Nov 18, 2009

Takahiro Kataoka (Graduate School of Health Sciences, Okayama University)

We previously reported that low-dose X-irradiation activates antioxidative function and alleviates ischemia-reperfusion injury. Ischemia-reperfusion injury is an inevitable result of liver transplantation and is closely related to the development of primary graft non-function. Activation of antioxidative function in grafts could potentially inhibit ischemia-reperfusion injury after transplantation. In the present day, we examined changes in antioxidative function induced in extirpated mouse liver by low-dose X-irradiation. Mouse livers were sham-irradated or were irradiated and stored in presearvation or saline at 4.The results show that the activity of superoxide dismutase (SOD) and catalase of mice livers kept in cold storage in saline solution after 0.5 Gy irradiation or in presearvation solution after 1.0 Gy irradiation was significantly increased. Following storage in presearvation solution, the activity of SOD and catalase of the 1.0 Gy irradiated group were singnificantly higher than those of the sham irradiated group. Moreove, lipid peroxide levels of the 0.5 Gy irradiated group were significantly lower than those of the sham irradiated group. These findings suggest that low-dose irradiation siginificantly activates antioxidative functions of liver grafts. Moreover, the dose at which enhancement of antioxidative function occurs in livers stored in presearvation solution, which contains glutathione, is higher than that in saline solution. The data presented in this study provide and essential basis for future studies aimed at determination of the possibility of remission of ischemia-reperfusion injury in livers after transplantation.

References:
[1] Kataoka et al. Basic study on active changes in biological function of mouse liver graft in cold storage after low-dose x-irradiation
J Clin Biochem Nutr. 2009 Sep;45(2):219-26
[2] Kataoka et al. Inhibitory effects of prior low-dose x-irradiation on ischemia-reperfusion injury in mouse paw
J Radiat Res (Tokyo). 2007 Nov;48(6):505-13
[3] Yamaoka et al. Activation of Antioxidant System by Low Dose Radiation and Its Applicable Possibility for Treatment of Reactive Oxygen Species-Related Diseases
J Clin Biochem Nutr. 2006 39(3):114-133

Jul 29, 2009

Mikio Shimada (Radiation Biology Center, Kyoto University)

Centrosomes function in proper cell division in animal cells. The centrosome consists of a pair of centrioles and the surrounding pericentriolar matrix (PCM). After cytokinesis, daughter cells each acquire one centrosome, which subsequently duplicates at the G1/S phase in a manner that is dependent upon CDK2/cyclin-E activity. Defects in the regulation of centrosome duplication lead to tumorigenesis through abnormal cell division and resulting inappropriate chromosome segregation. Therefore, maintenance of accurate centrosome number is important for cell fate. Excess number of centrosomes can be induced by several factors including ionizing radiation (IR). Recent studies have shown that several DNA repair proteins localize to the centrosome and are involved in the regulation of centrosome number possibly through cell cycle checkpoints or direct modification of centrosome proteins. Furthermore, it has been reported that the development of microcephaly is likely caused by defective expression of centrosome proteins, such as ASPM, which are also involved in the response to IR. The present review highlights centrosome duplication in association with genotoxic stresses and the regulatory mechanism mediated by DNA repair proteins.

References:
Shimada et al. Emerging Connection Between Centrosome and DNA Repair Machinery
J Radiat Res 50(4) 295-301 (2009)

Jun 10, 2009

Yi Shang (National Institute of Radiological Sciences)

Interleukin (IL)-2 family cytokine-mediated signal transduction plays important roles not only in normal development but also in the malignant transformation of lymphoid cells. However, little is known about the status of receptor activation and downstream signal transduction in primary lymphomas in vivo. In the current study, primary T-cell lymphomas (TL) of mice were induced by X-ray irradiation. Expression and activation of IL-2 family cytokine receptors and downstream Janus kinase (Jak)-signal transducers and activators of transcription (Stat) pathway were determined. As results, IL-9Ralpha was exceptionally highly expressed and phosphorylated in primary TL. IL-9Ralpha proteins in TL were heterogeneous due to different glycosylation. Downstream Stat3 and 5, but not Stat1, were also phosphorylated. There was a clear strain difference between susceptible C57BL/6 and resistant C3H mice in Stat3 and 5 activation and expression of Cyclin D1. Thus, aberrant expression, modification and activation of IL-9Ralpha and Stat proteins contribute to in vivo growth of TL in a manner linking to the genetic susceptibility to TL induction.

References:
Shang et al. Aberrant activation of interleukin-9 receptor and downstream Stat3/5 in primary T-cell lymphomas in vivo in susceptible B6 and resistant C3H mice
In Vivo 22(6) 713-720 (2008)

Jun 16, 2009

Kazuhiro Daino (National Institute of Radiological Sciences)

To better understand the molecular basis of radiation-induced osteosarcoma (OS), we performed global gene expression profiling of rat OS tumors induced by the bone-seeking alpha emitter Pu-238, and the expression profiles were compared with those of normal osteoblasts (OB). The expressions of 72 genes were significantly differentially expressed in the tumors related to OB. These included genes involved in the cell adhesion (e.g., Podxl, Col18a1, Cd93, Emcn and Vcl), differentiation, developmental processes (e.g., Hhex, Gata2, P2ry6, P2rx5, Cited2, Osmr and Igsf10), tumor-suppressor function (e.g., Nme3, Blcap and Rrm1), Src tyrosine kinase signaling (e.g., Hck, Shf, Arhgap29, Cttn and Akap12), and Wnt/beta-catenin signaling (e.g., Fzd6, Lzic, Dkk3 and Ctnna1) pathways. Expression changes of several genes were validated by quantitative real-time RT-PCR analysis. Notably, all of the identified genes involved in the Wnt/beta-catenin signaling pathway were known or proposed to be negative regulators of this pathway and were downregulated in the tumors, suggesting the activation of beta-catenin in radiation-induced OS. By using immunohistochemical and immunoblot analyses, constitutive activation of the Wnt/beta-catenin signaling pathway in the tumors was confirmed by observing nuclear and/or cytoplasmic localization of beta-catenin and a decrease in its inactive (phosphorylated) form. Furthermore, we found a significant reduction in the levels of glycogen synthase kinase 3beta (GSK-3beta) protein in the tumors relative to OB. Taken together, these findings provide new insights into the molecular basis of radiation-induced OS.

References:
Daino et al. Gene expression profiling of alpha-radiation-induced rat osteosarcomas: Identification of dysregulated genes involved in radiation-induced tumorigenesis of bone
Int J Cancer 125, 612-620 (2009)

Jun 11, 2009

Tatsuhiko Imaoka (National Institute of Radiological Sciences)

Ionizing radiation is the only firmly established human breast carcinogen. Rodent mammary cancer is unquestionably a useful model of human breast cancer. However, chemical induction models of rodent mammary cancer have been the central players in the field of experimental breast cancer research. To draw more attention to radiation induction models, discussions are made on similarities and dissimilarities between radiation and chemical models. The topics include: (i) pathology and natural history of cancer, (ii) genetic changes associated with induced cancer, (iii) indirect carcinogenic mechanisms, (iv) modification of cancer susceptibility by reproductive and age factors, and (v) the target cell type of carcinogens. Careful comparison of the two models shows some important differences such as genetic alterations of induced cancers and modification of susceptibility by age and reproductive factors. We propose that identification of the target cell type and relevant translational research for human risk management is an important issue that is addressed by the radiation carcinogenesis model.

References:
Imaoka et al.Radiation-induced mammary carcinogenesis in rodent models: what's different from chemical carcinogenesis?
J Radiat Res 50(4):281-93 (2009)

Jun 01, 2009

Kentaro Ariyoshi (National Institute of Radiological Sciences)

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging and caused by mutations of the WRN gene mapped at 8p12. Because WS is caused by a defect of the WRN gene, it is highly expected that all abnormal phenotypes observed in WS cells can be rescued by expression of the WRN gene. To examine functional complementation of WS phenotypes, we introduced a normal human chromosome 8 into a strain of WS fibroblasts (WS3RGB) immortalized by expressing a human telomerase reverse transcriptase subunit (hTERT) gene. Here, we demonstrate that the abnormal WS phenotypes including cellular sensitivities to 4-nitroquinoline-1-oxide (4NQO) and hydroxy urea (HU), and chromosomal radiosensitivity at G(2) phase are corrected by expression of the WRN gene mediated by introducing a chromosome 8. This indicates that those multiple abnormal WS phenotypes, i.e., the hypersensitivities to some types of DNA or chromatin lesion and DNA replication arrest, and also the abnormal response to ionizing radiation at G(2) phase, are derived from a primary, but not secondary, defect in the WRN gene.

References:
Ariyoshi et al. Introduction of a normal human chromosome 8 corrects abnormal phenotypes of Werner Syndrome cells immortalized by expressing an hTERT gene
J Radiat Res 50(3):253-9 (2009)

May 31, 2009

Mikio Shimada (Radiation Biology Center, Kyoto University)

Nijmegen breakage syndrome is characterized by genomic instability and a predisposition for lymphoma and solid tumors. NBS1, the protein which is mutated in these patients, functions in association with BRCA1 and ATR as part of the cellular response to DNA double strand breaks. We show here that NBS1 forms foci at the centrosomes via an interaction with gamma-tubulin. Down-regulation of NBS1 by siRNA induces supernumerary centrosomes, and this was confirmed with experiments using Nbs1 knockout mouse cells; the introduction of wt-NBS1 cDNA into these knockout mouse cells reduced the number of supernumerary centrosomes to normal levels. This phenotype in NBS1-deficient cells is caused by both, centrosome duplication and impaired separation of centriols, which have been observed in BRCA1-inhibited cells. In fact, supernumerary centrosomes were observed in Brca1 knockout mouse cells, and the frequency was not affected by NBS1 down-regulation, suggesting that NBS1 maintains centrosomes via a common pathway with BRCA1. This is consistent with findings that NBS1 physically interacts with BRCA1 at the centrosomes and is required for BRCA1-mediated ubiquitination of gamma-tubulin. Moreover, the ubiquitination of gamma-tubulin is compromised by either ATR depletion, or by an NBS1 mutation in the ATR interacting (FHA) domain, which is essential for ATR activation. These results suggest that, although centrosomes lack DNA, the NBS1/ATR/BRCA1 repair machinery affects centrosome behavior, and this might be a crucial role in the prevention of malignances.

References:
Shimada et al. Inactivation of the Nijmegen Breakage Syndrome gene leads to excess centrosome duplication via the ATR/BRCA1 pathway
Cancer Res 69:(5), 1768-75 (2009)

Jun 13, 2009

Genro Kashino (Research Reactor Institute, Kyoto University)

To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. METHODS AND MATERIALS: Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. RESULTS: More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BP1 foci were observed in nonirradiated, conditioned medium-treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells. CONCLUSIONS: Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation.

References:
Kashino et al. Induction of DNA double-strand breaks and cellular migration through bystander effects in cells irradiated with the slit-type microplanar beam of the spring-8 synchrotron
Int J Radiat Oncol Biol Phys. 74, 229-236 (2009)

Feb 23, 2009

Hiroshi Harada (Graduate School of Medicine, Kyoto University)

Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tumor radioresistance; therefore, it is recognized as an excellent target during radiation therapy. However, the inhibition of HIF-1 in unsuitable timing can suppress rather than enhance the effect of radiation therapy because its anti-angiogenic effect increases the radioresistant hypoxic fraction. In this study, we imaged changes of HIF-1 activity after treatment with radiation and/or an HIF-1 inhibitor, YC-1, and optimized their combination. Hypoxic tumor cells were reoxygenated 6 h postirradiation, leading to von Hippel-Lindau (VHL)-dependent proteolysis of HIF-1alpha and a resultant decrease in HIF-1 activity. The activity then increased as HIF-1alpha accumulated in the reoxygenated regions 24 h postirradiation. Meanwhile, YC-1 temporarily but significantly suppressed HIF-1 activity, leading to a decrease in microvessel density and an increase in tumor hypoxia. On treatment with YC-1 and then radiation, the YC-1-mediated increase in tumor hypoxia suppressed the effect of radiation therapy, whereas on treatment in the reverse order, YC-1 suppressed the postirradiation upregulation of HIF-1 activity and consequently delayed tumor growth. These results indicate that treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the therapeutic effect of radiation, and the suppression of the postirradiation upregulation of HIF-1 activity is important for the best therapeutic benefit.

References:
Harada et al. Treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the effect of radiation therapy
Br J Cancer, 10;100(5):747-57 (2009)

Feb 23, 2009

Hiroshi Harada (Graduate School of Medicine, Kyoto University)

Transcriptional activity of HIF-1 (hypoxia-inducible factor-1) has been reported to be up-regulated in solid tumors after ionizing radiation; however, the molecular mechanism underlying the response remains to be elucidated. In the present study, we performed a series of molecular imaging experiments using a HIF-1-dependent reporter gene, 5HREp-ODD-luc, and found an essential role of the Akt/mTOR pathway. Hypoxic tumor cells distant from blood vessels were dramatically reoxygenated at 24 h postirradiation, and HIF-1 activity increased as HIF-1a accumulated in the reoxygenated regions. The accumulation was inhibited with a nonmetabolizable glucose analog, 2-deoxy-d-glucose, through the suppression of radiation-induced phosphorylation of Akt in the reoxygenated regions. Akt knockdown and an mTOR inhibitor revealed the importance of the Akt/mTOR pathway in the postirradiation accumulation of HIF-1a. In vitro experiments confirmed that an increase in glucose availability induced Akt phosphorylation under reoxygenated conditions and consequently up-regulated HIF-1a translation. Moreover, both the accelerated translation and the previously reported reactive oxygen species-mediated stabilization of HIF-1a protein were essential to the activation of HIF-1. All of these results indicate that Akt/mTOR-dependent translation of HIF-1a plays a critical role in the postirradiation up-regulation of intratumoral HIF-1 activity in response to radiation-induced alterations of glucose and oxygen availability in a solid tumor.

References:
Harada et al. The Akt/mTOR Pathway Assures the Synthesis of HIF-1a Protein in a Glucose- and Reoxygenation-dependent Manner in Irradiated Tumors
J Biol Chem 284, 5332-5342 (2009)