Mizuho Aoki

Inactivation of Aerobic and Hypoxic Cells from Three Different Cell Lines by Accelerated 3He-, 12C- and 20Ne-Ion Beams

Abstract Furusawa, Y., Fukutsu, K., Aoki, M., Itsukaichi, H., Eguchi-Kasai, K., Ohara, H., Yatagai, F., Kanai, T. and Ando, K. Inactivation of Aerobic and Hypoxic Cells from Three Different Cell Lines by Accelerated 3He-, 12C- and 20Ne-Ion Beams. The LET-RBE spectra for cell killing for cultured mammalian cells exposed to accelerated heavy ions were investigated to design a spread-out Bragg peak beam for cancer therapy at HIMAC, National Institute of Radiological Sciences, Chiba, prior to clinical trials. Cells that originated from a human salivary gland tumor (HSG cells) as well as V79 and T1 cells were exposed to 3He-, 12C- and 20Ne-ion beams with an LET ranging from approximately 20–600 keV/μm under both aerobic and hypoxic conditions. Cell survival curves were fitted by equations from the linear-quadratic model and the target model to obtain survival parameters. RBE, OER, α and D0 were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/μm, then decreased with a further increase in LET. Clear splits of the LET-RBE or -OER spectra were found among ion species and/or cell lines. At a given LET, the RBE value for 3He ions was higher than that for the other ions. The position of the maximum RBE shifts to higher LET values for heavier ions. The OER value was 3 for X rays but started to decrease at an LET of around 50 keV/μm, passed below 2 at around 100 keV/μm, and then reached a minimum above 300 keV/μm, but the values remained greater than 1. The OER was significantly lower for 3He ions than the others.

Nitric oxide-mediated bystander effect induced by heavy-ions in human salivary gland tumour cells

Purpose : To investigate the signal factor and its function in the medium-mediated bystander effect during heavy-ion irradiation of human salivary gland (HSG) neoplastic cells. Materials and methods : Unirradiated recipient HSG cells were co-cultivated with HSG donor cells irradiated with 290 MeV/u carbon beams having different LET values. Cell proliferation and micronucleus (MN) induction in recipient cells with and without treatment of a NO scavenger (PTIO) were measured and the concentration of nitrite in the co-culture medium was detected. As a direct control, the effects of a nitric oxide (NO) generator (sper/NO) on cell proliferation and MN induction were also examined. Results : Increases in cell proliferation and MN induction were found in the recipient HSG cells as a result of co-culturing and cell proliferation was obviously enhanced during a further subculture. In comparison with 13 keV/ μ m, 100 keV/ μ m carbon-ion irradiation was found to be a more efficient inducer of the medium-mediated bystander effect. The treatment of cells by PTIO resulted in elimination of such effects, which supports a role for NO in the medium-mediated bystander effect. As an oxidization product of NO, nitrite was detected in the co-culture medium, and the dose-response for its concentration was similar to that of cell proliferation and MN induction in the recipient cells. When the HSG cells were treated by sper/NO with a concentration of less than 20 μ M, cell proliferation was enhanced, whereas MN increased along with sper/NO concentration. Conclusion : NO participated in the medium-mediated bystander effects on cell proliferation and MN induction, depending on the LET of irradiation.

Role of Gap Junctional Intercellular Communication in Radiation-Induced Bystander Effects in Human Fibroblasts

Abstract Shao, C., Furusawa, Y., Aoki, M. and Ando, K. Role of Gap Junctional Intercellular Communication in Radiation-Induced Bystander Effects in Human Fibroblasts. Radiat. Res. 160, 318–323 (2003). Involvement of gap junctional intercellular communication (GJIC) in bystander responses of confluent human fibroblasts irradiated with a carbon-ion beam was investigated. It was found that the lower the radiation dose, the higher the yield of radiation-induced micronuclei per nuclear traversal, suggesting the existence of bystander effects. This low-dose sensitivity was increased when GJIC was enhanced by treating cells with 8-Br-cAMP, but it was partly reduced by treating cells with DMSO, an effective scavenger of reactive oxygen species (ROS). Moreover, no low-dose sensitivity was observed when cells were treated with 100 µM lindane, an inhibitor of GJIC. The survival of irradiated cells was increased by DMSO but was not influenced significantly by cAMP or lindane. On the other hand, G1-phase arrest was detected in the irradiated cells, and it was enhanced by cAMP. In contrast, this arrest was reduced or almost eliminated by DMSO or lindane, respectively, even when cells were irradiated with such a high dose that each cell received five nuclear traversals on average. Thus the bystander responses occurred after both low-dose and relatively high-dose irradiation. Our results indicated that both GJIC and ROS contributed to the radiation-induced bystander effect, but gap junctional channels might play an essential role by modulating the release of radiation-induced signaling factors.

Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center

PURPOSE To assess the biologic effects of proton and carbon ion beams before clinical use. METHODS AND MATERIALS Cultured cells from human salivary gland cancer (HSG cells) were irradiated at 5 points along a 190 MeV per nucleon proton and a 320 MeV per nucleon carbon ion beam, with Bragg peaks modulated to 6 cm widths. A linac 4 MV X-ray was used as a reference. Relative biologic effectiveness (RBE) values at each point were calculated from survival curves. Cells were also irradiated in a cell-stack phantom to identify that localized cell deaths were observed at predefined depth. Total body irradiation of C3H/He mice was performed, and the number of regenerating crypts per jejunal section was compared to calculate intestinal RBE values. For carbon ion and referential 4 MV X-ray beams, mouse right legs were irradiated by four-fractional treatment and followed up for skin reaction scoring. RESULTS RBE values calculated from cell survival curves at the dose that would reduce cell survival to 10% (D10) ranged from 1.01 to 1.05 for protons and from 1.23 to 2.56 for carbon ions. The cell-stack phantom irradiation revealed localized cell deaths at predefined depth. The intestinal RBE values ranged from 1.01 to 1.08 for protons and from 1.15 to 1.88 for carbon ions. The skin RBE value was 2.16 at C320/6 cm spread-out Bragg peak (SOBP) center. CONCLUSION The radiobiologic measurements of proton and carbon ion beams at Hyogo Ion Beam Medical Center are consistent with previous reports using proton beams in clinical settings and carbon ion beams with similar linear energy transfer (LET) values.

Gene expression analysis in human malignant melanoma cell lines exposed to carbon beams.

Purpose: To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. Materials and methods: We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. Results: Twenty-two genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle – related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including the tumor protein p53 (p53) target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). Conclusion: Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle – related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.

Radiosensitization by Hyperthermia in the Chicken B-Lymphocyte Cell Line DT40 and its Derivatives Lacking Nonhomologous End Joining and/or Homologous Recombination Pathways of DNA Double-Strand Break Repair

Abstract Yin, H. L., Suzuki, Y., Matsumoto, Y., Tomita, M., Furusawa, Y., Enomoto, A., Morita, A., Aoki, M., Yatagai, F., Suzuki, T., Hosoi, Y., Ohtomo, K. and Suzuki, N. Radiosensitization by Hyperthermia in the Chicken B-Lymphocyte Cell Line DT40 and its Derivatives Lacking Nonhomologous End Joining and/or Homologous Recombination Pathways of DNA Double-Strand Break Repair. Radiat. Res. 162, 433–441 (2004). Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70−/−, DNA-PKcs−/−/−, RAD54−/− and KU70−/−/RAD54−/−. Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70−/−/RAD54−/−, which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70−/− and DNA-PKcs−/−/−, consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G1 and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G2, capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G1 and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.

Simultaneous exposure of mammalian cells to heavy ions and X-rays

Crews of space missions are exposed to a mixed radiation field, including sparsely and densely ionizing radiation. To determine the biological effectiveness of mixed high-/low-LET radiation fields, mammalian cells were exposed in vitro simultaneously to X-rays and heavy ions, accelerated at the HIMAC accelerator. X-ray doses ranged from 1 to 11 Gy. At the same time, cells were exposed to either 40Ar (550 MeV/n, 86 keV/micrometers), 28Si (100 MeV/n, 150 keV/micrometers), or 56Fe (115 MeV/n, 442 keV/micrometers) ions. Survival was measured in hamster V79 fibroblasts. Structural aberrations in chromosome 2 were measured by chemical-induced premature chromosome condensation combined with fluorescence in situ hybridization in isolated human lymphocytes. For argon and silicon experiments, measured damage in the mixed radiation field was consistent with the value expected using an additive function for low- and high-LET separated data. A small deviation from a simple additive function is observed with very high-LET iron ions combined to X-rays.

Sper/NO-induced reversible proliferation inhibition and cycle arrests associated with a micronucleus induction in HSG cells.

Nitric oxide (NO) is an important messenger molecule with multiple biological activities. In the present study, sper/NO, a NO generator, showed a biphasic effect on the proliferation of human salivary gland neoplastic (HSG) cells. Sper/NO of less than 20 micro M stimulated cells to depart from the G2/M phase and so enhanced cell division and cell proliferation. But sper/NO at higher concentrations restrained cell proliferation and blocked cell-cycle progression. Cells were mainly arrested in the G2/M phase and S phase when they were treated with 100-200 and 300-500 micro M sper/NO, respectively. A special S-phase peak was detected in a histogram of the cell-phase distribution of sper/NO-treated HSG. When the concentration of sper/NO increased, the S-phase peak shifted from early the G2/M-phase to later the G1-S-phase boundary. Sper/NO-induced cell-cycle arrests were reversible when the cells were released from NO stress for 48h and hence cell proliferation was recovered. In addition, micronucleus, but no apoptosis, was produced in the sper/NO-treated cells, and its yield tended to a saturation value with increasing concentrations of sper/NO. The sper/NO-induced effects were effectively eliminated or reduced by treating cells with PTIO, a NO-specific scavenger, indicating that NO is the main source of these effects.

Radioprotective activities of beer administration for radiation-induced acute toxicity in mice

BACKGROUND AND PURPOSE We previously found that drinking beer reduces chromosome aberrations in blood lymphocytes that were collected and irradiated in vitro. In this study, we investigated the radioprotective activities of beer-administration for bone marrow and intestine in mice. METHODS C3H/He female mice received an oral administration of beer, ethanol or saline at a dose of 1 ml/mouse 30 min before whole body irradiation with 137Cs gamma rays or LET 50 keV/microm carbon ions. Radioprotective activities were estimated using a LD(50/30) (The dose required to kill 50% of the mice within 30 days) and a microcolony technique for intestine. RESULTS The LD(50/30) for the beer-administered mice was significantly increased in comparison with saline administered mice. The LD(50/30) of gamma-ray was 7.8 Gy (p < 0.05), 7.6 Gy and 7.3 Gy for beer-, ethanol- and saline-administered group, respectively. The LD(50/30) of carbon ions was 6.6 Gy (p < 0.05), 6.2 Gy and 5.9 Gy for the beer-, ethanol- and saline-administered groups, respectively. The crypt survivals that were semi-logarithmically plotted against dose were well fitted to a linear regression line. The dose reduction factor (DRF) (D10) of beer- and ethanol-administered mice for gamma rays was 1.09 and 1.08, respectively. The DRF (D10) of beer- and ethanol-administered mice for carbon ions was 1.08 and 1.07, respectively. CONCLUSIONS The radioprotection by beer-administration is due to not only OH radical-scavenge action by the ethanol contained in beer.