Exposure to low priming doses of ionizing radiation is known to decrease the biological effects of a subsequent higher challenging dose. This adaptive response (AR) to low dose radiation was described in a variety of models, using various endpoints. In this study, we investigated the ability of low doses of X-rays to induce an AR to the biological effects of high-LET heavy-ion radiation (carbon-ion, neon-ion, 20 to 150 keV/micrometer), in cultured human lymphoblastoid cells TK6 (p53 +/+) and AHH-1 (p53 +/-). We observed that cells adapted by X-rays showed a reduced mutation frequency at HPRT locus after exposure to high-LET radiation at HIMAC (NIRS, Chiba, Japan). AR in our model was dependent on p53 status but linked to neither cell cycle effects nor modulation of radiation-induced apoptosis. The analysis of H2AX phosphorylation kinetics in adapted and non adapted cells suggested that modulation of DNA double-strand break repair activity may be involved in this phenomenon. Knowing that high-LET radiation produces non-randomly distributed DNA damage in the form of clusters, or locally multiply damaged sites (LMDS), it seems that triggering AR by exposing cells to low doses of ionizing radiation could protect cells against the detrimental effects of such damage.

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Radiation-responsive genes in environmental organisms have received increasing attention for their potential as the diagnostic tools for radiation hazard to environment. In the present study, we used high-coverage expression profiling (HiCEP), a method that requires no prior sequence knowledge, to search for radiation-responsive genes in Enchytraeus japonensis, a terrestrial earthworm. Many radiation-responsive genes and their dose-dependency were detected at effective doses for growth, and 11 transcript-derived fragments (TDFs) markedly up-regulated in response to radiation were sequenced and examined for the similarity with known genes from the other organisms. The only one TDF showed high resemblance to poly(ADP-ribose) polymerase (PARP) of Xenopus laevis, Mus musculus and Homo sapiens, but the other TDFs showed no similarity to any genes in the gene databases. The open reading frame of the TDF with high resemblance to PARP was deduced by 5 and 3 RACE, and it was shown that N terminal sequence of PARP from the earthworm resembled to that of the other organisms more than the other parts of PARP sequence. PARP gene expression was known to be up-regulated in Arabidopsis thaliana in response to ionizing radiation. On the other hand, there is no study that demonstrates radiation-induced enhancement of PARP gene expression in mammal. There is obviously difference among organisms in the regulation of PARP gene expression in response to ionizing radiation.

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Understanding of radiation effects and its molecular mechanisms in environmental organisms has been received increasing attention for the radiological protection of the environment. Comparative studies on radiation responses at a transcript level of diverse environmental organisms may lead to understanding of generality and diversity of radiation responses across species and mechanisms underlying species-specific susceptibility. However, little is known about radiation responsive genes in environmental organisms. Therefore, it is imperative to gather information of these genes in diverse species. Soils are of ecologically importance and harbour most animal taxa found on the terrestrial ecosystem. Springtails and earthworms are common soil animals and used as representatives in ecological impact assessment. In the present study, we identified radiation responsive transcripts in the springtail Folsomia candida and the earthworm Eisenia fetida using high-coverage expression profiling (HiCEP), and compared the transcript species between the soil animals. Identified transcripts in the springtail included the ones not known to be up-regulated in response to irradiation in mammals. Identified transcripts in the earthworm included the ones known to be up-regulated in mammals. Further comparative analyses are going on.

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Human glioblastoma is one of the most aggressive tumor cells in human cancers. Glioblastoma shows resistance to ionizing radiation, so if one could modify the radioresistance, it would lead to a better therapeutic gain. We investigated the effects of high LET heavy ion irradiation to human glioblastoma xenograft in mice using a tumor stem cell marker. Although a significant decrease in tumor volume by heavy ion radiation was observed, the cause of this phenomenon was not determined at this stage. We are investigating if this shrinkage is related to the control of tumor stem cells by high LET irradiation, and the experimental data on this issue will be presented.

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Increasing attention has come to be focused on the protection of the environment, i.e., non-human biota and ecosystems, from ionizing radiation, over the last decade. For example, the International Commission on Radiological Protection (ICRP) established a new committee for the environmental protection in 2005, and introduced a new chapter entitled "Protection of the Environment" in the ICRP recommendation in 2007. Although a lot of efforts have also been put on the collection of available scientific information mainly in European countries, the relevant data sets are still extremely limited. This paper summarizes recent progress of research activities in National Institute of Radiological Sciences (NIRS) for evaluation of the radiation effects on selected terrestrial and aquatic organisms as well as the ecosystems. Among organisms, conifers, earthworms, collembolans, algae, Medaka, etc. are presently selected to study. Transfers of radionuclides and related elements from medium to organisms are evaluated, for the estimation of possible radiation dose. Dose-effect relationships of acute gamma radiation on the survival, growth, and reproduction of selected organisms have been studied. Information of genome- and metagenome-wide gene expression has been collected. Studies on the effect of chronic gamma radiation at low dose rate were also started. In order to evaluate ecological effects of radiation, study methods by using three-species microcosm were established, and an index for the holistic evaluation of effects on various ecological parameters was proposed. Developments of more complicated and practical model ecosystems have been started. The Denaturant Gradient Gel Electrophoresis (DGGE) has been applied on soil bacterial community for evaluation of the radiation effects on soil ecosystems.

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Ionizing radiation produces short-lived radicals through hydrolysis of water molecules. Recently, it is suggested that radiation causes disturbance of intracellular oxidation control system in mitochondria. Because elevated intracellular oxidative stress is involved in various delayed effects, we examined delayed production of ROS in normal human cells exposed to gamma-rays. Involvement of delayed dysfunction of mitochondria was also investigated. Immortalized normal human diploid cells were used in this study. The amount of reactive oxygen species (ROS) was determined using aminophenyl fluorescein (APF) reagent. Levels of superoxide localized within mitochondria were measured by MitoSox Red reagent. Mitochondria were visualized by staining with MitoTracher reagent, and structural change was examined under fluorescence microscope. While transient increase in the ROS level was detected immediately after irradiation, it returned to the control level within a few hours. Then, the second increase became obvious 2 to 3 days after irradiation, which was accompanied by the increased level of mitochondrial superoxide. Interestingly, delayed morphological change was observed in mitochondria, suggesting that delayed dysfunction of mitochondria causes delayed oxidative stress.

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Mutation of p53 is the most common genetic alteration observed in human tumours. Mutation of p53 is reported to lead to variations in cell radiosensitivity. We investigated the mutant p53 signal pathway in human osteosarcoma cell line, Saos-2. We made 15 mutations of p53 gene, which were found in human tumours at various point of DNA binding domain, and introduced to Saos-2. The sensitivity of mutant p53 cells to ionizing radiation varies with the mutation point of the p53 gene. All hot spot mutations (175H, 245S 273H and 282W) conferred radioresistance. Then we investigate which gene expression may have a relation with the radiosensitivity in mutant p53 cells. We examined the expression of genes in mutant p53 cells irradiated at 3 and 6 Gy by DNA microarray of Affymetrix Gene Chip (Human Genome U133 Plus 2.0 Array). All mutant p53 cells lost the function of induction in may genes those were induced in wild p53 cells. On the other hands, many mutant p53 cells gained the function of induction in some genes those were not induced in wild p53 cells. Especially, 245S mutant cells induced many TNF associated-gene expressions by radiation. As TNF associated-gene expressions inhibit an apoptosis induction, this may explain a radioresistance of 245S mutant cells. We found some genes which may have some correlation with the radiosensitivity in mutant p53 cells. But there was no gene which directly determined the radiosensitivity of the cells. The radiosensitivity may be determined by several genes with collaboration.

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Purpose: This study is to asses the meaning of the number of NORs in tumor cells nuclei compared with other proliferative markers before and during chemoradiotherapy.
Material and Method: Forty six biopsy specimens from twenty three patient with cervical squamous cell carcinoma treated with chemoradiotherapy taken before radiotherapy and at 10 Gy were analyzed. The number of NORs was measured using the silver stained method. The MIB-1 LI and p53 labeling indexes (LI) were measured by using immunohistochemical method.
Result: After 10 Gy irradiation, the number of NORs (AgNOR score) was decreased from 4.93 to 3.39 (p<0.0001). MIB-1-LI and MI were increase from 25.55 to 39.05 (p<0.0001) and from 0.0059 to 0.0122 (p<0.0001), respectively. Before radiation therapy, we found positive correlation between AgNOR score and MIB-1-LI (p=0.0002), and MI (p=0.0017). At 10 Gy, there was no significant correlation among number of NORs, MIB-1-LI, MI and p53-LI.
Conclusion: The number of NORs can be used as proliferative marker for assessing tumor cell proliferative activity before chemoradiotherapy, but not during therapy.

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As a ubiquitous phenomenon, DNA synthesis activity will be decreased after X-ray irradiation in living cells. However, we found that fibroblast cells derived from Gorlin (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation (Fujii et al., Biochem. Biophys. Res. Commun. 240, 269–272, 1997). Furthermore, the induction of DNA synthesis after irradiation was suggested to be caused by down-regulation of SUMO-3 gene expression (Mutat. Res. 578, 327-332, 2005). In the present study, after X-ray irradiation, the increase of DNA synthesis activity was also found in HeLa cells with knockdown of SUMO-3 by its special siRNA. Hereby, we can use HeLa cells, instead of NBCCS cells with very low capacity of growth. In order to investigate the cellular mechanisms underlying the down-regulation of SUMO-3, we performed proteomic analysis using Two-dimensional (2D) electrophoresis and mass spectrometry, and then searched for the proteins whose expression levels change early after X-ray irradiation in HeLa cells with knockdown of SUMO-3. As a result, expression levels of some candidate proteins were altered, and therefore the relationship between candidate proteins and DNA synthesis induction will be discussed.

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Magnetic resonance imaging with high static magnetic fields (SMFs) has become widely used for medical imaging purposes because SMFs cause fewer genotoxic side effects than ionizing radiation (IR). However, the effect of exposure to high SMFs on global transcription is little understood. In this study, to elucidate the effects of SMFs or IR on individuals at the molecular level, we monitored the transcriptional alterations in C. elegans that were exposed to 3 or 5T SMF and 100Gy gamma-rays using the whole genome GeneChip array. In addition, we compared the effect of high SMFs with IR on genotoxicity, DNA double-strand break (DSB) formation and apoptosis in germline cells during gametogenesis. We demonstrate that genes involved in motor activity, actin binding, cell adhesion, cuticles and Ca²⁺ binding transiently and specifically induced following exposure to 3 or 5T SMFs. The small-heat shock protein gene hsp16 was also induced but to a much lesser extent, and the LacZ-stained population of hsp-16.1::lacZ transgenic worms did not significantly increase after exposure to SMFs with or without a second stressor, mild heat shock. Several genes encoding apoptotic cell-death activators and secreted surface proteins were upregulated after IR, but were not induced by SMFs. In addition, we constructed experimental systems using him-17 and abl-1 mutants to monitor genotoxicity of SMFs and IR. These results clearly indicate that exposure to high SMFs did not induce DSBs and induce apoptosis. Altogether suggests that the response of C. elegans to high SMFs is unique and capable of adjustment during long exposure, and that this treatment may be less hazardous than other therapeutic tools.

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p21^WAF1 is transcriptionally induced after exposure to ionizing radiation (IR) through the p53-dependent pathways, and plays critical roles in cell cycle checkpoint regulation. Elucidation of detailed mechanisms for p21^WAF1 gene induction after irradiation is an important issue to control the efficacy of cancer radiotherapy in cancer treatment. For this purpose, we here performed a comprehensive analysis of the p21^WAF1 gene promoter after irradiation with clinically relevant doses of IR. The sensitive reporter gene assay system by use of recombinant adeno-associated virus vectors was effectively utilized. It was demonstrated that the Oct-1 sites at –1.1 kb, –1.8 kb and –1.75 kb are involved in regulation of the p21^WAF1 gene promoter in response to 0.2-2.0 Gy of IR. Constitutive binding of Oct-1 to the sites at –1.1 kb, and –1.75 kb was demonstrated by an electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Functional involvement of Oct-1 was confirmed by use of Oct-1-specific siRNA, which suppressed both the basal and the IR-inducible components of the p21^WAF1 gene expression. Furthermore, we found that inducible binding of p53 to the site at –1.4 kb is indispensable for IR responsiveness, although it contains only one and a half repeats of the consensus recognition sequence. Based on the observation that binding of p53 to its canonical recognition sequence at –2.2 kb is constitutive, inducible binding of p53 at –1.4 kb site was suggested to play a major role in IR responsiveness of the p21^WAF1 gene.

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[Objective] X-irradiation generates cellular reactive oxygen species (ROS). Transcriptional factor NF-E2-related factor 2 (Nrf2) regulates the expression of multiple antioxidant enzymes, including NAD(P)H-quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1). This study investigated whether the induction of HO-1 and NQO1 via Nrf2 is associated with individual differences in the radiosensitivity of human hematopoietic progenitor cells (hHPCs).
[Material and Methods] Human peripheral blood mononuclear cells (hPBMCs) were separated from the buffy-coat by centrifugation on a cushion of Lymphosepar I. X-irradiated hPBMCs (0, 0.2, 0.5, 2 Gy) were cultured in a methylcellulose medium with a combination of cytokines (G-CSF, GM-CSF, IL-3, SCF and EPO). After 14 days of culture, the colonies containing ≥ 50 cells were counted as hHPCs. CD14⁺ cells (monocytes) were isolated from hPBMCs using an I-Mag separation kit. Total RNA was extracted from the X-irradiated CD14⁺ cells cultured with cytokines. The expression of HO-1 and NQO1 were assayed by quantitative RT-PCR.
[Results and Discussion] A significant correlation was observed between the HO-1 and NQO1 expressions in hPBMCs monocytes. A positive correlation was observed between the levels of HO-1 and the surviving fraction of hPBMCs exposed to 0.5 Gy. However, no correlation was observed in either the HO-1 expression in hHPCs exposed to 0.2 Gy and 2 Gy or in the levels of NQO1. These data suggest that the expression of Nrf2 depends on individual differences in the responses of hHPCs exposed to 0.5 Gy.

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G1 checkpoint is activated in radiation cells that have DNA double strand breaks. The ATM-p53-p21 pathway is critical for the initial activation of G1 checkpoint, however a role of the pathway for the maintenance of G1 arrest has not been examined. In this study, we hypothesized that there is signal stabilization mechanism to maintain G1 arrest. To prove this, normal human fibroblasts were synchronized in G0 phase, irradiated with 4 Gy of gamma rays, and released immediately following irradiation. After incubation of 4 hours, cells were treated with ATM kinase inhibitor KU55933. Then, cells were fixed at 24 hours after irradiation and percentage of cells in S phase was determined by immunofluorescence staining for Replication Protein A(RPA). We found that the percentages of RPA positive control cells 41 % was decreased to 16 % by 4 Gy irradiation, KU55933 treatment increased RPA positive cells(27 %), but 14 % of cells were still arrested in G1 phase. We also examined that the expression levels of p53, Ser15-phosphorylated p53 and p21 at 24 hours following irradiation and treatment with KU55933 after irradiation. Post-treatment of KU55933 decreased the level of p53 and Ser15-phosphorylated p53 to the control level, whereas expression level of p21 was unchanged. These results suggest that there is an ATM-independent signal stabilization mechanism of p21 protein, which could be involved in the maintenance of G1 arrest.

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Histone H2AX is phosphorylated by ATM with Mre11/Rad50/Nbs1 complex at the site of DNA double strand breaks (DSB). The phosphorylated H2AX (gamma H2AX) foci are thought to be a marker of DSB caused by radiation. However, recent research has revealed that nonDSB-causing chemicals and UV also make gamma H2AX foci, suggesting the presence of multi-pathways of H2AX phosphorylation. A purpose of our study is to elucidate which types of DNA lesions induce gamma H2AX foci and to classify the chemicals with types of H2AX phosphorylation pathways. DSB-inducers, X-ray and etoposide phosphorylated H2AX in all phases of the cell cycle. On the other hand, UV-induced pyrimidine dimers and benzo(a)pyrene- and 1,8-dinitropyrene-induced bulky adducts phosphorylated H2AX in the S-phase specifically. Methylating agents, methylnitrosourea and methylmethane sulfonate, and SSB-inducer, camptothecin, also phosphorylated H2AX in the S-phase specifically. These results suggest that arrest of DNA replication forks by various types of DNA lesions may be a general phenomenon, and clarification of this detailed mechanism is required to understand chemical carcinogenesis.

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In contradiction to classical paradigm, which assumes that radiation effects are directly proportional to energy deposit, numerous studies described the existence of radiation-induced adaptive response (AR), which is the phenomenon of priming low-dose irradiation-induced resistance to subsequent irradiation at higher doses. As investigations on the conditions essential for induction of these effects provide important scientific basis for radiation risk estimates and offer significant insight into the novel biological defense mechanisms regarding protection against radiation, study on AR is of great concern for both public health and academic research. In a series of study in progress, possible induction of adaptive response by high LET irradiation of accelerated heavy ions is to be tested both in vivo in young adult mice and in utero in fetal mice of C57BL/6J strain. Investigations are to verify 1) if the known X-ray-induced adaptive response could reduce any heavy-ion-induced detrimental effects such as growth retardation, death or malformation, 2) if the heavy ion irradiation at certain low doses could induce any adaptive response against the detrimental effects induced by high dose of X-irradiations, and 3) if the heavy-ion irradiation at certain low dose could induce any adaptive response against the detrimental effects induced by high dose of heavy ion irradiations. Three kinds of accelerated heavy ions are to be examined, carbon (290MeV/u) mono beams with a linear energy transfer (LET) value of about 15 keV/micro meter, silicon (490MeV/u) mono beams with a LET value of about 55 keV/micro meter, and ion (500MeV/u) mono beams with a LET value of about 200 keV/micro meter. In this report, we will present the data on the first question - if the known X-ray-induced adaptive response could reduce any heavy-ion-induced detrimental effects.

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We investigated the mechanism underlying the radioadaptive response that rescues low-dose preirradiated mice from hematopoietic failure after challenge irradiation. C57BL/6 mice were irradiated with low-dose acute X rays (0.5Gy) prior to a high-dose challenge irradiation. Bone marrow cells, erythrocytes and platelets in low-dose-preirradiated mice showed a rapid recovery following the challenge irradiation compared to those in mice subjected only to the challenge irradiation. This suggests that hematopoiesis following the challenge irradiation could be enhanced in the preirradiated mice. The rapid recovery of bone marrow cells following the challenge irradiation was consistent with the proliferation of hematopoietic progenitors expressing the cell surface markers c-kit⁺, Sca-1^- and Lin^- in low-dose-preirradiated mice. A subpopulation of myeloid (Mac-1⁺/Gr-1⁺) cells, which were descendants of c-kit⁺, Sca-1^- and Lin^- cells, more rapidly recovered in the bone marrow of low-dose-preirradiated mice. Cytokine profiles analysis by using antibody arrays and quantitative suspension arrays indicated that the concentrations of growth factors for myelopoiesis and of interferons after the challenge irradiation were considerably increased by low-dose preirradiation. The rapid recovery of erythrocytes and platelets suggests that low-dose preirradiation triggers the differentiation to myelopoietic cells. Thus the adaptive response induced by low-dose preirradiation in terms of the recovery kinetics of the number of hematopoietic cells may be due to the rapid recovery of the number of myeloid cells after high-dose irradiation.

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Purpose : Radioadaptive response (RAR) is one of the biodefensive responses by which cells acquire radioresistance after exposure to low dose radiation. RAR is an important biological phenomenon in regard to the accurate estimation of the low-dose radiation risk. We have previously suggested a distinct molecular mechanism of RAR in terms of mutation from that of chromosomal aberration. In addition, by analyzing the gene expression profile, we have demonstrated that the expression of genes involved in the Ras signaling pathway varies in correlation with induction of RAR. Based on these results, we attempted to identify the genes that function in RAR. Methods : At 3h, 6h, 9h, and 24 h after irradiation with 0.02 Gy of X-rays, total RNAs were purified from the human lymphoblastoid cells (AHH-1) that were unirradiated or irradiated. Then we examined the gene expression by Northern blot analysis or real-time PCR for the genes which we selected as genes potentially related to RAR based on the analysis of the gene expression profile. To generate knockdown cells in which these potential genes are specifically targeted, AHH-1 cells were stably transfected with respective shRNA-expression plasmids. Then, it was analyzed whether RAR could be induced in these knockdown cells. Results : We identified DIDO1, MAPK8IP1 and SOCS3, whose expressions were significantly altered after irradiation with 0.02 Gy by analysis of the gene expression profile. Alteration in expression of these genes was confirmed by Northern blot analysis or real-time PCR. In cells stably transfected with respective shRNA-expression plasmids, it was observed that the knockdown efficiency was up to 68%, 64%, and 71% for DIDO1, MAPK8IP1, and SOCS3, respectively. Relationship of these genes with RAR will be discussed.

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In the present study, CCD32SK diploid normal human fibroblasts were transiently transfected with short interfering RNA (siRNA) specific for human p53 (CCD/p53i). There was a small increase in the frequency of centrosome amplification in CCD/p53i cells without hyperthermia. In contrast, CCD/p53i cells after hyperthermia showed a marked increase in abnormal nuclear shapes and pronounced amplification of centrosomes. At 12 h after irradiation, irradiated CCD/p53i cells were arrested in G2 phase. On laser scanning cytometry, abnormal mitosis with amplified centrosomes was frequently observed in the accumulating G2/M population at 48 h after hyperthermia. In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by hyperthermia, efficiently induces centrosome amplification and nuclear fragmentation, but these phenomena were not observed with either siRNA-mediated silencing of p53 or irradiation alone.

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A medaka cultured cell line RIC1-e9 has defects in the pathways of apoptosis and cell cycle regulation (49th JRR). Comet assay revealed that RIC1-e9 cells required about 2h to repair the DSBs induced by IR, while in CAB cells the process was completed within 30min (13th ICRR). In this study, we established other cell lines (RIC1) to confirm that the defects are common characteristics of RIC1 cell lines. In 24h after γ-irradiation, 30% of the CAB cells were apoptotic. In contrast, apoptotic and fragmented RIC1-e42 cells and RIC1-e44 cells were very few. CAB cells and RIC1 cells showed mitotic inhibition immediately after irradiation, but same as RIC1-e9, both of RIC1-e42 and RIC1-e44 resumed cell division earlier than the CAB cells. In addition, in RIC1-e9 cells fewer H2AX were phosphorylated than in CAB-e3 cells after γ-irradiation. Comet assay and immunohistochemistry analysis with RIC1-e42 and RIC1-e44 ensure the relation between the intensity of fluorescence to phosphorylated H2AX and the time for repair.

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Because of an increased chance of children to be exposed to medical radiation, effects of radiation during developmental stage have attracted attention in recent years. However, there is very little information about this. We therefore started a study on radiation effects in children. The aim of this study is to demonstrate age dependency of radiation-induced apoptosis in intestine. First, we investigated normal development of intestine from fetal to adult stage histologically. Crypt formation was not found in fetus and newborn stage, and then small size of crypts formed clearly in 2-week-old mice. Thereafter, morphologically matured crypts were formed in 7-week-old mice. Next, we investigated the apoptic index in small and large intestine by immunohistochemistry using antibody of active-caspase 3 in 2-week-old and 7-week-old mice following whole body X-irradiation at 2 Gy. The preliminary data indicated that there were differences in the peak time of the apoptic index between 2-week- and 7-week-old mice.

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Accumulating evidence indicates that the biological effects of low dose and low dose-rate radiation are different from those of high dose and high dose-rate radiation. Radiation-induced bystander response is defined as a response in cells that have not been directly targeted by radiation but is in the neighborhood of cells that have been directly exposed. Thus, to elucidate the bystander reponse is important to evaluate the risk of low dose radiation. It has already reported many results of bystander response induced by high-LET charged particle radiations. On the other hand, it has not yet fully recognized that to low-LET photons.
The CRIEPI microbeam X-ray irradiation system is a powerful tool to investigate cellular response to low dose and low dose-rate radiation. X-ray microbeam irradiation system has been developed in March, 2007. Our system is characterized by (1) tabletop (2) X-ray focusing system using Fresnel zone plate (FZP), and (3) on-line confocal laser microscope. Characteristic X-ray of aluminum (1.49 keV) was generated by focused electron bombardment of aluminum target. The FZP was used to focus aluminum K-shell X-ray. The size of X-ray beam is 2-3 microns in diameter.
Using our irradiation system, we identified the dose response of X-ray-induced bystander cell killing of normal human WI-38 cells obtained using our system. In addition, we could observe the induction of complex clustered DNA damage by the high dose of X-ray microbeam irradiation. Recent cell irradiaion studies and the details of the performance of our system will be present.

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We have recently described a higher incidence of multiple primary cancers in A-bomb survivors. Inasmuch as a long-lasting radiation effect is considered to be a contributing factor in tumorigenesis in the survivors, the molecular mechanisms involved are not yet understood. The incidence of basal cell carcinoma (BCC) of skin was reported to be elevated in the survivors, suggesting a radiation etiology in skin carcinogenesis as well as ultraviolet. Any DNA damages can induce DNA damage response (DDR) leading to genomic instability (GIN), which allows the accumulation of genetic mutations that is implicated in both initiation and progression of cancers. This study evaluated the presence of GIN in the epidermis surrounding BCC from the survivors. A total of 146 BCC were identified in the survivors from 1968 to 1999. Among 146 BCC, 23 cases arose at nonexposed skin consisting of 9 proximal, 4 intermediate, and 10 distal distance groups. Total 19 epidermis surrounding BCC at the nonexposed sites from 7 proximal, 5 distal, and 7 control groups were tested for 53BP1 expression with immunofluorescence to analyze the level of GIN. Because one manifestation of GIN is the induction of endogenous DDR, the level of 53BP1-focus formation (DDR type) can be considered as a marker for GIN. In results, the incidence rate of BCC increased significantly as exposure distance decreased from the hypocenter. Of the 7 epidermis in proximal group, five cases predominantly expressed DDR and abnormal type. In contrast, 4 of 5 cases in distal group and all cases in control groups predominantly expressed stable type in the epidermis. This study demonstrated the sporadic activation of DDR in the epidermis surrounding BCC from the survivors of proximal group, suggesting the presence of a GIN in the survivors as a late effect of A-bomb radiation which can be a cause of cancer predisposition.

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Ionizing radiation is a carcinogenic agent. Radiation carcinogenesis is the results of a series of somatic mutations. Rev1 plays the central role on the translesion DNA synthesis known as error-prone DNA repair. DNA synthesis by such translesion polymerase can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. However, how Rev1 affects the mutagenicity and tumorigenicity in vivo after irradiation is still unclear. To address this issue, we have generated metallothionein promoter-Rev1 (MT-Rev1) transgenic mice that continuously express a Rev1 transgene in multiple tissues. Both Rev1-Tg and wild type mice (C57BL/6) were exposed to radiation and measured mutant frequency and micronucleus frequency. The detail will be discussed in this conference.

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Previously in this meeting, we reported that irradiation in young age induced delayed mutation, and that decrease function of p53 gene related to delayed mutation. In a word, in mice that were received a whole-body dose of 3 Gy at 8 weeks of age, T-cell receptor (TCR) mutation frequencies (MFs) increased from 60 weeks of age in p53(+/+) mice and from 40 weeks of age in p53(+/-) mice much higher than in the control mice. Also, we showed the decrease of apoptosis activity induced by radiation, the rise in translocation frequency in 11th chromosome and p53 genome mutation in CD3-CD4+ cell from spleen. In this study, we investigated that the protein expression of p53 and the related gene by western blot method and the p53 loss of heterozygosity (LOH) by PCR. p53(+/+) mice and p53(+/-) mice were received a whole-body dose of 3 Gy at 8 weeks of age. Mice were sacrificed at 10, 24, 56 (p53(+/-) mice only) and 72 (p53(+/+) mice only) weeks of age. The levels of p53 and p21 protein increased gradually with ageing. These protein levels in the irradiated group were smaller than that in the control group. The levels of p53ser15/18, ATM and ATR protein decreased in old mice and furthermore these protein levels in the irradiated group were smaller than that in the control group. Some old mice showed LOH of p53. We suggested that the decreased activity of ATM-p53 and ATR-p53 pathway and LOH of p53 were related to the delayed mutation after irradiation in young age.

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X-ray induces myeloid leukemia in C3H/He mice with the incidence of 20-25% at 3Gy. Mutation of Sfpi1 (PU.1) is crucial in the development of myeloid leukemia. The mutation, however, is mostly specific point mutations in the DNA-binding Ets domain of PU.1, and they are unlikely to be a direct consequence of radiation-induced DNA damage. To elucidate when and how often this type of mutation arises in hematopoietic cells, we have developed a technique that is capable of detecting a small number of mutated cells in the vast majority of non-mutated cells. The technique is based on the wild-type blocking PCR, in which LNA (locked nucleic acid) oligonucleotide blocks amplification of wild-type DNA during PCR while permitting amplification of mutants. The PCR followed by restriction enzyme digestion has enabled to detect mutant DNA in 10⁴ times amount of wild-type DNA. This technique is being applied to DNA samples from C3H/He mice that were exposed to 3Gy of x-rays and were maintained for more than a year. Among 12 exposed mice and 6 age-matched controls analyzed so far, the mutation has been detected in a sample from the spleen of an exposed mouse.

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We previously reported that mice continuously irradiated with gamma-ray at low-dose-rate (LDR) of 21 mGy/day for 400 days showed higher incidence of leukemia than non-irradiated mice. However, little is known of the mechanisms underlying development of leukemia induced by LDR-irradiation. In this study, we compared cell differentiation stage of leukemias induced by gamma-rays at three different dose-rates. In mice irradiated at three different dose-rates of gamma rays [high dose rate (HDR): 1.0 Gy/min (total dose of 3 Gy), medium dose rate (MDR): 400 mGy/day (4 Gy) and LDR: 20 mGy/day (8Gy)] and non-irradiated mice, 10, 6, 3 and 10 leukemias were, respectively found. Hematopoietic cell-differentiation stages were analyzed by FACS in the bone marrow and spleen cells from the mice bearing leukemias. The analysis showed that significant increases of LSK^- population with features of hematopoietic stem cell, lineage marker- (Lin^-), Sca1⁺, c-kit⁺ and CD34^- were found commonly in all groups of leukemia. However, leukemias from MDR and LDR-irradiated and non-irradiated mice showed increase of Lin^- Sca1^- c-kit⁺ CD34⁺ population, which is similar to common lymphoid progenitor, and their incidences were 80% (8/10 cases), 66% (2/3) and 66% (4/6), respectively. In contrast, HDR showed expansion of Lin^- Sca1⁺ c-kit^- CD34⁺ population resembling common myeloid progenitor and the incidence was 80% (8/10). Furthermore, when 100 cells at each different stage of cell populations of bone marrow cells from leukemic mice sorted by FACS were transplanted into a syngeneic mouse, CD45R/B220⁺ or Gr-1⁺ population in MDR, and LSK^- or common myeloid progenitor-like population in HDR had an overt potential to develop leukemia in recipient mice. These findings indicate a possibility that differentiation stages and patterns of leukemic stem cell are influenced by dose-rates of gamma-rays. This study was performed under contract with the Aomori Prefectural Government, Japan.

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In the previous report, transgenic mice with mutant p53 from a radiation-induced mouse skin tumor showed a 1.7 fold elevated fibrosarcoma incidence as compared to wild type mice after subcutaneous injection of 3-methylcholanthrene. These tumors were suppressed by treatment with promoter-responsive siRNA #220 mixed with atelocollagen, with a frequency equivalent to the increased fraction of tumor incidence. Present report provides further characterization of the siRNA#220-responding tumor transplant line TT18. TT18 showed a drastic apoptosis and loss of the fibrous structure after treatment with siRNA#220 (0.0145 mg in 0.2ml; 2 applications with 2 day interval) along the time course of its regression from the size 5 mm diameter. When siRNA#220 was applied 2 days after TT18 transplantation at the right groin and negative control siRNA at the left groin, in the same mouse, tumor grew to 1.1 cm in diameter in 17 days at the left groin, while no tumor was formed in the right groin, indicating the effectiveness of local treatment with siRNA#220. These results show that the mutant p53 is indeed the cause of tumors and that these tumors can be cured by suppressing expression of the mutant p53.

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Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. Here we examine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogenesis are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells.
In this study, we used the primary cells derived from 13-days embryo of C57BL mouse with or without p53 gene functions. Cells were subcultured every 5-day at 106 cells per 752cm culture flask. Both p53 (+/+) and p53 (-/-) cells were spontaneously immortalized, but only p53 (-/-) cells became tumorigenic. All of tumorigenic cells were aneuploid. Instability of chromosome of p53 (-/-) cells was greater than that of p53 (+/+) cells. On the other hand, non-tumorigenic cells were tetraploid, it was stable. Considering these results, we conclude that aneuploidy is the cause rather than a consequence of transformation.

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Our previous reports (Radiat. Res. 160: 376, 2003, & 167: 417, 2007) indicate that the life shortening due to early neoplastic death was observed in B6C3F1 mice after long-term, continuous irradiation with low-dose-rate (21 mGy/day) and high dose (8000 mGy) gamma-ray. Although many of neoplasms from irradiated mice were malignant lymphomas, their incidences were not different from those of non-irradiated mice. In this study, we investigated the global gene expression between 20 malignant lymphomas from irradiated mice (RL) and 20 from non-irradiated controls (CL). Comparative microarray analysis using Agilent 44k microarray identified 418 up-regulated genes and 406 down-regulated genes in RL as compared with CL. The Gene Ontology classifications of the up-regulated genes in RL were shown significant enrichment to "immune response" or "positive regulation of cell proliferation", while the down-regulated genes in RL were significantly enriched to "cell cycle" or "apoptosis". These results indicate a possibility that the different genes are associated with development of RL and CL. This study was performed under the contract with the Aomori Prefectural Government, Japan.

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Osteosarcoma (OS) is the most common primary tumor of bone in children and adolescents. It is highly aggressive and thought to arise primarily from osteoblasts (OB), bone-forming cells. Radiation exposure is the most significant environmental risk factor for OS. OS occurs as a secondary tumor after the radiotherapy, and is also known to be induced following the incorporation of bone-seeking alpha emitters. However, the genes and signaling pathways involved in radiation-induced OS are not fully understood. Using oligonucleotide microarrays, we profiled gene expression in rat OS tumors induced by the bone-seeking alpha emitter ²³⁸Pu and compared these expression profiles to those of normal OB. The expressions of 72 genes were significantly differentially expressed in the OS related to OB samples. These included genes involved in the cell adhesion, tumor-suppressor function, differentiation, developmental processes, Src tyrosine kinase and Wnt/β-catenin signaling pathways. Expression changes of several genes were validated by quantitative real-time RT-PCR analysis. Constitutive activation of the Wnt/β-catenin signaling pathway in the tumors was suggested by the transcriptome analysis and confirmed by observing nuclear and/or cytoplasmic localization of β-catenin in the tumors using immunohistochemistry. In addition, we found a decrease of the glycogen synthase kinase 3β (GSK-3β) and the inactive (phosphorylated) form of β-catenin in the tumor relative to OB samples by immunoblot analysis.

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Breast cancer ranks top in the incidence among the main sites of female cancer in developed countries. The epidemiological study on atomic bomb survivors has suggested that the excess absolute risk for breast cancer incidence is higher than any other sites. Little is known, however, about the molecular mechanisms of breast cancer induction by radiation. We focused on tumor suppressor genes p15INK4b, p16INK4a and p19ARF and analyzed these genes in radiation-induced rat mammary adenocarcinoma. Mammary carcinomas were induced by 2 Gy gamma irradiation of Sprague-Dawley (SD) rats at 3 or 7 weeks of age. Array CGH analysis revealed that homozygous and heterozygous deletions of the Cdkn2a and Cdkn2b region were observed in 2 of 14 mammary carcinomas. Cdkn2a encodes two transcripts, p16INK4a and p19ARF from overlapping reading frames, and Cdkn2b encodes p15INK4b. The expression of p15INK4b, p16INK4a and p19ARF, examined by quantitative RT-PCR, was downregulated in the carcinoma with homozygous deletion. Unexpectedly, upregulation of these genes was observed in the other mammary carcinomas compared with normal mammary tissues. No mutation was observed in these genes except for a single nucleotide polymorphism in p16INK4a. No correlation between the gene expression and protein expression of p15INK4b, p16INK4a and p19ARF was demonstrated. On the other hand, deletion of Cdkn2a and Cdkn2b region were absent in spontaneous mammary carcinomas. These results suggest that the deletion of Cdkn2a and Cdkn2b was associated with radiation exposure.

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