Effects of low-dose radiation have been evaluated in changes of molecular levels, however, relationship of its change with biological phenomenon such as adaptive responses, radioresistance or carcinogenesis remains unclear. We have previously reported that, in the presence of insulin, MCF7 cells (human breast cancer cells) appear to be sensitive to low-dose irradiation (0.25 Gy) in MTS assays, which is based on measuring the total viable cell number. Using colony-forming assays, however, no change in the cell survival was observed. These results suggest that insulin induces growth inhibition in the cells after low-dose irradiation. As phosphatidylinositol 3 (PI3)-kinase is a know participant in insulin-related signaling cascades, effects of a PI3 kinase inhibitor on the insulin mediation were evaluated. LY294002, one of PI3-kinase inhibitors sensitized to irradiation with 0.25 Gy in the absence of insulin, similarly to the insulin mediation. These results suggest that PI3-kinase works to maintain cell growth after low-dose-irradiation, and the function is possibly inhibited in the presence of insulin. In addition, microarray analyses in the presence or the absence of insulin were performed and three genes were identified as altered genes whose expressions were increased by more than two folds after irradiation in the absence of insulin and had almost no change after irradiation in the presence of insulin. Roles of PI3 kinase or the gene expression will be discussed in low-dose radiation-induced signaling cascades.

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We have examined the elevation of T-cell receptor (TCR) variant fractions in splenic T lymphocytes by beta-rays from tritiated water (HTO) with 3 Gy by a single i.p. injection and 137Cs gamma-rays with 3 Gy at a low dose rate mimicking internal expose (simulation-irradiation). In Trp53+/+ mice, although the TCR variant fraction did not increased at all by 137Cs gamma-rays, that by 3H beta-rays increased 19 day after injection. In this study, We analyzed the p53 activity in T cells from mice after whole body exposure by 3H beta-rays and 137Cs gamma-rays with 3 Gy.

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We reported that antioxidant activities in various tissues of mice were enhanced by low dose-rate (0.63 mGy/h) gamma ray irradiation. We furthermore found that Type II diabetes model mouse and human premature-aging syndrome model mouse (klotho) consecutively for the low dose-rate irradiation prolonged of their lifespan. Some researchers reported that the effects of lifespan prolongation were due to improve antioxidant functions or asthenia of insulin resistance. In addition, lifespan prolongation might be thought calcium metabolism contributed. Therefore we used female klotho mouse as primary study of the action that low dose-rate irradiation gave to lifespan prolongation effect, and measured change of hepatic antioxidant activities and plasma calcium concentration. The klotho mice irradiated from 28 days old at a source of gamma ray (137-Cs). The hepatic antioxidant activities and the plasma calcium concentration were measurements after appropriate irradiation time.
In the irradiated group, antioxidant activities slightly increased after the irradiation. The level of lipid peroxidation in the liver however was not related with the changes in the antioxidant activities. The plasma calcium concentration of irradiated group was less than that of the non-irradiated group.
These results indicated that the prolongation effect of lifespan of this mouse by the irradiation might be caused to increase in antioxidant activities, and controlled calcium metabolism.

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We have reported that low-dose irradiation enhanced antioxidative function and reduced oxidative damage in some organs of mice. Reactive oxygen species contribute to the formation of edema after cold injury. In the present study, we examined the change of brain edema of mice after low-dose X-irradiation. Mice were irradiated by sham or 0.5 Gy of X-ray. At 4 hr after irradiation, the right cerebral hemisphere was injured by placing a cooled metal probe on the skull surface. The water content of right hemisphere was measured at 0 (control), 1, 4, 24, and 48 hr after the cold injury. Histological observation (hematoxylin-eosin (HE) stain) and biochemical assays were performed. Results showed that the water content in the right hemisphere of the sham irradiated group were significantly increased at each time. On the other hand, those of the 0.5Gy irradiated group were lower than those of the sham irradiated group. The superoxide disumutase activities and the total glutathione contents in brains of the 0.5Gy irradiated group were greater than those of the sham irradiated group. Moreover these results were supported by the changes of cells in brains using the HE stain. These findings suggest that low-dose irradiation activates antioxidant function and inhibits the brain edema in mice. These results are similar to the previous report of the inhibition of ischemia-reperfusion injury in mouse paw.

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The catalase is an important component of the cellular defense system against oxidative damage induced by hydrogen peroxide. The catalase activities in blood and organs of the acatalasemic mouse of C3H strain are lower than those of the normal mouse. To elucidate the radiosensitivity of an acatalasemic mouse, we examined the time and dose-dependency in the survival rates, the lymphocytes and the intestinal epithelial cells, and the antioxidant function after 3.0 to 12.0 Gy whole-body irradiation. Results showed that no significant differences between acatalasemic mice and normal mice were observed in the survival rates and the histological changes in spleens and small intestine after each irradiation. The catalase activities in livers and spleens of acatalasemic mice were significantly lower than those of normal mice and the glutathione peroxidase activity in livers of acatalasemic mice was significantly higher than that of normal mice. At 10 days after 6.0 Gy irradiation, the catalase activities in livers of acatalasemic and normal mice and that in spleens of normal mice significantly decreased compared with no-irradiation, and there were no differences between those catalase activities. The total glutathione content in acatalasemic mice was significantly higher than that in normal mice at 10 days after 6.0 Gy irradiation. These findings suggested that the radiosensitivity of acatalasemic mice in terms of whole body irradiation does not significantly differ from that of normal mice, probably due to compensated sufficient contents of glutathione peroxidase and total glutathione in acatalasemic mice.

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We previously reported that radon inhalation enhanced antioxidative function in some organs of mice. On the other hand, acute alcohol injury is induced by reactive oxygen species in liver or brain. In the present study, we examined the effects of radon inhalation on acute alcohol injury in mice. The radon concentration in the air was regulated 4000Bq/m³ using the DOLL STONE. BALB/c mice were inhaled radon for 28 hr. The mice were received 30 % ethanol (6g/kg) by gavages every 12 hr three times. At 4 hr after the final ethanol dose, the livers, brains and blood in mice were obtained for biochemical assay and histological observation. Results show that the radon inhalation for 28 hr increased the catalase activities in liver and brain and decreased the lipid peroxide level in liver. Alcohol treatment tended to increase the lipid peroxide level in liver, but radon inhalation inhibited the level of that. The fat degeneration was exhibited in liver tissues after the administration of ethanol. These findings suggest that the radon inhalation activates the antioxidative function and alleviates the acute alcohol injury in mice. We similarly intend to discuss the levels of transaminase (liver function) in serum and acetaldehyde (oxidative damage) in liver.

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Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), which is clinically used for brain infarct in Japan from 2001, is a potent antioxidative medicine. Edaravone is also expected as a radioprotector for its high reactivity towards oxidizing radicals. Recently, some edaravone derivatives have been being investigated in order to study the Structure Activity Relationship (SAR) and to seek for more efficient radioprotector(s) other than edaravone. It was reported that a derivative with a pyridine ring as a substitute had much higher reactivity with hydroxyl radical than edaravone. However, the early processes of the reactions of edaravone derivatives are not fully understood and the quantitative data about their reaction rate have not been determined. In this study we investigated the reactions between some edaravone derivatives and hydroxyl radical and determined the rate constants of these reactions by pulse radiolysis technique. The rate constants were measured by three different methods. The first was direct measurement from the buildup kinetics of transient radicals. The second was competition method using carbonate ion as a competitor. The third was competitive measurement using DMPO, which is widely used spin-trapping agent for hydroxyl radical or hydrogen atom. The experimental results showed high reactivity of all derivatives investigated in this study. These reactions are near diffusion controlled. Moreover, the rate constants of derivatives with a pyridine ring by competition with DMPO were estimated twice as high as that by direct measurement. A possible interaction between the pyridine ring and DMPO is proposed.

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Natural flavonoids such as silybin (SLB), hesperetin (HESP), naringenin (NAN) and naringin( NAR) are believed to be active constituents of plants accounting for the reported chemoprevention of certain cancers. However, the molecular mechanisms by which the low concentrations of silybin and analogues in human can reduce the incidence are not clear. Using an in vitro plasmid DNA system and radiolytically generating reactive oxygen species (ROS) under constant scavenging conditions, we have shown that the four flavonoids are able to ameliorate free radical damage sustained by DNA. The dose modifying factor for 0.1 mM SLB for 50 percent protection of the plasmid DNA was found to be 8.9. SLB was found to be the most active among the tested flavonoids, with effects seen at micromolar concentrations. Combined fast-reaction chemistry studies support a mechanism of electron transfer (or H-atom transfer) from silybin to ROS-induced radical sites on the DNA. These results support an antioxidant role for silybin and analogues in the direct interaction with DNA radicals.

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We examined the mechanism for the radioprotective effect by DMSO treatment. When CB09 mice cells were treated with 2%DMSO 1 h before and during X-irradiation, radioprotective effects were clearly observed in survival assay and micronucleus assay. The suppressive effects of DSB by DMSO were also observed both 15 min and 2 h after irradiation. Interestingly, the ratio of suppression by DMSO in 2h after irradiation was higher than that in 15 min after irradiation. This result suggests that DSB repair is activated between 15 min and 2 h after irradiation by DMSO treatment. Moreover, little protective effects by DMSO were observed in DNA-PKcs deficient SD01 cells derived from scid mice. These results suggest that the radioprotective effect by DMSO is brought by activation of DNA-PK dependent repair, not by suppression of indirect action.

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The reaction between reactive nitrogen species (RNSs) such as peroxynitrite and nitrogen oxide (NO_x), and aromatic amino acids such as tyrosine and tryptophan results in a number of modifications, of which tyrosine nitration has gained much attention. 3-Nitrotyrosine (3-NT) was considered as an important biomarker of nitrative stress in vivo. In this work, the reaction rate constants of 3-NT, N-acetyl-3-nitrotyrosine ethyl ester (NANTE ) and 3-NT containing peptide Gly-nitroTyr-Gly reacting with azide radical (N3^•) at pH 6.0 were measured as 9.8±0.7x10⁸, 8.0±0.4x10⁸ and 9.1±1.0x10⁸ [L mol^-1 s^-1], respectively, which were almost one order magnitude higher than that of tyrosine and tyrosine containing peptides. The UV-visible absorption spectra of the transients of N3^• reacting with the above compounds were also investigated, bands of the transients located at a wavelength around 415 nm, which showed characteristic of tyrosyl radical. The absorption of the transients was pH dependent. Radiation-induced dimerization of 3-NT and N-acetyl-3-nitrotyrosine ethyl ester were identified and investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MSⁿ). The results implied the possibility of protein aggregation through covalent dimerization of 3-NT residue in vivo.

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Radiotherapy is now widely applied for the eradication of malignant tumors, however, the existence or outbreak of radioresistant cells is one of the major concerns in radiotherapy. In order to develop more effective radiotherapy, we have to understand the characteristics of radioresistant cells. Recently, we obtained a clinically relevant radioresistant cell line, HepG2-8960-R by exposure of HepG2 cells to long-term fractionated X-rays. These cells continue to proliferate even with the administration of daily exposure to 2 Gy of X-rays, that is, clinically relevant dose to the standard radiotherapy. In this study we examined the contribution of autophagy to radioresistance in vitro. After administration of fractionated 2 Gy of X-rays, autophagy was induced in HepG2 but not in HepG2-8960-R, suggesting that mTOR pathway is activated in radioresistant cells. Administration of mTOR inhibitor, rapamycin reverted radiosensitivity of HepG2-8960-R cells to the level of parental cells determined by high density survival assay. These strongly suggest that radiotherapy combined with the administration of rapamycin could be effective for the eradication of radioresistant cells.

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Activation of p53 is well known to induce apoptosis and cell growth arrest through the expression of downstream genes and to play an important role in the suppression of tumor growth. Mutated p53 (mp53) protein is defective in these functions due to conformational change of p53 protein. Our previous in vitro study using cultured human cancer cells showed that radiation treatment in the presence of a molecular chaperone, p53 C-terminal peptide (amino acid residues 361-382) is more effective for inducing p53-mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in mp53 cancer cells. In this study, we examined effects of p53 C-terminal peptides on tumor growth in in vivo experiments. p53 C-terminal peptide treatment to tumors induced growth delay in mp53 tumors transplanted with H1299/mp53 cells but not in p53-null tumors transplanted with H1299/neo cells. Tumor growth delay by X-rays was enhanced by p53 C-terminal peptide in the mp53 tumors. These results showed that the growth inhibition of the tumors after the combination treatment may be due to the increased apoptosis or cell growth arrest of H1299/mp53 cells. p53 C-terminal peptide might be available for p53-targeted radio-cancer therapy in patients with mp53 tumors.

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(Purpose) Sulphoraphane (SFN) is an isothiocyanate derived from broccoli and other cruciferous vegetables. It has been reported that highly effective in antitumor by SFN inducing apoptosis and cell cycle arrest. Our group has reported that the combination treatment of SFN and x-irradiation enhanced radiosensitivity. Our results demonstrated SFN led to radio-sensitization through the inhibition of DNA double strand breaks (DSBs) repair (NHEJ and HRR). Here, we report that SFN enhanced radiosensitivity in human cancer cells exposure to high LET irradiation (carbon irradiation). (Materials and methods) HeLa cells were used for this study. High LET radiation (carbon (290 MeV/n, 70 keV/um)) was used. HeLa cells treated with 20 uM SFN for 24 hours were irradiated. Cell survival level was monitored by colony forming assay. DNA DSB repair was measured by constant field gel-electrophoresis (CFGE) and immunofluorescence staining with gamma-H2AX (DSBs marker). We are also examining the possibility of activation of homologous recombination repair (HRR) pathway and non-homologous end joining (NHEJ) pathway using immunofluorescence staining experiments. We detected cell cycle distribution of cells treated with SFN and/or carbon irradiation by flow cytometry. We also monitored radiosensitivity by SFN in xenograft mouse model with HeLa cells. (Results and discussion) In the cells treated with combination treatment of SFN and carbon irradiation, survival level was significantly reduced as compared with the control populations. In vivo study, the combined treatment with carbon irradiation (4 Gy) and SFN (i.p. 300 umol/kg, 8 times) showed an efficient inhibition tumor growth. Our results demonstrated that SFN enhancing radioresitivity of tumor therapy in vitro and in vivo.

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Background: Brain irradiation is often performed in patients with brain tumors, however, this treatment leads to brain dysfunctions. We previously reported that 30 Gy X-irradiation did not induced the neural apoptotic death in mature neurons. Therefore, in the present study, we analyzed the effect of X-irradiation on dendritic spine-resident proteins immunocytochemically in mature neurons. Methods: We prepared primary neuronal cultures the hippocampi of fetal rats with Banker Methods. X-irradiation (30 Gy) was performed on the mature neurons at 21 days in vitro (DIV). After X-irradiation, the cells were immediately fixed with 4% paraformaldehyde and immunostained with antibody against to a presynaptic marker Synapsin I, and a postsynaptic actin-cytoskeleton regulator protein drebrin. Then the clusters of drebrin and Synapsin I were analyzed by MetaMorph software (Universal Imaging, West Chester, PA). The cluster of each protein was defined as a round staining region with a peak fluorescent level that was twofold greater than the averaged fluorescent level of the dendrites. Results: Thirty dendrites in each experiment were analyzed. The number of drebrin clusters along dendrites was 97.66 / 100 μm after 30 Gy X-irradiation, while the number of the control was 124.16 / 100 μm. X-irradiation decreased significantly the number of drebrin clusters (P=0.006). The number of synapsin I clusters along dendrites 113.14 / 100 μm after 30 Gy X-irradiation, while the number of the control was 101.23 / 100 μm. X-irradiation did not significantly decrease the number of Synapsin I clusters (P=0.091). Conclusions: These findings show that X-irradiation induces the change in the postsynaptic actin-cytoskeleton, and suggest the synaptic dysfunction based on morphological plasticity.

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[abstract] The number of HIV-1 infected patients is growing in Japan every year. Prognosis of the HIV-1 infected patients is improved dramatically by progress of drug treatment, but recently malignant tumors, in particular, malignant lymphoma becomes major problem for long-term prognosis. Primary Effusion Lymphoma (PEL) is a nonspecific disease relatively seen in the HIV infected patients and is well known that its stronger chemotherapy resistance makes the prognosis extremely poor. In this study, we examined the effectiveness of the radiotherapy for PEL using the culture cells and the Balb/c Rag-2/Jak3 double deficient mice models. [method] 1. We investigated radiosensitivity of the PEL cell strain (BCBL-1, BC-1, BC-3, TY-1). Using ¹³⁷Cs gamma rays (662 keV), the change of the cell counts after the radiation exposure by means of MTT method and a cell count measurement with Trypan Blue was analyzed and compared with other blood malignant tumors stock (Raji, Jurkat, K562). In addition, we confirmed whether or not these cells were induced apoptosis with DNA, ladder, and Annexin V dyeing. 2. We made a model mouse of PEL by transplanting PEL cell strain under the skin or in the abdominal cavity of severe immnodeficient mice (Balb/c Rag-2/Jak3 double deficient mice). After radiation exposure to these mice (whole body, 4 Gy*2), bone marrow transplantation was performed. [result] 1. The radiosensitivity of the PEL cell strain was significantly high in comparison with other blood malignant tumors stock. It was confirmed that PEL cell strains were induced to apoptosis with DNA, ladder, and Annexin V dyeing. 2. On the model of subcutaneous inoculation of BCBL-1, the non-irradiation group showed temporal tumor growth under the skin and all died 6 weeks after tumor implantation. However, in the irradiation group, such a tumor growth was not recognized 6 weeks after tumor implantation. In the model for the intraperitoneal injection of BC-3, the non-treatment group developed ascites, but in the irradiated treatment group the development of ascites was not recognized 6 weeks after tumor implantation. [conclusions] It was found that radiosensitivity of the PEL cell strain is significantly high. It was suggested that radiotherapy is very useful to PEL which has malignancy and resistance to chemotherapy.

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The microdosimetric quantities such as lineal energy y are better indexes for expressing the RBE of HZE particles in comparison to LET. However, use of the microdosimetric quantities in the computational dosimetry was severely limited because of the difficulty in calculating their probability densities (PDs) in macroscopic matter. We therefore improved the particle transport simulation code PHITS to be capable of estimating the microdosimetric PDs in macroscopic area, by means of incorporating a mathematical function that can instantaneously calculate the PDs around the trajectory of HZE particles with precision equivalent to a microscopic track-structure simulation. A new method for estimating biological dose, a product of physical dose and RBE, from charged-particle therapy was established, using the improved PHITS coupled with a microdosimetric kinetic model. The accuracy of the biological dose estimated by this method was reliably verified by comparing the calculated physical doses and RBE values with the corresponding data measured in a slab phantom irradiated with several kinds of HZE particles. The simulation technique established in this study will help to optimize the treatment planning of charged-particle therapy for maximizing the therapeutic effect on tumor while minimizing unintended harmful effects on surrounding normal tissues.

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(Purpose) Purpose of this study is to compile and analyze RBE data obtained by using Chiba-HIMAC synchrotron along with GSI-SIS synchrotron. Some data reported in past from Lawrence Berkeley Laboratory are also included. (Materials and Methods). A criteria for selection is made to so that only data satisfy the following conditions are used; LET below 100 keV/ microm and single dose irradiation. When authors describe RBE values along with dose response data, these values are employed. In case authors show dose responses but not cite any RBE values, an isoeffect line was drawn on a dose-response curve to read corresponding doses of ions and of reference photons. (Results) A total of 767 RBE values including 474 carbon-ion date were collected from 56 published papers. As RBE values of three cell lines that were extensively studied for a wide range of LET showed linear increase with an increase of LET up to 100 keV/ microm, a slope of regression line for RBE-LET (up to 100 keV/microm) relation was calculated for various biological endpoints. The shallowest slope among RBE data on cell lethality determined by colony formation was 0.011 keV/ microm for tongue tumor cells while the steepest slope of 0.33 keV/ microm was for Chinese hamster V79 cells. The slopes for chromosomal aberrations ranged widely in such that the smallest values of 0.014 keV/ microm was for lymphocytes and the largest value of 0.68 keV/ microm for malignant melanoma were obtained. This regression analysis was also applied to other endpoints including apoptosis, normal tissue/tumor response in animals, in vitro transformation, and also for fractionated irradiation. (Conclusion) Slopes of regression analysis widely distributed for cell lethality and chromosomal damage while slopes were relatively small for apoptosis and normal/tumor tissue response.

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Haplotypes of multiple SNPs in human PTTG1 and CD44 genes have been reported to show statistically significant association with severity of adverse effects induced by radiation therapy of breast cancer patients. In this study, a novel methodology of haplotype determination involving sensitive amplification of single chromosomes was developed. Limited dilution of chromosomes was performed within heated alkaline agarose solution to avoid extensive aggregation during dilution process. Aliquoted and solidified agarose gel pieces were then treated by exogenously provided Phi29 DNA polymerase and random hexamer oligonucleotides, yielding up to 100 000-fold amplification of immobilized chromosomes. The amplified materials were recovered in solution in PCR-ready form by simple heating to melt the gel, making them conveniently available for further multiple locus genotyping. With this amplification methodology, haplotypes of above radiation sensitivity-associated genes were successfully determined in individual breast cancer patients.

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High sensitivity Imaging Modality and CROSS(Correlation Response Observatory for Scintillation Signals) Project

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Recent studies have showed that the sharp of the LET-RBE curves of biological effects was different, when using different ion species even if similar LET values. We reported that the sharps of LET-RBE curves of biological effects reflected by repair process, such as cell killing, mutation induction and non-rejoining chromatin breaks were dependent on ion species. However, the biological effect without going through repair process, such as initially measured chromatin breaks was independent on ion species and LET values over 100keV/μm. In this study, we investigated whether repair kinetics of chromatin breaks induced by different heavy ions with a similar LET value were different or not. Normal human skin fibroblasts were irradiated with around 100keV/μm of carbon, neon and silicon ions generated by Heavy Ion Medical Accelerator in Chiba at National Institute of Radiological Sciences. Chromatin breaks were counted as excess fragments of prematurely condensed chromosomes using a technique of prematurely chromosome condensation (PCC).
The ratios of residual chromatin breaks in carbon and neon ions after 24h of post-irradiation incubation were different in silicon ions. Furthermore, the ratios of residual chromatin breaks in carbon ions after 0.5h of post-irradiation incubation was greater than those in neon and silicon ions. These results suggest that difference in the ratio of residual chromatin breaks after 24h of post-irradiation incubation reflected the results of both fast and slow repair process.

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Bystander effect observed in mouse splenocytes following cranial gamma-ray irradiation Yuko Kinashi, Hiroki Tanaka, Shinichiro Masunaga, Minoru Suzuki, Genro Kashino, Liu Yong, Koji Ono, Sentaro Takahashi Research Reactor Institute, Kyoto University The evidence of bystander effect was inspected in vivo study following the local radiation. The head of C3H mice were exposed to 10Gy of gannma ray, while the other part of body was shielded with 5cm thick lead block. The effective dose for reminder body except head was calculated 1.0-1.4Gy. The splenocytes were isolated 1-24 hours after the cranial irradiation and the apoptosis was measured with Elisa kit (Roche). The T-lymphocytes were isolated and the micronucleus induction was estimated by the cytokinesis-block micronucleus assay. The damage of splenocytes measured by these above two method was more excessive than that is prospected under the radiation dose of 1.0-1.4Gy. Our results suggested that bystander effect was observed in distant non-irradiated organ under the local irradiation.

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Fibroblast growth factor (FGF) family plays numerous roles in regulating physiological events through regulation of growth and differentiation of the cells, and is expected to be used as curatives for various health problems. We reported that FGF1 protects jejunum from radiation-induced injuries more effectively than does FGF7 or FGF10. However, the structural instability of wild-type FGF1 and its dependence on exogenous heparin diminish its potential for practical use. Here we developed an optimized FGF1:FGF2 chimeric molecule, FGFC, based on a chimeric molecule that had been created years ago. FGFC demonstrated higher stability in its biological activity, higher resistance to trypsin digestion, and slower decrease of its concentration in solution than FGF1 or FGF2. Fluorescence analysis suggested that these characteristics of the FGFC protein were the results of its folding stability. Evaluation of its receptor specificity revealed that FGFC activates all seven subtypes of FGF receptors, including FGFR2b that is not strongly activated by FGF2. FGFC stimulated proliferation of keratinocytes in the presence and absence of heparin, more strongly than FGF1 or FGF2. FGFC increased survival of jejunum crypt of the mice that had received total body gamma-irradiation at 10 Gy. These results prove FGFC to be a promising therapeutic agent for radioprotection, wound cure and other clinical applications.

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Radiation therapy using heavy ion beams has a merit compared to low LET radiation therapy using such as X-rays and gamma-rays that injury to normal tissues is reduced. However, it is still possible that high LET heavy ion radiation induces radiation injury along the beam at entrance side. Here we report LD50/30 value for haematopoietic death of mice after whole body carbon beam irradiation. The LD50/30 value was about 5.5 Gy. By comparing with the value of 6.6 Gy for X-ray irradiation, RBE value was calculated to be 1.2. Next, we examined effects of radiation modifiers (cysteamine, WR-2721, zinc-yeast, and gamma-TDMG) on the survival of mice. Mice were whole-body irradiated with the carbon beam at 6.0 Gy and 30-days survival was measured. All the modifiers examined showed significant increase of the survival rate similar to what was shown in X-ray irradiation experiments.

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Sanazole (AK-2123, 3-nitrotriazole derivative, N1-(3-methoxypropyl)-2-(3-nitro-1 H-1, 2, 4-triazol-1-yl)acetamide) has been tested clinically as a hypoxic cell radiosensitizer. The aim of this study was to examine whether sanazole enhances apoptosis induced by hyperthermia (HT) or ionizing radiation (IR) in air condition. The combined effects of HT (44 C, 20 min) or IR (X-rays, 10 Gy) and sanazole on apoptosis in human lymphoma U937 cells were investigated. When the cells were treated first 10 mM sanazole for 40 min, and exposed to HT or IR afterwards, a significant enhancement of HT- or IR-induced apoptosis was evidenced by DNA fragmentation and phosphatidylserine externalization at 6 h after treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to sanazole, and showed subsequent and transient increase in intracellular peroxide formation. Mitochondrial membrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combined treatment. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by HT or IR, was enhanced by sanazole. An increase in the intracellular Ca2+ concentration [Ca2+]i, externalization of Fas, and decrease in Hsp70 were observed following the combined treatment. These results indicate that the intracellular superoxide and peroxides generated by sanazole are involved in the enhancement of apoptosis through Fas-mitochondria caspase and [Ca2+]i-dependent pathways, and a decrease in Hsp70 also contributed to the enhancement of apoptosis.

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It is well known that hyperthermia of 42-43^oC shows higher lethal sensitivity to a certain specific cancer cell. This heat treatment does not have an influence on normal tissue very much from experience. This becomes a base of cancer therapy by cancer hyperthermia. However, it is not clear why hyperthermia sensibility is higher in cancer tissue than normal tissue. Researchers have reported several reasons for hyper-lethal sensitivity of cancer cells, such as anoxia, low pH, and deficiency of repair system of heat damage. Our previous results shows that target of hyperthermia is centrosome. There is no difference in heat sensitivity of centrosome between normal and cancer cells. Structural aberration of centrosome induces abnormal division in mitotic period, and the cell that caused abnormal division dies with high frequency. In other words, lethal sensitivity of cells is controlled by the frequency of centrosome aberration when cells are going into mitosis after heat-treatment. On the other hand, we have reported that low dose radiation exposure of a cGy level is not accompanied with derivation of p53 and promotes cellular proliferation potency. By taken together our results, it is expected that a cancer cell increase heat sensitivity than normal cells by combination of low dose radiation and hyperthermia. This time, I will report the possibility of combined treatment of hyperthermia and low dose radiation.

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Purpose The thermo-enhancement effects of Parthenolide (PTL), a principal ingredient of herb which target on NF-kappaB, transcript factor, were investigated in human prostate PC3 and DU145 cells. The expression of heat induced hsp72 proteins and p53 proteins, takes part in apoptosis induction, were examined. The thermo-enhancement effects of PTL on the signaling of NF-kappaB through the caspase-8 or -9 were analyzed. Materials & Methods PC3 and DU145, human prostate cells, were used in this study. The cytocidal effect or the thermosensitivity to PTL, hyperthermia or the combination therapy in both cells were estimated by colony forming assay. The kinetics induction of various proteins was analyzed by western blot analysis. Induction of apoptosis was analyzed by flow cytometry. Results The combination therapy with PTL and hyperthermia in PC3 and DU145 cells showed synergistic thermo-enhancement effects. After treatment with hyperthermia alone, the accumulated amount of hsp72 increased markedly, but with PTL alone did not increase. Both hsp72 and p53 proteins were not induced significantly after combined treatment with PTL and hyperthermia. Apoptosis after treatment with PTL showed remarkable incidences, while apoptosis after combination therapy showed significant incidences. Conclusion Thermosensitivity of PC3 and DU145 was enhanced synergistically in combination therapy with PTL. Apoptosis after combined treatment showed increase significantly. The thermo-enhancement effects of PTL were not due to inhibit the heat-induced hsp72, but induce apoptosis by blocking the activation of NF-kappaB. We are studying about the signaling of NF-kappaB through the caspase-8 or -9 on the induction of apoptosis.

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Hypoxia-inducible factor-1 (HIF-1) plays important roles in angiogenesis, invasion and metastasis of cancer cells, and change of metabolic process from oxidative phosphorylation to glycolysis in response to hypoxia. In addition, it was recently reported that the level of HIF-1 activity is a significant predictor for tumor recurrence after radiation therapy. However, the molecular mechanism underlying the HIF-1-mediated radioresistance remains to be elucidated. The purpose of this study is to approach to the problem. Growth delay assays showed that HIF-1-targeting treatment with a protein drug, TOP3, or with a gene therapy strategy, Ad/5HREp-BCD, increased the effect radiation therapy in vivo, while siRNA-mediated modulation of HIF-1 activity in vitro showed little effect on radiosensitivity of cultured cells. Based on these results, we hypothesized that HIF-1-active tumor cells are radiosensitive, but they have a property to facilitate the radioresistance of the other constituents of tumors. To examine the possibility, we established a unique system to tag the HIF-1-active cells in tumor xenografts and chased the fate of them after radiation therapy. The optical imaging experiment clearly showed that HIF-1-active cells were predictably radiosensitive and recurrent tumors were mainly composed of the tag-free tumor cells. We are now examining the next hypothesis that HIF-1-active cells protect endothelial cells from cytotoxic effect of radiation, because TOP3 dramatically decreased micro vessel density in tumor xenografts after radiation treatment. In the present study, we present our latest data in this field and propose a model underlying the HIF-1-mediated tumor radioresistance.

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In order to measure in vitro radiosensitivity, clonogenic assay has been the gold standard for evaluating overall consequences of early and late responses to radiation. Since clonogenic assay relies on the ability of cells to form viable colonies derived from "a single cell", it is not directly applicable to cells with very low plating efficiencies. Moreover, this assay ignores the cell-to-cell communication because cells are usually plated out at very low density to form colonies from single cells. To overcome these problems, the "high density survival" (HDS) assay was recently developed. In this study, we developed the modified HDS assay which is easier to be performed. We have recently defined and established radioresistant cells with clinical relevance. Those radioresistant cells continued to proliferate with daily exposure to 2 Gy of X-rays for more than 30 days. The modified HDS assay successfully detected clinically relevant radioresistance which is not always observed by colony assay or other ordinary assays for cell survival and death. Therefore, we think that the modified HDS assay in the present assay is a useful tool to predict radioresistance with clinical relevance.

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It has been reported that cancer radiotherapy occasionally results in invasion of survived tumor cells to surrounding tissues. This increased invasiveness seems to be a cellular response to radiation at sub-lethal doses; however, the underlying mechanisms have not been well addressed. To understand the effect of radiation on tumor cell invasion in view of cell motility, we irradiated human glioblastoma cell line A172 (p53 wild) with 3Gy of X-ray, corresponding to approximately 70% of cell survival, and evaluated the number of cells migrated toward laminin, a potent chemoattractant to these cells, through a micropore filter with 8μm pore. The results showed approximately two-fold increase in cell motility by the irradiation, associated with an increased expression of integrin α6β1, a laminin receptor as revealed by flowcytometry using a specific antibody. Furthermore, an increased and sustained phosphorylation of focal adhesion kinase (FAK), a signaling molecule downstream the interaction of integrin and laminin, was observed in irradiated cells. The expression profiles for cell adhesion molecules by microarray technology exhibited the specific upregulation of integrin a subunit and also collagen type 18. Interestingly, all of those cellular responses were suppressed or hardly observed in irradiated A172M (A172 bearing R248W p53 mutant) cells. These results suggest that X-ray irradiation at sub-lethal doses potentiates motility of glioblastoma cells possibly through the mechanisms involving p53-associated upregulation of the integrin-laminin interaction and their downstream signaling pathways.

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Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin. The total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^- cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 hr before cytokine stimulation, a similar degree of hematopoiesis obtained in the results of non-irradiated cells stroma-free culture was observed, thus suggesting the critical role of cell-to-cell interaction. Since the efficient expansion of stem cells derived from radiation casualties provides a suitable cell source for autologous cell therapy, the present results showed the efficacy of human CB-derived mesenchymal stem cell-like stromal cells for the regenerative expansion of hematopoietic stem/progenitor cells exposed to irradiation.

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Purpose: The treatment of radiation-induced intestinal injury is difficult and there is no effective treatment now. It is important to develop new treatments for radiation-induced intestinal injury. Previously, we reported that embryonic stem cells (ESCs) transplanted directly into the wall of irradiated intestine could colonize and differentiate. But the transplantation of ESCs showed no influence on the survival rate and the change of body weight of the mice with radiation-induced intestinal injury. In this study, we investigated whether the mesenchymal stem cells (MSCs) transplantation could prevent the radiation-induced intestinal injury. Methods: The intestines of female nude mice (ICR nu/nu) were irradiated at a single dose of 30 Gy (X-ray of 150 kV, 5 mA, with 0.5 mm Al filters, at a dose rate of 1.9 Gy/min), and were immediately transplanted with male MSCs (C57BL/6n) into the wall of the irradiated intestine by direct injection. For 13 days after irradiation, mice were weighed daily, and the survival was recorded. At 13 days to 27 days after irradiation, the intestines of mice were obtained to assay histological change by Haematoxylin-eosin staining. Results: The body weight of the irradiation and MSCs transplantation group increased significantly compared with the irradiation only group from 8 days after irradiation. In addition, the survival rate of the irradiation and transplantation group was significantly higher than that of the irradiation only group from 5 days after irradiation. In the histological observation, the intestines of MSCs transplanted mice were thick in the submucosa and muscle layer, and were recovered from the radiation-induced intestinal injury. Conclusions: These results suggest that transplanted MSCs may play important role to prevent radiation-induced injury. It may become a new treatment method to repair the radiation-induced intestinal injury. The protective mechanism of MSCs transplantation will be needed to study.

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Fibroblast growth factors (FGFs) play important roles in numerous biological events such as angiogenesis and wound repair.FGFs need heparan sulfate / heparin as a co-factor to activate FGF receptor (FGFR). FGF7, one of FGFR2b ligands, has been approved as a remedy for radiotherapy-induced oral mucositis. We previously studied the protective activity of FGF1, FGF7, and FGF10, as ligands for FGFR2b, and reported that FGF1 protects the jejunum from radiation-induced injuries more effectively than FGF7 and FGF10. However, the structural instability of wild-type FGF1 and its dependence on exogenous heparin diminish its potential for practical use. Although FGF2 is structurally related to FGF1, its activity is more independent on exogenous heparin than FGF1.We have created an FGF1:FGF2 chimera (FGFC) and found its activity in the absence of heparin was more potent than FGF1. This study aimed at evaluating the protective activity of FGFC against radiation-induced intestinal damage. Receptor specificity analysis showed that FGFC was able to activate all of the FGFR subtypes similar to FGF1. When FGFC was administered with heparin intraperitoneally to mice at 24 h before total body irradiation, it increased small intestinal crypts survival with a potency similar to FGF1. Whereas it was even superior to FGF1 when administered without heparin. Finally, the effectiveness of FGFC was also observed without heparin when it was administered 24 h after irradiation. These findings suggest that FGFC is useful in clinical applications for both prevention and post-treatment of radiation injuries.

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[Objective] To determine the potential of three different clinically available recombinant human granulocyte-colony stimulating factors (rhG-CSF: nartograstim, lenograstim, filgrastim) on the differentiation of human CD34⁺ cells to form mature neutrophils, the phagocytic ability of these neutrophils and the survival of X-irradiated granulocytic progenitor cells (CFU-G) were investigated. [Materials and Methods] CD34⁺ cells were purified by the EasySep human CD34⁺ cells isolation kit. Purified CD34⁺ cells were cultured for 14 days in serum-free medium supplemented with rhG-CSF. Harvested cells were analyzed for the induction rate to mature neutrophils and the phagocytic ability of control or X-irradiated cells (2Gy). A quantitative analysis of CFU-G and its surviving fractions were assayed by using a methylcellulose culture containing rhG-CSF. [Results and Discussion] The number of CD34⁺ CFU-G-derived colonies were increased with rhG-CSF dose dependently and reached a plateau at 50 ng/ml. The following experiments were performed with 100 ng/ml. Nartograstim significantly induced higher neutrophils in both cultures of non-irradiated or X-irradiated cells than lenograstim or filgrastim. However, no significant effects were observed in the phagocytic ability of non-irradiated or X-irradiated neutrophils and the surviving fraction of CFU-G by any rhG-CSF. The present results thus provide valuable information regarding the in vivo kinetics and function of neutrophils after the administration of each type of rhG-CSF.

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  Survival rate of mice are dramatically increased by the administration of radioprotectors such as heat-killed Lactobacillus casei (LBC) approx. 1 day before exposure of lethal dose of ionizing radiation. To reveal the mechanism, blood parameters that reflect the survival rate were examined. We found the relationship between the survival rate and blood interleukin-1beta (IL1b) level.
  Strong radioprotective effect of LBC was reconfirmed by the following experiments: All the C3H/He mice exposed at supralethal dose (8.0Gy) of X-ray were died within 19 days following the radiation. When the LBC was injected 16∼48 hours before the radiation, survival rate of the mice was 80% at Day-28. To reveal the systemic effects of the LBC-injection, we quantified the levels of cytokines in the circulation and found that IL1b level was drastically increased. Plasma IL1b level was increased at 4 hours, and reached to the maximal levels between 8 and 16 hours, then decreased to undetectable level (<10 ng/ml) at 48 hours. When glucocorticoids and their stimulator were injected with LBC, LBC-induced increases in the plasma IL1b level as well as survival rate were abolished. Co-administration of non-steroidal anti-inflammatory drugs with LBC have no effect on the IL1b levels and the survival rates. In contrast, administration of mineral corticoids with LBC enhanced the blood IL1b levels and survival rate. The data show that radioprotection by the stimulator of inflammation such as LBC are mediated by the elevation of blood IL1b.

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Basic fibroblast growth factor (bFGF) is the growth factor which accelerates cell proliferation differentiation, cell migration and angiogenesis. Previous studies have shown that administration of bFGF enhances epithelial cell proliferation and restitution as well as crypt stem cell survival after radiation injury to the intestine. In this study we examined the effect of bFGF on acute radiation induced injury in rat small intestine. Six-weeks male Wistar rats were administered 4 mg/kg bFGF intraperitoneally at 25 hours before irradiation. The jejunum and the colon tissue were removed at 3, 6, 16 hours, 1, 2, 3 and 5 days after 8 Gy whole-body irradiation. The sections were stained H&E. The length of villi and crypt in the jejunum were measured, and the number of apoptotic and mitotic cells per crypt were counted. We performed immunohistochemical staining against the Ki-67 antigen. The expression of PCNA, p53, p21 and Bax after irradiation were examined by Western blot analyses. The length of villi in the jejunum of control rats was decreased at 1, 2, 3 days after irradiation. In contrast, the length of villi of rat treated with bFGF was higher than that of control until 2 days after irradition. The length of crypt was also similar. bFGF treatment reduced the number of apoptotic cells per jejunum crypt at 3 and 6 h after irradiation to 29 % and to 11 % of the control values. bFGF treatment increased the number of mitotic cells at 2, 3 and 5 days after irradiation compared to control value. The Ki-67-positive cells in the jejunum of rat treated with bFGF were higher than in control rat. PCNA expression in rat treated with bFGF was significantly higher than in control at 2 days after irradiation. bFGF treatment resulted in decreases of p53 accumulation, p21 and Bax expression at 3 and 6 hours after irradiation. Our results suggested that bFGF has a protective effect against acute radiation-induced injury in rat jejunum by suppressing the apoptotic cells including the stem cells and accelerating the crypt cell proliferation.

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Introduction: Skeletal muscle is relatively stable tissue. However, growth and repair of skeletal muscle are carried out in response to damage or stretch. Skeletal muscle is relatively resistant to X-ray radiation, but the formation of multinucleated myotubes is delayed or suppressed by X-ray irradiation of myoblasts. In this study, we investigated whether insulin-like growth factor-1 (IGF-1), expression of which increases during generation and regeneration of skeletal muscle, improve the delay or suppression of myotube formation induced by X-ray irradiation.
Materials and Methods: Mouse derived myoblast, C2C12 cells, were seeded at a density of 4104 cells/cm2 on a 24-well celldesk plate (Sumitomo Bakelite). After an overnight culture in DMEM medium supplemented with 10% feral bovine serum (FBS), X-ray irradiation at 2 or 4 Gy using an X-ray generator (MBR-1520R; Hitachi Medical Corporation, at 90 cGy/min) was conducted, and then the medium was replaced with DMEM medium with 2% FBS and the culture was continued for 6 days to induce differentiation. Differentiation of cells into myocytes and myotubes was identified by fluorescence immunostaining with an anti-myosin (skeletal muscle) monoclonal antibody (Histofine).
Results and Discussion: Decreased myotube formation was observed after X-ray radiation of 2 Gy and this change was reversed by adding 5 ng/ml IGF-1. The decrease in the number of differentiated cells and reduction in myotube formation induced by X-ray radiation of 2 Gy were inhibited by IGF-1. The X-ray irradiation at 2 Gy is used in multi-fractionated irradiation in radiotherapy, and therefore IGF-1 may be useful for reduction of radiation damage.

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Sulphoraphane (SFN), an isothiocyanate devrived from broccoli and other cruciferous vegetables, is a positive regulator of Phase II detoxification enzymes. It is highly effective in affording protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN enhanced radiosensitivity in HeLa human cervix epithelium carcinoma cells by inhibiting double strand breaks (DSBs) repair pathways. HeLa cells stimulated with SFN for 24 hours were irradiated, and cell survival level was significantly reduced as compared with the control populations. DNA double strand breaks (DSBs) repair as measured by constant field gel-electrophoresis showed a clear inhibition of DSBs repair in cells with SFN, while little inhibition was observed in cells with negative control. Our immunofluorescence staining experiments revealed a significant delay in gamma H2AX (DSBs marker), Rad51 (homologous recombination repair (HRR) related protein) and phosphorylation of DNA-PKcs (a critical non-homologous end joining (NHEJ) protein) foci formation in cells with SFN when compared with the control populations. In addition, the combined treatment with radiation and SFN (i.p. 300umol/kg, 1 time/day) in xenograft model with HeLa cells showed an efficient inhibition in vivo tumor growth. Our results demonstrated SFN led to radio-sensitization through the inhibition of DSBs repair; a possibility of using SFN as an effective radiosensitizer in tumor radiotherapy may arise. To the best of our knowledge, the present study is the first published report about SFN enhancing radioresitivity of tumor therapy in vitro and in vivo.

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[Purpose] Aim of the present study was to clarify the hypoxic radiosensitizing effects of novel 2-nitoroimidazole acetamide (2NIA) derivatives on human lung cancer cells. [Materials and Methods] Human lung carcinoma A549 cells grown in exponential phase in alpha-MEM supplemented with 10% FBS were used throughout the experiments. The cells were trypsinized, suspended in complete medium which was supplemented with the sensitizers (1mM). The suspensions of 0.5 ml in a glass tube were flushed with humidified mixed gas (95%air+5% CO2 or 95%N2+5%CO2) at room temperature. The cells were irradiated with 6MV X-rays. Cell survival was obtained from the colony formation assay. The 2NIA derivatives (TX-2243, TX-2244, TX-2246) and etanidazole were used as the sensitizers. [Results and Discussions] Oxygen enhancement ratio of hypoxic cells exposed to X-rays without the sensitizer was 2.78 based on 10% survival dose (D10). Enhancement ratio (ER=(D10 c/o sensitizer) / (D10 c/ sensitizer)) in the presence of TX-2243, TX-2244, TX-2246 and etanidazole was 1.13, 1.50, 1.56 and 1.75, respectively. On the other hand, ER based on 50% survival dose (D50) was 1.50 for TX-2243, 1.54 for TX-2244, 1.59 for TX-2246 and 1.69 for etanidazole. ERs for TX-2244 and TX-2246 were small as compared with that of etanidazole, but was significant. Present study suggested that both TX-2244 and TX-2246 were useful as the hypoxic cell radiosensitizer for human lung cancer cells. Characteristics of radiosensitizing effect of TX2243 was unusual. For TX-2243, although the small radio sensitizing effect (ER=1.13) at D10 was observed, the large effect (ER=1.50) at D50 was mentioned. The mechanisms of hypoxic radiosensitization of 2NIA derivatives still remain to be investigated.

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[Purpose] Gliomblastoma is one of the most common tumor types resistant to radiation. Arsenite was reported to show synergistic radiosensitization effect in vitro and in vivo. But, the mechanism of radiosensitization is not well understood. Last year, we shown the induction of senescence-like growth arrest (SLGA) including heterochomatin formation by treatment of radiation or arsenite. This year, we have studied the difference of mechanism of SLGA between radiation and arsenite. [Results] Glioblastoma U87MG cells treated by irradiation or arsenite. It is kown as p21 or p16 relate to SLGA. U87MG lacks the p16 gene. Thus, we analysed the p21 by Westen blotting. The significant induction of p21 was observed by irradiation, but not by treatment of arsenite. Now, p21 dependency about SLGA by irradiation or treatment of arsenite are analysing by siRNA and the data will be presented.

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Isothiocyanates (ITC; chemical structure: R-N=C=S) are present in cruciferous vegetables such as broccoli, cabbage and cauliflowers. Some ITSs have been shown to be effective in preventing chemically induced cancer. Benzyl isothiocyanate (BITC), an ITC compound isolated from papaya, has been reported to prevent cancers through the induction of Phase II detoxification enzymes in animal models, and induce apoptosis in human maligmant cell lines. These findings suggest that BITC could be a useful chemopreventive and/or chemotherapeutic agent against human cancers. We investigated the effect of BITC on the radiosentitivity in two human panreatic cancer cell lines, MIA PaCa-2 and Panc-1 carrying mutant p53. BITC sentitized both cell lines to radiation, but potentiated X-ray sentitivity more in MIA PaCa-2 cells. A combination of BITC and radiation induced apoptosis synergistically and cleavage of PARP in MIA PaCA-2, but not Panc-1 cells. Next, we determined the effect of BITC treatment on the level of Apaf-1 protein, which plays an important role in regulation of apoptosis. The BITC-treated MIA PaCa-2 cells exhibited a marked increase in protein level of Apaf-1. These results indicate that BITC induces radiosentitization through the suppression of anti-apoptosis signal and increase of apoptosis promotion factor in human certain cancer cells. Thus, Isothiocyanate, iucluding BITC, may present a new class of radiosensitization in clinical radiation therapy.

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High atomic number materials, such as gold and platinum are known to be able to interact with X irradiation, resulting in the enhancement of the biological effect by Photoelectric and Compton effect when cells are X-irradiated in the kilo-voltage range. Herold et al (Int J Radiat Biol, 2000) demonstrated radiosensitization effects when mouse tumor EMT-6 cells were irradiated in the presence of the gold particles (3 μm diameter). If the diameter of the particles could be much smaller, the effective radiosensitization can be expected by the localization of gold-nanoparticles nearby the cell nucleus. In this study, we investigated whether nanogel reagent containing gold nanoparticles with a diameter of less than 10 nm enhanced the reproductive cell death in X-irradiated cells. Chinese hamster V79 cells, human lung adenocarcinoma A549 cells and mouse squamous cell carcinoma SCCVII were incubated with or without various concentration of the gold-nanogel reagent for 14 h. After the treatments of the reagent followed by X irradiation at 200 kV, the reproductive cell death was evaluated by a colony formation assay. The cytotoxity of the gold-nanogel reagent was not observed when V79 cells and A549 cells were treated with the gold-nanogel reagent in the concentration of 50 μg/ml and 30 μg/ml, respectively. The increases of the reproductive cell death by the X irradiation under the gold-nanogel reagent were observed in all cell lines. These results suggest a possibility that the gold-nanogel reagent can be an effective radiosensitizer. The detailed mechanism for the radiosensitization effect of gold nanoparticles will be discussed.

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We studied the localization of phosphorylated H2AX (γ-H2AX) in cultured human fibroblasts (NB1RGB) and EGFP-tagged rad51 in Chinese hamster CHO cells after irradiation with heavy ion beams. Asynchronous cells were irradiated with X-rays, carbon ion beam (LET is about 30 or 88 keV/μm), Si (220 keV/μm), Ar (95 keV/μm), and Fe ion beam (440 keV/μm) at room temperature. Gamma-H2AX in irradiated cells was detecteded by immuno-staining from 0 to 24 h after irradiation. Gamma-H2AX monitored by using flow cytometry increased just after irradiation of each radiation and reached maximum around 30 min. Gamma-H2AX was then decreased quickly for cells irradiated with X-rays but presented longer for Si and Fe beams. Comparing the induction of the amount of γ-H2AX at 30 min after irradiation, high LET Si and Fe were more effective than X-rays and C beams. Carbon ion beam with lower LET of 30 keV/μm induced similar to X-rays and C ion with 88 keV/μm was intermediate between High LET group and low LET group. Under the confocal microscopy, foci of γ-H2AX on cell nucleus was not visible just after X-irradiation, but obvious after Fe ion irradiation. Number of foci per nucleus was increased with increasing dose at 30 min after irradiation similarly for all radiation tested. On the other hand, rad51 foci were not visible just after irradiation even for Fe beams. They were observed about 1 h after irradiation, and were co-localized with γ-H2AX foci. They were visible at 20 h after irradiation both for heavy ions and X-rays.

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[Introduction] The mechanism by which heavy ions have greater effects than low-LET radiations is not completely understood. In this study, we investigated the cell-killing effect of heavy ions in normal human fibroblasts.
[Materials & Methods] Normal human diploid fibroblasts immortalized with the hTERT telomerase catalytic subunit gene (BJ-hTERT cells) were used. Confluent cell monolayer was irradiated with ⁶⁰Co γ-rays (LET=0.2 keV/μm), helium (17 keV/μm), carbon (70-212 keV/μm), neon (310 and 430 keV/μm) and argon ions (1320 and 1530 keV/μm). Cell survival was determined with a colony formation assay. The 10% survival dose (D₁₀) was calculated from survival curves and RBE based on the D₁₀ was obtained.
[Results & Discussion] D₁₀ values of γ-rays, helium, carbon, neon and argon ions were 4.3, 2.4, 1.0-1.5, 1.6-2.5 and 5.8-7.0 Gy, respectively. RBE reached the maximum at 70 and 110 keV/μm carbon ions. We will also discuss the DNA fragmentation by heavy-ion irradiation in normal human fibroblasts.

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High-LET heavy charged particle radiation has been used in radiotherapy and radiation breeding because of its greater biological effectiveness compared with low-LET radiation. The health risk assessment for astronauts due to space radiation during interplanetary manned missions is also an important issue concerning the biological effects of heavy-ion radiation. However, the many parts of mechanisms underlying its greater biological effectiveness still remain unclear. Localized irradiation of specific regions within organisms using heavy-ion microbeam systems provides an attractive means of investigating the mechanism of heavy-ion radiation action. Therefore, we had developed the heavy-ion microbeam system at TIARA of the Japan Atomic Energy Agency, and utilized for analyzing heavy-ion induced biological effects. Additionally, we have started developing another new focusing microbeam system for carrying out the experiments that cannot be archived by existing collimating microbeam system. The new system can provide finer microbeam by focusing heavy-ion beam from AVF cyclotron using a quadruplet quadrupole lens system. We are now developing a method for irradiating specific region of target sample using the cell targeting station installed last year. The collimating microbeam system was also improved for allowing a novel irradiation means that make a good use of its high fluence characteristics. For that purpose, the cell targeting system of collimating microbeam system was newly designed and updated.

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Malignant mesothelioma (MM) is a highly aggressive tumor arising from serosal surfaces of the pleura, peritoneum, and pericardium. MM, having three histological types, epithelioid, sarcomatoid and mixed/biphasic, is associated with previous asbestos exposure with a latency of 30–40 years, and its incidence is expected to increase. Current treatments usually include some form of surgery and may be combined with chemotherapy and/or radiation. Since the prognosis of patients with current therapy remains poor, it is important to develop new and more effective treatments. A previous in vitro study of MM cell has shown that relative biological effectiveness (RBE) of carbon-ion compared to X-ray was 2.9–3.4. Thus, we evaluated the efficacy of carbon-ion radiation therapy (CIRT) for MM in mouse models. We inoculated human epithelioid and sarcomatoid MM cells subcutaneously into a hind leg of nude mice. Xenografts were irradiated with 0, 2, 5, 10 and 15 Gy of carbon ions (290 MeV/u, a 6-cm SOBP), and 5, 15 and 30 Gy of X-ray (200 kVp, 20 mA). We measured the size of xenografts and body weight twice a week. The size of xenografts irradiated with 15 Gy of carbon ions were apparently reduced in both epithelioid and sarcomatoid models. Although the size of xenografts irradiated with 30 Gy of X-ray were also reduced, CIRT was more effective compared with conventional radiotherapy. The RBE values were calculated to be 2.2 and 2.0 for epithelioid and sarcomatoid models, respectively, compared with X-ray. These results suggest that CIRT could be more effective than X-ray treatment.

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The aim of this study is to clarify the time and dose dependency of cell cycle in tumor cells exposed to C-ions and X-rays. Six human malignant melanoma cell lines, 92-1, C32TG, Colo679, HMV-I, HMV-II and MeWo were used. Cells were exposed to X-rays or 290 MeV/u C-ions at the center of SOBP. The cell survival after irradiation was obtained using a colony formation method. The 10% survival dose (D10) were estimated from cell survival curves as iso-effect dose, and used for the time dependency analysis. The same physical dose of C-ions and X-rays (1-8Gy) were used for dose dependency analysis. After irradiation, the cells were trypsinized and re-seeded to dishes in appropriate concentration, and cultured in CO2 incubator. Cells were collected at adequate time point (6-204 h after re-seeding), dyed with PI, fixed with Ethanol, and observed by the flow cytometry technique. It was suggested the melanoma cell lines used in this study were arrested in G2/M or G0/G1 phase after C-ions. Cells exposed to X-rays were accumulated in S or G0/G1 phase. Additionally, it was clarify the cells exposed to C-ions still existed in the G2/M phase at a high rate compared to non-irradiation samples 130 hours later. On the other hand, the percentage of cells arrested in G2/M was increased depending on physical dose in C32TG, HMV-I, HMV-II, and MeWo after C-ions irradiation. However the cells were accumulated in G0/G1 in 92-1, and the cell cycle was not changed in Colo679 depending on dose increasing. It has been understood that the behavior at the cell cycle depend on the cell lines, quality of radiation, time course, and irradiation dose. In addition, it was suggested even if the number of cells that dies finally is the same, the process of cell death is quite different between C-ions and X-rays, and it was clarified the transient prolonged G2/M arrest was specific phenomenon induced by heavy-ion beams.

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Purpose: Malignant pleural mesothelioma(MPM) is an aggressive tumor arising from serous surfaces and often related to asbestos exposure. MPM is resistant to various forms of therapy, such as radiotherapy, surgery or chemotherapy, and only slightly improve prognosis. So far, no effective therapeutics including chemotherapy or radiotherapy has been established for the disease-advanced case. The present study was carried out in order to examine the radiosensitivity of MPM cell lines by X rays and carbon-ion beams.Materials and methods: Officially distributed 6 kinds of MPM cell lines were used and irradiated with X-ray or carbon-ion beams (13keV/&mu and approximately 80keV/&mu) at the Heavy Ion Medical Accelerator in Chiba. Cell-killing effect was detected using a colony-formation assay. Result: The relative biological effectiveness (RBE) values calculated by the D10,relative to X-ray, range from 1.25 to 1.62 for 13keV/&mu ion beam and from 2.42 to 3.15 for approximate 80keV/&mu ion beam irradiation on each cell line . Conclusion: The data of previous reports using carbon ion beams, Suzuki et al. showed that RBE values for 16 different human cell lines were about 1.06 to 1.33 for 13keV/&mu and from 2.00 to 3.01 for 77keV/&mu beam. The RBE values of 6 cell lines of MPM in this study for the carbon beams are almost consistent with that report using carbon ion beams with lower (13keV/&mu) and higher(approximate 77keV/&mu) LET values. This result suggest that high LET carbon ion irradiations are also significantly more cell killing effective for the 6 kinds of MPN cell lines.

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It is known that high LET heavy-ion radiation have a high RBE compared to low LET radiation such as X-ray and proton for the cell death, mutation induction and chromosome damage. Recently, it has been shown that heavy-ion radiation is effective in the treatment of recurrent colon cancer. However, it is still unclear what is the difference in the gastroenterological tumor control and the mechanisms between heavy-ion and X-ray radiation therapy. In the present study, we examined effects of carbon-ion and X-ray on the treatment of transplantable human colon cancer in Balb/c-nu/nu mice. HCT116 (8x104) and SW480 (7x105) cells were inoculated into right hind legs of mice and were irradiated with 27, 30, 33 Gy carbon-ion (C290, 50keV/um, SOBP) or X-ray when the xenograft tumors grew to a certain size. We found that both X-ray and carbon-ion radiation effectively suppressed tumor growth with dose-related manner. However, the tumors were re-grew in the X-ray irradiated mice after 2 weeks, in contrast, all the tumors were repressed and consequently the tumor size was remarkably decreased or completely disappeared without any relapse or re-growth. Tumor-supplying vessels were also reduced in carbon-ion irradiated mice compared to those of X-ray irradiated mice when examined one month later. Taken together, heavy-ion irradiation can radically control tumor growth compared to X-ray. Further studies on the influences of heavy-ion on cancer stem cells and the molecular pathological mechanisms need to be done.

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Micronuclei of the onion root tip cells irradiated by accelerated heavy ions showed upward convex bell shaped dose-effect relationships. The shape can be accounted for by a simple mathematical model. The analysis with the model implies that the micronuclei and the damages to induce interphase death or division delay were both formed averagely proportionally to the square of LET. To evaluate the interphase death, we investigated shortening of the root growing. We expected the shortening to be proportional to the interphase death. But the root length L(D) with absorbed dose D follows the expression L(D)= L(∞)−Aexp(-αD), and the growth did not stop even with extremely high absorbed dose. LET dependence of α existed, but it was slower than the micronuclei induction.

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The purpose of present study is to clarify experimentally significance of tumor heterogeneity. Two sarcomas of # 6107 and #9037 were transplanted into syngeneic C3H male mice. Single cell suspensions for each tumor were mixed together at various ratios just prior to transplantation. When tumors grew to reach 7.5-8.0 mm in diameter, leg tumors were locally irradiated with either Cs-137 gamma rays or 290 MeV/n carbon ions at an LET of 74 keV/microm. Tumor growth (TG) time was obtained by calculating days required for a tumor to reach 5 times initial volume, and Tumor Growth Delay time was used to obtain an isoeffect dose. Tumor control probabilities were calculated by counting number of mice free of tumors 120 days after irradiation. TG time of tumors was identical to each other when the mixture ratio was varied by 11 steps. TG time after transplantation of 100 % #6107 cells was 36 days, and shortened to 28 or 20 days after that of 90 or 50 %. respectively. Tumors after transplantation of lower than 50% #6107 cells showed TG time identical to those after transplantation of 100% #9037 cells. Gamma ray radiosensitivity got higher with an increase of the mixture ratio of #6107 cells. Isoeffect doses for TGD 20 days increased from 15 to 32 Gy when the mixture ratio increased from 0 to 100%. This dose increase was less prominent for TCD50 (50% tumor control dose). We concluded that tumor heterogeneity affected more prominently growth delay than tumor control.

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