Oct 23, 2010

Yoshihiro Fujii (University of Tokyo)

We studied the effects and mechanisms of ascorbic acid as a radiation protector. Cell survival, repair of DNA double strand breaks (DSBs), and sister chromatid exchanges (SCEs) were examined in normal human fibroblasts irradiated with X-rays and heavy ions. Post-irradiation treatment with 5mM ascorbic acid for 24 h in plateau phase (non-cycling) cells enhanced cell survival and DNA double strand break repair, and reduced SCEs after X-rays irradiation. On the other hand, only reduced SCEs were observed after heavy ion exposure such as to carbon ions. Judging from our data, it is possible that the radioprotective action of ascorbic acid would be effective in non-complex type DNA damage such as induced by X-rays. These findings provide new insight into the mechanism of DNA damage and repair produced by heavy ion irradiation.

References:
Fujii et al. Ascorbic acid gives different protective effects in human cells exposed to X-rays and heavy ions.
Mutat Res. 699(1-2):58-61 (2010)

Oct 23, 2010

Yoshihiro Fujii (University of Tokyo)

Inhibition of heat shock protein 90 (Hsp90) is an attractive modality for cancer therapy. Recent studies presented that an Hsp90 inhibitor, 17AAG (17-allylamino-17-demethoxygeldanamycin), enhanced tumor radio-sensitivity, while this was not observed in normal cells. One of the studies reported that the effect of this drug was only observed in tumor cells carrying the wild-type p53 gene, thus demonstrating p53-dependent tumor radio-sensitization by 17AAG. We have now tested the effects of 17AAG on two human lymphoblastoid cell lines from the same donor, TK6 cells with the wild-type p53 gene and WTK1 cells with the mutated p53 gene. The effects of 17AAG were tested at concentrations of 10 and 100 nM on various parameters, including growth inhibition of the cells, enhancement of radio-sensitivity by colony formation assay, apoptosis and chromosomal radio-sensitivity and abrogation of radiation induced G2/M checkpoint. When 100 nM 17AAG was applied, all of these parameters were enhanced in a similar fashion in both cell lines, indicating that the drug effect is p53-independent. Our results suggest that 17AAG is likely to be an effective sensitizer for radiotherapy, even on tumors with mutated p53.

References:
Fujii et al. p53 independent radio-sensitization of human lymphoblastoid cell lines by Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin.
Oncol Rep. Jan:23(1):199-203 (2010)

Sep 25, 2010

Yuka ISHIDA (National Institute of Radiological Sciences)

Accurate cancer risk assessment of low-dose radiation poses many challenges that are partly due to the inability to distinguish radiation-induced tumors from spontaneous ones. To elucidate characteristic features of radiation-induced tumors, we analyzed 163 medulloblastomas that developed either spontaneously or after X-ray irradiation at doses of 0.05-3 Gy in Ptch1 heterozygous mice. All spontaneous tumors showed loss of heterozygosity in broad regions on chromosome 13, with losses at all consecutive markers distal to Ptch1 locus (S-type). In contrast, all tumors that developed after 3 Gy irradiation exhibited interstitial losses around Ptch1 with distal markers retained (R-type). There was a clear dose-dependent increase in the proportion of R-type tumors within the intermediate dose range, indicating that the R-type change is a reliable radiation signature. Importantly, the incidence of R-type tumors increased significantly (P = 0.007) at a dose as low as 50 mGy. Integrated array-comparative genomic hybridization and expression microarray analyses demonstrated that expression levels of many genes around the Ptch1 locus faithfully reflected the signature-associated reduction in genomic copy number. Furthermore, 573 genes on other chromosomes were also expressed differently between S-type and R-type tumors. They include genes whose expression changes during early cerebellar development such as Plagl1 and Tgfb2, suggesting a recapitulation of gene subsets functioning at distinct developmental stages. These findings provide, for the first time, solid experimental evidence for a significant increase in cancer risk by low-dose radiation at diagnostic levels and imply that radiation-induced carcinogenesis accompanies both genomic and gene expression signatures.

References:
Ishida et al. Genomic and gene expression signatures of radiation in medulloblastomas after low-dose irradiation in Ptch1 heterozygous mice.
Carcinogenesis. 31, 1694-1701 (2010)

Sep 25, 2010

Akinori MORITA (Faculty of Science and Technology, Tokyo University of Science)

We recently reported a novel suppressive effect of sodium orthovanadate (vanadate) on the DNA-binding activity of p53. Here, we showed that vanadate had a more potent antiapoptotic activity than three other chemical p53 inhibitors, including pifithrin-α (PFTα). Although the other agents inhibited p53’s transcriptional activity, they did not suppress p53-dependent apoptosis in irradiated MOLT-4 cells. To investigate the cause for the different effects of vanadate and the other inhibitors, we chose PFTα and PFTμ (an inhibitor of the p53-mediated transcription-independent apoptotic pathway), as references, and determined their and vanadate’s effect on p53-mediated apoptosis, with a focus on the transcription-independent pathway. We found that vanadate suppressed the p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and p53’s interaction with Bcl-2. Vanadate also suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of the SaOS-2 cell line. Finally, we tested vanadate’s potential as a radioprotector. Vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Our data demonstrate that vanadate can suppress both the transcription-dependent and the transcription-independent p53 pathways, and suggest that both pathways must be inhibited to completely block p53-mediated apoptosis.

References:
Morita et al. Sodium orthovanadate inhibits p53-mediated apoptosis.
Canser Res. 70, 257-265 (2010)

Sep 25, 2010

Daisuke IIZUKA (Research Institute for Radiation Biology and Medicine, Hiroshima University)

Chromosomal amplifications and deletions are thought to be important events in spontaneous and radiation-induced carcinogenesis. To clarify how ionizing radiation induces mammary carcinogenesis, we characterized genomic copy number aberrations for gamma-ray-induced rat mammary carcinomas using microarray-based comparative genomic hybridization. We examined 14 carcinomas induced by gamma radiation (2 Gy) and found 26 aberrations, including trisomies of chromosomes 4 and 10 for three and one carcinomas, respectively, an amplification of the chromosomal region 1q12 in two carcinomas, and deletions of the chromosomal regions 3q35q36, 5q32 and 7q11 in two, two and four carcinomas, respectively. These aberrations were not observed in seven spontaneous mammary carcinomas. The expression of p16Ink4a and p19Arf, which are located in the chromosomal region 5q32, was always up-regulated except for a carcinoma with a homozygous deletion of region 5q32. The up-regulation was not accounted for by gene mutations or promoter hypomethylation. However, the amounts of Rb and its mRNA were down-regulated in these carcinomas, indicating a disruption of the p16Ink4a/Rb pathway. This is the first report of array CGH analysis for radiation-induced mammary tumors, which reveals that they show distinct DNA copy number aberration patterns that are different from those of spontaneous tumors and those reported previously for chemically induced tumors.

References:
Iizuka et al. DNA copy number aberrations and disruption of the p16Ink4a/Rb pathway in radiation-induced and spontaneous rat mammary carcinomas.
Radiat Res. 174(2):206-15 (2010)

Sep 25, 2010

Mikio Shimada (Radiation Biology Center, Kyoto University)

Centrosomes are important cytoplasmic organelles involved in chromosome segregation, defects in which can results in aneuploidy, and thus contribute to tumorigenesis. It is known that DNA damage causes the supernumerary centrosomes by a mechanism, in which centrosomes continue　to duplicate during the cell cycle arrest at checkpoint. We showed here that radiation (ionizing radiation) induces the over-duplication of centrosomes in a dose-dependent manner, and that the level of over-duplication is pronounced in BRCA1- and NBS1-deficient cells, even though their checkpoint control is abrogated. Conversely, marginal increase in the over-duplication was observed in Ku70- and DNA-PKcs-deficient cells, which are intact in checkpoint control. The frequency of radiation-induced over-duplication of centrosomes might be associated with DNA repair, since it was decreased with reduced cell killing after protracted exposures to radiation. As a result, when the frequency of radiation-induced centrosome over-duplication was plotted against radiation-induced cell killing, similar curves were seen for both protracted and acute of exposures in wild-type cells, Ku70- and DNA-PKcs-deficient cells, indicating a common mechanism for centrosome over-duplication. However, the absence of either BRCA1 or NBS1 enhanced radiation-induced over-duplication frequencies by 2-4-fold on the basis of the same cell killing. These results suggest that radiation-induced centrosome over-duplication is regulated by at least two mechanisms: checkpoint-dependent pathway involved in wild-type cells, Ku70- and DNA-PKcs-deficient cells, and checkpoint-independent pathway as observed in BRCA1- and NBS1-deficient cells.

References:
Shimada et al. Differential role of repair proteins, BRCA1/NBS1 and Ku70/DNA-PKcs, in radiation-induced centrosome over-duplication.
Cancer Sci. 2010 Aug 2. [Epub ahead of print]

Aug 11, 2010

Yoshikazu KUWAHARA (Institute of Development, Aging and Cancer, Tohoku University)

Radiotherapy and radiochemotherapy are well-established treatment modalities for many types of tumor. Recently, in order to understand the mechanisms of tumor radioresistance and develop more effective tumor radiotherapy, we established clinically relevant radioresistant cell lines, HepG2-8960-R from HepG2 and SAS-R from SAS. These cells continue to proliferate with daily exposure to 2 Gy of X-rays for more than 30 days. However, we failed to detect the cellular radioresistance of these cells by clonogenic assay. The high-density survival (HDS) assay was originally elaborated to assess cancer cell responses to therapeutic agents under the influence of intercellular communication. Here, we simplified the original high-density survival assay by scoring the total number of surviving cells after exposure to X-rays, and studied its applicability for the detection of cellular radioresistance. The modified HDS assay successfully detected cellular radioresistance of HepG2-8960-R and SAS-R. Therefore, we believe that the modified HDS assay presented in this study is a powerful tool to predict the effectiveness of fractionated radiotherapy against malignant tumors.

References:
Kuwahara et al. The modified high-density survival assay is the useful tool to predict the effectiveness of fractionated radiation exposure.
J Radiat Res. 51 (2010) 297-302.

Jul 31, 2010

Yuichiro YOKOTA (Japan Atomic Energy Agency)

Ionizing radiation-induced genomic instability has been documented in various end points such as chromosomal aberrations and mutations, which arises in the descendants of irradiated mammalian or yeast cells many generations after the initial insult. This study aimed at addressing radiation-induced genomic instability in higher plant tobacco cells. We thus investigated micronucleus (MN) formation and cell proliferation in tobacco cells irradiated with g-rays and their descendants. In g-irradiated cells, cell cycle was arrested at G2/M phase at around 24 h post-irradiation but released afterward. In contrast, MN frequency peaked at 48 h post-irradiation. Almost half of 40 Gy-irradiated cells had MN at 48 h post-irradiation, but proliferated as actively as sham-irradiated cells up to 120 h post-irradiation. Moreover, the descendants that have undergone at least 22 generations after irradiation still showed a two-fold MN frequency compared to sham-irradiated cells. This is the direct evidence for radiation-induced genomic instability in tobacco cells.

References:
Yokota et al. Enhanced micronucleus formation in the descendants of g-ray-irradiated tobacco cells: Evidence for radiation-induced genomic instability in plant cells.
Mutat Res-Fundam Mol Mech Mutagen. 691, 41-46 (2010)

Jul 28, 2010

Nobuyuki HAMADA (Central Research Institute of Electric Power Industry)

Excellent biological effectiveness and dose conformity represent a rationale for heavy-ion therapy that has heretofore achieved good cancer controllability while sparing critical normal organs. Soon after irradiation, heavy ions produce dense ionization along their trajectories, cause irreparable clustered DNA damage, and alter cellular ultrastructure. As a result, these ions inactivate cells more effectively with less cell-cycle and oxygen dependence than conventional photons. Apoptosis, necrosis, autophagy, premature senescence, accelerated differentiation, delayed reproductive cell death of progeny cells, and bystander cell death are the proposed modes of heavy ion?induced cell death/inactivation. Here we briefly overview the current knowledge of the biological aspects of heavy-ion therapy, with a focus on our recent findings. The topics include (i) repair mechanisms of heavy ion?induced DNA damage, (ii) superior effects of heavy ions on radioresistant tumors/cells (intratumor quiescent cell population, p53-mutated and Bcl-2-overexpressing tumors), (iii) novel capacity of heavy ions in suppressing cancer metastasis and neoangiogenesis, and (iv) potential of heavy ions to induce secondary (especially breast) cancer.

References:
Hamada et al. Recent advances in the biology of heavy-ion cancer therapy.
J Radiat Res. 51, 365-383 (2010)

Apr 15, 2010

Kensuke OTSUKA (Central Research Institute of Electric Power Industry)

Hematopoietic stem cells are supposed to repopulate and maintain long-term regeneration of the lethally irradiated recipient's bone marrow and peripheral blood. We evaluated the regeneration capability of Lin?/c-Kit+/Sca-1+ (LKS) cells, the putative hematopoietic stem cells, after radiation exposure at graded doses, for long-term regeneration (~ 270 days) of peripheral blood in lethally irradiated recipients. Differential regeneration capability of LKS cells irradiated in vitro at graded increased doses showed a dose-dependent suppression of regeneration of peripheral blood in the recipient mice as compared with LKS cells without radiation exposure, but it was partially maintained 270 days after transplantation. The amount of intracytoplasmic ROS in LKS increased slightly after radiation exposure. However, the radiation-induced ROS was estimated small in the cells contributed to long-term regeneration of peripheral blood. These results indicated that progenitor cells regenerating peripheral blood cells 270 days after transfusion were assumed to be anaerobic and more immature and radioresistant than those on day 35 or day 90.

References:
Otsuka et al. Regeneration capability of Lin?/c-Kit+/Sca-1+ cells with or without radiation exposure for repopulation of peripheral blood in lethally irradiated mice monitored using Ly5.1 isotype on days 35, 90, and 270 after transplantation.
Experimental Hematology. 38, 417-425 (2010)