gross or microscopic pathological evidence of tumors. Male and female mice receiving 1.0 Gy TBI showed radiation-induced life shortening after 120 days that was decreased by MnSOD-PL administration and that was not associated with an increase in rate of tumor-associated death. Therefore, systemic MnSOD-PL radioprotective gene therapy is not associated with a detectably higher incidence of late carcinogenesis.

Zong, Z-W., Cheng, T-M., Su, Y-P., Ran, X-Z., Shen, Y., Li, N., Ai, G-P., Dong, S-W. and Xu, H. Recruitment of Transplanted Dermal Multipotent stem Cells to the Sites of Injury in Rats with Combined Radiation and Wound Injury by Interaction of SDF-1 and CXCR4. Radiat. Res. 170, 444–450 (2008).

Systemic transplantation of dermal multipotent stem cells has been shown to accelerate both hematopoietic recovery and wound healing in rats with combined radiation and wound injury. In the present study, we explored the mechanisms governing the recruitment of dermal multipotent stem cells to the sites of injury in rats with combined injury. Male dermal multipotent stem cells were transplanted into female rats, and using quantitative real-time PCR for the sex-determining region of Y chromosome, it was found that the amounts of dermal multipotent stem cells in irradiated bone marrow and wounded skin were far greater than those in normal bone marrow and skin (P < 0.01). However, incubation of dermal multipotent stem cells with AMD3100 before transplantation, which specifically blocks binding of stromal cell-derived factor 1 (SDF-1) to its receptor CXCR4, diminished the recruitment of dermal multipotent stem cells to the irradiated bone marrow and wounded skin by 58 ± 4% and 60 ± 4%, respectively (P < 0.05). In addition, it was confirmed that the expression of SDF-1 in irradiated bone marrow and wounded skin was up-regulated compared to that in their normal counterparts, and in vitro analysis revealed that irradiated bone marrow and wounded skin extracts had a strong chemotactic effect on dermal multipotent stem cells but that the effect decreased significantly when dermal multipotent stem cells were preincubated with AMD3100 (P < 0.05). These data suggest that transplanted dermal multipotent stem cells were recruited more frequently to the irradiated bone marrow and wounded skin than normal bone marrow and skin and that the interactions of SDF-1 and CXCR4 played a crucial role in this process.

Fujiwara, S., Suyama, A., Cologne, J. B., Akahoshi, M., Yamada, Y., Suzuki, G., Koyama, K., Takahashi, N., Kasagi, F., Grant, E. J., Lagarde, F., Hsu, W. L., Furukawa, K., Ohishi, W., Tatsukawa, Y. , Neriishi, K., Takahashi, I., Ashizawa, K., Hida, A., Imaizumi, M., Nagano, J., Cullings, H. M., Katayama, H., Ross, N. P., Kodama, K. and Shore, R. E. Prevalence of Adult-Onset Multifactorial Disease among Offspring of Atomic Bomb Survivors. Radiat. Res. 170, 451–457 (2008).

The first study to examine whether parental radiation exposure leads to increased heritable risk of common adult-onset multifactorial diseases (i.e., hypertension, diabetes mellitus, hypercholesterolemia, ischemic heart disease, and stroke) was conducted among 11,951 participants in the clinical examination program out of a potential of 24,673 mail survey subjects who were offspring of survivors born from May 1946 through December 1984. Logistic regression analyses demonstrated no evidence of an association between the prevalence of multifactorial diseases in the offspring and parental radiation exposure, after adjusting for age, city, gender and various risk factors. The odds ratio (OR) for a paternal dose of 1 Gy was 0.91 [95% confidence interval (CI) 0.81–1.01, P = 0.08], and that for a maternal dose of 1 Gy was 0.98 (95% CI 0.86–1.10, P = 0.71). There was no apparent effect of parental age at exposure or of elapsed time between parental exposure and birth, but male offspring had a low odds ratio (OR = 0.76 at 1 Gy) for paternal exposure, but cautious interpretation is needed for this finding. The clinical assessment of nearly 12,000 offspring of A-bomb survivors who have reached a median age of about 50 years provided no evidence for an increased prevalence of adult-onset multifactorial diseases in relation to parental radiation exposure.

Pignalosa, D., Bertucci, A., Gialanella, G., Grossi, G., Manti, L., Pugliese, M., Scampoli, P. and Durante, M. Chromosome Inter- and Intrachanges Detected by Arm-Specific DNA Probes in the Progeny of Human Lymphocytes Exposed to Energetic Heavy Ions. Radiat. Res. 170, 458–466 (2008).

We measured residual cytogenetic damage in the progeny of human peripheral blood lymphocytes exposed to 1 GeV/ nucleon iron ions or γ rays. Arm-specific DNA probes for chromosome 1 were used to detect aberrations as a function of dose in cells harvested 144 h after exposure. In addition, arm-specific mFISH was applied to samples exposed to a single dose of 2 Gy. These methods allowed the detection of interarm intrachanges (pericentric inversions) in addition to interchanges. The ratio of these types of aberrations (F ratio) has been proposed as a fingerprint of exposure to densely ionizing radiation. The fractions of aberrant cells in the progeny of cells exposed to iron ions were similar to those in the population exposed to γ rays, possibly because many rearrangements induced by heavy ions ultimately lead to cell death. Simple inter- and intrachanges were also similar, but more complex rearrangements were found in cells that survived after exposure to iron ions. We did not find a significant difference in the ratio of simple interchanges to simple intrachanges for the two radiation types. However, iron ions induced a much higher frequency of events involving both inter- and intrachanges. We conclude that these complex rearrangements represent a hallmark of exposure to heavy ions and may be responsible of the decrease of the F ratio with increasing LET reported in the literature in some in vitro and in vivo experiments.

Anzenberg, V., Chandiramani, S. and Coderre, J. A. LET-Dependent Bystander Effects Caused by Irradiation of Human Prostate Carcinoma Cells with X Rays or Alpha Particles. Radiat. Res. 170, 467–476 (2008).

Radiation-induced bystander effects have been demonstrated in both normal and tumor cells using a variety of different radiation qualities. Literature reports are contradictory, however, on whether there is an LET dependence of the bystander effect. This study investigated the ability of DU-145 human prostate carcinoma cells irradiated with either α particles or 250 kVp X rays to cause medium-mediated bystander effects in unirradiated populations of DU-145 cells or in AG01522 human fibroblasts. The end points measured in both of the bystander cell lines were micronucleus formation, γ-H2AX focus induction, and the surviving fraction. The incidence of micronuclei increased 1.5–2.0-fold in both tumor and fibroblast bystander cells after 4 h of co-culture with DU-145 tumor cells that had been directly irradiated with either α particles or X rays. Only the AG01522 fibroblasts showed bystander effects for the γ-H2AX focus (a 1.5-fold increase) and surviving fraction (a decrease to 0.8) end points when co-cultured with X-irradiated tumor cells. Alpha-particle irradiation of DU-145 tumor cells produced no decrease in the surviving fraction and no increase in γ-H2AX focus induction in co-cultured bystander cells of either cell line. These results indicate that there are LET-dependent differences in the signal released from DU-145 human prostate carcinoma cells and that, for some end points, bystander AG01522 fibroblasts and bystander DU-145 prostate carcinoma cells respond differently to the same medium-mediated signal.

Jiang, H., Xu, Y., Li, W., Ma, K., Cai, L. and Wang, G. Low-Dose Radiation Does Not Induce Proliferation in Tumor Cells In Vitro and In Vivo. Radiat. Res. 170, 477–487 (2008).

We have demonstrated that exposure of mice to low-dose radiation (75 mGy) stimulated bone marrow cell proliferation and peripheral mobilization (Li et al., Exp. Hematol. 32, 1088– 1096, 2004). It is unclear whether such stimulating effects induced by low-dose radiation can also occur in tumor cells. In the present study, cells of two leukemia cell lines and five solid tumor cell lines together with four normal human cell lines were used to determine whether exposure to low-dose radiation (25 to 200 mGy X rays) can cause a stimulating effect on cell proliferation. A stimulating effect was found in the normal cell lines but not in the two leukemia and five solid tumor cell lines in response to low-dose radiation exposure in vitro. Examination of cell cycling changes and cell death for these cells by flow cytometry at different times after low-dose irradiation did not identify any changes attributable to the distinct effects of low-dose radiation on cell proliferation between tumor and normal cells. To provide further evidence for the absence of low-dose radiation-induced stimulating effects in tumor cells in vivo, cells of two solid tumor cell lines were implanted in nude mice. Exposure of tumor cells in vitro before implantation in nude mice or of tumor-bearing mice to low-dose radiation (75 mGy X rays) did not stimulate tumor growth compared to the tumor-bearing mice without low-dose radiation exposure. These results suggest that low-dose radiation stimulates growth of normal cells but not of leukemia and solid tumor cells in vitro and also does not stimulate growth of solid tumor cells in vivo.

Franzellitti, S., Valbonesi, P., Contin, A., Biondi, C. and Fabbri, E. HSP70 Expression in Human Trophoblast Cells Exposed to Different 1.8 GHz Mobile Phone Signals. Radiat. Res. 170, 488–497 (2008).

The heat-shock proteins (HSPs) are important cellular stress markers and have been proposed as candidates to infer biological effects of high-frequency electromagnetic fields (EMFs). In the current study, HSP70 gene and protein expression were evaluated in cells of the human trophoblast cell line HTR-8/SVneo after prolonged exposure (4 to 24 h) to 1.8 GHz continuous-wave (CW) and different GSM signals (GSM-217Hz and GSM-Talk) to assess the possible effects of time and modulation schemes on cell responses. Inducible HSP70 protein expression was not modified by high-frequency EMFs under any condition tested. The inducible HSP70A, HSP70B and the constitutive HSC70 transcripts did not change in cells exposed to high-frequency EMFs with the different modulation schemes. Instead, levels of the inducible HSP70C transcript were significantly enhanced after 24 h exposure to GSM-217Hz signals and reduced after 4 and 16 h exposure to GSM-Talk signals. As in other cell systems, in HTR-8/SVneo cells the response to high-frequency EMFs was detected at the mRNA level after exposure to amplitude-modulated GSM signals. The present results suggest that the expression analysis for multiple transcripts, though encoding the same or similar protein products, can be highly informative and may account for subtle changes not detected at the protein level.

Wrzal, P. K., Bettaieb, A. and Averill-Bates, D. A. Molecular Mechanisms of Apoptosis Activation by Heat Shock in Multidrug-Resistant Chinese Hamster Cells. Radiat. Res. 170, 498–511 (2008).

Multidrug resistance (MDR) is a major obstacle to the success of chemotherapy in cancer treatment and is associated with overexpression of P-glycoprotein. MDR cells, aside from resistance to chemotherapy, might also inhibit apoptosis at various levels in the death signaling pathways. Currently, hyperthermia is used in cancer treatment to sensitize tumor cells to radiation and/or chemotherapy. This study investigated the induction of death receptor and mitochondria-mediated signaling pathways of apoptosis by hyperthermia (41–43°C) in MDR CH^RC5 cells compared to drug-sensitive AuxB1 Chinese hamster ovary cells. In the receptor-mediated pathway, CH^RC5 cells exhibited higher levels of c-FLIP and lower caspase 8 and caspase 10 activation in response to hyperthermia. In the mitochondria-mediated pathway of heat-induced apoptosis, CH^RC5 cells showed higher mitochondrial levels of Bax and tBid, more pronounced mitochondrial membrane depolarization, and increased Apaf-1. Similar levels of caspase 3 activation and cleavage of caspase substrates occurred, showing that overall, CH^RC5 cells are not resistant to hyperthermia-induced apoptosis compared to AuxB1 cells. This study reveals for the first time the molecular mechanisms of hyperthermia-induced apoptosis in MDR cells overexpressing P-glycoprotein. CH^RC5 and AuxB1 cells showed similar clonogenic survival responses to heat, which implies that hyperthermia could be a promising strategy for eradicating MDR tumor cells in the clinic.

Wu, W., Wang, M., Mussfeldt, T. and Iliakis, G. Enhanced Use of Backup Pathways of NHEJ in G₂ in Chinese Hamster Mutant Cells with Defects in the Classical Pathway of NHEJ. Radiat. Res. 170, 512–520 (2008).

In higher eukaryotes DNA double-strand breaks (DSBs) are repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) using DNA ligase III and PARP1. We have reported previously that mouse embryo fibroblasts (MEFs) defective in D-NHEJ show enhanced repair of DSBs in G₂ not reflecting a contribution of HRR. Here we extend these studies to Chinese hamster mutant cells with defects in the DNA-PKcs, Ku80 or XRCC4 components of D-NHEJ or in the XRCC2 and XRCC3 components of HRR. Using cell sorting to separate cells at defined times after irradiation, we measure repair of DSBs with pulsed-field gel electrophoresis in unperturbed G₁- and G₂-phase cells. Wild-type cells and mutants of XRCC2 and XRCC3 repair DSBs with similar efficiency in G₁ and G₂. Mutants of DNA-PKcs, Ku80 and XRCC4 show more pronounced repair in G₂ than in G₁. These and previously published results provide support for the notion that the increased efficacy of DSB repair in G₂ reflects the enhanced function of B-NHEJ, which may be a general feature of rodent cells that also holds for human cells.

Yamashita, S., Katsumura, Y., Lin, M., Muroya, Y., Miyazaki, T., Murakami, T., Meesungnoen, J. and Jay-Gerin, J-P. Water Radiolysis with Heavy Ions of Energies up to 28 GeV. 3. Measurement of G(MV^· ) in Deaerated Methyl Viologen Solutions Containing Various Concentrations of Sodium Formate and Monte Carlo Simulation. Radiat. Res. 170, 521–533 (2008).

Formation yields of methyl viologen cation radicals G(MV^· ) (100 eV)⁻¹ have been measured in deaerated aqueous solutions of 0.25 mM methyl viologen (MV² ) containing various concentrations of formate anion (0.01–2 M) after irradiation with six different ion beams (⁴He² , ¹²C⁶ , ²⁰Ne¹⁰ , ²⁸Si¹⁴ , ⁴⁰Ar¹⁸ and ⁵⁶Fe²⁶ with incident energies varying from 0.6 to 28 GeV) provided by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. The sample solutions are irradiated at the incident energy of the ions using 1-cm irradiation cells. Corresponding LET values cover the range from 2.2 to 185 eV/nm. G(MV^· ) increases with increasing formate concentration. In ⁴He² radiolysis, it increases from 5.7 to 7.1 as the concentration of formate is increased from 0.01 to 2 M, while in ⁵⁶Fe²⁶ radiolysis, the MV^· yield value changes from 2.2 to 4.1. The other values lie between the yields for ⁴He² and ⁵⁶Fe²⁶ . In addition, G(MV^· ) decreases with increasing LET. In the case of ¹²C⁶ radiolysis, G(MV^· ) increases with increasing energy of the carbon ions from 135 to 400 MeV/nucleon, i.e., with decreasing LET from 21 to 11 eV/nm. In parallel to the above measurements, Monte Carlo simulations of the radiolysis of the MV² /formate solutions have been performed. Ionic strength effects on reactions between charged species are taken into account. To reproduce the experimental results, previously unreported reactions such as e⁻_aq MV^· , MV^{· ·}OH and ^·COO^{− ·}OH have been introduced in the reaction scheme. After optimization, the rate constants of these latter two reactions are determined to be (3 ± 0.5) × 10¹⁰ and (5 ± 0.5) × 10¹⁰ M⁻¹ s⁻¹, respectively. By contrast, the reaction between e⁻_aq and MV^· is too slow to affect G(MV^· ). On the basis of these calculations, characteristics of intratrack reactions induced by heavy-ion beams are discussed in reference not only to the scavenger method used for measurement of water decomposition product yields but also to the differences in the relative spatial distribution of the reactants as well as the places where their intratrack reaction occurs within the geometry of the ion track structure.

Igarashi, K. and Miura, M. Inhibition of a Radiation-Induced Senescence-Like Phenotype: A Possible Mechanism for Potentially Lethal Damage Repair in Vascular Endothelial Cells. Radiat. Res. 170, 534–539 (2008).

The well-established process of potentially lethal damage (PLD) repair enhances plateau-phase cell survival after exposure to ionizing radiation. PLD repair requires that confluent cells be incubated prior to plating for a colony-forming assay rather than being plated immediately. Enhanced double-strand break (DSB) repair during this incubation period has been implicated in the enhanced survival, but the precise molecular mechanism and its biological significance remain largely unclear. Radiation has been recently reported to induce premature senescence, and increasing evidence suggests that DSBs commonly mediate cellular senescence. Here we successfully related these two biological phenomena using bovine aortic endothelial cells, and propose that enhanced DSB repair during the plateau-phase incubation prevents expression of the radiation-induced senescence-like phenotype, eventually leading to an enhanced colony-forming ability. This could be a novel biological interpretation of PLD repair.

Cassatt, D. R., Kaminski, J. M., Hatchett, R. J., DiCarlo, A. L., Benjamin, J. M. and Maidment, B. W. Medical Countermeasures against Nuclear Threats: Radionuclide Decorporation Agents. Radiat. Res. 170, 540–548 (2008).

Exposure to radionuclides disseminated by a radiological dispersion device or deposited as fallout after a nuclear power plant accident or detonation of an improvised nuclear device could result in internal contamination of a significant number of individuals. Internalized radionuclides may cause both acute and chronic radiation injury and increase an individual's risk of developing cancer. This damage and risk can be mitigated by the use of decorporation agents that reduce internal contamination. Unfortunately, most effective agents decorporate only a limited range of radionuclides, and some are formulated in ways that would make administration in mass casualty situations challenging. There is a need for new radionuclide decorporation agents, reformulations of existing agents, and/or expansion of the labeled indications for existing treatments. Researchers developing novel or improved decorporation agents should also understand the regulatory pathway for these products. This workshop, the first in nearly half a century to focus exclusively on radionuclide decorporation, brought together researchers and scientific administrators from academia, government and industry as well as senior regulatory affairs officers and U.S. Food and Drug Administration personnel. Meeting participants reviewed recent progress in the development of decorporation agents and contemplated the future of the field.