[Purpose] Extracellular materials of cancer cells play crucial roles leading to development of cancers and resistance to anticancer treatment. Pancreatic carcinoma is resistant to almost all chemotherapeutic agents. In this study, we searched for candidates involved in resistance to X ray or anticancer drugs among proteins released from cancer cells into extracellular space. [Methods] Medium from 5-h culture of various cancer cells was collected. Proteins in culture medium were detected by molecular mass analysis and immunoblotting. Sensitivity of cells preincubated with or without recombinant annexin II (rANX II) to X ray and various anti-cancer drugs were measured using crystal violet assay and colony survival assay. Apoptosis-related molecules were analyzed by immunoblotting. [Results and Conclusions] Annexin II was released from pancreatic cells into condition medium. Addition of rANX II in condition medium of non-cancerous cells and cancer cells resulted in suppression of cell death induced by X ray and cisplatin. Suppression of cisplatin-induced cell death by rANX II supplementation, associated with suppression of caspase-3 activation and increase of Bcl-2/Bax ratios, was canceled by inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) signal pathways. The current study is the first report to demonstrate that supplementation of rANX II into condition medium increased resistance to X ray and anticancer drugs in human cells. rANX II exerts cell death-suppressive function by anti-apoptotic effect against cisplatin, possibly via PI3K and MEK signal pathways.

View full abstract

In apoptotic cells, XRCC4 is cleaved by caspase-3 or -7 to form a 35 kDa-fragment (pN35) which is composed of the N-terminal 265 residues. This fragment includes the DNA ligase IV binding domain, but not the nuclear localization signal, of XRCC4. To investigate a role of XRCC4 cleavage in apoptosis, we examined the effect of caspase-resistant XRCC4 on staurosporine (STS)-induced apoptosis. Apoptosis was assessed by immunoblotting analysis of the cleaved-form of caspase-3, and the TUNEL assay which detects the internucleosomal DNA fragmentation. We established a XRCC4-deficient cell line M10, which is derived from a murine lymphoma cell line L5178Y. Expression in M10 cells of wild type XRCC4, but not caspase-resistant XRCC4 (XRCC4D265A), was required for induction of apoptosis by STS. Furthermore, more cleaved-forms of caspase-8, and -9, the upstream caspases of caspase-3 in the caspase-activation cascade, were detected in cells expressing wild-type XRCC4 than cells expressing caspase-resistant XRCC4 after treatment of cells with STS. Expression of the pN35 in M10 cells did not enhance STS-induced apoptosis. However, expression of the pN35 fused to the nuclear localization signal (pN35-NLS) in M10 cells enhanced the STS-induce apoptosis. In both wild-type and caspase-resistant XRCC4-expressing cells, localization of XRCC4 and DNA ligase IV changed from nucleoplasm to cytoplasm during the progression of apoptosis. These results revealed that pN35 augments apoptosis when it is located in the nucleus, and suggested that pN35 activates cleavage of both caspase-8 and -9.

View full abstract

P53 gene is well known as a tumor suppressor gene which plays an important role in conserving stability by preventing genome mutation and inducing apoptosis. Fishes also have p53 gene as mammal. Reports showed that induction of apoptosis by γ irradiation in zebrafish is suppressed in p53 mutant embryos, and cancer occurred in p53-/- zebrafish (Berghmans S, Proc Natl Acad Sci U S A. 2005 Jan 11). And similar reports about medaka showed that, in p53-/- cells apoptosis is suppressed observed by time lapse, and cancer occurred in medaka (Genome Biology 2006, 7:R116). In our study, to make the role of medaka p53 gene clear, we compared the phenotype of medaka wild type cells and p53-/- cells, and transfected p53 over-expressed plasmid into p53-/- cells for rescue experiments. At first, we analyzed apoptosis occurred in p53-/- cells. As a result, the proportion of apoptotic cells in p53-/- cells is much smaller than in wild type cells. We also added Pifithrin-α. Hydrobromide, a p53 gene inhibitor of mice, to wild type cells, and found that the proportion of apoptotic cells decreased significantly, the p53 gene inhibitor of mice is also effective in medaka. To investigate the relationship between p53 gene and DNA repair, we used comet assay to compare the DSBs repair induced by γ ray irradiation between wild type cells and p53-/- cells. As a result, we found that the tail moment which values the ability of DNA repair reduced significantly after few hours passed irradiation in wild type cells, while there is no significant change in p53-/- cells. So we get a conclusion that the ability of DNA repair in p53-/- cells is weaker than wild type cells. In order to investigate the relationship between p53 and the first stage of DNA repair, we used γ H2AX focus assay to compare the focus formation in wild type cells and p53-/- cells after irradiation. At result, there is no significant difference between normal cells and p53-/- cells. At present, we are using p53 gene transfected p53-/- cells to investigate the resumption of the functions lost in p53-/- cells.

View full abstract

Spontaneous germ cell death is a common cellular process in mammalian testis and p53 seems to play a role in germ cell quality control by causing cell death. Taniguchi et al. (2006) isolated p53 (-/-) mutant of Medaka (Oryzias latipes) by screening of ENU induced mutations in TILLING (Targeting Induced Local Lesions In Genome) library. We introduced p53 nonsense mutation into HdrR inbred strain by crossing. In testis of p53 (-/-) mutant, there are several abnormal cells with large and less electron-dense nucleus and distinct nucleolus. The number of these cells increased from 3 days after 1-5Gy gamma-irradiation, and their sizes were also increased. However, such histological change in testis became non-irradiated level until 30days after irradiation. The morphology of the abnormal cells looks like testis-ova induced by endocrine disruptors, and shows high expression of 42Sp50 gene that express in previtelogenic oocytes. We examined testis structure at 7days after irradiation using electron microscopy and meiosis specific synaptonemal complex but could not observed lampbrush chromosomes. The apoptotic cells with nuclear condensation at 1 day after irradiation could be observed in wild type testis but it could not be detected in p53 (-/-) mutants. However, at 7days after irradiation, many testis-ova with activated caspase 3 that suggests that there is a p53 independent pathway to eliminate testis-ova induced by irradiation.

View full abstract

There are two p53-mediated apoptotic pathways which are transcription-independent pathway and transcription-dependent pathway. Transcription-dependent pathway is activated by the transcription of p53-targeted factor such as PUMA and Noxa. Transcription-independent pathway is activated by a direct interaction between accumulated p53 and mitochondrial Bcl-2 family members, including Bcl-2, Bcl-xL. However, the contribution ratio of both pathways in the cells and organs is not clear. To clarify the contribution degree to either pathway, it is essential to develop a compound which specifically inhibits either pathway. It is reported that p53 inhibitor pifithrin-µ (PFTµ) specifically suppresses transcription-independent pathway out of two p53-mediated apoptosis pathways and protects mice from bone marrow lethal dose irradiation. However our studies demonstrated that PFTµ cannot protect mice from total-body irradiation, which suggested that it is not a useful tool to examine whether the inhibition of transcription-independent pathway is important to radiation protection. A part of Zinc chelator acts with zinc ion coordinated to p53 DNA binding domain, and inactivates p53, resulting to inhibition of apoptosis. We found 5,7-bis(N,N-dimethylaminosulfonyl)-8-quinolinol (bis(DMAS)-QOH) as the zinc chelator which suppresses the transcription-independent pathway without effecting p53 target-gene expression. Moreover we showed that bis(DMAS)-QOH has lower toxicity and higher cell death inhibitory effect than PFTµ. These results indicate that bis(DMAS)-QOH can be a inhibitor of transcription-independence pathway superior to PFTµ and will be a useful tool to reveal the role of the divergence pathway. We intend to examine the radioprotective effect of bis(DMAS)-QOH in a mouse model and reveal the mechanism for the specificity of it to the transcription-independent pathway.

View full abstract

We have demonstrated that sodium orthovanadate (vanadate) is the first inhibitor that can protect death from radiation-induced gastrointestinal syndrome in mice by blocking both transcription-dependent and transcription-independent p53 apoptotic pathways. In this study, we initially found that vanadate has a unique activity in inducing a denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α and pifithrin-µ. We therefore postulated that the activity might be associated with the potent radioprotective activity of vanadate, and searched for a new p53 inhibitor that could induce p53 denaturation. On the other hand, it is known that p53 denaturation is induced by dissociation of a zinc ion, which is coordinated to metal ion binding site of p53, and hence we evaluated some zinc chelators for inhibition of p53-dependent apoptosis of irradiated MOLT-4 cells. As a result, two out of five zinc chelators suppressed the apoptosis. Especially, bispicen, having the highest efficacy in inhibition of the apoptosis, shows the effects on p53 denaturation as well as on inhibition of both transcription-dependent and -independent apoptotic pathways, the similar effects to vanadate. In addition, we revealed that the suppressive effect of bispicen on apoptosis is specifically mediated by p53 using p53-knockdown MOLT-4 transformants, p53 mutant cells, and p53-null cells. Our findings indicate that using zinc chelation would be a new approach to inhibition of p53-dependent apoptotic pathways. We are currently characterizing a new compound that is a hybrid compound that consists of bispicen and a p53-specific peptide, which is synthesized in order to provide the specificity for p53.

View full abstract

To examine the effects of carbon ion and gamma ray irradiation on cancer-induced osteoclastogenesis, mouse calvarias MC3T3-E1 cells were cultured with conditioned medium from irradiated and non-irradiated MCF7 human breast cancer cells. We examined RANKL and OPG mRNA expression in osteoblastic MC3T3-E1 cells following treatment with conditioned MCF7 medium. Co-cultured MC3T3-E1 and bone marrow cells treated with conditioned medium from irradiated MCF7 cells showed decreased numbers of osteoclasts assessed using TRAP staining. Conditioned medium from control MCF7 cells elevated the RANKL/OPG mRNA ratio in MC3T3-E1 cells; this effect was suppressed by carbon ion irradiation of the MCF7 cells. These data demonstrate that indirect interactions between breast cancer cells and MC3T3-E1 cells induce osteoclastogenesis in vitro through modulation of RANKL expression and that this process is suppressed by carbon ion irradiation.

View full abstract

Radioresistance is thought to be caused by intrinsic characteristics of cancer stem cells (CSCs) due to their unique DNA damage response (DDR), efficient DNA repair and resistance to apoptosis. Repopulation of surviving CSCs is associated with recurrence of tumors after radiotherapy (RT). Therefore, eradication of CSCs is important to improve the efficacy of RT. We have established long-term fractionated radiation (FR) cells from HepG2 and A172 by exposure of X-rays every 12 hours for 82 days (82FR-31NR cells). 82FR-31NR cells exhibited CSC like properties, such as overexpression of CSC marker CD133 and the ABC transporter (MDR1 and BCRP1). Furthermore, 82FR-31NR cells have the tumotigenic capacity when the lower number of cells was implanted into nude mice. Thus, we successfully isolated CSCs from HepG2 and A172 by repeated FR for a long-time. In 82FR-31NR cells, the AKT pathway was activated after 5Gy-irradiation but not in parental cells. Inhibition of the AKT pathway by either an AKT inhibitor, API-2 or a Cdk4 inhibitor could be suppressed radioresistance of 82FR-31NR cells. Similarly, knockdown of cyclin D1 or Cdk4 by its siRNA was sufficient to suppress radioresistance of 82FR-31NR cells. These results demonstrated that the AKT/cyclin D1/Cdk4 pathway is important for radioresistance of CSCs. In conclusion, we would present the newly discovered DDR of CSCs, and propose a strategy for eradication of CSCs using fractionated RT combined with the AKT inhibitor.

View full abstract

17-N-Allilamino-17-demethoxygeldanamycin (17-AAG) is a low-toxic derivative of geldanamycin and a potent anticancer drug. Also, 17-AAG is known as a potent radio-sensitizer. On the other hand, we have reported that Serine Threonine Kinase 38 (STK38) is stimulated by oxidative stresses including X-irradiation, and that activation of STK38 is necessary for protection of oxidative stress-induced cell death. Here, we found that STK38 interacts with HSP90. Inhibition of HSP90 by treatment with 17-AAG resulted in reduction of STK38 expression level. 17-AAG also decreased stk38 gene expression level. Knockdown of STK38 enhanced X-ray-induced cell death. Together, 17-AAG radiosensitized cells through down-regulation of STK38.

View full abstract

Senescence-like growth arrest is now recognized as the major cell death pathway in human solid carcinomas treated with ionizing radiation, however, its process has not been fully described yet. This study was designed to analyze cell cycle during senescence-like growth arrest of MCF-7 mammary carcinoma exposed to X-rays. Using live-cell imaging following 10 Gy of X-irradiation, we newly found cell cycle transition from G2 to G1 phase without mitosis in 17.8% of G1-irradiated cells and in 69.4% of G2-irradiated cells. Entry to G1 phase was confirmed by nuclear accumulation of cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin or CENP-F. The data indicate that ionizing radiation triggers mitotic skipping which is represented as cell cycle transition from G2 to G1 phase without mitosis. More than 90% of cells skipping mitosis was persistently arrested in G1 phase and showed positive staining for the biochemical senescent marker, which is senescence-associated ß-galactosidase, elucidating induction of senescence-like growth arrest accompanied by mitotic skipping. Furthermore, transduction of shRNA for p53, which efficiently suppressed p21 induction, significantly reduced mitotic skipping. The data suggest that ionizing radiation-induced mitotic skipping is associated with the presence of functional p53. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry particularly following G2-irradiation.

View full abstract

53BP1 is a mediator of DNA double-strand break response including DNA damage checkpoint and DNA repair. Previously, we identified 53BP1 as a protein binding to tumor suppressor p53, but it is not clear whether 53BP1 is involved in p53 function. To elucidate this, we analyzed activation of p53 after X-irradiation using human cancer cell lines with functional p53, in which 53BP1 were depleted by RNAi. The result exhibited that depletion of 53BP1 down-regulated p53 activation but not ATM activation. Thus, our data indicate that 53BP1 functions upstream of p53 and downstream of ATM in the signaling pathway of p53 activation.

View full abstract

Cataracts are opacities of the transparent lens. Mounting epidemiological evidence has indicated that lens opacification results from radiation exposure at lower dose and/or dose rates than previously thought. However, the biological mechanism behind radiation cataractogenesis is not known. We thence set out to examine the radiation response of mouse lens and human lens epithelial cells, and here shall present its preliminary results.

View full abstract

It is well established that the biological consequences of radiation exposure basically depend on the total dose of irradiation. While many studies on the biological effects of acute irradiation have been conducted, fewer data are available on chronic irradiation studies. In addition, the molecular mechanisms underlying the dose-rate effect on various biological responses to radiation have been remained to be resolved. In this study, in order to better understand the biological effects of chronic radiation exposure and the molecular mechanisms underlying the dose-rate effects, the cellular responses induced by chronic low-LET irradiation at different dose-rates were investigated from different aspects.
Various types of cultured human cell lines, including normal primary diploid fibroblasts, hTERT-immortalized fibroblasts, and a variety of tumor cell lines, were acute irradiated with different doses of ¹³⁷Cs γ-rays at a dose-rate (1.0 Gy/min) or chronically irradiated at different dose-rates (0.007-0.694 mGy/min). The cellular responses to radiation under different irradiation conditions are analyzed using various approaches. The clonogenic survival curves to acute γ-irradiation shown that the analyzed all cell lines had a similar sensitivity to γ-rays. However, significantly different growth inhibitory effects were observed in each cell lines under chronic radiation exposure. Clonogenic assays under chronic conditions demonstrated that there is a dose-rate threshold between reversible and irreversible growth inhibitory effects of radiation. Furthermore, transcriptome analysis by using next-generation sequencing exhibited dose-rate dependent alterations in mRNA expression levels in cells under chronic irradiation conditions. It is noteworthy that the p53 pathways largely contribute to the cellular responses to chronic irradiation in a dose-rate dependent manner.

View full abstract

Radiotherapy improved the local control by the development of instrumentation, machine software and diagnostic techniques. Despite these technical improvements, the satisfactory results are not yet provided about the regulation of tumor cell migration and invasion. Recently, although it is reported that radiation accelerates cell motility, little is known about molecular mechanisms of radiation-induced cell migration and/or invasion. Hence, in this study, we investigated to clarify the molecular mechanisms of radiation-induced cell migration.
Human glioblastoma CGNH-PM and U251 cells were exposed to X-rays (200 kVp, 14.6 mA). Cell migration was evaluated by wound-healing assay 24 h after X-ray irradiation. Cell migration was accelerated by X-ray irradiation in dose-dependent manner. The amount of glutamate released in culture medium was also increased in dose-dependent manner. Antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor suppressed radiation-induced cell migration.
These results suggest that glutamate is involved in radiation-induced cell migration. A further investigation about correlation between cell adhesion molecule and X-ray-induced cell migration via glutamate is being analyzed.

View full abstract

Pulmonary fibrosis is one of the most severe late-onset lung injuries resulting from radiation therapy. We previously reported a strain difference in the incidence rate of pulmonary fibrosis induced by carbon-ion irradiation in the mouse model. Determining the cause of this difference could aid the development of prevention and treatment strategies that consider the individual variations in pulmonary fibrosis induced by carbon-ion therapy. Although collapse of the expression balance among MMP2, MMP9, and TIMP1 has been reported in a bleomycin-induced fibrosis model, the roles of these factors in radiation-induced fibrosis are not fully understood. In this study, we analyzed the expression of MMP2, MMP9, and TIMP1 in pulmonary fibrosis induced by carbon-ion irradiation. The lungs of two mouse strains (C57Bl/6J and C3H/He) were irradiated by carbon-ion irradiation at 10 Gy. Pulmonary fibrosis was observed in all C57Bl/6J mice at 24 weeks after the irradiation and was exacerbated at 28 weeks. On the other hand, pulmonary fibrosis was also observed in only three of five C3H/He mice at 24 weeks, with none apparent at 28 weeks. These results suggested that pulmonary fibrosis continuously progressed in the C57Bl/6J mice, but that this injury could be improved in the C3H/He mice. We then investigated the expression of MMP2, MMP9, and TIMP1 in the cases of pulmonary fibrosis by real-time PCR and immunohistochemistry. The levels of MMP9 expression in the fibrotic area differed significantly between the two strains, although the expression of MMP2 and TIMP1 was upregulated throughout the lungs of both strains. Herein we present our comparative analysis of the observed strain difference in both MMP expression and progression of pulmonary fibrosis induced by carbon-ion irradiation.

View full abstract

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. In this study, we assessed in vitro the ability of priming low doses (0.01-0.1 Gy) of X-rays and heavy-ion radiation to induce an AR to a subsequent challenging dose (1-4 Gy) of high-LET IR (carbon-ion: 20 and 40 keV/µm, neon-ion: 150 keV/µm) in cultured lymphoblastoid TK6, AHH-1 and NH32 cells. Pre-exposure of p53-competent cells (both with low-dose X-rays and high-LET IR) resulted in decreased mutation frequencies at Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon was independent of radiation-induced apoptosis or cell cycle effects. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms. Even though the cells directly hit by heavy-ion beams (even at low doses) are likely to suffer significant damage, our results constitute the first report to date indicating that low doses of high-LET radiation can nevertheless induce protective effects against subsequent high-LET irradiation. Taking inter-individual variability into account, these results might have interesting implications for high-LET radiation therapy and space research.

View full abstract

In general, transforming growth factor-beta-activated kinase 1 (TAK1) plays a role to inhibit apoptosis in response to multiple stresses. To assess the role of TAK1 in X-irradiated cells, cell death and cell cycle distributions were investigated in TAK1 knockdown and its parental HeLa cells. Furthermore, changes of gene expressions were also examined to elucidate the molecular mechanisms. After X-irradiation, radiosensitivity estimated by colony formation assay increased in the TAK1 knockdown cells. Apoptosis induction indicated by caspase-3 cleavage also supported the enhanced radiosensitivity in the TAK1 knockdown cells. Simultaneously, irradiated cells in G2/M phases decreased and those in S and SubG1 phases increased by TAK1 depletion, indicating loss of cell cycle checkpoint regulation promotes radiosensitivity in the TAK1 knockdown cells. However, we could not identified significant difference of increased NF-kappa B, p38 MAPK and ERK phosphorylation, major downstream molecules of TAK1, between TAK1 knockdown cells and its parental control cells after irradiation. Instead, global gene expression analysis revealed the differentially expressed genes after irradiations and bioinformatics analysis identified a genetic network associated with cell cycle regulation. Several genes associated with cell cycle in the genetic network showed different expression patterns between TAK1 knockdown and its parental cells after X-irradiation. Especially, CDKN1A (coding p21WAF1), which was central in the identified network, was up-regulated in control cells but not in TAK1 knockdown cells after X-irradiations. RNA interference of p21 decreased cells in G2/M phases and increased cells in S and SubG1 phases after X-irradiations. In conclusion, these findings showed the role of TAK1 in cell cycle regulation and apoptosis in a manner independent on NF-kappa B, p38 MAPK, and ERK phosphorylations but dependent on p21 induction, at least in part.

View full abstract

[Objective] Reoxygenation of hypoxic region in tumor has been widely recognized as a typical response of tumor tissue toward ionizing radiation. However, the mechanism of the radiation-induced tumor reoxygenation has not been fully elucidated. Therefore, we aimed in this study to evaluate the fluctuations in tumor oxygenation status after ionizing radiation and determine the factors responsible for radiation-induced tumor reoxygenation.
[Methods] As an animal model, we used a transplanted tumor of murine squamous carcinoma SCCVII cells in C3H mice. To detect histological change of hypoxic region after X-irradiation, we performed immunohistochemical (IHC) analysis with two different hypoxia probes (pimonidazole and EF5). To examine the change in partial pressure of oxygen (pO₂) in tumor, in vivo electron spin resonance (ESR) technique with LiNc-BuO as an oxygen sensitive probe was utilized. To investigate the involvement of nitric oxide (NO˙) in radiation-induced tumor reoxygenation, we tested the effect of a nitric oxide synthase inhibitor, L-NAME.
[Results] Double-stained images of hypoxia revealed that primarily existing hypoxic regions were completely diminished at 24 h after X-irradiation, followed by the recovery at 48 h. The result of ESR oxymetry showed that tumor pO₂ was time-dependently increased and peaked at 24 h after X-irradiation, and then decreased and kept at higher level than the pre-irradiation up to 120 h. Administration of L-NAME inhibited the first phase of reoxygenation. These results indicated that X-irradiation induced two-phase reoxygenation and NO˙ was suggested to be involved in the first phase of this reoxygenation.

View full abstract

MicroRNAs (miRNAs) are small noncoding RNAs of about 19-22 nucleotides that regulate protein expression by posttranscriptional silencing. Recent investigations have suggested that miRNA play important roles in cellular processes, including differentiation, cell proliferation and cell death. In 2008, Lawrie et al. showed the correlation between the serum levels of miR-21and relapse-free survival in patients with diffuse large B-cell lymphoma, suggesting that miR-21 may be a potential diagnostic biomarker for the disease. The aim of this study is to identify radiation exposure specific miRNAs in mouse plasma. We analyzed the expression profiles of 4 Gy of gamma-irradiated mouse plasma using TaqMan Array MicroRNA Card (ABI). Several miRNAs were increased or decreased compared with control mouse plasma. The dose response and time course of these miRNAs will be shown.

View full abstract

Ataxia-telangiectasia (AT) is a highly radiosensitive genetic disorder caused by mutational inactivation of the ATM gene. ATM is activated by DNA double-strand breaks and induces DNA repair and cell cycle arrest. Recently, it has been reported that hydrogen peroxide (H₂O₂) induces ATM activation, and that ATM deficiency sensitizes the cells to H₂O₂. The activation of ATM by H₂O₂ is reported to involve cell surface PDGF receptor in human umbilical vein endothelial cells (HUVECs). In this study, we initially found that ATM is activated by H₂O₂ treatment in human T-cell leukemia cell line MOLT-4, and aimed to assess the role of ATM and p53 in H₂O₂ signaling compared with ionizing radiation signaling. The level of H₂O₂-induced apoptosis in MOLT-4 cells reaches a plateau at 200 µM, which is almost the same level as the apoptosis induced by 5 Gy gamma-ray. We next revealed that H₂O₂-induced apoptosis is specifically mediated by p53 using p53-knockdown MOLT-4 transformants. Furthermore, the apoptosis was highly correlated with the phosphorylation of p53 Ser-15 or ATM autophosphorylation, and less correlated with p53 protein accumulation. Further investigations are needed to determine whether the damaged cell membrane or DNA initiates the signaling.

View full abstract

Superoxide dismutases (SODs) are antioxidant proteins converting superoxide to hydrogen peroxide. In vertebrate cells, SOD1 is mainly present in the cytoplasm, and small portions in nucleus and mitochondrial inter-membrane space and SOD2 is present in the mitochondrial matrix. Previously we have conditionally disrupted SOD1 or SOD2 gene in DT40 cells, respectively. SOD1 depletion caused lethality, while depletion of SOD2 showed growth defect. Our intriguing observation from previous work is that lethality observed in SOD1-depleted cells is completely rescued in the presence of ascorbic acid. In this study, we confirmed ascorbic acid offset growth defect observed in SOD2 depleted cell and completely normalized increased mitochondrial superoxide levels in either SOD1 or SOD2 depleted cells. Moreover intracellular oxidative stress reflected by increase of either mitochondrial or cytoplasmic superoxide levels was reduced by ascorbic acid treatment. All the data so far in this study suggested ascorbic acid could be antioxidant which is mimic of either cytoplamic or mitochondrial superoxide dismutase.

View full abstract

Purpose: Previously, we reported that irradiation in young age induced delayed mutation, and that decrease function of p53 gene related to delayed mutation. In this study, we investigated the p53 gene function and mutations at old age in p53+/- mice after irradiation at several points. Methods and Materials: p53+/- mice were received a whole-body dose of 3 Gy at 28 and 40 weeks of age. We evaluated TCR MFs after irradiation to 60 weeks of age, and apoptosis, loss of p53 alleles, p53 gene methylation, and protein expression of p53, p53ser15 and p21 at 60 weeks of age. Results: We previously reported that in p53+/- mice that were received a whole-body dose of 3 Gy at 8 weeks of age, T-cell receptor (TCR) mutation frequencies (MFs) reached at a peak level at 10 weeks of age, decreased at spontaneous level at 20 weeks of age and re-increased from 40 weeks of age. At 60 weeks of age, TCR MFs and p53 gene methylation were higher in mice after irradiation at 40 weeks of ages compared to mice after irradiation at 28 weeks of ages. On the other hand, at 60 weeks of age, apoptosis and protein expression of p53, p53ser15 and p21 were lower in mice after irradiation at 40 weeks of ages compared to mice after irradiation at 28 weeks of ages. Conclusion: We concluded that, because p53 would damage with age gradually, the delayed effect of irradiation was heavier when the mice received irradiation at older age.

View full abstract

Purpose: Aneuploidy occurs in 90% of solid human tumors but a role of aneuploidy in carcinogenesis was unclear. We examined the role of aneuploidy in carcinogenesis.

Methods: To isolate the diploid, the triploid and the tetraploid clones, 9 clones were cloned from p53 (-/-) mouse embryonic cells and the chromosome number of these clones was counted. These cells were cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum. To study whether aneuploidy contributed to carcinogenesis, we examined (1) anchorage-independent growth, (2) tumorigenicity (3) micronuclei frequency, (4) a number of co-localized p-H2AX and 53BP1 foci, (5) chromosome structural aberration, (6) reactive oxygen species (ROS) levels using the fluorescent dye DCFH-DA, (7) cell growth and (8) the gene expression profiles by microarray analysis.

Results: Anchorage-independent growth and tumorigenicity were observed in triploid clones but not in both diploid and tetraploid clones. Micronuclei frequencies, the number of foci containing co-localized p-H2AX and 53BP1 and chromosome structural aberration were increased in the triploid clones compared with the diploid clones. There were no differences in the diploid clones and the tetraploid clones. The triploid clones contained much lower levels of ROS than both the diploid clones and the tetraploid clones. Number of genes 10-fold altered compared with the diploid clones in the triploid clones was markedly increased. Despite lower levels of ROS, both chromosome structural aberrations and DNA double strand breaks occurred in triploid clones.

Conclusion: Triploidy resulted in chromosome and DNA instability, and disruption of global gene expression, finally gave rise to cancer.

View full abstract

In human epidemiological studies, a significant increase in the cancer incidence rate has been reported by exposure to 100 mSv or higher doses of ionizing radiation. However, the relationship between lower doses of ionizing radiation and cancer incidence is still unclear. In general, oxidative DNA damage is closely related to cancer generation. We studied the oxidative DNA damage elicited by low dose ionizing irradiation. Mice were irradiated with X-rays, and the 8-hydroxydeoxyguanosine (8-OHdG) levels in the tissue DNA and urine were measured by HPLC-ECD. The γ-ray irradiation of a dG or DNA solution caused a linear increase in the 8-OHdG levels, in the range of 20 – 300 mGy. Thus, 8-OHdG seems to be a good marker of the oxidative DNA damage caused by ionizing radiation. In contrast, in the case of the whole body irradiation of mice, the 8-OHdG levels in liver DNA and urine increased from about 0.5 Gy irradiation. These results indicate that living organisms have a defense mechanism against the oxidative damage caused by ionizing radiation. Considering the 8-OHdG level as an ionizing radiation effect marker for living organisms, a threshold level of irradiation seems to exist for oxidative damage and tumorigenesis. Detailed results with lower irradiation doses will also be discussed.

View full abstract

Radiation effect has been studied on mouse small intestine that contains stem cells and progenitors in the crypt. The stem cells are classified into two types of cells, one of which is label-retaining cells at position+4 (+4 LRCs) and the other is Lgr5⁺ CBC cells at the crypt base. The transcription factor Bcl11b is a tumor-suppressor and is expressed in these cells. Here we show that loss of a Bcl11b allele affects the regeneration of IECs after γ-irradiation. We investigated the migration rate of IECs along the crypt-villus axis by BrdU pulse chase. The rate of neither migration nor proliferation did not differ between Bcl11b^KO/+ and Bcl11b^+/+ IECs. In contrast, γ-irradiation gave differences in these rates; the distribution of BrdU⁺ cells was more lowered in Bcl11b^+/+ mice than in Bcl11b^KO/+ mice. Also, BrdU incorporation at 16 h after irradiation was more in Bcl11b^KO/+ CBC cells, indicating that Bcl11b^KO/+ CBC cells were more refractory to radiation-induced cell cycle arrest. p53, which is central to the DNA damage checkpoint, was less activated in crypt cells of Bcl11b^KO/+ after irradiation. These results suggest that the loss of a Bcl11b allele promotes proliferation of stem cells by elevating radio-resistancy or enhancing transient activation after irradiation.

View full abstract

Mutations or deletion of BCL11B gene were found in 16% of human T-cell acute lymphoblastic leukemias (T-ALLs), and also in a half of γ-ray induced mouse thymic lymphomas. We previously showed that loss of one Bcl11b allele plays a key role in clonal expansion of premalignant thymocytes in irradiated Bcl11b^KO/+ mice. However, it remains open whether the premalignant thymocytes originate from differentiated cells in the thymus or progenitor cells in bone marrow. In order to address this issue, we developed Lck-Cre;Bcl11b^flox/+ mouse model, in which Bcl11b heterozygosity occurs only in cells at a specific developmental stage in the thymus. Those mice were subjected to γ-irradiation of 3Gy at 8 weeks of age and their thymocytes were characterized of cell number, clonal expansion and differentiation. We found clonal expansion of premalignant thymocytes after irradiation and premalignancy started mostly in thymocytes at a mature type of CD8 single-positive cells bearing highly expressed TCRβ. These results suggest that cells of origin in thymic lymphomas are differentiated cells in the thymus. However, these premalignant thymocytes tended to have characteristics of more immature thymocytes. Our results also raise a possibility that differentiated thymocytes can be the radiation target leading to malignancy.

View full abstract

Although radiation-induced cancer, life-shortening and chromosomal abnormality are known to be influenced by the age of individual when the radiation is exposed, little is understood for the mechanism responsible for these phenomena. Here, we asked if the efficiency of mutation induction changes depending on age at exposure using lacZ-transgenic mice. We examined liver because it is shown to display strong age-dependency for tumorigenesis (S. Sasaki, J Radiat Res, 32, (Suppl. 2), 73-83).
Muta-mice containing lacZ gene were irradiated with 0, 10 and 20 Gy of X-rays (0.72 Gy/min) at the following ages; 15.5 d embryo, postnatal 2 days, 7 days, and 2 months. Liver was dissected 3 days later and genomic DNA was extracted. Mutant frequencies in the lacZ gene were determined by retrieving lacZ-containing lambda DNA into phages followed by the determination of the total number of phages and that of mutated lacZ-containing phages.
The dose-responses of mutation induction were almost linear. Thus we calculated the slopes to estimate the efficiency of mutation induction. It was 0.507 x 10^-5/Gy at 15.5 d embryo and 1.32 x 10^-5/Gy at 2 months of age. The efficiencies at 2 days and 7 days after birth showed intermediate values. The difference could be explained if error-free homologous recombination repair is dominant in embryo stage for radiation-induced DNA double strand breaks and if error-prone non-homologous end joining becomes dominant after maturation of liver. The results indicate that the age-dependency of liver tumor induction would not be explained by a difference in mutation induction.

View full abstract

Radiation carcinogenesis in human is results from combined exposure to numerous environment factors. However, little is known about its molecular mechanisms. In order to determine if the combined exposure activates the specific carcinogenic pathway or enhances each one, we scrutinized extensively the frequency and spectrum of mutations of the genes, which are critically important for T-cell lymphomagenesis, i.e., Ikaros and p53 as tumor suppressor gene, Notch1 and K-ras as oncogene, in mice thymic lymphomas (TL) induced synergistically by simultaneous combined exposure to X-rays and N-ethyl-N-nitrosourea (ENU). Molecular analyses identified that Ikaros alteration frequency, especially point mutation frequency, was increased significantly in combined exposure (47.2%) compared with X-ray- (12.9%) or ENU-exposure (0%)(P<0.01). Our previous study demonstrated that point mutations of Ikaros are characterized by accompanying LOH in X-ray-induced TL, but not in ENU-induced TL. Here, a half of point mutations in combined exposure were associated with LOH, and remaining without LOH. Interestingly, some were accompanied by concomitant frameshift mutation or altered splicing in another allele. Thus, Ikaros inactivation was stimulated by mutation induction from both X-rays and ENU in combined exposure. The frequency of Notch1 abnormality was high in all treatments (over 58%), suggesting a mandatory step in T-cell lymphomagenesis. In contrast, mutation frequencies of p53 and Kras were low in all treatments (<23%). In conclusion, Ikaros is a critical mutational target for enhancement and acceleration of lymphomagenesis after combined exposure to X rays and ENU.

View full abstract

The mean cause of lung cancer is tobacco, which contains numerous alkylating agents. Recently, increase in the chest CT, especially pediatric CT, is also paid rapid attention for the future risk of lung cancer. Therefore, it is a great concern about the risk of lung cancer by childhood exposure of ionizing radiation followed by adulthood exposure to lung carcinogenic chemicals. We examined combined effects of X-rays and an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), on pulmonary tumorigenesis in juvenile and adult rats. Female 1-, 5- and 22-week-old Wistar rats were irradiated locally on the thorax with X-rays (3.18 Gy), and/or were given BHP (1 g/kg body weight) intraperitoneally 1 week after each X-rays or 23 weeks of age, and sacrificed at 90 weeks of age.
We found that (i) the induction of lung tumors (both adenoma and carcinoma) increased as a function of age at X-ray-exposure, but decreased at BHP administration, (ii) combined effect of X rays, either exposure at 1, 5 or 22 weeks of age, with BHP at 23 weeks of age induced adenoma and carcinoma in an additive and synergistic fashion, respectively, (iii) the longer interval between two treatments reduced the carcinogenic effects. Immunohistochemical analysis indicated that most of tumors were originated from SP-A-positive alveolar type II cells and that activation of ERK pathway was limited in this rat model.
In conclusion, the combined effect of X rays with adulthood exposure to BHP on lung tumorigenesis, especially malignant carcinomas, was manifested in a synergistic manners, and the carcinogenic events of juvenile exposure lasted up to adult stage.

View full abstract

The potential role of fetal exposure in the increase of childhood cancers is still a matter of debate. Environmental and genetic factors, or combined effects of radiation with these factors need to be considered. We studied the effect of in utero exposure on lymphoma development using Mlh1-deficient mice, which are genetically prone to lymphoma. A germline mutation in MLH1, one of the DNA mismatch repair (MMR) genes associated with hereditary nonpolyposis colorectal cancer, causes childhood T- and B-cell leukemia, when homozygously defective. The tumors obtained from non-irradiated and in utero irradiated groups were diagnosed either thymic lymphoma or splenic lymphoma based on lymphoma-appearing anatomical site. According the cell-surface marker status, splenic lymphomas were further divided into two groups, T-cell origin (TCR+, CD4+ and CD8¬–) and B-cell origin (IgH+). Mutation analysis of Ikaros and Trp53 revealed that these genes were frequently mutated by one base insertion or deletion at mononucleotide repeat sequences, resulting in a frameshift mutation. Ikaros frameshift mutations were only found in thymic lymphomas, which were characterized TCR+, CD4+ and CD8+, indicating an immature T-cell. Trp53 frameshift mutation was found both T-cell and B-cell splenic lymphomas, the mutation was occurred about 50 % in each lymphoma with a heterozygous status. Radiation exposure increased point mutations in these genes in both thymic and splenic lymphomas. Interestingly, in utero exposure significantly shortened the latency of B-cell origin splenic lymphomas only, but did not affect those of thymic lymphomas and T-cell origin splenic lymphoma. Our data demonstrated that B-cell lymphoma development is accelerated by in utero exposure in Mlh1-deficient mice. Hence, in utero exposure should be paid more attention especially to persons with MMR gene-deficient background.

View full abstract

We have been developing new assay systems that use GFP gene expression for mutant detection. The principle of the approach is to make the cells fluorescent when mutation occurs at specified gene loci in the genome. One example is to make partial duplications of endogenous gene, in which the distal segment of the duplicate has a GFP gene tag. In this system, reversion from the duplicate makes the mutant cells fluorescent. The other example is to introduce cells co-expression of two vectors, one produces GFP constitutively and the other produces repressor protein to silence the expression of GFP gene. In this system, any kinds of forward mutation in the repressor gene in the targeted allele allow GFP gene transcription from null state, which make the mutant cells green. Both of the knock-in mice were generated. We report the current status of the mouse analyses.

View full abstract

We have reported that radon inhalation activates antioxidative functions in some organs of mouse. We previously reported that prior radon inhalation inhibits oxidative damage such as carbon tetrachloride (CCl₄)-induced hepatopathy. However, there is no comparative study on effect of prior or post radon inhalation on oxidative damages. We examined the effects of prior and post radon inhalation on CCl₄-induced oxidative damage in brain, heart, lung, liver, and kidney of mouse. BALB/c mice (7 weeks old age) inhaled radon of 18,000 Bq/m³ for 6 hours before or after CCl₄ administration. In result, the relative increases in lipid peroxide (LPO) levels in lung, liver, and kidney of mouse treated with prior or post radon were significantly smaller than those of mice treated with sham. In addition, there were no significant differences in the LPO levels of lung, liver, and kidney between prior and post radon inhaled mouse. The relative decreases in total glutathione (t-GSH) content in liver of mice treated with prior radon were significantly smaller than those of mice treated with post radon. The relative decreases in superoxide dismutase (SOD) activities in Brain of mice treated with post radon were significantly smaller than those of mice treated with prior radon. These results suggested that prior or post radon inhalation activate antioxidative functions and inhibit CCl₄ induced oxidative damage, the degree differ according to organs and its susceptibility to oxidation.

View full abstract

We have conducted radon exposure experiments with mice to clarify the mechanism of radon therapy. Radon, an inert gas, is mostly exhaled even if inhaled, but radon progeny of solids is deposited on respiratory airways. In the present study, we calculated fractional deposition and absorbed dose of inhaled radon progeny in mice lung. The results were compared with those of rats and humans. A lung model of mice (Oldham et al., Anat. Rec., 2007) divided a lung into 23 airway generations. The generation 1 (trachea) to 16 is a tracheobronchial (TB) region, and the other generation are an alveolar region (from generation 17 to 23). First, we calculated inhalability and deposition in the nasopharyngeal (NP) region. Factional depositions in mice lung were then computed, taking into account the deposition mechanism of inertial impaction, gravitational sedimentation and diffusion. Finally, absorbed doses were assessed for after calculating the particle clearance and absorbed fraction of alpha-particle energy emitted in target cells. The condition of radon progeny was assumed as follows: equilibrium factor 0.4, unattached fraction 0.01, particle-size distribution of geometric mean diameter 250 nm (geometric standard deviation 2.5) for attached radon decay products and of geometric mean diameter mean 1 nm (geometric standard deviation 1) for unattached ones. There were two peaks of fractional depositions at a particle size of 20 nm to 5 μm in the tracheobronchial region, and the two peaks also occur at 80 nm and 3 μm in the alveolus region. Absorbed dose rates in the tracheobronchial airway and alveolus region was 51.6 and 4.6 nGy/h/(Bq/m³)), respectively, which was comparable to 35.9 nGy/h/(Bq/m³)) in the whole lung. Here, this concentration means the radon equilibrium equivalent radon concentration. The same dosimetry was performed for rats and humans. As a result, absorbed doses in mice were two and seven times higher than those in rats and humans, respectively.

View full abstract

In 1980s, Russell et al. studied the genetic effects of low-dose-rate (LDR) irradiation on male mice using phenotypes such as coat color of mice, however effects at LDR was not observed. Recent advances in genome analysis technology, has enabled more thorough analyses of genomic effects. To clarify incidence of copy number aberrations (CNAs) on the genome of progeny mice, progeny was obtained from C57BL/6J male mice exposed to LDR gamma rays (20 mGy/22 h/day; 0.91 mGy/h) for 400 days (total dose : 8000 mGy) from 8 weeks of age. Using oligo-microarray CGH (Agilent Technologies), we have so far analyzed a total of 107 genomes from mice (32 progenies from six pairs of parents in LDR-irradiated group and 49 progenies from seven pairs of parents in non-irradiated group). In the LDR-irradiated group, four mice had one CNA each, and four other mice had multiple (3~35) CNAs. On the contrary, only five mice from the non-irradiated group had one CNA each. Present results indicate that LDR-irradiated mice had significantly higher frequencies of genomic aberrations than non-irradiated mice (2.09 loci/genome vs 0.10 loci/genome). This study was performed under contract with the Aomori Prefectural Government, Japan.

View full abstract

Biological effects of internal radiation exposure have not been well evaluated as compared to the effects of external exposure. As a preliminary study for establishment of the in vitro model for risk estimation by internal exposure, we exposed the cultured H1299 cells to I-125 deoxyuridine (81.4TBq/mmol) or H-3 deoxyuridine (740TBq/mmol) which were present inside (intracellular exposure) or outside (extracellular exposure) the cells for up to 8 weeks in frozen condition. The intracellular irradiation with I-125 exhibited a rapid lethality and reached approximately 40% viability in one week exposure (10.94 total decays/cell), which was comparable to the lethal effect by 4Gy irradiation with external Cs-137source. Phosphorylations of p53, ERK-MEK, and p38 were also observed in cells after the intracellular exposure to I-125. The extracellular irradiation with I-125 caused a gradual decrease in cell viability to approximately 70% of unirradiated cells in 8 weeks. In contrast, no lethality was observed by irradiation with H-3 either in intracellular or in extracellular condition. Based on the methodology shown here, we are about to examine the effects of internal irradiation with I-131 in cultured thyroid cells.

View full abstract

The dose-rate effect (DRE) is very important phenomenon to estimate the radiation risk for human health. However the mechanisms of DREs have not been clarified. We thought that dose-rate was fractionated irradiation per unit time, and hypothesized that the DREs might be induced by the accumulation of unrepaired DNA damages. It is necessary to examine the time course of radiation-induced initial DNA damage in detail to prove our hypothesis. First, we investigated the time course analysis of radiation–induced initial DNA damage based on 53BP1 forms discrete foci in primary human fibroblasts (MRC-5). The result showed that the numbers of unrepaired DNA damage were 1.3 per cell at 0 min, 1.33 per cell at 3 min, 1.57 per cell at 5 min, 2.62 per cell at 15 min, 1.2 per cell at 30 min after 20 mGy. Next, we investigated whether unrepaired DNA damages are accumulated in MRC-5 irradiated with 2x20 mGy per 3-10 min. The numbers of unrepaired DNA damage were 2.5-2 per cell after the second irradiation. However, when MRC-5 were irradiated with 2x20 mGy per 15-30 min, the numbers of unrepaired DNA damage were 1.8-1.6 per cell after the second irradiation. This result indicated that unrepaired DNA damage was not accumulated by fractionated irradiation per 15-30 min. Thus, we found that the mechanism of DREs is not able to explain from DNA damage and repair.

View full abstract

The effects of low-dose heavy ions on the prenatal development of animals as well as of cells are not well elucidated. The neural crest-derived melanocyte is a good model to study the effects of heavy ions on the proliferation and differentiation of cells, since it possesses a higher proliferation rate in the embryonic stage and visible pigment melanin as a differentiation marker. In 18-day-old murine skin, the accumulation of melanoblast, precursors of melanocyte, and the initiation of differentiation of melanocyte are observed in the epidermis, in addition, hair bulb melanocytes are already colonized with the initiation and progression of the development of hair follicles. Therefore, in this study, 18-day-old murine skin was selected to study the effects of low-dose heavy ions. Pregnant females of C57BL/10J mice at 9 days of gestation were whole-body irradiated with a single acute dose of heavy ions (0.1, 0.25, 0.5, and 0.75 Gy). The effects were studied by scoring changes in the prenatal development of mice as well as of epidermal melanoblasts/melanocytes and hair bulb melanocytes. Although the frequency of developmental anomalies such as bent tails, short tails, microphthalmia and anophthalmia is increased as dose and LET increased, the frequency of developmental anomalies such as cacomelia and hemorrhage showed no change by dose and LET. The number of epidermal melanoblasts/melanocytes and hair bulb melanocytes in the dorsal and ventral skins significantly decreased even at 0.1 Gy-treated embryos, and gradually decreased as dose increased. The effects in the ventral skin were greater than those in the dorsal skin. The effects of heavy ions on the number of hair bulb melanocytes were increased as LET of heavy ion species increased, whereas the number of epidermal melanoblasts/melanocuytes showed no difference between heavy ions and gamma-rays. These results suggest that heavy ions cause cell death or inhibition of the differentiation of melanoblasts/melanocytes and other cells even at the low dose in a tissue-specific manner. Their effects in the ventral skin appear to be greater than those in the dorsal skin.

View full abstract

Alzheimer's disease (AD) is the most common form of dementia while its cause and progression are not well understood. The possible cognitive and behavioral consequences induced by low-dose radiation are of great concern as humans are exposed to ionizing radiations from various sources including medical diagnosis. A recent study in mice reported early transcriptional response in brain to low-dose X-rays (0.1Gy) suggesting alterations of molecular networks and pathways associated with cognitive functions, advanced aging and AD. To investigate the cognitive and behavioral consequences induced by low doses, C57BL/6J mice were whole-body irradiated with either X-rays (0.1Gy) or accelerated carbon ions (0.05 Gy or 0.1 Gy). The hippocampus was collected and the expression of AD-related genes was analyzed. Morris Water maze test was applied to the measurement of the learning ability and memory of the animals. Amyloid imaging with positron emission tomography (PET) were performed to detect the accumulation of fibrillary amyloid beta peptide, and characteristic pathologies of AD were examined with immunohistochemical staining of amyloid precursor protein (APP), amyloid beta peptide, tau and phosphorylated tau. Results showed significant acute transcriptional alterations in several AD-related genes at 4 h after irradiation while no marked changes 1 yr later. No significant difference on learning ability and memory was observed 1 yr after irradiation. Imaging and immunohistochemical staining showed that no change in the accumulation of fibrillar amyloid and the expression of APP, amyloid beta peptide, tau, phosphorylated tau were not detectable in the animals 4 m and 2 yr after irradiation. These findings suggest that low-dose irradiations could induce acute transcriptional alterations in several AD-related genes without significant late consequences on behavioral and AD-related pathological changes in mice.

View full abstract

Recently, we reported that radon inhalation activates antioxidative functions in some organs and inhibits oxidative damage such as carbon tetrachloride-induced hepatopathy. In this study, we examined the inhibitory effects of radon inhalation on carrageenan-induced inflammatory paw edema in mice using our new radon exposure system. Carrageenan was dissolved physiological saline solution (50μl of 1% v/v) and injected to the right hindpaw of female ICR mice (8 weeks old age). Prior to carrageenan injection, mice inhaled approximately 2000 Bq/m³ radon for 24 hours. In result, carrageenan injection induced paw edema, and inflammatory leukocytes were observed in paw. The tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) levels in serum significantly increased and superoxide dismutase (SOD) and catalase activities in paw significantly decreased following carrageenan injection. However, the relative increase in serum TNF-α levels of radon-treated mice were significantly smaller and the number of inflammatory leukocytes of radon-treated mice were significantly fewer than those of sham-treated mice. In addition, the relative decreases in paw SOD and catalase activities of radon-treated mice were significantly smaller than those of sham-treated mice. These findings suggested that radon inhalation activates antioxidative functions and inhibits carrageenan-induced inflammatory paw edema in mice. Our data show that radon therapy has a possibility for treating inflammation as a new indication.

View full abstract

Radom therapy is clinically used for intervention of rheumatoid arthritis. The mechanism for the reduction of pain has not been clarified. In the present study, we investigated the relieving effect of radon inhalation on inflammatory pain. ICR mice (male and female, age: 8-9 weeks) inhaled radon at a concentration of 1000 Bq/m³ or 2000 Bq/m³ for 24 hours. Twenty μl of 0.5 % formaldehyde was injected into the hindpaw of the mice immediately after radon inhalation. Formalin injection induces a transient biphasic inflammatory pain. We examined the licking response time induced by formalin. In results, the first phase began immediately after formalin injection and lasted for 5 minutes. The second began 10 minutes after formalin injection and lasted for 20 minutes. Radon inhalation at a concentration of 1000 Bq/m³ or 2000 Bq/m³ did not inhibit the first phase of pain, suggesting that radon inhalation did not show the effects of narcotic analgesics such as morphine. However, radon inhalation at a concentration of 2000 Bq/m³ inhibited the second phase of pain, suggesting that radon inhalation show the effects of analgesic effects like nonsteroidal anti-inflammatory drugs (NSAIDs). We also found the migration of inflammatory leucocytes by formalin injection. However, radon inhalation inhibited the migration of inflammatory leucocytes. This report will be mentioned other changes in the characteristic of pathological observations, antioxidant function, or inflammatory mediators.

View full abstract

We have reported that radon inhalation activates of antioxidative functions and inhibits carbon tetrachloride (CCl₄)-induced liver damage. It is also reported that ascorbic acid inhibits liver damage induced by CCl₄. In the present study, we examined the inhibitory effect of radon inhalation or ascorbic on liver damage induced CCl₄. ICR mice were subjected to intraperitoneal injection of CCl₄ after inhaling approximately 1000 or 2000 Bq/m³ radon for 24 hours or immediately after intraperitoneal injection of ascorbic acid (100, 300, or 500 mg/kg). In results, the activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) of radon inhalation or ascorbic acid treated groups were lower than those of CCl₄ treated group, suggesting the inhibition of CCl₄ induced liver damage. In the radon irradiated groups, the relative decrease in the activities of superoxide dismutase (SOD) and catalase was smaller than those of ascorbic acid treated groups. However, no significant differences in lipid peroxide levels in liver were observed between radon inhalation group and ascorbic acid treated group. In addition, the radon inhalation has an effect similar to treatment of ascorbic acid (300 mg/kg).

View full abstract

Radioresistant cells are one of the obstacles of tumor radiotherapy. In order to understand the molecular mechanisms of radioresistance and develop more effective tumor radiotherapy, clinically relevant radioresistant (CRR) cells were established from several cancer cell lines. Those cells continue to proliferate under exposure to 2 Gy/day of X-rays for more than 30 days, a standard protocol for tumor radiotherapy. Our previous study indicated that CRR cells are resistant to anticancer drug docetaxel (DOC). One of the factors of cellular DOC resistance is thought to be the resistance against hydrogen peroxide because DOC produce hydrogen peroxide in the cells. It is well known that X-ray exposure generates reactive oxygen species (ROS) in cells and inadequate removal of ROS results in oxidative stress that leads to damage to biological macromolecules; the products of lipid peroxidation can cause DNA damage leading to cell death. Therefore, we hypothesized that more efficient removal of ROS in CRR cells would be the reason for DOC resistance and radioresistance. Both the modified high density survival assay and MTT assay showed that all clinically relevant radioresistant cells are resistant to hydrogen peroxide. We think that DOC resistance of CRR cells is due to the resistance against hydrogen peroxide.

View full abstract

There are many kinds of flavonoid (over than 7000) derived from plants. However, their functions are not fully understood. We previously performed screening of about 40 kinds of flavonoids to find novel radiosensitizers derived from plants. From the screening, we found Flavonoid A (tentative name) has the faint cytotoxicity and definitive radiosensitization action in cancer cells. The present study compared the effects of Flavonoid A on radiosensitivity in normal cells and cancer cells. We used human lung normal fibroblasts (HFL-III) and mouse normal fibroblasts (C3H/10T1/2), and human lung cancer cells (H1299) and mouse cancer cells (C3H/MCA Cline 15) derived from C3H/10T1/2. Colony formation assay showed that Flavonoid A sensitized radiosensitivity more effectively in cancer cells compared with normal cells. Flavonoid A is regarded as an efficacious radiosensitizer because of its faint cytotoxicity and preferential sensitization to cancer cells.

View full abstract

[Objective] The unfolded protein response (UPR) is an intracellular signal pathway for responding to endoplasmic reticulum (ER) stress. Whereas hypoxia, nutrient starvation and other conditions causes ER stress and activates UPR to up-regulate survival signal in tumor cells, excess ER stress stimulates the apoptotic signal pathway. Although UPR could be a promising target for a novel anticancer strategy, the precise mechanism underlying ER stress cascade remains elusive. In this study, we examined whether ER stress affects DNA repair machinery and enhances radiation-induced cell death in solid tumor cells.
[Methods] Human lung adenocarcinoma A549 cells were treated without or with ER stress inducers, tunicamycin (TM) or thapsigargin (TG). The expression of DNA-repair-related proteins was examined by Western blot analysis and semi-quantitative RT-PCR analysis. Reproductive cell death was assessed by clonogenic assay.
[Results] TM clearly suppressed the protein levels of Rad51, which is the key protein of the DNA homologous recombination repair pathway. However, mRNA of Rad51 was not affected by TM treatment. This down-regulation of Rad51 protein induced by TM or TG was inhibited by a proteasome inhibitor MG132. In clonogenic cell survival assays, the pretreatment of TM showed a significant radiosensitizing effect. These results suggested that ER stress induced Rad51 degradation through the ubiquitin-proteasome pathway and enhanced radiosensitivity by suppressing of DNA repair capacity.

View full abstract

It is well established that interactions between tumor cells and the host tissue stroma play a key role in tumor progression. However, the contribution of the different stromal cell components to tumor growth remains to be clarified after radiation therapy. We report here the antitumor characteristics of tumor-infiltrating neutrophils (CD11b⁺Gr-1^+high cells), which were recruited to irradiated tumor in LLC-OVA (OVA-transfected LLC)-bearing mice. We observed that the number of them peaked at 36 h after 11 Gy irradiation. Surprisingly, the therapeutic effect of irradiation was attenuated in the mice depleted of both CD11b+Gr-1+high cells and CD8+ cells before irradiation compared to in the mice depleted only CD8⁺ cells. Although CD11b+Gr-1+high cells isolated from tumors both 36 h post-irradiated mice and un-irradiated mice had a neutrophil-like morphology, most of them in irradiated tumors were more hyper-segmented. Coinjection of LLC-OVA with radiation-induced CD11b⁺Gr-1^+high cells resulted in a dramatic decrease in tumor growth. They expressed higher levels of pro-inflammatory cytokines and lower levels of VEGF and MMP9 than control cells. Thus, our findings strongly suggest that radiation-induced CD11b⁺Gr-1^+high cells may have the antitumor activity.

View full abstract

[Purpose] Recently, there are several papers reporting that the mitochondrial reactive oxygen speices (ROS) produced by X-irradiation influences cellular radiosensitivity. Therefore, we hypothesized that drugs, which activate oxidative phosphorylation in cancer cells, enhanced radiation-induced cell death by stimulating cellular energy production and mitochondrial ROS production. To test this hypothesis, we examined the effect of methyl-pyruvate (MP), a membrane-permeable pyruvate derivative, on radiation-induced cell death. [Materials and Methods] The mouth squamous carcinoma SCCVII cells and human lung carcinoma A549 cells were used in this study. Cells were treated with 30 mM or 50 mM MP and X-irradiation. After 24 h replaced by freash medium, the radiation-induced reproductive cell death was evaluated by clonogenic assay. ROS derived from mitochondria and mitochondrial membrane potential of cells treated with MP and X-irradiaton were evaluated with the specific fluorescent probes MitoSOX™ Red and tetramethylrhodamine methyl ester (TMRM), respectively. [Results] The treatment of subtoxic 50 mM MP significantly enhanced radiation-induced reproductive cell death in SCCVII cells and A549 cells. While X-irradiation after 24 h increased mitochondrial membrane potential and mitochondrial ROS production, the addition of MP caused the further enhancement of them. To examine whether this increase of ROS production contributed to the cell death caused by the combination of MP and X-irradiation, we tested the effect of an antioxidant ascorbic acid in SCCVII cells. The treatment of ascorbic acid partially protected SCCVII cells from the cytotoxic effect of the combination of MP and X-irradiation. These results suggested that the exposure of MP combined with X-irradiation enhanced oxidative phosphorylation and induced mitochondrial ROS production, leading to the enhancement of the radiation-induced cell death.

View full abstract

Purpose It has been postulated that COX-2 is related to metastasis, neovascularization and radiosensitivity of cancer cells. In addition, COX-2 over-expression is correlated to low radiosensitivity and high malignancy in neoplastic cells. Especially, high grade gliomas are known to be hypervascular and radioresistant, because of their hypoxic environment. Thus, in this study, a selective COX-2 inhibitor, celecoxib was tested to determine whether it has synergistic effects on the radiosensitivity of malignant glioma cells under hypoxic conditions. Materials and Methods Materials are human glioblastoma cell line A172 and mouse glioma cell line GL261. Cells were cultured under hypoxic conditions using gas pack pouches. Celecoxib ( Pfizer, US ) was used as the selective COX-2 inhibiting drug. 137Cs gamma cell was used for gamma irradiation. Various densities of celecoxib were added to the cells, and all were irradiated with 5Gy of gamma rays. After irradiation, antitumor effects were evaluated by proliferation control examination and colony formation assay, and the appearance of COX-2 and endoplasmic reticulum (ER) stress related proteins was evaluated by western blotting. Results Although low dose (<10 micro-M) celecoxib suppressed the expression of COX-2, significant growth suppression was not observed. High doses (>30 micro M) of celecoxib itself induced ER stress. A significant growth delay was seen in cells treated with gamma ray + celecoxib as compared to gamma ray alone. These effects were observed not only in normoxic but also in hypoxic conditions. The effect of gamma ray irradiation under the existence of celecoxib did not depend on the state of oxygen of the cell, and the growth of malignant brain tumor cells was significantly suppressed. Conclusions and discussion Celecoxib has been reported to upregulate ER stress related protein and induce apoptosis. On the other hand, radiation also induces ER stress. In the present study, we were able to confirm radiosensitizing effects of celecoxib in glioblastoma cells in hypoxic states as well as normoxic states. From these results, we conclude that celecoxib is a promising drug for increasing the radiosensitivity of malignant glioma cells.

View full abstract

Boron Neutron Capture Therapy (BNCT) is now currently one of the increasing radiation therapies in Japan. In BNCT borate-ions selectively accumulated in tumor an following neutron capture reactions emit secondary particles,mainly primarily 1.4MeV alpha and 0.84MeV lithium ion particles. These low energy particles deposit their energies in tumor effectively, and as such.This method can be used to that treat those tumors, which cannot be treated by X-ray method. However, there is not enough experimental data to evaluate BNCT biological effectiveness from clinical and research experiments. And therefore the micro-dosimetry techniques are often used instead. In our presentation, we introduce results on the research focused on evaluation of the biological effectiveness of the production of DNA strand breaks during BNCT. In order to evaluate number and space distributions of DNA strand breaks we simulate frequency of direct attacks and indirect attacks by radicals generated in ionization or excitation of water, and we also estimate local energy deposition from low energy particles emitted in BNCT, by using Monte Carlo track simulation code. This presentation at first shows the results of simulation models and their comparison with the exiting experimental results. The compared results are those of energy deposition at displacement volumes by particles (linear energy deposit), and those of the concentration of OH radicals produced by indirect attacks. In addition to this comparison, the presentation also shows results of simulation as compared with experimental data on the yields of DSB or SSB in changing concentrations of borate-ions or DMSO in plasmid DNA. With this research we aim to simulate those missing fundamental data on BNCT, which are difficult to get from experiments. Finally we also plan to conduct DNA strand break simulations under conditions as those in actual treatments.

View full abstract

Micro-planar beam radiation therapy has been developed with synchrotron radiation. The method is single irradiation of one direction or orthogonal two directions at high dose up to 600 Gy using an array of narrow planar beam (width of about 20-50μm) with the center-to-center distance of about 10 times of the beam width, resulting in an increase in life span of tumor baring animals with high recovery ratio of normal tissue. The energy of X-rays for the animal study has been reported below 300 keV with synchrotron radiation.
In the present study, beam profiles at high energy up to 1 MeV was calculated by Monte Carlo simulation with the code system of PENELOPE-2008*. The model arrangement of the system was as follows: Thickness of 10cm collimator made of tungsten having 5 slits of 20μm beam width and 200μm center-to-center distance, air layer of 1mm, and water phantom of 20cm. Beam profiles were obtained for the relative absorbed dose in 1cm depth at 10cm to 11cm in water. The results show that peak-to-valley ratio of 100 keV, 200 keV, 500 keV and 1 MeV were 150, 332, 2.37 and 1.62, respectively. Possibility of micro-planar beam radiation therapy at high energy will be discussed.
*F. Salvat, J.M. Fernández-Verea and J. Sempau, "PENELOPE-2008: A Code System for Monte Carlo Simulation of Electron and Photon Transport" (OECD Nuclear Energy Agency, ISSY-les-Moulineaux, France, 2008).

View full abstract

Abstract Purpose: The healing mechanism of tumors after local radiotherapy is closely related to cellular immune response of hosts. In this study, we evaluated the modulation of the expression of immunological molecules in tumor tissue, the immunological escape phenomenon, and quality and quantity of tumor infiltrating immune cells after radiation with different doses and time points. Materials and methods: We established syngeneic subcutaneous tumor bearing mouse model inoculating the GL-261 and SCC-7 cells to C57BL/6J and C3H/HeN mice, respectively. Irradiation to the tumors was performed using 130kV X-ray. Mice were divided into 4 groups; control, 17.5Gy/single fraction, 24Gy/2 fractions and 32Gy/4 fractions. After each radiation, tumor tissue was fixed at appropriate times and immunohistochemical analyses were performed using anti-mouse CD3, CD8, CD4 and FoxP3 antibodies. Also, the expression of MHC-I on tumor cell surface was evaluated. We compared the X-ray results with those of 155 MeV proton beams irradiation. Results: Radiation induced tissue necrosis became visible in the middle region of tumor tissue from day-1 to 4 after irradiation. Hematoxylin eosin (HE) staining showed lymphocyte infiltration in marginal regions of the necrotic foci from day-4. After day-7, lymphocytes infiltrated over the entire tumor tissue, particularly in tissues irradiated with 32Gy/4 fractions. Precise evaluation of the occurrence of tumor specific immune response after local tumor irradiation is necessary to optimize the effect of local irradiation and tumor specific immunotherapy.

View full abstract