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Multiple epidemiological studies have shown that obesity is a serious risk factor for cancer development. While the underlying mechanisms between obesity and cancer are still unknown, obesity disrupts the role of adipocytes in energy homeostasis, and the alteration of adipokine, insulin and sex steroid signaling. Recently, it has been identified that adipose tissue-derived exosome-like vesicles (ELVs) regulate metabolic homeostasis. In this study, we collected ELVs from adipose tissue of an obese mouse (ob/ob) strain and control mouse (C57BL/6) strain, and checked whether adipose ELVs influence radiation-induced cell death on mouse fibroblast cells (m5S). Furthermore, we analyzed the micronucleus (MN) frequency in survived cells after radiation exposure to investigate the effect of ELVs on radiation-induced genomic instability. We first observed that ELVs from control and obese mice showed enhanced colony forming ability in un-irradiated m5S cells. However, enhanced survival was observed only in 3 Gy-irradiated m5S cells with obese ELV treatment. Despite no ELV effect on colony size, interestingly, the frequency of MN in survived m5S cells after 3 Gy irradiation was elevated when treated obese ELVs compared to control ELVs. These results suggested that obese mouse adipose ELVs could enhance the survival of irradiated cells harboring increased radiation-induced genomic instability.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 μGy/d (Hirosaki), 14.4 ± 2.4 μGy/d (Tanashio), 208.8 ± 31.2 μGy/d (Ide), 470.4 ± 93.6 μGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones.Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas.Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

Several past studies using a mouse model of radiation-induced AML (rAML) have shown that hemizygous deletion of the Sfpi1 gene (HDSG) is an initiating event for the development of rAML. In this stu...

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in Japan on March 11 2011, the surroundings became contaminated with radionuclides. To understand the possible biological effects af...

PURPOSE Cytokinesis-block micronucleus (CBMN) assay in cytogenetic biodosimetry uses micronucleus (MN) frequency scored in binucleated cells (BNC) for dose estimation. Cell-cycle progression parameters of nuclear division index (NDI) and percentage of BNC (% BNC) are also evaluated. Whole blood (WB) or peripheral mononuclear cells (PBMCs) isolated from WB can be used for lymphocyte culture. Previously, 2 Gy PBMCs showed higher NDI and lower MN frequency than WB in 15 ml polypropylene tube single cultures. In this follow-up study, we wanted to assess if soluble factors present in WB but absent in PBMCs could increase MN frequency or decrease NDI in PBMCs co-cultured with WB. MATERIALS AND METHODS Peripheral blood from four healthy donors (two males: 25, 51; two females: 23, 26 years old) was irradiated with X-ray at 1 Gy/min. CBMN assay was performed with different combinations of 0 and 2 Gy WB and PBMC (WB, WB-IR, PBMC, PBMC-IR) mono- and co-cultures in a polystyrene six-well plate. Co-cultures were separated by 0.4 µm transwell inserts. Log2 fold changes and values of NDI, % BNC and MN frequency analyzed by three scorers were obtained. RESULTS As upper and lower wells of the same culture condition showed some significant differences, wells of the same level were compared. NDI of PBMCs increased when PBMC or PBMC-IR was co-cultured with WB or WB-IR, respectively, as compared to mono-cultures. There was no increase in PBMC-IR's MN frequency when co-cultured with WB or WB-IR. MN frequency was consistently higher in WB-IR than PBMC-IR in both mono- and co-cultures. NDI, % BNC and MN frequency were similar when WB or PBMC were co-cultured with PBMC-IR or WB-IR, respectively. Significantly lower NDI and % BNC, and higher MN frequency were also seen in some conditions of 15 ml cultures than six-well mono-cultures. CONCLUSIONS Instead of the hypothesized decrease in NDI and increase in MN frequency, our co-culture set-up showed that in the absence of direct cell-cell interaction, soluble factors in WB increased NDI but not MN frequency in PBMCs. Moreover, radiation-induced bystander effects could not be observed. As the type of cell culture (WB, PBMC) and culture vessels could influence NDI and MN frequency, CBMN culture protocols should be kept consistent for dose-response calibration curve construction and dose estimation.

In order to establish suitable protocols of blood culture to obtain sufficient numbers of metaphases for dicentric chromosome assay (DCA), we have examined the effect of storage temperature, storage time, and anticoagulant type.Peripheral blood was collected from five healthy donors with lithium heparin and ethylenediaminetetraacetic acid dipotassium salt (EDTA-2K). These samples were irradiated with X-rays at 3 Gy or sham; the samples were further divided into groups that were either stored at room temperature (RT) or 5.2 ± 1.0 °C. After 6, 24, 48, 72, and 168 h of storage, both blood counts and the mitotic index (MI) were analyzed.Heparinized blood samples stored under cold conditions exhibited low white blood cell, lymphocyte, and platelet counts. EDTA-treated blood samples did not show such obvious changes in cell counts. After 6 h of storage, heparinized blood samples stored at RT had MI of 21.5-29.3%. Similar MI was obtained in the EDTA-washed group stored for 6, 24, 48, and 72 h.Our study confirms that heparinized blood samples should be stored at RT to get sufficient metaphases for DCA, and that EDTA blood samples also can be used for blood culture after washing and storage under 5.2 ± 1.0 °C.

Hirosaki University has been designated by the Nuclear Regulation Authority as an Advanced Radiation Emergency Medical Support Center and as a hospital which accepts radiation emergency medical patients in Japan. One of the efforts of the Advanced Radiation Emergency Medicine Support Center is cytogenetic biodosimetry in radiation emergency medicine. We are working on validating all the steps from blood collection and storage to DNA damage evaluation. We are also improving various methodologies, such as the cytokinesis-block micronucleus (CBMN) assay. In our improved CBMN harvest protocol to store fixed isolated PBMCs (peripheral blood mononuclear cells), a high frequency of scorable binucleated cells can be obtained with routine biodosimetry reagents and equipment. In addition, we are strengthening infection control measures and working to establish a system that enables safe and reliable dose estimation of exposed patients during the COVID-19 pandemic. Furthermore, we have developed on-campus education system for undergraduate and graduate students, and programs for off-campus trainees, such as "International Training Program for Radiation Protection" to develop human resources responsible for cytogenetic biodosimetry. In this time, I would like to introduce the efforts of the cytogenetic biodosimetry laboratory in Hirosaki University, Japan.

In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods.Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy - background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated.94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures.This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (137 Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

Several past studies using a mouse model of radiation-induced AML (rAML) have shown that hemizygous deletion of the Sfpi1 gene (HDSG) is an initiating event for the development of rAML. In this study, we examined the difference in frequency of HDSG in hematopoietic stem cells (HSCs) Rich hematopoietic Cell population (HRCs) from bone marrow (BM) and spleen of C3H mice irradiated with 3 Gy X-rays.8-weeks old male C3H mice were irradiated 3Gy of whole body X-ray (1 Gy/min) and mice were sacrificed at 1, 4, 8, and 26 weeks. Then, HSPCs were isolated from BM of femur and spleen, the frequency of HRCs with Sfpi1 gene deletion was analyzed by fluorescence in situ hybridization (FISH).The frequency of HRCs with HDSG in both BM and spleen was increased 1 week after X-irradiation. Then, the frequency of HRCs with HDSG in BM showed a gradual decrease from 4 to 26 weeks, whereas HRCs with HDSG in spleen remained high, even at 26 weeks after X-irradiation. HDSG is less likely to be eliminated, particularly in the spleen, after X-irradiation. The spleen as well as BM of the femur may be major sites of rAML development.

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in Japan on March 11 2011, the surroundings became contaminated with radionuclides. To understand the possible biological effects after chronic low dose-rate radiation in contaminated areas of Fukushima, we assessed the effects in large Japanese field mice (We collectedAlthough the mice in the contaminated areas were chronically exposed, there was no radiation-specific chromosome aberrations observed, such as dicentric chromosomes and rings. Some structural aberrations such as gaps and breaks were observed, and these frequencies decreased annually in mice from Namie Town.These findings suggest that chromosome aberration analysis is useful to evaluate and monitor radiation effects in wild animals.

Although evidence suggests that ionizing radiation can induce the bystander effect (radiation-induced bystander effect: RIBE) in cultured cells or mouse models, it is unclear whether the effect occurs in cells of wild animals. We investigated medium-mediated bystander micronucleus (MN) formation and DNA damage in un-irradiated cells from a large Japanese field mouse (Apodemus speciosus). We isolated four clones of A. speciosus embryonic fibroblasts (A603-1, A603-2, A603-3, and A603-4) derived from the same mother, and examined their radiation sensitivity using the colony-forming assay. A603-3 and A603-4 were similar, and A603-1 and A603-2 were highly sensitive compared with A603-3 and A603-4. We examined RIBE in the four clones in autologous medium from cell cultures exposed to 2 Gy X-ray radiation (irradiated cell conditioned medium: ICCM). We only observed increased MN prevalence and induction of DNA damage foci in A603-1 and A603-3 cells after ICCM transfer. The ICCM of A603-3 (RIBE-induced) was able to induce MN in A603-4 (not RIBE-induced). To assess the possible contribution of reactive oxygen species (ROS) or nitric oxide (NO) in medium-mediated RIBE, dimethyl sulfoxide (DMSO; a ROS scavenger) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; an NO scavenger) were added to the medium. A suppressive effect was observed after adding DMSO, but there was no effect after treatment with c-PTIO. These results suggest that an enhanced radiosensitivity may not be directly related to the induction of medium-mediated RIBE. Moreover, ROS are involved in the transduction of the RIBE signal in A. speciosus cells, but NO is not. In conclusion, our results suggest that RIBE may be conserved in wild animals. The results contribute to better knowledge of radiation effects on wild, non-human species.

Primary hepatocyte culture is a crucial tool for investigations of liver function and for evaluating the toxic effects of drugs. In addition, chromosomal analysis of hepatocytes could also prove useful for understanding the mechanisms of hepatocarcinogenesis. However, cultivation of primary hepatocytes for chromosome analysis has been hampered by the specific equipment and skill required to perform the in situ perfusion step necessary for isolation of primary hepatocytes. In the present study, we aimed to establish a simple and efficient method of isolating hepatocytes suitable for chromosome analysis. We performed hepatocyte isolation without using collagenase perfusion, instead digesting liver tissues using collagenase in tubes. In addition, we examined hepatocyte and bone marrow cell (BMC) co-culture and cultivation of hepatocytes with medium containing BMC culture medium supernatants. We found that hepatocyte viability and attachment rate were significantly improved, both by co-culture with BMCs and medium containing BMC culture media supernatants, with the latter also significantly increasing the mitotic index. Using this simple method of isolation and cultivation, we could successfully perform chromosomal analysis of mouse primary hepatocytes. This method has the potential to help understand the mechanisms underlying chromosomal instability-mediated hepatocarcinogenesis.

Exosome-like vesicles (ELV) are involved in mediating radiation-induced bystander effect (RIBE). Here, we used ELV from control cell conditioned medium (CCCM) and from 4 Gy of X-ray irradiated cell conditioned medium (ICCM), which has been used to culture normal human fibroblast cells to examine the possibility of ELV mediating RIBE signals. We investigated whether ELV from 4 Gy irradiated mouse serum mediate RIBE signals. Induction of DNA damage was observed in cells that were treated with ICCM ELV and ELV from 4 Gy irradiated mouse serum. In addition, we treated CCCM ELV and ICCM ELV with RNases, DNases, and proteinases to determine which component of ELV is responsible for RIBE. Induction of DNA damage by ICCM ELV was not observed after treatment with DNases. After treatment, DNA damages were not induced in CCCM ELV or ICCM ELV from mitochondria depleted (ρ0) normal human fibroblast cells. Further, we found significant increase in mitochondrial DNA (mtDNA) in ICCM ELV and ELV from 4 Gy irradiated mouse serum. ELV carrying amplified mtDNA (ND1, ND5) induced DNA damage in treated cells. These data suggest that the secretion of mtDNA through exosomes is involved in mediating RIBE signals.

Age at exposure is a critical factor that influences the risk of radiation-induced leukemia. Accumulating evidence suggests that ionizing radiation can induce genomic instability and promote leukemogenesis in hematopoietic stem cells (HSCs); however, the influence of age on this phenomenon has not been elucidated. In this study, infant (1-week-old) or adult (14-week-old) C3H/He mice received sham or 4 Gy whole-body irradiation, and bone marrow cells were transplanted to recipients at day 1 or 60 postirradiation. Twelve days after bone marrow transplant, we analyzed the radiation-induced genomic instability by scoring the frequency of DNA damage and micronucleus formation in colony-forming units-spleen (CFU-Ss). We observed significant increases in DNA damage and micronucleus formation in CFU-Ss of the 4 Gy irradiated adult cells transplanted at day 1 or 60 postirradiation. However, the frequency of DNA damage focus and micronucleus formation in CFU-Ss of 4 Gy irradiated infant cells transplanted at day 1 or 60 postirradiation was relatively decreased. Quantitative differences in the reactive oxygen species and cells expressing inducible nitric oxide synthase in CFU-Ss suggested that age-dependent radiation-induced genomic instability may result from chronic oxidative stress by pro-inflammatory states in HSC descendants after radiation exposure.

Radiation-induced acute myeloid leukemia (rAML) in C3H mice is commonly developed through inactivation of PU.1 transcription factor encoded in Sfpi1 on chromosome 2. PU.1 inactivation involves two steps: hemizygous deletion of the Sfpi1 gene (DSG) and point mutation of the allele Sfpi1 gene (PMASG). In this study, we investigated the dose-rate dependence of the frequency of both DSG and PMASG in hematopoietic stem cells (HSCs) of C3H mice that received a total of 3 Gy gamma-ray exposure at dose rates of 20 mGy/day, 200 mGy/day or 1,000 mGy/min. All mice were followed for 250 days from start of irradiation. Fluorescent in situ hybridization of the Sfpi1 gene site indicated that frequency of HSCs with DSG was proportional to dose rate. In cell surface profiles, PU.1-inactivated HSCs by both DSG and PMASG were still positive for PU.1, but negative for GM-CSF receptor-α (GMCSFRα), which is transcriptionally regulated by PU.1. Immunofluorescent staining analysis of both PU.1 and GM-CSFRα also showed dose-rate-dependent levels of PU.1-inactivated HSCs. This study provides evidence that both DSG and PMASG are dose-rate dependent; these experimental data offer new insights into the dose-rate effects in HSCs that can lead to radiation-induced leukemogenesis.

Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

We developed a neutron irradiation facility, neutron exposure accelerator system for biological effect experiments (NASBEE) for biological studies in National Institute of Radiological Sciences, Japan. Irradiation field of 2 MeV average neutrons generated by a Be(d–n)B reaction is established. Dose uniformity of 240 mm in diameter irradiation field is producible within ±2.5% with a dose rate of 0.87 Gy/h at sample target distance of 1170 mm. Two irradiation rooms, a specific pathogen-free (SPF) conditioned one and a conventional, are now available. Irradiation protocols for in vitro experiments are now established and demonstrated by obtaining a relative biological effectiveness (RBE) of cell inactivation measured to be 3.54 with 10% survival dose ( D 10 ).

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging and caused by mutations of the WRN gene mapped at 8p12. 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 G2 phase are corrected by expression of the WRN gene mediated by introducing a chromosome 8. This indicates that those multiple abnormal WS phenotypes are derived from a primary, but not secondary, defect in the WRN gene., Journal of radiation research, 50(3), pp.253-259; 2009

Evidence has been accumulated to challenge the mutation theory for carcinogenesis, a major idea about a cause of cancer. The mutation theory starts from initiation, a mutation of a critical gene, being followed by promotion, which stimulates proliferation of the initiated cells. A majority of tumors, especially solid tumors, exhibits chromosomal instability characterized by random structural aberrations and ploidy changes. So far, no responsible gene has been found to account for chromosomal instability. Instead of the mutation theory, we introduce the telomere dysfunction theory for radiation carcinogenesis. In this model, a mutation of a critical gene is not presumed for initiation. Here, we demonstrate that radiation contributes to the induction of telomeric instability, which may lead to breakage–fusion–bridge cycle that potentially drives genome rearrangements. We propose that telomere dysfunction initiates and promotes chromosomal instability that plays a crucial role in an early step in radiation carcinogenesis.

Chromosome Instability/DNA Double-strand Breaks/Werner Syndrome/Premature senescence/Oxidative stress/Genomic instability. Werner syndrome (WS) is a premature aging syndrome caused by mutations of the WRN gene. Here, we demonstrate that a strain of WS fibroblast cells shows abnormal karyotypes characterized by several complex translocations and 50-fold more frequency of abnormal metaphases including dicentric chromosomes without fragments than normal cells when examined at a similar culture stage. Further, telomere fluorescence in situ hybridization indicates that the abnormal signals, extra telomere signal and loss of telomere signal, emerge two- to three-fold more frequently in WS cells than in normal cells. Taken together, these results indicate that chromosome instability including dysfunction of telomere maintenance is more prominent in WS cells than in normal cells. In addition, the accumulation of DNA double-strand breaks (DSBs) at the G1 phase, including those at telomeres, detected by phosphorylated ATM (ataxia telangiectasia mutated) foci is accelerated in WS cells even at a low senescence level. The increased accumulation of DSBs in WS cells is reduced in the presence of anti-oxidative agents, suggesting that enhanced oxidative stress in WS cells is involved in accelerated accumulation of DSBs. These results indicate that WS cells are prone to accumulate DSBs spontaneously due to a defect of WRN, which leads to increased chromosome instability that could activate checkpoints, resulting in accelerated senescence.

Item does not contain fulltext Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype.