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Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. We demonstrated the existence of this phenomenon, called adaptive response (AR), in mice during late organogenesis. Molecular mechanisms underlying AR in this model were investigated. Using DNA microarrays, AR-specific gene modulations were identified. Our results suggested the involvement of signal transduction and p53-related pathways in the induction of AR. In order to validate microarray results and to gain better insights into molecular mechanisms of AR in fetal mice, we analyzed the expression kinetics of several target genes by real-time quantitative fluorescence RT-PCR in different organs of irradiated embryos. Whole-body expression of Csf1 gene was significantly different after exposure to AR-inducing and non AR-inducing priming radiation. Comparative gene expression in adapted and non-adapted fetuses will be presented. In an in vitro model of cultured limb bud cells from mouse fetuses, we checked p53 protein expression and phosphorylation levels on Ser18 following priming and challenging irradiations. After exposure to challenging dose alone or to 0.5 Gy priming dose (not efficient for inducing AR) before challenging radiation, p53 protein expression and phosphorylation, initially at low levels, peaked 4 to 6h after exposure to challenge dose. When priming dose was performed in AR-inducing conditions, these values followed similar time course, but reached lower peak level. The involvement of p53 protein in AR in this model will be discussed., New Nuclear Research Symposium "Biological Responses to Low Dose Radiation"

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were treated either with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or its combination with NNK at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks (DSBs) in DNA., New Nuclear Research Symposium "Biological Responses to Low Dose Radiation"

Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. This phenomenon, called radiation-induced adaptive response (AR), has been described in a wide range of biological models. We previously demonstrated the existence of AR in mice during late organogenesis. For better understanding of molecular mechanisms underlying AR in this model, we performed a global analysis of transcriptome regulations in cells collected from whole mouse fetuses, after in utero exposure to priming irradiation. Several combinations of radiation dose and dose-rate were applied to induce or not an AR in our system. While many genes were not modulated at all after radiation exposure and several genes were deregulated for all conditions, we identified a panel of genes (so called AR genes) showing significantly different expression ratios in AR and non AR-inducing conditions. Since AR genes were implicated in a variety of functions and cellular processes, we applied a functional classification algorithm which clustered genes in a limited number of functionally related groups. We also established that AR genes are significantly enriched for specific keywords. Our results suggested the involvement of signal transduction and p53-related pathways in the induction of AR, as well as possible consequences for subsequent developmental process. Even though no evidence of specific DNA repair modulation at the transcriptional level was observed in adapted cells, we studied H2AX phosphorylation kinetics following challenging irradiation in adapted and non-adapted fetal liver cells and observed that H2AX phosphorylation peak appeared earlier in adapted than in non adapted cells (while disappearance rates were similar), which could indeed suggest a possible modulation of DNA repair induced by priming radiation exposure. However, no modulation of radiation-induced cell death by AR was described in fetal liver cells. Taken together, our results present the first evidence of molecular mechanisms modulation by AR in utero, which may have potentially important consequences for further developmental processes, and could even influence tumorigenesis. The late effects occurring in mice adapted in utero, such as chromosomal aberrations or cancer initiation) therefore are a promising and interesting area for future research., New Nuclear Research Symposium on Biological Response to Low Dose Radiation- New Aspects of Low Dose Radiation Effects

Risk assessment of low-dose rate radiation is an important issue. Tanaka et. al. have previously reported the late effects of chronic exposure to low-dose rate gamma rays as assayed by life span using 4000 mice (Rad. Res. (2003)160, 376-379). Statistical analyses showed that the life spans of mice of both sexes irradiated with 16uGy/min(P < 0.0001) and of females irradiated with 830nGy/min(P < 0.05) were significantly shorter than those of the control group. In this study, we investigate the molecular mechanisms of this shortening of life span under continuous low-dose rate irradiation in mice. We followed experimental conditions of Tanaka et. al. C57BL/6J mice were exposed to gamma rays at the low-dose rates of 32nGy/min (21mGy), 650nGy/min (420mGy), 13uGy/min (8.0Gy) continuously. RNA was extracted from kidney of irradiated or on-irradiated mice, and gene expression profiles were determined using Illumina Sentric Mouse-6 microarrays. The 54 genes have been extracted with a difference in expression by 1.6 times or more after irradiation. The pathway analysis was done for the gene of each 54. In the irradiated groups at 650 nGy/min (total dose 420 mGy) and 13 uGy/min (total dose 8 Gy), 4 genes categorized to the mitochondrial oxidative phosphorylation pathway were activated. These results strongly suggest one possibility that the reactive oxygen produced by activated mitochondrial oxidative phosphorylation after continuous low-dose rate irradiation is a cause for the shortening of life span., 13th International Congress of Radiation Research

A radiation-induced adaptive response, which was judged as reduction of both fetal death and digital malformation, was demonstrated in fetal mice by priming low-dose radiation on E11 prior to a high-dose irradiation on E12. Suppression of the digital defects was correlated to the inhibition of radiation-induced apoptosis. The priming dose was mouse strain-related. Both Trp53 alleles were essential for induction of the adaptive response. Furthermore, existence of the dose-rate effect was verified for delivery of the priming dose. There was a high postnatal mortality in the offspring that survived from prenatal death. Postnatal retardation, alteration in adult behavior, and life span shortening were observed in the survivals. In addition, a significantly increased radiosensitivity to the killing effect was also observed in the postnatal survivals. These results indicate that radiation-induced adaptive response in fetal mice is due to a complex interplay between dose, dose rate and animal factors such as the strain, developmental stage, and gene background. Though the priming dose could rescue some mice from high dose irradiation, the survivals are not healthy as alterations in development, behavior and radiosensitivity were observed. This work was supported in part by the Budget for New Nuclear Crossover Research from the Ministry of education, Culture, Sports, Sciences and Technology, Japan., 13th International Congress of Radiation Research

Objectives: In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (, 2nd International Congress of Molecular Medicine

Radiation-induced adaptive response is the phenomenon of priming low-dose irradiation-induced resistance to subsequent irradiation at higher doses. As investigations on adaptive response provide important scientific basis for radiation risk estimates and offer significant insight into the novel biological defense mechanisms regarding protection against radiation, study on radiation-induced adaptive response is of great concern for both public health and academic research. It is demonstrated that the radiation-induced adaptive response exists in both procaryotic and eucaryotic cells. For the research work on mammalian systems, most of the studies were performed using cultured cells in vitro. Only a few works were done at the whole body level. Since the data of epidemiological studies in humans are insufficient, the results from animal experiments are extremely important. The in vivo studies using either mouse pre-implantation embryos or rat fetal brain have failed to show any positive data. Using fetal mice in utero, we demonstrated the existence of radiation-induced adaptive response and characterized the experimental conditions for the successful induction. To date, it is known that a variety of physical and biological conditions with respect to both irradiation and irradiated subjects are crucial to the induction of adaptive response. The biological endpoints employed are also of importance to confirmation of the existence or a judgment of the successful induction of adaptive response. Radiation induces high incidences of abnormalities and neonatal death in late organogenesis in mice. As gross malformations in limbs and digits are easy to be judged, prenatal fetal death and digital defects were used as the endpoints in our study. Thus, the radiation-induced adaptive response was judged as a priming low dose radiation-induced reduction of a subsequent high dose radiation-induced fetal death and prenatal digital defects. As the developing limb system in mice is widely used for correlating the initial pathological changes in early limb buds with final digital defects and digital defects were due to an excess induction of p53 dependent apoptosis by the high dose of irradiation, to study the cellular response under an adaptive response, investigation was conducted for the correlation between both the p53 gene status and the radiation-induced apoptosis with the induction of digital defects in fetal mice. In a series of our studies in fetal mice, existence of the radiation-induced adaptive response was demonstrated in both ICR and C57BL strain fetal mice by priming low dose radiation on gestation day 11 prior to a high dose irradiation on gestation day 12. It was found that suppression of the digital defects was correlated to the inhibition of radiation-induced apoptosis in the predigital regions. The efficient priming dose was mouse strain-related, namely, the efficient priming dose for ICR fetal mice was 0.3 Gy while either 0.05 Gy or 0.3 Gy was efficient for induction of an adaptive response in C57BL fetal mice. An adaptive response could not be induced in p53 heterozygous (p53+/-) fetal mice under the same conditions as those used in the p53 wild type (p53+/+) fetal mice. This indicated that both p53 alleles were essential for a successful induction of adaptive response in fetal mice. Furthermore, existence of the dose-rate effect was verified for delivery of the priming dose in ICR fetal mice. Adaptive response was observed within two dose-rate ranges for the same dose of priming irradiation, namely, 0.3 Gy was delivered either from 0.18 to 0.98 Gy/min or from 3.5 to 4.6 Gy/min on gestation day 11, for reduction of the detrimental effect induced by a challenging dose of 3.5 Gy on gestation day12. In the follow-up study on the offspring that survived from high dose induced prenatal death, a high postnatal mortality was observed. In addition, detrimental effects such as postnatal retardation, alteration in adult behavior, and life span shortening were also observed in these survivals. Furthermore, using 30-day survival test and radiation treatment conditions for induction of adaptive response in vivo in adult mice, the responses of the postnatal survivals to the lethal irradiation and to the adaptive response induction were investigated in C57BL mice. A significantly increased radiosensitivity to the killing effect was observed and the adaptive response could not be induced in these offspring. These findings suggested that some tissue damage, which was caused by fetal high dose irradiations, could not be completely repaired although the animals may survive. In summary, the results obtained in this experimental system indicated that radiation-induced adaptive response in fetal mice is due to a complex interplay between dose, dose rate and animal factors such as the strain, developmental stage, and genetic background. A priming low dose of irradiation, if delivered at certain restrictive conditions, i.e., at the efficient dose and dose rate, and on the specific developmental stage, could rescue some fetal mice from prenatal death induced by the subsequent high dose of irradiation, while the postnatal survivals are not as healthy as those non-irradiated controls, showing significant alterations in physiological maturation, neuroreflex, behavior, and lifespan. The postnatal survivals did not response to radiation the same as the control non-irradiated animals. This work was supported in part by the Budget for New Nuclear Crossover Research from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan., NIRS International Symposium on Visualization of Radiation Response at the Molecular and Cellular Levels

Risk estimation of ionizing radiation in the middle to high dose range has been mainly based on the epidemiological studies on Hiroshima and Nagasaki A-Bomb survivors. However, the importance of molecular and cellular studies on mechanisms for the biological effects should be emphasized due to the inherent limitation of the epidemiological studies at low levels of radiation exposure and risk. An increasing number of low-dose ionization radiation-responsive genes are being repoted in mouse and human cells. Analyses of these genes are considered to provide clues to the mechanisms for low-dose radiation effects, because the major ionizing radiation-protection mechanisms, including cell cycle checkpoint regulation and apoptosis, involve the regulation of gene expression in response to ionizing radiation. Radioactive response is a biological response, by which individuals/cells acquire radiation-resistance after exposure to low-dose radiation. This is observed only after exposure to low-dose radiation, and is therefore considered to be an important determination of low-dose radiation risk. In this paper, authors describe the regulation of gene expression in low dose range. Especially, transcriptional regulation involved in the radioadaptive response will be discussed., 20th CODATA International Conference

Health effects of low-dose-rate or low-dose radiation should be evaluated in combination with chemicals as humans are exposed to a variety of chemicals. Here, we report combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed at the molecular level. Female gpt delta transgenic mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. The NNK treatments enhanced the frequency of base substitutions significantly, but no obvious combined effects of irradiation were observed. In contrast, the NNK treatments appeared to suppress radiation-induced large deletions. The frequency of large deletions more than 1 kb in size increased in a dose-dependent manner of gamma irradiation. When NNK treatments were combined, the dose-response curve became bell-shaped where the frequency at the highest radiation dose decreased substantially. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized., 20th CODATA International Conference

Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. This phenomenon, called radioadaptation (or adaptive response - AR), has been described in a wide range of biological models. In a series of studies, we demonstrated the existence of a radiation-induced AR in mice during late organogenesis. For better understanding of molecular mechanisms underlying AR in our model, we performed a global analysis of transcriptome regulations in cells collected from whole mouse fetuses. Using cDNA microarrays, we studied gene expression in these cells after in utero priming exposure to irradiation. Several combinations of radiation dose and dose-rate were applied to induce or not an AR in our system. Gene regulation was observed after exposure to priming radiation in each condition. Student's t-test was performed in order to identify genes whose expression modulation was specifically different in AR-inducing and non-AR-inducing conditions. Genes were ranked according to their ability in discriminating AR-specific modulations. Since AR genes were implicated in a variety of functions and cellular processes, we applied a functional classification algorithm, which clustered genes in a limited number of functionally related groups. We established that AR genes are significantly enriched for specific keywords. Our results show a significant modulation of genes implicated in signal transduction pathways. No AR-specific alteration of DNA repair could be observed. Nevertheless, it is likely that modulation of DNA repair activity results, at least partly, from post-transcriptional regulation. One major hypothesis is that deregulations of signal transduction pathways and apoptosis may be responsible for AR phenotype. In previous work, we demonstrated that radiation-induced AR in mice during organogenesis is related to Trp53 gene status and to the occurrence of radiation-induced apoptosis. Other work proposed that p53 may contribute to AR induction by shifting DSB end-joining activity from illegitimate error-prone rejoining to error-free direct ligation. For all these reasons, particular attention was given to the transcriptional behavior of p53-related genes in our model. Although average gene regulation fold was relatively small, a significant proportion of p53-related genes showed AR-specific modulation, indicating a role for directly and indirectly p53-related pathways in the induction of AR. Even though no evidence of specific DNA repair modulation at the transcriptional level was observed in adapted cells, a large amount of data suggested that induction of AR may result from modification of DNA repair activity. We studied H2AX kinetics following challenging irradiation in adapted and non-adapted cells. H2AX phosphorylation increased shortly after irradiation, and then decreased progressively during the subsequent 24 hours. No significant difference was observed between the rate of disappearance of H2AX in adapted and non-adapted cells, indicating that AR in this model seems not to be related with a modulation of DNA break rejoining speed. However, the peak of H2AX phosphorylation appeared slightly earlier in adapted than in non-adapted cells. In accordance with other data, these results are compatible with the hypothesis that the low adapting dose could induce a repair mechanism that, if activated at the time of challenging exposure with high doses of radiation, would lead to less residual damage. In conclusion, we observed transcriptional deregulations resulting from exposure to low dose X-rays. Our results highlighted some specific transcriptional response of cells when the characteristics of irradiation were efficient for inducing AR, indicating that AR may result from specific molecular mechanisms induced by exposure to priming dose of irradiation. To our knowledge and to date, this is the first report of AR-specific transcriptional deregulations in fetal mouse., The 6th Japan-France Workshop on Radiobiology and Isotopic Imaging

Radiation has been reported to induce low-dose-specific effects on cells. Adaptive response and low-dose hypersensitivity observed in some kinds of cell types suggest that low-dose irradiation induces cell survival relating repair processes. However the relationship between cellular effects and molecular processes remains unknown in low-dose-specific effects. Here we tried to investigate low-dose-specific cell survival induction using the human breast cancer cell line (MCF7). Five hours after the inoculation of MCF7 cells, the cells were irradiated at 0.1, 0.25 0.5 and 1Gy. The cell survival rates of the irradiated cells decreased dose-dependently at 0.1, 0.5 and 1Gy. However, at 0.25Gy the cell survival rate increased relatively in comparison with the other points. Using kinase-specific substrate antibodies, changes in signaling cascades were studied as a phosphoproteome analysis in cell survival induction. Signaling pathways in low-dose-specific cell survival induction will be discussed with the data showing culture condition dependency., Keystone Symposia

For a cancer gene therapy, control of anti-tumoric transgene expression in radiation field by use of ionizing radiation-inducible promoters is one of the approaches for the tumor-specific gene delivery. The most widely utilized promoter for this purpose is that of the early growth response 1 gene (Egr-1). However induction of the Egr-1 promoter requires a high dose of radiation, and physical burden for the patients imposed by a substantial dose of radiation is inavoidable. Although tumor suppressor protein p53 is induced by low doses (, ヨーロッパ遺伝子治療学会第12回年会

Studies on adaptive response have been of great concern for both public health and academic research, which could bring significant insight into the biological defense mechanisms and offer critical scientific basis for radiation risk estimation and protection. In a series of our recent studies, the existence of radiation-induced adaptive response was revealed in late organogenesis in mice. Characterization of the essential conditions for the successful induction indicated that this phenomenon was due to a complex interplay between dose, dose rate, mouse strain, fetal developmental stage, and mouse gene background. Since the discovery of radiation-induced adaptive response by a cytogenetic study on chromosome aberrations in vitro, investigations to date have completed a large amount of cytogenetic data. However, for most of the reports claimed as the in vivo cytogenetic work, the challenging irradiation was usually performed in vitro to the collected cells that were exposed to a low dose or a priming irradiation in vivo. Such studies could only verify if these cells were capable of bringing into an adaptive response in vitro when receiving a challenging irradiation. Thus these studies are not actually in vivo works in the strict sense of the word. In fact in the last one and half decades, little has been done in the cytogenetic studies in vivo. In addition, most of these rare data were published in non-English language journals, which made the limited information even hard to be accessed to. No cytogenetic data are available on the acute changes in fetuses and late consequences in mammalians survived a challenging high dose of prenatal irradiation by virtue of an adaptive response. In the coming Crossover Research Project, a cytogenetic study both on prenatal mice irradiated with/without conditions to induce the adaptive response using fetal hepatocytes, and on postnatal survival mice using splenocytes, will be carried out. This study would provide some basic cytogenetic data on in vivo adaptive response in fetal mice. The cytogenetic data on the early chromosome damage and their timely progress from the fetal mice could contribute important information to possible differences in the occurrences of chromosome damage between the adapted animals and the non-adapted animals, and possible differences in the repair of chromosome damage in the finally survived animals as well. The postnatal cytogenetic data could also be of interest to comprehensively evaluate the life quality as well as to make risk estimation for the animals survived the lethal irradiation by the adaptive response., International Workshop on Biological Responses to Low Dose Radiation

Activation of tumor suppressor protein p53 following DNA damages results in different cellular responses depending on the cell type. For example, hematopietic cells were observed to undergo p53-dependent apoptosis more easily than fibroblasts [1]. Fei et al. [2] indicated that the selective regulation of p53 target gene could be a determinant of whether cells undergo cell cycle arrest or apoptosis. We consider that a similar selective regulation of p53 target genes may play an important role also in cellular response to low dose radiation. It has been revealed that low dose radiation causes distinct biological responses, such as adaptive responses, bystander effects and induction of genomic instability, which could not be observed in experiments using lethal dose of radiation. There is evidence for a pivotal role played by p53 in these low dose radiation-distinct responses [3-5]. As p53 is activated as a transcription factor by a wide range of radiation doses, it is considered that the mechanism for low dose radiation-distinct biological responses involve, at least in part, molecular consequences of selective regulation of p53 target genes. The selective regulation of the p53 target genes should be attributed to their promoter structure. We consider that several ways of distinguishing the target gene promoter by p53 are possible. First, transcription factor(s), that can cooperate or modify the p53 function, may be activated by low dose radiation and recognize a specific set of p53 target gene promoters. This is an analogy to the observation that p63 and/or p73 is necessary for regulation of proapoptotic genes, but not cell cycle arrest-related genes, by p53 after DNA damage [6]. In addition, it should be noted that a lot of transcription factors have been demonstrated to associate with p53, modulating its function in regulation of the downstream genes. For examples, transcription factors Sp1 [7, 8], gut-enriched kruppel-like factor (GKLF) [9], Ets1 [10], and interferon regulatory factor-1 (IRF-1) [11] have been shown to be involved in the p53-dependent transcriptional activation after DNA damage or p53-overexpression. It is interesting to examine if some of transcription factors may be activated to facilitate the selective regulation by p53 after low dose radiation. Second, p53 may be post-translationally modified by low dose radiation, so that the binding affinity to a specific set of p53 target gene promoters may be selectively changed. This is thought to be a possible way of selection of the target gene promoter, because the p53 consensus sequence has a remarkable redundancy. There is some evidence that phosphorylation of p53 on serine 46 causes the selective regulation of the p53 target gene by directing p53 towards activation of proapoptotic genes including p53Aip1 after DNA damage [12]. Alternatively, post-translational modification of p53, such as phosphorylation, acetylation, sumolation, ubiquitination, may enhance interaction with nuclear factors that facilitate selective gene regulation. It was revealed that BRCA1 stabilizes p53, causing upregulation of cell cycle arrest genes and DNA repair genes, but not proapoptotic genes [13]. We have developed sensitive methods for analyzing the promoter of the p53 target genes, i.e. the comprehensive EMSA [14] and the adenoassociated virus-based reporter vector. The comprehensive EMSA enables us to detect an alteration of DNA/protein interaction in the promoter after irradiation with low dose radiation. And the adenoassociated virus-based reporter vector responds to low dose radiation with a high sensitivity, and can be used to reveal the functional DNA elements by a site-directed mutagenesis analysis. In the Crossover Research Project, we will attempt to identify transcription factors involved in low dose radiation-response of various p53 target genes in various types of the cell by use of these methods. In addition, we will examine whether exchange of p53 recognition elements between separate p53 target genes would influence selective gene regulation. On the basis of the data obtained from these experiments, we aim at providing an important insight into the selective regulation of p53 target genes by low dose radiation., International Workshop on Biological Responses to Low Dose Radiation

Elucidation of how living organisms respond to ionizing radiation of doses lower than 0.5Gy is critical for establishment of a scientifically-justified risk assessment. It has been revealed that low-dose radiation causes distinct biological responses, such as adaptive responses, bystander effects and induction of genomic instability, which were not observed in experiments using lethal dose of radiation. There is accumulating evidence showing that p53 plays a pivotal role in these low-dose radiation-distinct responses. In fact, p53 is activated by a faint damage of the genome, and its target genes, such as p21WAF1/CIP1, GADD45a and MDM2, are induced by low-dose radiation. We have observed 8-fold increase in p21 gene transcription and 5-fold increase in GADD45a gene transcription after 0.5Gy of X rays in human myeloblastic leukemia cell line ML1. However activation of p53 by radiation is not limited in a low-dose range. Therefore, as yet determined mechanisms involving p53 must be operating in inducing the low-dose radiation-distinct biological responses. In this regard, it is noteworthy that a lot of nuclear factors have been demonstrated to associate with p53, modulating its function in regulation of the downstream genes. For examples, transcription factors Sp1, gut-enriched kruppel-like factor (GKLF), Ets1, and interferon regulatory factor-1 (1RF-1) have been shown to be involved in the p53-dependent transcriptional activation. BRCA1 has also been shown to play a role in both p53-dependent and p53-independent transcriptional activation of the GADD45a gene. Histone acetyl transferase p300/CBP and PHD-finger motif proteins ING family are the cofactors, modulating p53 function through chromatin remodeling. Based on these observations, it may be hypothesized that a specific combination of cooperating transcription factors and cofactors is activated by low-dose radiation, and it causes a distinct biological response. We attempt here to review recent data, including ours, concerning nuclear factors participating in transcriptional regulation by p53, The Fifth Japan-France Workshop on Radiobiology and Medical Imaging

Induction of adaptive response (AR) by the high LET accelerated heavy ion irradiations (HI) is being attempted both in young adult female mice and in fetal mice of C57BL/6J strain, using mainly thirty-day survival in vivo and prenatal death & malformation in utero as endpoints. In fiscal year 2011, using carbon, silicon, neon, and iron HI and X-rays, we demonstrated that 1) in the in utero studies, the priming low dose neon-ion irradiations could not induce any protective effects against the detrimental effects from the challenging irradiations from X-rays; 2) in the in vivo studies, no AR was observable in the following combinations for the priming and challenging irradiations: silicon plus carbon, iron plus carbon, and iron plus silicon. Thus we have finished all the radiation combinations in the in utero studies. The findings in the in utero studies indicate that induction of AR by X-rays against high LET HI is an event depending on the LET or/and species of the ions, and it seems hardly to induce AR by high LET HI against X-rays. For the in vivo studies, most radiation combinations are finished or under investigation, and we expect to complete the remaining combination in the fiscal year 2012.

Several stress-responsive genes, including p53-target genes, are induced by low-dose of ionizing radiation ranging from 0.02 to 0.5 Gy. We show here that 0.5 Gy of X-rays increase the transcription rate of the GADD45 gene, with maximum induction at 0.5 to 1 h after irradiation, much earlier than the maximum accumulation of stabilized p53 protein. It could be suggested that some transcription factors cooperate with p53 in regulating the GADD45 gene at an early time after low-dose irradiation. This idea is supported by the studies that showed GKLF and Sp1 are required for p53-dependent transcriptional activation of the p21WAF1/Cip1and BAX genes, respectively. To examine the possible involvement of cooperating transcription factors in regulation of the GADD45 gene after low-dose radiation, we attempted a comprehensive EMSA, in which 136 species of double-stranded DNA probes were used to identify X-ray-inducible factor-bindings to the upstream and the third intron regions of the gene after exposure to 0.5 Gy of X-rays in human myeloblastic leukemia ML-1 cells. Several X-ray-inducible DNA-protein complexes were observed. The factors related to forkhead transcription factors, POU domain transcription factors and kruppel-like factors were putatively identified by the competition assay. It is possible that these factors cooperate with p53 to mediate the transcriptional regulation of the GADD45 genes after low-dose irradiation., 低線量放射線の生物影響に関する国際シンポジウム-放射線による細胞応答と癌発生の分子機構

Reactive oxygen species (ROS) play a critical role in the onset of apoptosis induced by various extracellular stimuli, including ionizing radiation. Therefore active regulation of ROS-metabolizing enzymes may be one response to an apoptotic stimulus. In this regard HP100 cells, H2O2-resistant variants derived from human leukemia HL60 cells, display an interesting phenotype wherein the activity of catalase is constitutively high, while its mRNA is reduced after X ray-irradiation. In the present study, we investigated the molecular mechanisms underlying this phenomenon. By combining analyses from nuclear run-on, reporter gene transient transfection, genomic footprinting, site-directed mutagenesis, EMSA, and Western blotting experiments, we found that constitutively elevated catalase expression is strongly regulated at the transcriptional level by both Sp1 and CCAAT-recognizing factors, and that much higher levels of nuclear Sp1 and NF-Y are present in HP100 as compared to HL60 nuclei. In addition, we demonstrated an X ray-inducible association of a WT1/Egr-related factor with an overlapping Sp1/Egr-1 recognition sequence located within the core promoter of the catalase gene. This association may lead to inactivation of the promoter by disturbing, or competing with, the transactivating ability of Sp1., 41st Annual Meeting, The American Society for Cell Biology

urpose: Alzheimer's disease (AD) is the most common form of dementia, while its cause and progression are not well understood [ 1]. The possible cognitive and behavioral consequences induced by low-dose radiation are of great concern as humans are exposed to ionizing radiations from various sources including medical diagnosis [ 2]. A recent study in mice reported early transcriptional response in brain to low-dose X-rays (0.10 Gy) suggesting alterations of molecular networks and pathways associated with cognitive functions, advanced aging and AD. The present study is to investigate the late pathological, cognitive and behavioral consequences induced by low-dose radiation. Materials and methods: C57BL/6J mice were total-body irradiated with an acute dose from X-rays (0.10 Gy) or carbon ions (0.05 or 0.10 Gy). The hippocampus was collected and the expression of 84 AD-related genes was analyzed. Morris water maze test was applied to the measurement of the learning ability and memory of the animals. Amyloid imaging with positron emission tomography were performed to detect the accumulation of fibrillary amyloid β peptide (Aβ), and characteristic pathologies of AD were examined with immunohistochemical staining of amyloid precursor protein (APP), Aβ, tau, and phosphorylated tau. Results: For the transcriptional studies, results showed that a few genes out of 84 AD-related genes were significantly up-regulated at 4 h after irradiation and the other genes had no marked change; on the other hand, a few other genes showed a significant down-regulation, while the other genes had no marked change at 1 year after irradiation. For the behavioral studies, no significant difference on learning ability and memory was observed at 1 and 2 years after irradiation. Imaging and immunohistochemical staining showed no change in the accumulation of fibrillar amyloid and the expression of APP, Aβ, tau and phosphorylated tau were detectable in the animals 4 months and 2 years after irradiation. Conclusion: These findings suggest that total-body irradiation at a dose of 0.10 Gy could hardly induce significant early or late transcriptional alterations in most of the AD-related genes in the hippocampus, learning disability and memory impairment, and AD-like pathological change in the brain in mice [ 3].