Cell survival study is fundamentally important in radiation biology. However, the survival rate cannot be measured by the conventional technique used for uniformly irradiated population, since irradiated and non-irradiated cells are located in the same dish in the microbeam experiments. Therefore, we have tried to establish a method to measure the survival fraction appropriate for the microbeam experiments. Clear difference was observed between the histograms of the number of cells per colony for irradiated (6 to 12 Gy) cells and for non-irradiated cells after 60 hr incubation. Therefore, the threshold to determine live or dead was set between the peaks of the histogram. Survival fractions thus determined was found to depend upon the beam size of microbeam X-rays at the same exposure time. Studies with broad-beam are now under way for comparison. It is also necessary to consider how to evaluate the absorbed dose in microbeam experiments. [J Radiat Res 44:444 (2003)]

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For the performance test of the synchrotron X-ray microbeam system, especially accuracy and repeatability of the positioning in the system, the cell irradiation experiments were performed and microbeam-irradiated cells was observed after immunostaining with gamma-H2AX antibody. The dishes specially designed for microbeam experiments are composed of a thin Mylar bottom and a set of solid frames of stainless steel. Human fibroblast cells were inoculated to the dish and incubated to attach the surface of Mylar bottom. Before irradiation cell nuclei were stained with Hoechst33258 and identified with the fluorescence microscope equipped in the system. Selected cells were individually irradiated with X-ray microbeam and the positions of the irradiated cells were automatically memorized. After immunostaining treatment, the dish was set again to the system to revisit at the poison of the irradiated cells. All the irradiated-cells could be observed at the revisited position, and could be distinguished from surrounding unirradiated cells by their high yield of fluorescence. [J Radiat Res 44:444 (2003)]

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Involvement of protein kinase C (PKC) was assessed in radiation-induced apoptosis, using 3SBH5 cells, one of the radiation sensitive thymic lymphoma cells. It was demonstrated that Gö6976, a specific inhibitor of PKC (α or βI), promoted radiation-induced apoptosis in 3SBH5 cells and that the Ca²⁺-dependent PKC involving PKC (α or βI) was activated by irradiation with 50cGy. During the apoptosis process, PKC (α or βI) distribution did not change in the cells. Treatment with Gö6976 20min after irradiation had no effect on the radiation-induced apoptosis. These results suggest that PKC (α or βI)-mediated survival signal is induced not through translocation of the PKC in the early period after irradiation. On the other hand, it was observed that the distribution of PKCδ was different between 3SBH5 and the radioresistant subclone XR223. In addition, rottlerin, a PKCδ inhibitor, suppressed the radiation-induced apoptosis of 3SBH5 cells. Functions of PKC subtypes will be discussed in the regulation of radiation-induced apoptosis. [J Radiat Res 44:444-445 (2003)]

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Differential display analysis was used to search for novel radiation-inducible transcripts in human cells. Normal human or radiosensitive ataxia-telangiectasia (AT) derived fibroblasts were X-ray (6 Gy)-irradiated and total RNA was extracted 3 hours later. For differential display analysis, three oligo dT-based anchor primers and 20 arbitrary primers were used. A transcript whose expression was significantly elevated after irradiation in normal cells but not in AT cells was excised from the gel, PCR-amplified and cloned in pGEM-T Easy vector. The kinetics of transcriptional response was analyzed further by quantitative PCR method using the Light Cycler apparatus. The increase of the transcript was seen around 1 hour after 6 Gy exposure, and the increase was detectable as low as 0.5 Gy. The ATM status may not be the sole factor for this response, because some ATM-proficient tumor cell lines lacked this response. The transcript was derived from a gene located on human chromosome 12. Similar sequence was not found within the homologous region on mouse chromosome, suggesting this gene may have a recent evolutionary history. [J Radiat Res 44:445 (2003)]

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Cyclin kinase inhibitor p21 retard progression of G1/S, S and G2/M in response to DNA damage. To analyze the regulation of the association of p21-Cyclin A, we constructed the eukaryotic expression of full length(1-164), 1-147(delCy2), 21-164(delCy1), 21-147(delCy1,Cy2) and 49-71 deletion(delCdk2) and examined its CyclinA binding after gamma irradiation. On response to irradiation, CyclinA binding increased in delCy1 and delCy2, while diminished in delCdk2. To assess the role of CDK2 binding region on CyclinA binding, we tested the significance of phosphorylation of 49-71 region. Threonine 57 is reported to be a substrate of MAPK p38 or GSK3-beta. We examined the CyclinA binding of unphosphorylatable T57A or phospho-mimic T57D. The association of CyclinA on either of T57A or T57D in response to irradiation was similar to that of wild type full length p21, suggesting at least the phosphorylation at T57 is not the regulatory factor. Taken together, 49-71 region may have another regulatory function rather than phosphorylation, or CyclinA selectively associates with CDK2 binding region by itself or with CDK2. [J Radiat Res 44:445 (2003)]

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Ionizing radiation is known to cause cell-cycle arrest at the G2/M phase in various tumor cells. Recently, it becomes clear that abrogation of the G2 checkpoint by the treatment of cells with drugs such as 7-hydroxystaurosporine (UCN-01) and caffeine leads to a marked increase in their sensitivity to ionizing radiation. In this study, we focused on Cdc2, one of the G2 checkpoint regulators, and examined how inhibition of Cdc2 kinase activity by Purvalanol A, a specific inhibitor of Cdc2 kinase, influenced X rays-induced apoptosis. MKN45 (p53 wild type) and MKN28 (p53 mutation) cells were exposed to X rays with or without Purvalanol A. For measurements of apoptosis, apoptotic morphological changes of nuclei were accessed by fluorescence microscopy. Cell-cycle was observed by using flow-cytometry. When cells were exposed to X rays alone, G2/M arrest occurred. Co-treatment of X rays with Purvalanol A rendered the decrease in G2/M fraction and the increase in subG1 fraction. These results indicated that Purvalanol A sensitized X rays-induced apoptosis through abrogation of cell-cycle arrest. [J Radiat Res 44:445 (2003)]

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The c-Jun N-terminal kinases (JNKs) are a subfamily of MAPKs. The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create ten JNK isoforms that are either p46 or p54 in size. We found that the p52 form of JNK emerged in MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK, while, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 JNK also paralleled the appearance of the active form of caspase-3, and was suppressed by a caspase-inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant JNK1 beta 2 was cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1 beta 2 to alanine abolished the cleavage. Our results demonstrated that p54 JNKs were new selective targets of caspases, and suggested that the p52 form could serve as an apoptotic marker. [J Radiat Res 44:445 (2003)]

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Apoptosis triggered by DNA damage is known to be predominantly mediated by mitochondrial factors which lead to the activation of caspase cascade. We examined the activation of apoptosis signaling originating from the mitochondria in Jurkat cells after exposure to 10 Gy γ-rays or 15 J/m² UV light. As reported previously, exposure of Jurkat cells to UV resulted in a large amount of cytochrome c being released, and a clear laddering pattern of DNA fragments was observed within 3 h of incubation after irradiation. Simultaneously, activation of caspase-9 and its downstream caspases was detected. In contrast, γ-irradiated cells showed extensive release of cytochrome c and caspase-9 and -3 activation at 24 h or longer incubation periods after exposure. A broad spectrum caspase inhibitor, Z-VAD-FMK, suppressed the production of DNA fragmentation in Jurkat cells irradiated with either γ-rays or UV light. These results suggest that delayed or rapid form of apoptosis depending on radiation is due to the existence of cytosolic factors regulating the release of cytochrome c from mitochondria. [J Radiat Res 44:446 (2003)]

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DNA double strand break (DSB) is the most critical damage that are induced by radiation. Many DSB repair proteins have been identified using several approaches such as positional cloning and phenotype analysis of knockout cells. Among these, Nbs1, the underlying gene for Nijmegen breakage syndrome, forms a complex with Mre11/Rad50 and is essential for homologous recombination (HR) and S-phase check point. To clarify the role of Nbs1 protein on DSB responding pathways, we analyzed apoptosis induction in Nbs1 deficient chicken DT40 cells after irradiation. In sharp contrast to wild type DT40 cells, NBS1 deficient cells did not display apparent induction of apoptosis after exposure to 4 Gy of ¹³⁷Cs gamma-rays. Because DT40 cells are not expressing p53, which acts in apoptosis induction after DNA damage, this result suggests that Nbs1 possesses a critical role on p53 independent apoptosis induction. [J Radiat Res 44:446 (2003)]

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Apoptotic cytochrome c (Cytc) release follows the transcription dependent and independent pathways that p53 activates expression of proapoptotic BH3-only Noxa and BH1-4 Bax proteins, and p53 DBD binds to antiapoptotic Bcl-XL in the mitochondria (MT), respectively. The proposed Cytc-releasing pore models (Bax oligomers, Bax/VDAC complex, supramolecular pore) remain controversial. We studied the Bax (Bak)/VDAC complex formation and its role in Cytc release after 5 Gy in p53/Bax-expressing apoptosis-sensitive cells and Bak/Bcl-2-expressing resistant cells. The protein crosslinking approach for isolated MT showed time-dependent formation of Bax di- and tetramers as well as Bax/VDAC di- and tetramers. By immunofluorescent microscopy, clotorimazol which displaces hexokinase recruited Bax to VDAC in MT and released Cytc. Further, DIDS as a VDAC inhibitor blocked the clustering of Bax on MT, Cytc release and apoptosis after IR. Contrary, although Bak/Bcl-2-expressing IR apoptosis-resistant cells formed the Bak/VDAC heterodimer independently of post-IR time, they released no Cytc. Thus we suggest the Bax/VDAC channel, most likely its heterotetramer, may be responsible for Bax-mediated Cytc release from cellular MT in the p53/Bax-mediated apoptosis. [J Radiat Res 44:446 (2003)]

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Ataxia Telangiectasia (AT) is genetic disorder caused by lack of ATM (ataxia telangiectacia mutated) gene. ATM modulates DNA repair, cell cycle arrest, and apoptosis induced by ionising radiation. AT cell line has a deficiency of DNA repair and radiation induced cell cycle arrest, however, relationship between ATM and radiation induced apoptosis is still unclear. Moreover, apoptosis of AT cell line induced by low LET irradiation is well studied whereas by high LET irradiation such as heavy-ion irradiation is not. Here we reported heavy-ion induced apoptosis in human ATM deficient lymphocyte cell line GM01525 comparing with normal lymphocyte cell line GM14511. As irradiation sources, C, Si, Argon, Fe ion beams were used. For counting, we mainly used fluorescence staining method to see chromatin condensation as apoptotic cells. As a result, apoptosis in normal lymphocyte was occured with about 100keV/microm of LET irradiation, whereas apoptosis in AT lymphocyte was not. This suggests that ATM gene has a function to increase apoptotic cells in human lymphocyte cell line. [J Radiat Res 44:446 (2003)]

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Apoptosis is a gauge for radiation and hyperthermia therapy. Ultrasound (US) has been used in cancer therapy as a heating device; while recent study showed that nonthermal US is also capable of inducing apoptosis. However, yield is low and cell lysis is predominant. To search for an optimal apoptosis (highest apoptosis/lysis ratio), we applied different US conditions by varying the intensity, duty factor (DF), pulse repetition frequency (PRF) and duration of exposure. Sonicated U937 cells were incubated at different time intervals before measuring the apoptosis and its signal transduction. Apoptosis was assessed by morphological changes, DNA fragmentation and phosphatidylserine externalization. Decrease in MMP was also determined. Optimal apoptosis (70.0+13.8%) was attained at 1 MHz, 0.3 W/cm², 10% DF of 100 Hz PRF for 1 min. Apoptosis peaked at 12 hr after irradiation. This level of apoptosis is comparable to apoptosis induced by X-irradiation above 20 Gy, suggesting that US is competitive in terms of apoptosis induction. [J Radiat Res 44:447 (2003)]

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A metabolite of folic acid, 6-formylpterin (6-FP), is able to induce intracellular hydrogen peroxide (H₂O₂) production involved with induction of apoptosis. In this study, we investigated radiation-induced apoptosis and its possible enhancement in the presence of 6-FP in U937 cells. The agent enhances radiation-induced apoptosis significantly, as determined by nuclear morphological changes, phosphatidylserine externalization and DNA fragmentation. Formation of intracellular H₂O₂, decrease of mitochondria transmembrane potential (MMT), release of cytochrome c from mitochondria, down regulation of Bid, activation of caspase-8 and caspase-3 were enhanced after the combined treatment. Remarkable activation of PKC delta and its translocation from cytosol to mitochondria, increase of intracellular Ca²⁺ concentrations were also observed. JNK activation was not enhanced in the combined treatment and AIF remained unchanged even after 3 hr. These results indicate that 6-FP enhances radiation-induced apoptosis via the mitochondria-mediated caspase-dependent pathway, with the involvement of PKC delta activation. [J Radiat Res 44:447 (2003)]

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The aim of the study is to clarify injury and recovery process of irradiated taste bud in comparison with the intestine and bone marrow. The tongues of rats received 15 Gy, and immunohistochemistry for BrdU, p21, p53, and TUNEL were performed as well as HE. On HE, the number of taste bud decreased from the 4th day and became the lowest on the 6th day. On the 8th day, the epithelium showed marked thickening with increase of the epithelium cells without taste bud. Immature taste bud re-appeared at ten days. BrdU labelling revealed that DNA synthesis arrested as early as 10 hours after irradiation. Cells in the basal layer expressed p21 four hours after irradiation. Expression of p21 was no longer detectable on the sixth day or later, and DNA synthesis resumed on the sixth day. On the eight day, the number of BrdU-positive cells is 3-5 times of control, and then became normal thereafter. No apoptosis was detected at any time. Radiation damage of the taste bud and its recovery are caused by p21-regulated cell cycle arrest and resume, not by apoptosis. [J Radiat Res 44:447 (2003)]

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We examined the effects of an inhibitor (LY294002) of PI3K on radiation or hyperthermic sensitivity. We used two human non-small cell lung cancer cell lines (H1299/wtp53 and H1299/mp53) transfected with wild and mutant p53 genes. We measured the sensitivity to X-ray and heat by colony formation assay and frequency of apoptosis by Hoechst staining and accumulation of apoptosis-related proteins by Western blotting analysis. LY294002 (final concentration at 40 mM) was added to the culture medium at 1 hr before X-ray or hyperthermia treatment. The addition of LY294002 enhanced the sensitivity of X-ray or hyperthermia and accumulation of survivin independently on p53 genotype. The induction of hsp70 after hyperthermia was inhibited by LY294002. These results suggested that LY294002 sensitized radiosensitivity and thermosensitivity through the depression of survivin and hsp70-induction. Since we demonstrated that another PI3K inhibitor of wortmannin did not sensitize the thermosensitivity, on the contrary, it is suggested that there are some differences in the modality of protein phosphorylation between radiosensitivity and thermosensitivity. [J Radiat Res 44:447 (2003)]

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Effects of p53 inhibitor pifithrin-alpha and general caspase inhibitor z-VAD-FMK on radiation-induced teratogenesis and adaptive response were studied in ICR mouse fetuses. Both inhibitors markedly postponed and suppressed early apoptosis in limb buds of E12 fetuses irradiated with 3.5 Gy of X-rays, while neither of them affected incidences of digital defects, prenatal and postnatal death. A priming dose of 0.3 Gy on E11 resulted in an adaptive response that dramatically reduced those detrimental effects of the challenging dose of 3.5 Gy on E12. Both inhibitors significantly suppressed the priming radiation-induced apoptosis, however, they did not suppressed the apoptosis induced by the challenging dose. The adaptive response was totally vanished with administration of either inhibitor. These findings indicate that temporary delay and suppression of apoptosis do not lead to the relief of late teratogenesis, while suppression of the priming radiation-induced apoptosis interrupt the induction of adaptive response. [J Radiat Res 44:448 (2003)]

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We have previously reported that irradiated MOLT-4 cells developed a new protein p41 on 2-D PAGE dose and time dependently and that p41 was generated by the caspase-cleavage of p42/SETbeta, while sodium orthovanadate (vanadate) treatment inhibited the p41-induction. In the present study, we investigated mechanism(s) involved in the inhibition of radiation-induction of p41. Immunoblotting analysis and in vitro assays of caspases revealed that the inhibitory effect of vanadate was due to the suppression of caspase-activation. We further investigated the effect of vanadate on the course of caspase-activation and the apoptotic cell death of irradiated MOLT-4. As a result, we found that vanadate suppressed the cell death as well as the process related to the caspase-activation such as releases of cytochrome c and Smac/DIABLO from mitochondria, loss of mitochondrial membrane potential, and conformational change of Bax. These results indicated that vanadate acted on upstream event(s) of these releases, causing suppression of radiation-induced apoptotic cell death of MOLT-4. [J Radiat Res 44:448 (2003)]

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DNA damage induces cell death by apoptosis. Several recent studies have suggested that radiation could also generate signals at the cell membrane that led to apoptosis. To elucidate those radiation-induced apoptosis in detail, we investigated the induction of apoptosis by selective irradiation with non-DNA damage using the penetration controlled irradiation apparatus. Accelerated ion of 1.5 MeV/u ¹²C was provided by the tandem accelerator at TIARA JAERI-Takasaki. To evaluate radiationi-nduced DNA damage, comet assay was applied. Apoptosis fraction was detected by the TUNEL assay after irradiation for 72 hr. When CR-39 plates were irradiated at the distance of 13, 17 and 18mm from the beam window, etched pits were observed in the distance of 13 and 17 mm. Radiationi-nduced DNA damage was detected only at the distance of 13 mm. On the other hand, apoptosis fraction of 17 mm-irradiated cell was higher than that of non-irradiated control. This result indicates that one of the signal transductions of radiation-induced apoptosis do not mediate DNA damage. [J Radiat Res 44:448 (2003)]

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Ionizing-irradiation induces remarkable apoptosis in peritoneal resident macrophages (PRM) of the C3H mouse. A series of experiments performed by using various radical scavengers suggested that superoxide but not the other reactive oxygen species was responsible for the apoptosis. In the present study, superoxide dismutase (SOD) or catalase were introduced into PRM with high efficiency by means of HVJ (Hemagglutinating Virus of Japan) envelope vector kit (GenomONE^TM) purchased from Ishihara Sangyo, Ltd.(Osaka, Japan). Irradiation-induced apoptosis in C3H mouse PRM was significantly suppressed by the introduction of SOD but not catalase. The result confirmed that the apoptosis was induced by superoxide but not hydrogen peroxide. It also showed that HVJ envelope vector kit was a very useful tool for introducing large molecules such as proteins and DNA into cells. [J Radiat Res 44:448 (2003)]

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In the clinic, interindividual differences in normal tissue response to radiotherapy have been observed and genetic factors are suggested to be responsible for this variation. Murine models have proven to be useful for the study of cytotoxic agents including radiation on the jejunum. We here report three murine strain differences in the levels of radiation-induced apoptosis in jejunal crypt cells. The apotosis positivity cell in jejunum carried out apoptosis index comparatively, and radiation susceptibility of strains was evaluated. Apoptosis index in C3H/Hem was consistently lower than in A/J, or C57BL/6J at various times up to 24 hr after irradiation with 0.5 Gy. There was no difference among three strains after irradiation with 5 Gy. In the frequency of appearance of an apoptosis cell, C57BL/6J and A/J were radiation quantity susceptibility as compared with C3H/Hem. [J Radiat Res 44:449 (2003)]

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Proteases have received attention as important cellular components responsible for stress response in humans. However, little is known of proteases which play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa cells after X-ray irradiation. The activity was identified as fibrinolytic one, using ¹²⁵I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa cells showed an increased level of protease activity 10min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with X-ray susceptibility of cells. Leupeptin inhibited the protese activity in samples obtained from X-ray-irradiated RSa cells. Furthermore, treatment of RSa cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. [J Radiat Res 44:449 (2003)]

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Following exposure of p53 (-/-) and p53 (+/+) mice to 0.5 or 1 Gy of X-rays, micronucleus (MN) frequency was measured in the reticulocytes sampled at multiple time points after exposure. In either strain of mice, MN frequency increased over the spontaneous level as the time elapsed after exposure to a peak at 24 h and then decreased time-dependently to the spontaneous level at 120 h after exposure, irrespective of the dose used; the frequency averaged for the sampling times from 12 to 96 h, F (%), followed a linear equation of F=a+bD, where a is spontaneous MN frequency (%), b induced rate of MNs (% Gy⁻¹) and D dose (Gy). Experimentally obtained a values for p53 (-/-) and p53 (+/+) mice are 0.24±0.04 and 0.71±0.08, respectively; the b values are 1.1±0.1 and 2.3±0.3, respectively. These results suggest that normal p53-gene function is involved in apoptotic elimination of DNA damage induced, either spontaneously or after radiation, in erythroblasts of mice. [J Radiat Res 44:449 (2003)]

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The survival strategies used by the preimplantation embryos in response to sperm DNA damage are not completely understood. Our earlier observation has demonstrated the functioning of p53 dependent S-checkpoint in the zygotes fertilized with irradiated sperm, which suppressed pronuclear DNA synthesis. Interestingly, these zygotes with sub optimal DNA content progressed beyond implantation but a large number of the fetus have undergone post-implantation demise. These observations made us to undertake the present study to find out whether apoptotic machinery functions in the embryos and also whether developmental potential is altered in the embryos fertilized with X-irradiated sperm. Here we found that, the embryos derived from sperm irradiation had developmental delay and genomic instability at the compaction stage between day 2.5 and 3.5 post fertilization. The apoptotic response was detected only at peri-implantation period specifically after blastocyst formation. We further demonstrate that in vitro developmental potential was compromised in these embryos. [J Radiat Res 44:449 (2003)]

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Human leukemic MOLT-4 cells undergo apoptosis after X-irradiation through p53-dependent pathway. In MOLT-4 cells, p53 (wild type) is stabilized and increased after X-irradiation. We have studied the detection and the measurement of p53 accumulation per individual cell by flow cytometry. Irradiated cells were fixed, permeabilized, and analyzed on FACSCalibur (Becton Dickinson). Cells were stained with anti-human p53-PE (clone DO-7). The quantification of immunofluorescence was performed using Quantum Simply Cellular (Bangs Laboratories). Irradiated MOLT-4 cells were monitored for p53 at 1-6 h after irradiation by flow cytometory. The molecules per cell increased after 1h of postirradiation incubation after exposure to 9 Gy, and reached to 3.5 times after 5 h relative to control. Strong correlation was observed between the time course of the cell death induction and the increase in p53 per cell when p53 was quantified in terms of number of molecules per cell and the number was chosen at the peak position of the p53 profile. The method is useful for further study in a cell during the course of developing apoptosis. [J Radiat Res 44:450 (2003)]

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It have been reported that malignant melanomas (MM) are resistant to conventional radiotherapy with low-LET radiations, but surprisingly responsible to the carbon ion-beam therapy at HIMAC. On the other hand, it is also known that squamous cell carcinomas (SCC) are resistant to both radiation therapies. To make clear the difference and utilizing the heavy-ion radiotherapy, the difference in the survival curves between the cell lines of MM and SCC were studied. Measurements of the radio-responses for the MM cells were started in the previous year, and that for SCC were added. The MM and SCC cell lines have exposed to carbon-ion beams (290 MeV/u, at the center of 6 cm-SOBP) at HIMAC. alpha/beta ratio and other survival parameters of the survival curves were analyzed. In case of MM, alpha/beta ratio increased when carbon beam was used compared with that for X-ray. But, in case of SCC, no clear difference was observed between carbon and X-rays. It means that carbon beam has advantage for MM on radiotherapy. [J Radiat Res 44:450 (2003)]

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Radiation sensitivities of the cultured cell lines established from 31 human esophageal squamous cell carcinoma (ESCC) and 20 other human cancer tissues were investigated with the colony-formation assay. There was a large variation in radiosensitivity among the 51 cell lines. MRE11 and BRCA1 genes were involved in the homologous recombination (HR) pathway. Radiosensitivities of BRCA1- or MRE11-defective cell lines were higher than those of other cell lines. Twenty-six of 28 ESCC lines tested had mutations in the p53 gene. Variance in radiosensitivity was not explained according to the status of p53. Unusual radiosensitivity was observed in one ESCC cell line. DNA-PKcs protein had low levels and was not phosphorylated after X-irradiations. DNA-PKcs is involved in the non-homologous end-joining (NHEJ) pathway. Our data suggest that genes which are involved in the HR or NHEJ pathway on DSBs are mutated in cells having unusual radiosensitivity. [J Radiat Res 44:450 (2003)]

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Betel quid chewing is very common in India and other Southeast Asian countries, and it is an important risk factor in oral carcinogenesis. To know the underlying mechanism and the response to radiotherapy, we have analyzed 50 primary oral squamous cell carcinomas for mutations in the p53 gene (exons 4-8) and bak gene (exons 3,4,6) by PCR-Cold SSCP and direct sequencing. The incidence of p53 mutation was 58% (29 of 50), while it was only 0-10% in betel quid-unrelated oral cancer. Most mutations(86%) were transition mutations. Furthermore, mutations were found predominantly in codon 250 and codon 286 (15%, and 13%, respectively), and 35% of the amino acid changes were Pro to Ser. Mutations were also found in the bak gene, and poor prognosis was observed in cancer patients with bak mutation, although there were no differences between p53 mutation (+) and (-) patients. [J Radiat Res 44:450-451 (2003)]

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Purpose: Chemoradiation therapy for head and neck cancer may make some normal tissue toxicity and affecting quality of life of patients during and after treatment. Method: We prospectively studied 40 patients with advanced head and neck cancers who were received concurrent chemoradiotherapy. Patients were divided into two groups consecutively: BRK group received concurrent chemoradiotherapy with 1000 mg of EF-2001 three times per day during whole treatment schedule of concurrent chemoradiation therapy. Results: 33 patients were evaluated. There was no statistical difference comparing BRK group against NBRK group at first treatment week. The most remarkable change during the treatment is QOL score of the gastrointestinal symptom and the appetite in BRK group was reported. Conclusion: The use of EF-2001 as nutritional supplement may be proposed as a means of improving the worsening in quality of life for head and neck cancer patients during chemoradiation treatment. [J Radiat Res 44:451 (2003)]

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For conducting radiotherapy properly, accurate does distributions in a patient body is inevitable. Therefore, it is desired that basic technology for evaluating accurate dose applicable to versatile cases in order to promote the quality of radiotherapy generally. Further, it is important to reduce the load and time consumed in advanced therapy like IMRT. We started a project to develop a system for providing accurate patient doses utilizing a sophisticated human model and the Monte Carlo calculation. In the system, CT pictures and therapy procedure data are sent from the hospital to the dose calculation center through a network; a human model is constructed rapidly; the accurate dose distribution is calculated on parallel computers and sent back to the hospital. [J Radiat Res 44:451 (2003)]

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Therapeutic carbon ions and target nuclei are broken up into fragment particles when colliding with each other. The biological effect to a cell depends not only on dose, but also the beam quality, i.e., the LET and particle species. At NIRS, the beam is treated as a cohort of monoenergetic carbon ions that have sole dose-averaged LET value. The response of HSG cells to such monoenergetic carbon ions is applied for the estimation. On the other hand, GSI uses the Local Effect Model that takes radial dose structure of each ion and resultant deposited local dose to a CHO-K1 cell nucleus into account for the estimation. The comparison of the two models was carried with elemental LET spectra of therapeutic carbon beam to make an intercomparison of both clinical results possible. It was found that both models produced almost identical biological dose distribution; however, NIRS scheme shows slightly shallower biological effect around the distal edge of SOBP region. [J Radiat Res 44:451 (2003)]

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We have been studying LET and ion species dependence of RBE values in cell killing and mutation induction. Normal human fibroblasts were irradiated with heavy-ion beams, such as carbon (290Mev/u and 135Mev/u), neon (230Mev/u and 400Mev/u), silicon (490Mev/u) and iron (500Mev/u) ions, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). Cell killing effect was detected as a reproductive death using a colony-formation assay. Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies.The RBE values of cell killing increase with increasing in the atomic number of ion species. The RBE-LET curve of mutation induction for carbon-ion beams showed a peak at the LET of around 100keV/micron, while it showed a plateau without a remarkable peak ranging from 100 to 150 keV/micron for silicon ion beams.There is evidence that the RBE-LET relationship for mutation induction is not the same with different ion species. [J Radiat Res 44:451 (2003)]

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Clinical trials indicate that the RBE of high LET radiation is large for slow-growing tumors. We here used 9 transplantable tumors with different growth time for each, and compared RBE values of 290 MeV/u carbon ions (74 keV/micrometer). NFSa fibrosarcoma spontaneously arisen in C3H mice and other 8 radiation-induced tumors were transplanted in hind legs, and irradiated with either Cs-137 gamma rays or 290 MeV/u carbon ions. Tumor growth delay time and specific growth delay were calculated from tumor growth time and used to obtain isoeffect doses. TG time for un-irradiated control ranged from 3 days to 9.6 days. RBE of carbon ions ranged from 2.1 to 3.4. It is concluded that tumor growth rate is less important than anticipated for a RBE determinant of carbon-ion radiation. [J Radiat Res 44:452 (2003)]

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[Purpose] We study in an anti-cancer effect and mechanism, and there are these studies for the purpose of offer to data in fundamental researches of ECT. [Methods] We divided it into control group, tumor with1 c group, tumor with 5 c and 10 c group with an ICR mouse and a C3H mouse and tested it. Subcutaneous in Sarcoma180, SCC-7 cell 2 × 10⁵ in right upward department of a right foot of a C3H mouse inoculated it. [Results] A tumor growth measurement study by Scc-7, that a meaningful difference of statistics was recognized in more than 5c group in comparison with control group. [Conclusion] We think an effect by obstruction of nutritional support to a tumor organization having been insufficient by an effect of generated gas and a change of pH, the blood clot formation because we have a short the time that did ECT with 100sec in 1c group. [J Radiat Res 44:452 (2003)]

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This study evaluated the ability of ¹⁴C-Thymidine and ¹⁸FDG for detecting the early effects of irradiation on tumor proliferation-rate. NFSa fibrosarcoma cells were transplanted into the right hind legs of syngeneic C3H male mice. Conditioning irradiation with 290 MeV/u carbon ions delivered to the tumors at 7.5 mm diameter. To measured ¹⁴C-Thymidine and ¹⁸FDG uptake, mice were intravenously injected with both tracers. The LET and dose-related decrease in ¹⁴C-Thymidine in NFSa tumor were observed 12 hours after carbon-beam irradiation. Furthermore, the reduction in ¹⁴C-Thymidine uptake at 12 hours was correlated with tumor-volume at 14 days after irradiation. In contrast, ¹⁸FDG uptake did not significantly decrease at 12 hours and 14 days. These findings demonstrated that the thymidine uptake could be an appropriate marker for investigating tumor proliferation-rate after radiotherapy of malignant diseases. [J Radiat Res 44:452 (2003)]

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1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (TAS106) is a newly developed anti-tumor agent targeting RNA synthesis. In this study, we examined whether the exposure of gastric tumor MKN45 cells to X-rays in the presence of ECyd at the low concentrations induced apototic. We report here that a low dose of ECyd radiosensitizes caspase-dependent-apoptosis in gastric tumor cell line MKN45. This radiosensitization was linked to the abrogation of the X-ray-induced G2/M checkpoint. Western blot analysis showed that X-rays increased the expression of cyclin B1, phospho-Cdc2 and Wee1, whereas co-treatment with X-rays and TAS106 decreased the expression of these signaling molecules associated with G2/M arrest. Furthermore, TAS106 was shown to decrease the expression of survivin. These results indicated that TAS106 sensitized X-ray-induced apoptosis through the down-regulation of survivin and abrogation of the cell cycle machinery. [J Radiat Res 44:452 (2003)]

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Biological effectiveness of carbon ions was compared between an experimentaltumor (NFSa fibrosarcoma) and skin of syngeneic C3H mice. 290 MeV/u carbon ions of LET 42 and 77 keV/micrometer, but not 14 or 20 keV/micromete, showed larger RBE (biological effectiveness relative to gamma rays) values in retarding tumor growth than causing skin reaction after daily-fractionated irradiation. Single doses failed to show therapeutic gain for any LET of carbon ions. RBE values of the two tissues for 14 and 20 keV/micrometer ranged from 1.2 and 1.7. The alpha term increased with an increase in LET irrespective of tumor or skin, while the beta term of skin, but not of tumor, increased with LET. It is implied that high-LET radiotherapy could achieve large therapeutic gain comparing to photon therapy when a fractionation scheme consisting of a large dose with small number of fractionation is used. [J Radiat Res 44:453 (2003)]

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