A critical accident happened at JCO nuclear fuel conversion facility in Tokai-mura on September 30, 1999. After the accident, two workers died due to high dose exposure of gamma-rays and neutrons. Dose estimation for gamma rays was carried out using tooth enamel ESR measurement and the estimated dose was about 12 Gy. And also, the amalgams and gutter-percha which were filled in the teeth of the worker were collected and used to estimated beta- and gamma-ray dose from the induced activities of ⁶⁴Cu, ¹⁰⁸Ag, ¹¹⁰Ag, ^110mAg, ¹⁹⁷Hg and ²⁰³Hg. However, neutron dose estimations was not carried out using the induced activities in these samples. We determined specific activity of ^110mAg and ⁶⁵Zn in the samples measureing gamma rays with Ge detector at the Ogoya under ground laboratory, Kanazawa University. From the result of the measurement, specific activity of ^110mAg/Ag:51(+/-)0.7Bq/g, and specific activity of ⁶⁵Zn/Zn:38(+/-)11Bq/g was obtained Neutron dose was evaluated based on the neutron spectrum which was previously obtained simulating neutron released from a conversion tank at the time of the criticality accident as well as a Monte Carlo technique using a mathematical head phantom and MCNP code. The neutron dose was estimated to be 3(+/-)0.2Gy from Ag sample measurements, while Zn-samples gave a larger value of result as 21(+/-)7Gy with large uncertainty.

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We assessed individual doses of inhabitants living in high-level natural radiation areas (HLNRAs) in Ramsar. Each of 15 inhabitants in HLNRAs and 10 inhabitants in a control area carried about an electronic personal dosimeter (EPD) for one day, twice in 2005. In addition, their individual doses were estimated from ambient radiation dose rates determined with a NaI(Tl) survey meter and occupancy factors. Results of personal dosimetry carried out twice were essentially similar, and a good correlation existed between the dose rate values obtained through estimation and personal dosimetry. Each of the dosimetric subjects carried about also an optically stimulated luminescence dosimeter (OSLD) for about one month, but a few values obtained by these measurements deviated widely from those obtained by one-day measurements with EPDs and those estimated by the above method. This deviation would be ascribed to the fact that these OSLDs were left behind somewhere in houses, since it was too troublesome for a few inhabitants to always wear dosimeters for a month. As a result, observed dose values depended heavily on the place where dosimeters had been left, because of the non-uniform distribution of Ra-226 contained in materials of their houses built in the HLNRAs. For this reason, one-day measurements with sensitive dosimeters would produce more reliable results than a-few-month measurements, if the former measurements are repeated every season and the results are confirmed by comparison with those estimated from ambient radiation dose rates.

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To detect expression of DNA repair genes of the tumor clonogens after carbon-ion or X-ray irradiation in relation to early and rapid survival recovery (PLD repair) occurring after irradiation. At first, we performed colony assay after irradiation of 5Gy of carbon-ion or 9 and 12Gy of X-ray to compare time-dependent cell survival of the IA cells after each irradiation. Second, to search the genes causing PLD repair after irradiation of 5Gy of carbon-ion or 12Gy of X-ray, we evaluated gene expressions by using semi-quantitative RT-PCR on the selected 33 genes reportedly related to DNA repair. PLD repair was found only after X-ray irradiation. The RT-PCR results were as follows. In this IA cells that had point mutation of p53, gene expressions of TP53, TP53INP1 and CDKN1A were not significantly vary. We think 10 genes that show a significant difference between X-ray and carbon-ion beam exposure are candidates associated with PLD repair in this IA cells .

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HiCEP (high-coverage expression profiling) enables to detect any altered gene expression among 60-70% of all the actually transcribed genes in any eukaryotic cells and tissues. We previously applied it to a primary culture of normal human fibroblasts and observed gene expression responding to X-ray at the very low dose (10 mGy). As the result of screening for approximately 23,000 transcripts, we have identified a set of CXC chemokines up-regulated by the 10 mGy X-rays in the normal human fibroblasts. Those genes have hardly been expected from the previous studies using the higher (>100 mGy) dose of radiations. Our observation indicated that different molecular mechanisms are involved in the response to ionizing radiation with different doses /dose rates, and suggested that different cellular responses are induced by ionizing radiation with different LET. Accelerated heavy ion particle (high LET) provides promising effects for radiotherapy of a certain type of malignancy, however, the molecular bases of its advantage to gamma rays is not fully understood. In this time, we applied our new expression assay (HiCEP) to the normal human fibroblasts (HFL III) irradiated with a high-LET which was generated by HiMAC in our institute. More than 40 genes were found to be unregulated in the irradiated cells by >3 folds within 4 hrs post-irradiation of carbon ion at 2Gy (~70 keV/micro m). Those included the DNA damage-inducible genes (CDKN1A, MDM2, Gadd45), and also some unexpected genes, both predicted and unpredicted from the current public databases.

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High-LET charged particle radiations are applied to cancer therapy because of its excellent dose distribution. However, charged particle therapy has not been applied in veterinary clinics even though it is obviously useful. On the other hand, the radiation exposure of the blood cells including lymphocytes cannot be completely avoided. Damaged cells distribute to whole body, and cause the systemic reaction. Therefore, the evaluation of the sensitivity of lymphocytes to charged particles will be indispensable information to decide the dose of particle radiation therapy for veterinary application. This study aims to compare the radiosensitivities of feline T-lymphocytes (FeT-J) between low-LET ⁶⁰Co γ-rays and high-LET charged particles such as proton, helium ion and 2 kinds of carbon ions. The radiosensitivity was evaluated in terms of clonogenicity and apoptosis incidence. The clonogenicity of FeT-J decreased in the same physical dose accompany with increasing the LET. RBE and inactivation cross sections had increased as LET dependency. However, there was no difference of apoptosis induction rate at the exposure of isosurvival dose that gave 10% survival in each radiation source. In conclusion, no change of cell killing process is generated in lymphocytes by exposure to isosurvival dose of varied particle radiations. This study indicated the basic index of radioresponses of cats' cells for particle therapy applied for veterinary medicine, and the selectivity of charged particles for animal therapy would be expanded not only carbon ions.

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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 also before reported that the sharp of LET-RBE curves of cell killing was different, when using different ion species such as carbon neon silicon and iron ions. This study, we investigated LET- and ion-species dependence on deletion pattern of exons in mutation induction at the hprt locus. Normal human skin fibroblasts were irradiated with carbon-, neon-, silicon- and iron- ion beams generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). The 6-thioguanine resistant colonies were isolated and extracted genomic DNA from the colonies. The mutation spectrum of the deletion pattern of exons was analyzed by multiplex polymerase chain reaction (PCR). The deletion pattern of exons was different in induced mutants among different ion species. Furthermore, the mutation spectrum in mutants induced by both carbon- and neon- ion beams showed LET dependent manner and was different when using different accelerated energies even if ion species were the same. These results suggested that the differences in the track structure of energy deposition with different ion species and energies could have an effect on mutation induction.

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NASBEE stand for Neutron Exposure Accelerator System for Biological Effect Experiment that had been settled at NIRS in 2004. The system consisted of two parts, inline type tandem accelerator with multi-cusp ion source (HVEE) and irradiation system (NEC Tokin). The system has dedicated to high flux neutron irradiation for biological specimens, especially Specific Pathogen Free (SPF) environment. The system had a Nuclear and Industrial Safety Agency Nuclear and Industrial Safety Agency license in December 2005. At beginning of beam tuning, we suffered a lot of ion source problem, ex; filament terminal melt down etc. we set ion source current limitation up to 0.4mA at 4MeV beam energy for deuteron. The neutrons are produced from a water cooled 2mm beryllium target. Neutron irradiation experiments on a mouse an embryo and a rat mammary gland are conducting since March 2006. This report is intended to present an accelerator and maintenance troubles. We will also present up to date neutron biological experiments and neutron exposure filed measurements.

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The present study intends to analyze internal radiation field of a typical mouse with voxel-based model and radiation transport code. A mouse was imaged by using the dedicated small-animal CT scanner, in which slice pitch was set at 0.1 mm. Each image with the resolution of 0.02 mm was segmented to construct a voxel-based model by using computer tools, which have been applied to process human-head images for 3-dimensional dosimetry in BNCT treatment at the Japan Atomic Energy Agency. Input files for particle and heavy ion transport code system (PHITS) were prepared from the constructed 3-dimensional voxel-based image data. Absorbed dose distributions and LET spectra in the mouse body were calculated by PHITS on the segmented images.

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Estimation of the specific energy distribution in a human body exposed to complex radiation fields is of great importance in the planning of long-term space missions and heavy ion cancer therapies. With the aim of developing a tool for this estimation, the specific energy distributions in liquid water around the tracks of several HZE particles with energies up to 100 GeV/n were calculated by performing track structure simulation with the Monte Carlo technique. In the simulation, the targets were assumed to be spherical sites with diameters from 1 nm to 1 um. An analytical function to reproduce the simulation results was developed in order to predict the distributions of all kinds of heavy ions over a wide energy range. The incorporation of this function into the Particle and Heavy Ion Transport code System PHITS enables us to calculate the specific energy distributions in complex radiation fields in a short computational time.

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Medaka ric1 homozygous mutant was isolated by large scale ENU mutagenesis screening. It showed high incidence of malformation and lower hatchability after ionizing irradiation at early embryonic stages and has a defect in the repair of DNA double-strand breaks in embryonic cells though it is highly fertile and easy to breed like wild-type fish under non-irradiated condition (Aizawa et al. 2004 Mech. Dev. 121: 895-902). Spermatogonial stem cells in ric1 fish are much more sensitive to ionizing radiation than wild-type fish and show delayed apoptosis, which suggests that the ric1-dependent DNA repair pathway plays a critical role in the SSCs. Using transgenic medaka Olvas-GFP strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the vasa gene (Tanaka et al. 2001 PNAS. 98: 2544-2549), we established Ric1Olvas-GFP strain with germ-cell-specific expression of GFP which can be visualized germ cells in living ric1 embryos. Embryonic germ cells in ric1 are sensitive to non-lethal dose of irradiation at stage 7 (32 cells ) and stage 33 (notochord vacuolation). The proliferating female germ cells are more sensitive than male germ cells at stage 33. It is also suggested that ric1 dependent maternal DNA repair pathway plays a major role in radiosensitivity of the PGC at stage 7 and at stage 24. These data suggests that ric1 mutant strain is a valuable model for germ-line mutagenesis, radiation biology, teratogenesis, carcinogenesis, aging and environmental monitoring

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Thorium-227 (t_1/2 = 18.72 d) is a promising alpha-emitting radiotherapeutic nuclide for treatment of bone metastases. The daughter nuclide ²²³Ra, which have been used in clinical study for human, also decays via several alpha-emitting nuclides to stable ²⁰⁷Pb. The decay and growth patterns of radioactivity of ²²⁷Th and ²²³Ra after accumulation in bone are specific for each nuclide. We attempt here to evaluate and compare the myelotoxicities of ²²⁷Th-EDTMP and ²²³Ra in rats. Seven-week-old Sprague-Dawley rats were injected ²²⁷Th-EDTMP or ²²³RaCl₂. The body weight and blood cells (RBCs, WBCs, platelets) of each rat were counted before injection and more than 8 weeks after injection. A rat injected 8 MBq/kg b.w. of ²²³Ra died 9 day postinjection. The rats injected more than 250 kBq/kg b.w. of ²²³Ra showed decrease of WBCs and platelets. In the case of ²²⁷Th injection, a rat injected 8 MBq/kg b.w. died 8 day postinjection. On the other hand, the rats injected 4 and 2 MBq/kg b.w. died 4 and 6 weeks postinjection, respectively. The rats injected more than 500 kBq/kg b.w. showed weight loss and decrease of RBCs, WBCs, and platelets. The myelotoxicity of ²²⁷Th-EDTMP showed specific pattern and may correlate to the decay and growth pattern of ²²⁷Th and its progeny radioactivity.

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High dose exposure to radiation causes gastrointestinal tract (GIT) injuries. GIT injury is one of serious problems in accidental exposure to high dose radiation and also in radiation therapy. However, the mechanism(s) is not fully understood and its treatment is unknown. Stem cells in crypt differentiate into mature cells and migrate upward from the base of the crypt toward the villus tip. Differentiated cells rapidly lose their proliferative ability and then undergo apoptosis. In order to study the mechanisms of GIT injury by radiation, we investigated that effect of radiation on protein expression of mouse intestinal epithelial cells along the crypt-villus axis. Epithelial cells were sequentially isolated cells from the villus tip to the crypts of mouse small intestine by the Mariadason method. We compared levels of protein by western blot analysis. Expression of proliferating cell nuclear antigen (PCNA) and Bax was expressed in the crypt and their levels were reduced toward the villus axis. On the other hand, the expression of active form of caspase 3 was accumulated in cells of the villus tip. Radiation reduced levels of PCNA. Radiation increased levels of Bax and activated caspase 3 in the crypt but not in the villus tip. The p21 protein was not detected in intestinal epithelial cells. However, radiation increased the level in cells toward the crypt. Our results suggest that the cell proliferation and response to radiation is differently regulated in the crypt from the villus.

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When evaluating effects of radiation, one area of emphasis is the effects of maternal exposure to radiation. Teratogenesis, tumorigenesis, and mental development delay are among those observed. Compared to subjects such as microcephaly and CNS malformation, however, little has been reported on neurocristopathy and congenital cardiovascular anomalies following maternal exposure. To study prevention and safety regarding radiation, unique biological effects of neutron and other radiation need to be evaluated. We report on relationship between maternal exposure to full body neutron irradiation and embryonic lethality and external as well as visceral malformations, especially of cardiovascular nature. We observed a high frequency of teratogenesis, especially cardiofacially, in the irradiated groups. This indicates fetuses' sensitivity to neutrons in forming neurocristopathy-induced cardiofacial syndrome, especially riding aorta, Tetralogy of Fallot, double outlet right ventricle, transposition of the great arteries, and aortic arch anomalies. The types and frequencies of these anomalies are similar to those in human, suggesting natural radiation and environmental factors may play a large role in human neurocristopahty and cardiovascular anomalies, termed DiGeorge-Velocardiofacial and Alagille Syndromes. Twenty years after Chernobyl, it is essential to consider effects of the nuclear fallout on these syndromes when evaluating the aftermath. This animal model should aid in investigating such human teratogenesis, and we intend to discuss their relative biological effectiveness.

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In past research, we found that carbon ion irradiation increased bone volume in rats, and a significant amount of cartilage remained inside the carbon ion-irradiated trabeculae. In the present study, we investigated the expression of Cbfa1 (core binding factor 1, Runx2), an osteoblast and chondrocyte maturation factor, and of the cartilage matrix absorption enzyme MMP-13, which is regulated by Cbfa1, in irradiated and non-irradiated bone. The amounts of Cbfa1 and MMP-13 mRNA in osteoblast-like MC3T3-E1 cells tended to decrease after carbon ion irradiation. The level of MMP-13 mRNA in non-irradiated cells was stable during the experimental period, but in gamma ray-irradiated cells it tended to increase. When localization of MMP-13 in locally irradiated experimental rats was investigated, it was found in the marginal trabeculae in both non-irradiated and gamma ray-irradiated animals. MMP-13 was detected in osteoid and neogenetic bone in the trabecular surface. The trabeculae in carbon ion-irradiated bone remained cartilaginous. Carbon ion-irradiated rats exhibited weak expression of MMP-13 around the cartilage inside the trabeculae. We conclude that carbon ion irradiation reduced expression of MMP-13, thus suppressing both chondrocyte maturation and cartilage resorption. Increases in hyperplasia of the bone trabeculae and of bone volume were caused by ongoing bone addition and calcification in the absence of sufficient cartilage resorption.

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In the present study, we examined in vivo radioprotection activity of Zn-, Mn-, Cu-, or Se-containing heat-treated Saccharomyces serevisiae yeast. The mineral-containing yeast powder was suspended in a 0.5% methyl cellulose solution. The suspension was administered i.p. to C3H mice before or after 7.5 Gy whole body X-irradiation. The 30-day survival of mice without injection of mineral yeast was about 7%. The survival of mice to which mineral-containing yeast was injected at 30 min before irradiation was 65-90%. The survival of mice was about 75%, when Se- or Mn-containing yeast was injected immediately after the irradiation, while it was more than 90% when Zn- or Cu-containing yeast was injected. Even the yeast containing no mineral showed significant radioprotection (70-80%). The Zn-containing yeast showed radioprotection when injected at 60 min post-irradiation (>80%) and even at 24 h post-irradiation (about 30%). The DRF of Zn-containing yeast (100 mg/kg, i.p. immediately after irradiation) was about 1.2. Bio-modulation is a possible mechanism for the radioprotection, since yeast without mineral is effective, it is effective for the radiation dose of bone marrow death, and it is effective for post-irradiation. In addition, other mechanisms may be involved since Zn- or Cu-containing yeast showed higher radioprotection than yeast without mineral.

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Quantitative evaluation of biological responses to gamma radiation at low doses or at low dose rates is not sufficiently investigated. We acquired iterative experimental data of biological reaction in cell level to gamma radiation of low dose/low dose rate, and analyzed them using meta-analysis to evaluate quantitatively the effects. Cytotoxic T cells were induced in C57BL/6 mice by intraperitoneal injection of P815 cells (2x10⁷ cells/mouse). Mice were killed 10 days after the immunization, and antigen-specific cytotoxic T cell activity in the spleen was determined as percent lysis (effector to target ratio = 40), lytic unit (LU) per 10⁴ splenocytes, or LU per mouse. One LU was defined as cytotoxic activity giving 10 % percent lysis. Mice were continuously irradiated at 4, 21 or 174 uGy/h for 17 days, from 7 days before the immunization throughout the experiment. These data were statistically and quantitatively analyzed by meta-analysis. Synthesized effects of the experiments which were obtained by fitting a fixed-effects model to the data considering their variances showed no significant difference between irradiated samples and control samples. However, the data showed heterogeneity according to the radiation doses.

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Ionizing radiation evokes biological responses against oxidative stress as well as those against DNA break and oncogenesis, through the interaction with intracellular molecules. Because the stress responses are regulated by the system involved in metabolic homeostasis, it is possible that ionizing radiation can be used for the therapy of human metabolic syndrome. We have found that continuous gamma-irradiation at low dose rate ameliorates type II diabetes in db/db mice genetically lacking leptin receptors, and elongates their life span. Here we studied mechanism of the amelioration of Type II diabetes by low dose rate gamma-irradiation. Eleven-wk female db/db mice were continuously irradiated at 174 microGy/h for 2-3 weeks in the irradiation room bearing 1.8PBq cobalt-60 as a radiation source, and found that the treatment significantly improved diabetes in glucose tolerance test. Reduction of blood insulin and degeneration of pancreatic islets, characteristics in chronic phase of type II diabetes, were significantly suppressed by continuous low dose rate irradiation, suggesting that it protects pancreatic islets from glucose toxicity. The irradiation suppressed apoptosis in pancreatic islets. Enhancement of SOD-2 expression was demonstrated by immunohistochemisty and RT-PCR in pancreas. These results suggest that continuous low dose rate gamma-irradiation ameliorates type II diabetes through the transcription activation of host surveillance system against oxidative stress.

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SCC VII tumor-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of tirapazamine (TPZ) in combination with conventional dose-rate irradiation (CDRI) or reduced dose-rate irradiation (RDRI) using gamma-rays or thermal neutrons. After irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumor cells was determined using tumors that were not pretreated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, especially Q cells. Further, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumor control as a whole, including intratumor Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumor cells caused by the decrease in irradiation dose rate in vivo.

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The dependence of the initial production of DNA damages on radiation quality was examined by using a proposed new model on the basis of target theory. For the estimation of DNA damage-production by different radiation qualities, five possible modes of radiation action, including both direct and indirect effects, were assumed inside a target the molecular structure of which was defined to consist of 10 base-pairs of DNA surrounded by water molecules. The induction of DNA damage was modeled on the basis of comparisons between the primary ionization mean free path and the distance between pairs of ionized atoms, such distance being characteristic on the mode of radiation action. The OH radicals per average energy to produce an ion pair on the nanosecond time scale was estimated and used for indirect action. Assuming a relation between estimated yields of DNA damages and experimental inactivation cross sections for AT-cells, the present model enabled the quantitative reproduction of experimental results for AT-cell killing under aerobic or hypoxic conditions. The results suggest a higher order organization of DNA in a way that there will be at least two types of water environment, one filling half the space surrounding DNA with a depth of 3.7 – 4.3 nm and the other filling all space with a depth 4.6 – 4.9 nm.

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The hormetic effect, which extends the lifespan by intermittent exposure to hyperoxia, is confirmed in Caenorhabditis elegans (C. elegans). To determine the mechanism of this hormesis, the mitochondrial superoxide radical (O₂^-) level in various age-related strains were measured using a chemiluminescent probe. Hence, the O₂^- level significantly decreased depending on the intermittent exposure to hyperoxia in a strain, which is activated by an insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway such as the age-1 mutant. Furthermore, the expression of some sod-genes was induced to the hormesis. These results suggest that a decrease in the mitochondrial O₂^- levels by activation of the antioxidant systems was associated with hormesis.

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[Purpose] It is known that electromagnetic waves, particularly ionizing radiation and UVB, have significant effects of immune system. This study was designed to evaluate the effect of electromagnetic waves on autoimmune reaction using a mouse Graves' model. [Methods] Graves' hyperthyroidism was induced by immunizing mice with adenovirus expressing TSHR 2x at 3 wk intervals, and hyperthyroidism was confirmed by measuring T₄ using sera obtained 2 wks after 2nd immunization. Electromagnetic waves were given to mice at a single dose (0.5 Gy or 10 kJ/m²) 1 wk before or after 1st immunization, or at fractionated doses (0.1 Gy or 1 kJ/m², 2x/wk for 6 wks). TSHR Abs were determined with FRTL5 cells (for stimulatory Abs) or CHO cells expressing TSHR (for total Ab titers). IFN-γ production from SPC was measured with ELISA kit. T cell subpopulations in SPC including CD4⁺CD25⁺ regulatory T cells (Treg) were evaluated by flow cytometry.[Results] Only a single administration of UVB after 1st immunization suppressed T₄ and stimulatory Ab titers, and IFN-γ production in immunized mice as compared to non-irradiated, immunized mice. The titers of total TSHR Abs were not decreased. The number of Treg was significantly increased by fractionated, but not a single dose of, UVB. [Conclusion] UVB irradiation after the immunization significantly suppressed development of Graves' hyperthyroidism without affecting systemic immune function. Involvement of Treg in this suppression is however unlikely.

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Structural information about DNA damage site is important to clarify the repair processes. Particularly, the damage induced by ionizing radiations is expected to be multifarious, so this has prevented us to discover the upper stream of the radiation biological effects. Here, we propose a novel method to obtain qualitative information as well as quantitative one using enzyme kinetics. To characterize the cleavage sites, digestion velocity of the irradiated DNA pretreated with or without calf intestinal alkaline phosphatase by snake venom phosphodiesterase (SVPD) was measured. SVPD is an exonuclease producing a deoxynucleoside-5'-monophosphate one after another from a 3'OH terminus. The alkaline phosphatase treatment enables some of the SVPD-resistant sites to be digestive by SVPD. The initial digestion velocity is expected to give us information about the cleavage sites such as the amount, the chemical structure In this presentation, we will discuss variations of DNA cleavage produced by ionizing radiations, suggested by the experiments based on enzyme reaction kinetics.

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Higher eukaryotes cell nucleus has a compartmentalized structure built up by chromosome territories (CTs) and an interchromatin compartment (IC). The IC expands both between neighboring CTs and into the interior of indivisual CT s and is lined by the surfaces of smaller and larger chromatin domains. Howe ver, little is known about the dynamic organization of higher order nuclear architectures including chromatin and non-chromatin nuclear domains after in duction of DNA damage. To examine the dynamic organization of higher order nuclear architecture for DNA repair, we have developed a system for local ir radiation of cell nuclei using a focused UV-laser (laser-UV-microirradiation ). Using this techniques, we have revealed that DNA repair related proteins, such as gamma H2AX, MRE11, RAD51 and PML, accumulated at site of containing DNA damage at various times after induction of DNA damage. To examine the t opological and chronological relationships between these non-chromatin nucle ar domains associated with DNA repair in a single cell, we have developed a multicolor immunofluorescence staining system. Using this method, we have re vealed that the dynamics of 53BP1, a protein involved in the signaling of DN A damage, was differently regulated after low and high dose gamma irradiatio n, respectively. Dynamic organization of higher order nuclear architecture f or DNA repair will be discussed.

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The time courses of the development and disappearance of gamma-H2AX foci after irradiation was measured in the nuclei of non cycling G0 human fibroblasts, and in the same cells after induction of premature chromosome condensation. The purpose was to investigate possible connections between the rejoining and mis-rejoining of DNA DSBs and the early formation of chromosome aberrations. For gamma-H2AX foci assayed in G0 nuclei after irradiation of G0 human fibroblasts, a maximum of about 30 foci per cell was observed after 1 Gy in agreement with estimation of the number of DNA DSBs expected for this dose. About three times the number of foci were seen in dose of 3 Gy after induction of premature chromosome condensation. For both assays the number of foci then decrease at about the same rate when foci were measured at various times after irradiation. gamma-H2AX foci in the prematurely condensed chromosomes were after seen at the broken ends of PCC fragments, and instances were also seen of gamma-H2AX foci at the junction point of translocation. Lesions allowing expression gamma-H2AX foci persist even after irradiated G0 cells are subcultured and allowed to progress to mitosis, but incubation of G0 cells up to for 12 hours after irradiation before subculture resulted in the disappearance of these foci on mitotic chromosomes. Numerous gamma-H2AX foci are seen in the nuclear of unirradiated S phase cells, and an enormous phosphorylation of H2AX was seen in unirradiated S cells but not G0 cells after fusion with mitotic HeLa cells.

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We investigated the effects of oxidative DNA lesion on transcription elongation by pol II using an oligo(dC)-tailed templates with a oxidative DNA lesion on the transcribed strand such as an 2-hydroxyadenie (2-OH A), 8-oxoadenine (8-oxoA), 8-oxoguanine (8-oxoG) or thymine glycorl (Tg). The results showed that pol II stalled at all types of oxidative DNA lesions. This indicates that pol II by itself stalls at the non-bulky and non-helix-distorting lesions without any factors. Next, we purified TFIIS (SII) which is a transcription elongation factor that binds and assists pol II to stimulate the transcription elongation, and performed transcription elongation assays using oligo(dC)-tailed templates with an oxidative DNA lesion and the SII. Interestingly, there is no blockage of transcription elongation by pol II at 8-oxoG lesions in the presence of SII, while there is the blockage at 2-OH A, 8-oxoA or Tg lesions. It is suggested that polII with SII bypasses 8-oxoG lesions. From these findings, here we propose two possible models for avoiding effects of oxidative DNA lesions. First, oxidative DNA lesions are subject to repair by the TCR pathway. Second, genes containing 8-oxoG lesions are transcribed by polII in the presence of SII.

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The structure and the function of telomere are controlled by various proteins. Although DNA-PK primarily plays an important role in rejoining DNA broken ends caused by DNA damaging agants, in telomere region, it has been thought to protect telomere DNA double strand ends from mis-rejoining. Previous study showed that DNA-PKcs, a component of DNA-PK, is important for telomere stability, however, it is still unknown whether the kinase activity is required for telomere maintenance. In this study, we investigated the effects of NU7026, a specific inhibitor of DNA-PK activity, on telomere stability. The inhibitory effects of NU7026 were examined by immunostaining using anti-phosphorylated-DNA-PKcs and anti-phosphorylated- histone H2AX in 1Gy-irradiated AT2KY cells. The most induced foci disappeared in cells treated with 10 uM of NU7026, indicating that the DNA-PK activity was completely suppressed by NU7026 at the concentration of 10 uM. Then, NU7026 was continuously exposed to normal human cells, and the cell growth was decreased to approximately 1/2 with 20 uM NU7026. Because abnormal cell divisions were observed in treated cells, we analyzed metaphase chromosomes in cells cultured in 10 uM and 20 uM NU7026. Telomere associations, telomere end fusions, both chromosome- and chromatid-type, were observed more frequently than in the untreated cells, which may result in defective growth of treated cells. These results indicate that the kinase activity of DNA-PK is essential for the stability of telomere DNA double strand end.

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In eukaryotic cells, including mammal, DNA double strand breaks (DSBs) are repaired mainly through two pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA-PK complex, composed of DNA-PKcs, Ku86 and Ku70, and XRCC4/DNA ligase IV complex have been considered key molecules in NHEJ but new molecule, XLF/Cernunnos was found very recently. It is largely unknown how these proteins are recruited to DSB sites and assembled into repair machinery. Another mystery is the role of protein phosphorylating function of DNA-PK. For our understanding of DSB repair mechanism through NHEJ, it is critically important i) to visualize and analyze the recruitment process of NHEJ molecules to DSBs and ii) to elucidate the genuine target of DNA-PKcs and the physiological significance of phosphorylation. Regarding this, we have reported the detection and characterization of DNA-PKcs foci induced by ionizing radiation and, this time, we successfully detected the binding of XRCC4 to damaged chromatin DNA. Additionally, we have identified the phosphorylation site of XRCC4 by DNA-PKcs, which is phosphorylated in living cells after irradiation and disruption of which resulted in elevated radiosensitivity with partially impaired DNA repair capability. Based on these results, the process of recruitment and assembly of NHEJ machinery and the role of DNA-PK phosphorylation therein will be discussed.

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Many types of DNA damage block replication because replicative DNA polymerases are unable to bypass altered bases. To overcome this, cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. The TLS polymerases are also characterized in terms of their low-fidelity on undamaged DNA, leading to the production of mutation. One of the most interesting studies for TLS polymerases is to know their role on biological consequence at tissue or whole-body level. For this purpose Medaka is most suitable because both of classical and modern molecular genetics are available. Recently a general reverse genetics method was reported, which can identify mutations in genes that are known only by their sequence. The method, called TILLING (Targeting Induced Local Lesions IN Genome), includes random mutagenesis, followed by screening for induced mutations in target genes at the genomic DNA level. Adult male Medaka were mutagenized with ENU and then outcrossed with female to generate F1 progeny for the library. We established 5771 ENU-mutagenized F1 male fishes. To construct a library, genomic DNA and testis samples were isolated and cryopreserved from each F1 fish. A pilot screening revealed that the average per-base mutation frequency was 1 in 350kbp. Rev1 protein is an essential player in the production of both spontaneous and DNA damage-induced mutations. We have identified 17 mutations in Rev1 gene, which include 2 nonsense and 15 missense mutations. Summary of the screening and the phenotype of these mutants will be described.

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Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by hypersensitivity to ionizing radiation, chromosomal instability, and a high risk of cancer. A responsible protein NBS1 has been shown to interact with gamma-H2AX, and is essential for homologous recombination repair. However, the mechanism of DNA dsb repair has not yet been fully defined. Recently, mediator proteins such as TopBP1, MDC1 and 53BP1 draw attention to understanding in DNA repair pathway. TopBP1 shares sequence homology with fission yeast Cut5/Rad4, suggesting its role in damaged DNA repair. It was reported that TopBP1 form nuclear foci with gamma-H2AX, NBS1, PCNA and BRCA1 after DNA damage. In this study, we show that TopBP1 interacts with NBS1. We also found that NBS cells which express NBS1 mutant with defective FHA/BRCT domain showed abnormal formation of TopBP1 foci after DNA damage. TopBP1 knockdown cells showed a decrease of homologous recombination, and an increased frequency of premature chromatin condensation (PCC), which is characteristic for ATR-defective Seckel syndrome cells. It has recently been reported that TopBP1 directly activates ATR. These results suggested that TopBP1 is recruited to damaged DNA sites in an NBS1-dependent manner, and is involved in homologous recombination repair.

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Proliferating cell nuclear antigen (PCNA) is monoubiquitinated in a RAD18/RAD6-dependent manner at stalled replication forks caused by DNA lesions. PCNA monoubiquitination mediates a polymerase switch from replicative to translesion polymerase to continue replication.Therefore, RAD18-deficient cells become sensitive to various DNA damaging agents.However, PCNA was not monoubiquitinated after X-irradiation in human cell line HCT116, although it's RAD18 deficient mutant was sensitive to X rays and defective in repair of X ray-induced chromosome aberrations.The mutant cells were hypersensitive to topoisomerase I inhibitor camptothecin that generated single strand breaks (SSBs) but not to topoisomerase II inhibitor etoposide that generated double strand breaks. Thus, human RAD18 is required for the repair of chromosomal SSBs and PCNA monoubiquitination is not necessary for the repair process.

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Although phosphorylation of histone H2AX is known to be a sensitive index of radiation exposure, most of the data are derived from cultured cells and little is known for different tissues in vivo. We analysed dose-response and time course of radiation-induced phosphorylation in the three tissues of mouse; spleen, liver and brain. We used Western blot analysis to quantitate the degree of phosphorylation. Almost linear dose responses were observed in the dose range of 0.5 to 20Gy when judged at 1 hr after irradiation. The induction in liver seemed less efficient. The induced phosphorylated H2AX disappeared at one day after irradiation. Thus, the response in mouse seems to be tissue-specific.

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We found highly unstable DNA sequences (AT-rich) during the study of genetic effects of radiation on mouse by 2D genome scanning. Among 368 F1 mice, we found 18 mutations after screening 514 paternal BALB/c spots per mouse (³²P labeled DNA at AflII (C/TTAAG) sites). Among the 18 mutations, 7 occurred at the same s754 spot as a result of three independent events, which gave the spontaneous mutation rate of 0.8% per generation. The s754 is one of the spots appearing on a same horizontal line on a 2D pattern. These spots were highly polymorphic among strains of laboratory mouse, and 40 spots differed already between the two closely related C57BL/6N and /6J strains. Three of these spots were cloned and found that the sequence was identical or almost identical to s754 sequence from BALB/c mouse. 2D Southern analysis using the s754 DNA as a probe showed spots at identical positions as we see on the 2D DNA gel in C57BL/6N, C57BL/6J, BALB/c and C3H mice. Thus, these aligned spots belong to the same family. Ordinary 1D Southern analysis using the same probe revealed the same size of bands on 17 M.mus DNA samples but no bands were detected on the samples from M.spretus, rat, or human. Although BLAST search revealed two BAC clones that contained the s754 sequences, no exactly matched sequences were found in the mouse draft sequences. This implies that the s754 sequences are in the gap of the draft sequences. The s754 sequence is specific to M.mus but the origin and the reasons for the instability remain unknown.

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We studied a mechanism of the p53 gene that protects against radiation-induced teratogenesis using p53(-/-) mice, p53(+/-) mice and p53(+/+) mice. In p53(+/+) mice, p53-dependent and independent DNA repair mechanisms combined to restore damaged DNA, and unrestorably damaged cells were effectively removed by p53-dependent apoptosis after low dose rate irradiation. In this model, the teratogenic rate is controlled. In contrast, in p53(-/-) mice, p53-independent DNA repair mechanisms worked, but p53-dependent DNA repair mechanisms and apoptosis did not work. Therefore, the teratogenic rate does not decrease to a controlled level, even at low dose rate irradiation in p53(-/-) mice. Irradiation-induced DNA damage is thought to provoke similar mechanisms in the processes of carcinogenesis and teratogenesis.
In this study we investigated whether the p53 gene functions to remove damage not only via DNA repair mechanisms, but also via apoptosis in the carcinogenesis process. In p53(+/+) mice, if repair of DNA damage and removal of damaged cells by apoptosis is complete after repetitive low dose irradiation, then cancer may not arise. Under the same experimental conditions, if the cancer appears in p53(-/-) mice, perhaps there is a threshold dose of radiation that induces the carcinogenesis process.
Method: Seven-week-old mice were used. The backs of the mice were irradiated with beta-rays three times a week throughout the life of the mice.
Source: ⁹⁰Sr-⁹⁰Y disk source, 1.85GBq, 15Gy/min.

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Deficiencies in DNA mismatch repair (MMR) result in replication errors that cause frameshift mutations and/or point mutations within key tumor suppressor genes or oncogenes. Heterozygous germline mutations in MMR genes are the cause of hereditary nonpolyposis colorectal cancer (HNPCC). In these cancers, mutations in mononucleotide repeat sequences in TGFbRII or BAX have been frequently reported. Homozygous germline mutations of MMR genes are also manifested by an early onset of childhood T- or B-cell leukemias, but the target gene was not identified yet. We previously showed the lymphomas of Mlh1-deficient mice have normal type of TgfbrII, although insertional mutation in this gene has been reported in Msh2-deficient mouse lymphomas. In this study, we focused on Ikaros as a target gene of Mlh1-deficiency and elucidated the frequency and spectrum of the mutation.
Most of the spontaneously induced lymphomas lacked Ikaros protein at the frequency of 71%. This was resulted from frameshift mutations, most of which were one-base deletion in a mononucleotide repeat sequence that created a stop codon. These mutations were found in 86% of Mlh1-deficient lymphomas, but rarely found in radiation- and chemical-induced mouse lymphomas or in human leukemias. Thus, Ikaros is a most likely target for MMR deficiency-mediated mutation during the development of lymphomagenesis.

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[Background] Low dose radiation risk is difficult to assess because expected excess cancer incidence is much smaller than the background, spontaneous incidence. It is hence anticipated that information of molecular characteristics of radiation-associated cancers would aid to improve low dose risk assessment by distinguishing radiogenic cancers from spontaneous ones. We aimed to distinguish between them by means of gene expression profiling method in rat mammary cancer.
[Method] Female rats were either irradiated with 1 Gy of gamma rays or left untreated. Mammary adenocarcinomas and normal mammary tissues were subjected to microarray analysis (Affymetrix GeneChip®; ~30,000 probe sets). Total RNA was extracted for labeled cRNA synthesis, followed by hybridization to microarrays and scanning. Real-time RT-PCR was performed for selected genes which showed differential expression.
[Result and Conclusion] Hierarchical cluster analysis separated a single cluster of spontaneous carcinomas, whereas radiation-induced cancers were heterogeneous and did not form a single cluster. We extracted 66 genes that showed differential expression between the two etiological groups, based on fold difference and P values of t-test. Real-time RT-PCR analysis on selected genes confirmed that 14 genes showed differential expression even after new tissue samples were included. These results strongly support the possibility to distinguish between radiogenic and spontaneous cancers based on gene expression and suggest a difference between their mechanisms of development.

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A p53 gene function controls cell cycle progress and apoptotic process at the site of DNA lesion as a genomic guardian. It is thought that p53 gene product inhibits the process that a cell transforms. However, intervention to a p53 gene function and malignant transformation is not clear. Therefore, we planned this study using embryonic cells derived from a p53 knockout mouse to clarify how a p53 function was concerned with cellular immortalization and malignant transformation. In this study, we inoculated 106 cells into a T75 flask with MEM culture medium containing 10% FBS, and passaged them every 5th day and examined expression of malignant transformation character. As a result, degradation of cell growth was seen at passage 7-9, but all cells acquired immortality regardless of presence of a p53 gene, and the p53 knockout cell acquired tumorigenicity easily. Aneuploid was seen with both cells with manifestation of cancer character, especially p53 knockout cells became aneuploid at two or three passage. By these results, I will discuss how p53 genes contributes to cellular transformation.

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Tumor induction with MCA was followed in transgenic mice with mutated p53 gene carrying a 9 bp deletion in exon 6. The mutated p53 cDNA was obtained from radiation-induced mouse tumors and ligated to the expression vector pCXN2. The construct, pTE50, was introduced into mouse embryos to generate pTE50(+/-) mice. The mice were injected subcutaneously with 0.02 mg MCA dissolved in olive oil. Tumor incidence by May 2006 was 64% (32/52) in pTE50 (+/-) mice and 41 % (20/49) in wild-type mice, indicating a substantial increase in tumor incidence (p<0.05). Treatment of autochthonous MCA-induced tumors of 5 mm in diameter with siRNA, that was designed from the promoter sequence and was confirmed to suppress the expression of the mutated p53 mRNA, was effective in yielding a complete cure in 2 and growth inhibition in 2 out of 17 in pTE50(+/-) mice, while for tumors in wild-type mice, siRNA was not effective in all 4 cases. For transplanted pTE50(+/-) tumors of the MCA-induced origin, siRNA treatment yielded a complete cure in 2 and growth inhibition in 3 out of 14 cases, while for transplanted wild-type tumors, siRNA was ineffective in all of 3 cases.

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Thorotrast (Th) is the trade name for 25% colloidal suspension of radioactive 232ThO2 that naturally emits alpha particles, and was used as a radiographic contrast agent in the World WarII. After intravascular injection, more than 50% of the total amount of Th is located in the liver and is known to induce liver angiosarcoma (AS) and cholangiocarcinma (ICC) several decades after injection. We performed genome-wide scan of loss of heterozygosity (LOH) in Th AS and compared LOH profile with that of non-Th AS and Th ICC. Over all LOH frequency was higher in Th AS than in non-Th AS but frequent LOH at 2q33, 8q23 and 20p13 was commonly obserbed. Frequent LOH at 8q and 13q was observed in Th-AS and Th-ICC. These results suggest that exposure to alpha-particles causes random genetic changes, however, crucial steps to develop tumors are histological differentiation specific but not carcinogen specific.

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Hematopoietic cells of mice have been examined for the evidence of in vivo delayed effects long time after radiation exposure. Ten-week male C3H/HeN mice were exposed to 3 Gy x-rays and kept for up to one year. Parts of them were sacrificed periodically, and bone marrow cells were collected. The cells were spread onto clean slides followoing the hypotonic treatment and fixation, and foci of phosphorylated histone H2AX were detected by the immunofluorescence microscopy. Another batch of mice were also exposed to x-rays, and bone marrow cells of them were served for the CFC assay and the CAFC assay to analyze the cellular composition of the hematopoietic system. The number of phosphorylated histone H2AX rapidly decreased after irradiation, and became comparable to those of the control mice after two months. At 6 months post irradiation, however, the foci were about one and half times as many as the unexposed, and the high level was retained even one year after irradiation. The assays of hematopoietic cells showed that CFU-C (myeloid progenitors) had reached to the almost normal level within a month while CAFC-d14 (multipotent progenitors) and CAFC-d28 (very primitive progenitors) had not recovered even at 5 months post irradiation. These phenomena could be manifestations of delayed radiation effects in murine hematopoietic system. The relevance to radiation leukemogenesis is also discussed.

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A Novel Phenomenon "Delayed Division Delay": Evidence for Delayed DNA Double-Strand Break and Rejoining in the Clonogenic Progeny of HeLa Cells Surviving Alpha or X Irradiation Hiroshi SASAKI (retired from The Dept. Exp. Radiol., Kyushu Univ.) Lethal sectoring is the process for survival by which lethal damage remaining in irradiated cells is eliminated as offspring without reproductive integrity, and occurs through the postirradiation 1st-4th divisions with the accompanying appearance of a clonogenic progenitor harboring no lethal damage. However, nonlethal damage remaining in the clonogenic progenitors led to an elevated incidence of delayed cell death in their progeny. From an analysis of the pedigrees of cells surviving alpha (0.45 Gy) or X irradiation (3 Gy) (20% survival dose), it became clear that the mean incidence was higher for alpha-particle (16.3%) than X-ray survivors (8.3%), indicating the greater potentiality for genomic instability by alpha particles. Evidence is available to suggest the association of misrepaired clustered DNA damage with alpha particles.
A novel phenomenon "delayed division delay (DDD)" was noticed, though occasionally (ca.10% per cell; ca.30% per pedigree), with the clonogenic progeny during the postirradiation 1st-3rd generations. DDD was much longer in alpha- (mean: ca.11 h) than X-irradiated cells (ca.4 h), and might be triggered by delayed DNA double-strand breaks at the fragile break-misrejoining sites. This demonstrates the usefulness of DDD as a new endpoint of radiation-induced genomic instability.

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The Long Evans Cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is also a model for hepatocellular carcinogenesis. We developed congenic LEC rat lines (recipient strain, Fischer 344 (F344)) and performed genome-wide genotyping to identify radiation susceptibility genes. We mapped seven positional candidate genes to an approximately 1.2-Mb region located between loci D4Got85 and D4Got148 on chromosome 4. None of the seven genes has been reported to be associated with radiation susceptibility. Comparison of the coding sequences for these seven genes in F344 and LEC rats showed no changes in deduced amino acid sequences. We also constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three three expressed sequence tags (EST) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.

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DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand breaks (DSBs) repair, and it consists of Ku70, Ku80 and DNA-PKcs. It has been shown that the promoter regions of these three genes have Sp1 binding sites, and the expression levels are correlated with that of Sp1. The purpose of this study is to clarify the contribution of Sp1 to radiation sensitivity of cells through the transcriptional regulation of DSBs-repair genes. We investigated whether Sp1 affects the protein and mRNA levels of Ku70, Ku80, DNA-PKcs, XRCC4, NBS1, MRE11 and MDC1. In addition, we examined the DSBs-repair, DNA-PK activity, cell cycle, and radiation sensitivity in Sp1-dawn-regulated cells. A human transformed kidney cell line 293T was transfected with siRNA vector targeting Sp1 (Sp1-siRNA) or control vector. The vector-transfected cells were selected with G418. After 7 days selection, protein and mRNA levels were evaluated by Western blotting and RT-PCR, respectively. The protein and mRNA levels of Sp1, Ku70, Ku80, DNA-PKcs, XRCC4, NBS1, MRE11 and MDC1 were down regulated by the Sp1-siRNA treatment. The DSBs-repair after 80 Gy irradiation and DNA-PK activity were suppressed by the Sp1-siRNA treatment. The surviving fraction after irradiation was also suppressed by the Sp1-siRNA treatment. However, cell cycle was not affected by the Sp1-siRNA treatment. These results suggest that Sp1 regulates the radiation sensitivity by the transcriptional regulation of DSBs-repair genes.

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Clonogenic assay is regarded as the golden standard to evaluate the radiation sensitivity. However, there is controversial point about the evaluation of radio sensitivity by the clonogenic assay, because the results of the clonogenic assay are dependent on the plating efficiency and/or growth rate of the cells examined. In this study, we tried to establish the radio resistant cells that could proliferate under the condition of 2 Gy/day X-ray exposure as the model of radio resistant cells evoked during the cancer radiotherapy. In order to establish the radio resistant cells, HepG2 cells established from human hepatoblastoma were exposed to 1 Gy/day of X-rays for more than 4 years (total dose; 1,700 Gy) and we established 4 cell lines, HepG2-400, -89, -A and -8960. Those cells showed slightly radio-resistant compared with the HepG2 cells evaluated by clonogenic assay. Interestingly, no statistical significance was detected by the clonogenic assay with regard to radio resistance between HepG2 and newly established 4 cell lines, the growth rate of HepG2-8960 cells didn't change after the administration of 2 Gy/day of X-rays for 30 days. We think that HepG2-8960 cells are truly the radio resistant cells. In this study, we indicated that radio resistant cells would appear after the long-term X-ray exposure and we have to reconsider the validity of the clonogenic assay to evaluate the radiosensitivity.

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Angiogenesis is essential for solid tumor growth and is comprised of multiple steps including the migration, invasion, and cell proliferation of vascular endothelial cells. Although radiotherapy is an important cancer treatment modality, the effects of ionizing radiation(IR) on tumor angiogenesis remain unclear; different groups of researchers have reported that IR either inhibits or activates angiogenesis. In this study, we found that IR(8Gy) induced a high frequency (-90%) senescence-like phenotype rather than apoptosis in exponentially growing endothelial cells. Thus, we investigated the angiogenic activity of such surviving senescent cells to determine the effects of irradiation on angiogenesis. Our results showed that senescence-like endothelial cells were growth-inhibited, which could be a major cause of angiogenic inhibition. Furthermore, other angiogenic processes -such as invasion- were also significantly inhibited. In conclusion, we have implicated the senescence-like phenotype of endothelial cells as a possible mechanism for IR-induced inhibition of angiogenesis.

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We have developed a gene expression profiling method called "high-coverage expression profiling" or HCEP. Using this method we have analyzed radiation response genes using cultured cell models. In contrast to DNA microarray technology, HiCEP is based on AFPL technique. Therefore it has excellent sensitivity and reproducibility for gene expression detection and could still be a powerful tool for gene expression profiling analysis of clinical specimen.
We analyzed gene expression profiles of breast tissues obtained from breast cancer patients. Using HiCEP we did comparative analysis of gene expression for 28,000 transcripts between metastatic and non-metastatic breast cancer tissue. As a result, 20 transcripts that remarkably overexpressed or underexpressed were detected and 17 of them were sequenced. Fourteen of 17 transcripts were categorized into known mRNA in the public database and 6 of 14 showed sequence similarity to cancer-related genes. Moreover, this cancer-related group was found to contain 2 alternative splicing variants of erbB2 (HER2) gene, which is used as a diagnostic marker to predict clinical outcome of breast cancer.
In this study we showed the effectiveness of the HiCEP gene expression profiling using clinical specimen. Further study would reveal radiation response or cancer-related marker genes from clinical materials.

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Radiotherapy is widely and successfully applied to treat many malignant tumor types. In some patients, however, radiotherapy causes unacceptable levels of damage to normal tissues. The number of effective agents available for preventing or reducing radiation-induced injury is limited, and thus additional radiation modulators are required. The identification of genes involved in radiation-induced responses will aid the development of new radiation modulators for both normal tissues and tumors. We previously performed large-scale expression profiling having varying degrees of susceptibility to ionizing radiation. We then constructed a small-scale RNAi library of these genes. We screened this library using a 96-well format and measuring the cell survivals after X-irradiation. Then we identified 12 radiation susceptibility genes, among which three known and nine are unknown. Eight of these are associated with cell cycle progression. For the remaining for genes, we analyzed cell cycle progression in knockdown cells using two distinct RNAi against each of other four genes following X-irradiation. A gene might be involved in the G2/M checkpoint. Our results indicate that our high-throughput method may be useful for large scale screening to identify novel radiation susceptibility genes. These 12 genes may constitute potential targets for new drugs that modulate the effects of radiation exposure on tissues or potentiate tumor radiotherapy.

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To understand the genetic mechanisms associated with heterogeneity in radioresistance among human tumor cells, gene expression profiles of a set of tumor cell lines previously characterized for radiosensitivity were examined. Because there may be combinations of genes that are activated on irradiation after the cell acquires resistance to radiation, DNA microarray technology was used to study the total effects of multiple radioresistance genes.
Focusing on D₁₀, a total of 65 genes were identified that may be involved in cellular reproduction after irradiation. Relatively radioresistant cell lines (D₁₀ > 5Gy) were clearly distinguished from nonresistant cell lines (D₁₀ < 4Gy) at both 1 and 3 h after irradiation. Furthermore, using genes selected for altered transcription levels at both the 1 and 3 h time points after irradiation, the correct classification of another four radioresistant cell lines (5.95 < D₁₀ < 7.21 Gy) was confirmed.
Genes related to the inhibition of cell death and the promotion of proliferation were preferentially activated in radioresistant cell lines, whereas those associated with pro-apoptotic or cell communication functions were generally transcribed at a higher level in radiosensitive cell lines.

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We have reported that purvalanol A, an inhibitor of Cdc2 kinase, sensitized X-ray-induced apoptosis by the inhibition of Cdc2 kinase activity and through the abrogation of G2/M arrest. But the interaction between the abrogation of G2/M arrest and induction of apoptosis remains unclear. In this study, we investigated the expression of anti-apoptotic proteins, i.e., survivin, XIAP, Bcl-2 and Bcl-XL, at sensitization to radiation-induced apoptosis by purvalanol A. Western blot analysis showed that purvalanol A inhibited the radiation-induced upregulation of Bcl-2 and Bcl-XL. The expression of survivin and XIAP were not increased by X irradiation, but were also inhibited by purvalanol A. On the other hand, pro-apoptotic protein, Bax, did not increased by X irradiation but slightly decreased by purvalanol A. semi-quantitative RT-PCR analysis showed that the expression of mRNA of Bcl-2 and survivin were inhibited by purvalanol A but not Bcl-XL and XIAP. These results indicated that the sensitization to radiation-induced apoptosis by purvalanol A was occurred by downregulation of these anti-apoptotic proteins.

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X-chromosome-linked inhibitor of apoptosis protein (XIAP) is known to be an inhibitory factor against caspase-3. We examined radiation sensitivity of human cancer cells is enhanced by small interference RNA (siRNA) targeted for XIAP (XIAP-siRNA) through increase of caspase-3 activity. We used a human cultured non-small cell lung cancer cell line (H1299) lacking a tumor suppression gene of p53 gene. We prepared wild-type p53 cells (H1299/wtp53) and mutated p53 cells (H1299/mp53) having identical genetic background except for p53 gene. When 21-mer XIAP-siRNA was transfected into these cells with liposome, we observed the suppression of the constitutive expression of XIAP protein. Radiation sensitivities of wtp53 and mp53 cells were enhanced by XIAP-siRNA. In accordance with this, radiation-induced activation of caspase-3 was enhanced by XIAP-siRNA. These results suggest that XIAP-siRNA is a possible candidate for radiation sensitizer in radio-cancer therapy.

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Cyclin kinase inhibitor p21 inhibits or arrests the cell cycle progression in response to DNA damage. In addition, p21 regulates the progression of apoptosis negatively or positively depending of the situation. The stability of p21 is regulated by its phosphorylation or binding with partner molecules. When cells grow without DNA damage, p21 is degraded with proteasome. In this study, we analyzed the mechanism to stabilize p21. The transient expression of various p21 deletion mutants revealed that the mutant with deletion of 15-48aa was extremely unstable. And the proteasome inhibitor, lactacystin, stabilized the del15-48. The del15-48 was unstable in the cells stably expressing 1-60aa region, indicating 1-60 did not function in trans. Fusion of the 1-60 fragment to N terminal of del15-48 stabilized the product, indicates 1-60 region in the molecule is effective for the stabilization. We next constructed the cells stably expressing del15-48. The del15-48 was unstable but stabilized by lactacystin. The gamma irradiation (6 Gy) enhanced the expression of del15-48 without elevation of mRNA level, and increased the binding with cyclinA or CDK2. Taken together, we hypothesize two mechanisms for the stabilization of p21. First, DNA damage stabilizes the p21 by phosphorylation or binding with partner molecules. Second, for the basal expression of p21, 15-48 region is essential to prevent the degradation by the proteasome.

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