<Purpose> It has not been elucidated whether heavy-ion beam irradiation modulates the biology of glioblastoma at the cellular level. Our project is designed to investigate whether heavy-ion beam exposure is effective on glioblastoma cells. We examined a human glioblastoma cell line (CGNH89) by morphological and immunohistochemical techniques after X-ray or heavy-ion beam exposure.
<Method> For X-ray and heavy-ion beam, CGNH89 cells were divided into 3 groups; control, 5Gy and 10Gy exposure. For each group, cells were fixed at the same time-points. Hematoxylin-Eosin (HE) and immunohistochemical staining were done in formalin-fixed cells. For electron microscopy, cells were fixed in fixative (1% glutaraldehyde, 10% formaldehyde). These samples were postfixed with osmium and embedded in Quetol 812. Ultrathin sections were examined under a tansmission electron microscope.
<Results and Discussion> After the exposure, both living cells and mitotic cells decreased in cell number. In the HE staining, nuclear condensation and apoptotic bodies increased after exposure. MIB-1 labeling index decreased in the early stage after exposure, but increased in later stages. It is suggested that cell cycle arrest and induction of apoptosis was induced by X-ray and heavy-ion beam exposure in CGNH89 cells. Ultrastructurally, the nuclei and cytoplasm after exposure showed a tendency to increase in size. Cellular swelling seemes to depend on the swelling of mitochondria, increase of cellular organelles, cytoskeleton, and secondary lysosome. However we did not find out either apoptotic body or necrotic change. We conclude that X-ray and heavy-ion beam exposure might trigger the same morphological changes in the surviving CGNH-89 cells.

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PURPOSE: The adhesion of inflammatory cells to endothelial cells has been reported to be involved in radiation-induced fibrosis. In this process, intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-β1) are suggested to play important roles. We investigated about the radiation-induced ICAM-1 expression to elucidate the relative biological effectiveness (RBE) of carbon-ion beam to X-ray with or without an inhibitor of TGF-β1 receptor kinase (SB431542).MATERIALS AND METHODS: Cell cultures of human umbilical vein endothelial cells (HUVEC) were irradiated with single doses of 1-10Gy of the 140 KV X-ray and 0.1-5.0Gy of 220MeV carbon-ion beams (108 keV/μm). Fluorescence-activated cell sorting analysis was used to quantify ICAM-1 expression at 24 and 48 hours after irradiation with or without SB431542.RESULTS: The expression of ICAM-1 was increased after X-ray and carbon-ion beam irradiations. The expression of ICAM-1 was significantly increased with the stimulation of TGF-β1, and decreased significantly by addition of SB431542 after carbon-ion beam irradiation. The expression of ICAM-1 increased by 6 folds with carbon-ion beam irradiation of 0.1-5Gy as compared with untreated control cells, while X-ray irradiation increased by 2.3 folds. The ICAM-1 expression was about 3 times greater in carbon-ion beam irradiation than that in X-ray irradiation.CONCLUSION: The present results suggested that radiation-induced ICAM-1 expression on HUVEC was regulated by TGF-β1. The RBE of carbon-ion beam to X-ray was about 3 for the expression of ICAM-1.

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Background
Experiments in mammalian cells suggested that ceramide acts as a signaling molecule in radiation-induced apoptosis, however, it is not well understood how the ceramide synthesis is regulated.
Methods
Quantitative analysis of ceramide: Lipids were extracted from Schneider line 2 (SL2) cells according to Bligh & Dyer method and analyzed by Thin-Layer Chromatography. Caspase 3/7 assay: DEVD-luciferin and luciferase was added to the cell lysate followed by measurement with luminometer.
RNA interference experiment: Double strand (ds) RNA was added to SL2 and incubated for 3 days to allow for turnover of the protein. To induce apoptosis cells were irradiated with UV-C.
Results
Three-fold increase in ceramide was observed 5 hours after irradiation. Cell permeable ceramide, C2 ceramide, can mimic the effect of irradiation and induces Caspase 3/7 activity. Overexpression of ceramidase that hydrolyzes ceramide causes reduction of Caspase 3/7 activity. In RNA interference experiments, Caspase 3/7 activity is decreased only by adding the ds RNA for neutral sphingomyelinase homolog. Other ds RNAs including acid sphingomyelinase homolog, ceramide synthase, and sphingolipid Δ4-desaturase have no significant effect.
Conclusion
In SL2, ceramide have an important role in radiation-induced apoptosis and neutral sphingomyelinase homolog might affect the regulation of ceramide synthesis.

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Potential advantage of greater biological effectiveness and excellent dose distribution over low-LET radiations represents the rational for the clinical usage of high-LET heavy ions, which are shown to effectively overcome the radioresitance of tumor cells. It has recently been reported that HA14-1, an inhibitor of Bcl-2, sensitizes radioresistant tumor cells, which overespress Bcl-2, to low-LET radiations. In this study, we tested whether HA14-1 exacerbates heavy ion-induced killing of Bcl-2 overexpressing tumor cells. In the absence of HA14-1, HeLa cells which overexpress Bcl-2 (HeLa/bcl-2) were more resistant to cobalt-60 gamma-rays than the cells which express only neomycin resistant gene (HeLa/neo), but the sensitivity of HeLa/bcl-2 cells to carbon ions (108 keV/μm) was comparable to that of HeLa/neo cells. Interestingly, our preliminary data show that treatment with HA14-1 1 hr prior to carbon ion irradiation sensitized both cells, such that HeLa/neo cells were sensitized more dramatically than HeLa/bcl-2 cells. This result suggests that HA14-1 enhances the killing effect of heavy ions in tumor cells in a manner that depends on the expression level of Bcl-2.

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Chromatin organization in nucleosomes and higher order structures plays an important role in DNA transcription, replication, recombination, and repair. Among proteins involved in chromatin reorganization, TIP60 histone acetylase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response to DNA damage is largely unknown. To better understand the mechanism of TIP60 complex in DNA repair, TIP60 complex purifies from chromatin soluble fraction after DNA damage. As a result, the TIP60 complex is associated with histone H2AX, a variant form of H2A, after DNA damage. Furthermore, DSBs facilitate the association of TIP60 with the ubiquitin-conjugating enzyme UBC13. The TIP60-UBC13 complex regulates the acetylation and ubiquitination of H2AX following the formation of DSBs. The DSB-induced acetylation of H2AX is required for this ubiquitination and occurs independently of the phosphorylation of H2AX. We also show that damage-induced acetylation and ubiquitination provoke the release of H2AX from chromatin immediately after induction of DSBs. Interestingly, the release of H2AX depends not on phosphorylation, but rather on acetylation and ubiquitination by TIP60. We conclude that sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promotes enhanced histone H2AX dynamics, which in turn stimulates a DNA damage response.

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DNA double-strand breaks (DSBs) are the major DNA lesions induced by ionizing radiation. Unrepired and/or misrepaired DSBs cause genome instability and leads to the onset of cancer. DNA-dependent protein kinase complex (DNA-PK), consisting of Ku70 and Ku80 heterodimer and DNA-PK catalytic subunit (DNA-PK_CS), is the key component of the non-homologous end-joining pathway of DNA DSB repair. To elucidate the mechanisms by which DNA-PK complex is recruited to DSBs in vivo and the temporal relationship with other DNA repair factors, we utilize a UV microbeam laser system to generate DSBs and live cell imaging approach to analyze protein dynamics at the laser induced DNA damage sites in live cells. Combination with genetics analysis and live cell imaging techniques, we are able to determine the temporal relationship among Ku, DNA-PKcs, XRCC4 and XLF at DSB sites upon laser damage. Further more, the use of photobleaching techniques (FRAP) has allowed us to determine that phosphorylation status of DNA-PK_CS influence the stability of its binding to DNA ends. This allowed us to determine that disabling the kinase activity and phosphorylation status of DNA-PKcs leads to a more rigid binding of DNA-PK_CS to DNA ends, which may interfere with effective ligation.

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The elevated risk of leukemia among atomic bomb survivors appeared from 1947, reached its peak around 1952, and continued until 1960s. By contrast, recent studies suggested that increased incidence of myelodysplastic syndrome (MDS), so-called preleukemia or smoldering leumkemia, continues even 60 years after the bombing. It is generally accepted that the early onset leukemia are caused primarily by chimeric genes as a result of chromosomal translocations, whereas point mutations and/or chromosomal deletions likely contribute to the late onset MDS.
Two genes that would be involved in the development of radiation-associated MDS were identified recently in our laboratory. First, mutations of AML1/RUNX1, a transcription factor indispensable for the definitive hematopoiesis, were found in approximately 20% of sporadic MDS. AML1 mutations were found especially at high frequencies (40-50%) in MDS patients among atomic bomb survivors in Hiroshima, as well as in radiation-associated MDS/AML patients. Moreover, mutations in radiation-associated MDS were limited to the runt-homology domain, which mediates DNA binding, while those in sporadic MDS patients distributed throughout AML1 protein. Second, we isolated a candidate gene, Miki(mitotic kinetics regulator) from the long arm of chromosome 7, which is frequently deleted in radiation-associated MDS and AML (up to 50%), as well as in sporadic cases (5-20%). Downregulation of Miki induced severe mitotic defect and abnormal morphology similar to those in bone marrow cells of MDS patients, suggesting that deletion of Miki gene would contibutes to development and/or progression of MDS.

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In the framework of the 21st Century COE Program “International Consortium for Medical Care of Hibakusha and Radiation Life Science”, we intend to establish a pivotal center for education/research, with close focus on local populations in Chernobyl and Semipalatinsk, as well as on Atomic bomb survivors. Moreover, in the context of social issues, including the Nuclear Power Plant construction rush in Asia, radioactive waste disposal and expanding medical exposure, the newly established Global COE Program will emphasize education/research in the three disciplines: (1) international radiation health sciences; (2) A-bomb disease medicine, and (3) radiation basic life sciences. Interdisciplinary approaches will be employed to enhance the quality of individual educational/research projects and to integrate basic and clinical research. Specifically, we will link bench work at laboratories with world-scale fieldworks unique to Nagasaki University; that is, medical cooperation and academic joint research on the radio-contaminated area and the radiation-exposed populations, with the aim of benefiting the public from the research outcomes. To this end, it is critical to establish a discipline of “Radiation Health Risk Control” based on research in the world's radiation-exposed populations, and to develop “human resources” who will become leading figures in the field of radiation medical sciences worldwide in cooperation with the research institutes in the US and Europe, WHO, etc. The COE finally seeks to make Japan-driven, creative, social and international contributions.

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Although radiation-induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation, the mechanism of this phenomenon is not known. The bystander observations have been made with both sub-confluent and confluent cultures as well as in three dimensional tissue cultures in a variety of endpoints. There is evidence that reactive radical species, cytokines and gap junctions are critical in mediating the bystander events. Monoclonal inhibitory antibody against TNF-a substantially suppressed cyclooxygenase 2 (COX-2) expressions in bystander cells via inhibition of the NF-kB pathway. Likewise, Bay 11-7082, a pharmacological inhibitor of NF-kB activation, demonstrated a similar effect in suppressing COX-2 activities and the bystander effects in human fibroblasts. Since COX-2 plays an important role in arachidonic acid metabolism and implicates the involvement of reactive radical species, mitochondrial DNA depleted (rho0) cells were used to illustrate the importance of mitochondrial oxidative metabolism in the bystander process. The bystander observations imply that the relevant target for various radiobiological endpoints is larger than an individual cell. A better understanding of the cellular and molecular mechanisms of the bystander phenomenon together with evidence of their occurrence in vivo will allow us to formulate a more accurate model in assessing the health effects of low doses of ionizing radiation.

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After the Chernobyl reactor accident on April 26th 1986, the incidence of thyroid cancer in children and adolescents living in contaminated areas of the Ukraine and Belarus increased significantly. Totally, between 1986 and 2002, 4.600 cases of thyroid cancer have been observed among those aged 0-18 years at the time of the accident. It is estimated, that approximately 40% of those cases are associated with radiation exposure and 60% are spontaneous cases. Starting in 1993, different projects on screening and treatment of radiation induced thyroid cancer have been launched by German institutions and the Center for Thyroid Tumors and the Institute for Radiation Medicine in Minsk, Belarus. Some of those projects have been carried out in the framework of international collaborative actions. In this symposium, the results of medical screening of thyroid glands and of clinical/therapeutic follow-up for childhood thyroid cancers will be summarized. A strict follow-up protocol for young females after high-dose radioiodine therapy has been developed together with the newly installed Belarussian-German Fonds “Arnica”. In the framework of radiation induced thyroid cancer after Chernobyl, the issue of the application of stable iodine for blockade of radioiodine uptake in the case of a radiation emergency has been studies extensively. For the future, a concerted European action to develop an agenda for research on Chernobyl health is planed under the leadership of Dr. Elisabeth Cardis from IARC/WHO with participation of many experts form Europe, the United States and Japan.

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