Optimizing chemical-induced premature chromosome condensation assay for rapid estimation of high-radiation doses.

In the event of exposure to high doses of radiation, prompt dose estimation is crucial for selecting appropriate treatment modalities, such as cytokine therapy or stem cell transplantation. The chemical-induced premature chromosome condensation (PCC) method offers a simple approach for such dose estimation with significant radiation exposure, but its 48-h incubation time poses challenges for early dose assessment. In this study, we optimized the chemical-induced PCC assay for more rapid dose assessment. A sufficient number of PCC and G2/M-PCC cells were obtained after 40 h of culture for irradiated human peripheral blood up to 20 Gy. By adding caffeine (final concentration of 1 mM) at 34 h from the start of culture, G2/M-PCC index increased by 1.4-fold in 10 Gy cultures. There was also no significant difference in the G2/M-PCC ring frequency induced for doses 0 to 15 Gy between our 40-h caffeine-supplemented chemical-induced PCC method and the conventional 48-h PCC assay.

Manual Scoring with Shortened 48 h Cytokinesis-Block Micronucleus Assay Feasible for Triage in the Event of a Mass-Casualty Radiation Accident.

The cytokinesis-block micronucleus (CBMN) assay in cytogenetic biodosimetry uses micronucleus (MN) frequency scored in binucleated cells (BNCs) to estimate ionizing radiation dose exposed. Despite the faster and simpler MN scoring, CBMN assay is not commonly recommended in radiation mass-casualty triage as human peripheral blood is typically cultured for 72 h. Furthermore, CBMN assay evaluation in triage often uses high-throughput scoring with expensive and specialized equipment. In this study, we evaluated the feasibility of a low-cost method of manual MN scoring on Giemsa-stained slides in shortened 48 h cultures for triage. Both whole blood and human peripheral blood mononuclear cell cultures were compared for different culture periods and Cyt-B treatment [48 h (24 h at Cyt-B); 72 h (24 h at Cyt-B); 72 h (44 h at Cyt-B)]. Three donors (26-year-old female, 25-year-old male, 29-year-old male) were used for dose-response curve construction with radiation-induced MN/BNC. Another 3 donors (23-year-old female, 34-year-old male, 51-year-old male) were used for triage and conventional dose estimation comparison after 0, 2 and 4 Gy X-ray exposure. Our results showed that despite lower percentage of BNC in 48 h than 72 h cultures, sufficient BNCs were obtained for MN scoring. Triage dose estimates of 48 h cultures were obtained in 8 min in non-exposed donors, and 20 min in 2 or 4 Gy exposed donors with manual MN scoring. One hundred BNCs could be scored for high doses instead of 200 BNCs for triage. Furthermore, observed triage MN distribution could be preliminarily used to differentiate 2 and 4 Gy samples. The number of BNCs scored (triage or conventional) also did not affect dose estimation. Dose estimates in 48 h cultures were also mostly within ±0.5 Gy of actual doses, thus showing the feasibility of manual MN scoring in the shortened CBMN assay for radiological triage applications.

Exosome-like vesicles released from ob/ob mouse adipose tissue enhance cell survival of cells with radiation-induced genomic instability.

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.

Activation of recombinational repair in Ewing sarcoma cells carrying EWS-FLI1 fusion gene by chromosome translocation.

Chromosome translocation (TL) is an important mode of genomic changes underlying human tumorigenesis, the detailed mechanisms of which are, however, still not well understood. The two major modalities of DNA double strand break repair, i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ), have been hypothesized. In a typical TL+ human neoplasm, Ewing sarcoma, which is frequently associated with t(11;22) TL encoding the EWS-FLI1 fusion gene, NHEJ has been regarded as a model to explain the disease-specific TL. Using comprehensive microarray approaches, we observed that expression of the HR genes, particularly of RAD51, is upregulated in TL+ Ewing sarcoma cell lines, WE-68 and SK-N-MC, as in the other TL+ tumor cell lines and one defective in DNA mismatch repair (MMR). The upregulated RAD51 expression indeed lead to frequent focus formation, which may suggest an activation of the HR pathway in these cells. Furthermore, sister chromatid exchange was frequently observed in the TL+ and MMR-defective cells. Intriguingly, ionizing irradiation revealed that the decrease of 53BP1 foci was significantly retarded in the Ewing sarcoma cell lines, suggesting that the NHEJ pathway may be less active in the cells. These observations may support an HR involvement, at least in part, to explain TL in Ewing sarcoma.

Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability.

PURPOSE: 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 (Apodemus speciosus). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

Detailed chromosome analysis of wild-type, immortalized fibroblasts with SV40T, E6E7, combinational introduction of cyclin dependent kinase 4, cyclin D1, telomerase reverse transcriptase.

Cell immortalization enables us to expand the cultured cell infinitely. However, the process of immortalization sometimes changes the nature of the original cell. In this study, we established immortalized embryonic fibroblasts with oncogenic SV40T and human papilla virus-derived E6E7, combinational expression of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomerase reverse transcriptase (TERT) from identical primary wild-type human embryonic fibroblasts (HE16). After the establishment of immortalized cells, we compared the details of chromosome condition with the G-banding and Q-banding methods. There is no example of detailed analysis so far about chromosome abnormalities, such as trisomy, ring chromosome, reciprocal translocation, and dicentric chromosomes. The detailed chromosome analysis revealed that immortalized cells with SV40T and E6E7 showed intensive chromosome abnormalities, such as gain or loss of the chromosomes all through the genome. Furthermore, we detected that the incidence of chromosome abnormities in the immortalized cell with the combinational introduction of R24C mutant of CDK4, cyclin D1, and TERT is almost identical to that of wild-type cell. Furthermore, short tandem repeat analysis demonstrated that the origin of K4DT cell is primary HE16. These results showed that cellular immortalization with CDK4, cyclin D1, and TERT is more advantageous in keeping the chromosome's original condition than oncogenic immortalization methods.

Cytokinesis-block micronucleus assay performed in 0 and 2 Gy irradiated whole blood and isolated PBMCs in a six-well transwell co-culture system.

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.

Extracellular vesicles released from irradiated neonatal mouse cheek tissue increased cell survival after 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.

Precision of scoring radiation-induced chromosomal aberrations and micronuclei by unexperienced scorers.

Purpose: Dose assessment plays an important role in case of radiological accidents and can be performed by scoring structural changes of chromosome morphology induced in cells by ionizing radiation. The results of such a test are biased by scorer experience, therefore, simple to learn assays are recommended to be used when fast analysis of a large amount of data is needed. The aim of this study was to compare the performance of two radiobiological assays - chromosomal aberrations and micronuclei - by unexperienced scorers with the reference values generated by an expert. Materials and methods: Each participant of an EU-funded two-week radiobiology course was asked to score Chinese hamster ovary cells exposed to gamma radiation up to 4 Gy. The congruence of students' and expert's scores at each dose and the coherence of the dose-response curve parameters between the students were investigated. Results: Micronucleus test tended to be faster and easier to learn than scoring chromosomal aberrations. However, both assays carried out by inexperienced students showed reasonable dose-response curves. Conclusions: In the case of a large radiological accident involving many casualties, the unexperienced scorers would support the process of biodosimetric triage by cytogenetic biological dosimetry.

Age Dependence of Radiation-Induced Genomic Instability in Mouse Hematopoietic Stem Cells.

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 bystander effect in large Japanese field mouse (Apodemus speciosus) embryonic cells.

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.

Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells.

B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.

A specific mode of microsatellite instability is a crucial biomarker in adult T-cell leukaemia/lymphoma patients.

PURPOSE: Microsatellite instability (MSI) has been a long-standing biomarker candidate for drug resistance in tumour cells. Despite numerous clinical studies, the data in the literature are not conclusive. The complexity of the MSI phenomenon in some malignancies may, at least partly, account for the discrepancy. In addition, methodological problems are also pointed out in the assay techniques. We previously established a unique fluorescent technique in which the major methodological problems in conventional assays are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. More importantly, we demonstrated that Type A MSI is the direct consequence of defective DNA mismatch repair (MMR) that causes cellular resistance against antineoplastic agents. METHOD: We first applied this technique to adult T-cell leukaemia/lymphoma (ATLL). RESULTS: The MSI phenomenon was indeed observed in ATLLs (4/20, 20%). Intriguingly, the observed microsatellite alterations were invariably Type A, which implies that the tumours were MMR-defective. Indeed, clinical outcomes of patients with these MSI+ tumours were significantly worse. Furthermore, multivariate analysis revealed that Type A MSI is an independent prognostic factor. CONCLUSION: These observations strongly suggest the possibility of Type A MSI as a prognostic and potentially predictive biomarker in ATLL.

Induction of genomic instability and activation of autophagy in artificial human aneuploid cells.

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.

Increase in dicentric chromosome formation after a single CT scan in adults.

Excess risk of leukemia and brain tumors after CT scans in children has been reported. We performed dicentric chromosome assay (DCAs) before and after CT scan to assess effects of low-dose ionizing radiation on chromosomes. Peripheral blood (PB) lymphocytes were collected from 10 patients before and after a CT scan. DCA was performed by analyzing either 1,000 or 2,000 metaphases using both Giemsa staining and centromere-fluorescence in situ hybridization (Centromere-FISH). The increment of DIC formation was compared with effective radiation dose calculated using the computational dosimetry system, WAZA-ARI and dose length product (DLP) in a CT scan. Dicentric chromosome (DIC) formation increased significantly after a single CT scan, and increased DIC formation was found in all patients. A good correlation between the increment of DIC formation determined by analysis of 2,000 metaphases using Giemsa staining and those by 2,000 metaphases using Centromere-FISH was observed. However, no correlation was observed between the increment of DIC formation and the effective radiation dose. Therefore, these results suggest that chromosome cleavage may be induced by one CT scan, and we recommend 2,000 or more metaphases be analyzed in Giemsa staining or Centromere-FISH for DCAs in cases of low-dose radiation exposure.

A novel parameter, cell-cycle progression index, for radiation dose absorbed estimation in the premature chromosome condensation assay.

The calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method for assessing the cell-cycle distribution in cells, since calyculin A induces chromosome condensation in various phases of the cell cycle. In this study, a novel parameter, the cell-cycle progression index (CPI), in the PCC assay was validated as a novel biomarker for biodosimetry. Peripheral blood was drawn from healthy donors after informed consent was obtained. CPI was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60)Co-gamma rays: ∼0.6 Gy min(-1), or X ray: 1.0 Gy min(-1); 0-10 Gy) model. The calyculin A-induced PCC assay was performed for chromosome preparation. PCC cells were divided into the following five categories according to cell-cycle stage: non-PCC, G1-PCC, S-PCC, G2/M-PCC and M/A-PCC cells. CPI was calculated as the ratio of G2/M-PCC cells to G1-PCC cells. The PCC-stage distribution varied markedly with irradiation doses. The G1-PCC cell fraction was significantly reduced, and the G2/M-PCC cell fraction increased, in 10-Gy-irradiated PBL after 48 h of culture. CPI levels were fitted to an exponential dose-response curve with gamma-ray irradiation [y = 0.6729 + 0.3934 exp(0.5685D), r = 1.0000, p < 0.0001] and X-ray irradiation [y = -0.3743 + 0.9744 exp(0.3321D), r = 0.9999, p < 0.0001]. There were no significant individual (p = 0.853) or gender effects (p = 0.951) on the CPI in the human peripheral blood ex vivo irradiation model. Furthermore, CPI measurements are rapid (< 15 min per case). These results suggest that the CPI is a useful screening tool for the assessment of radiation doses received ranging from 0 to 10 Gy in radiation exposure early after a radiation event, especially after a mass-casualty radiological incident.

High-incidence spontaneous tumors in JF1/Ms mice: relevance of hypomorphic germline mutation and subsequent promoter methylation of Ednrb.

PURPOSE: JF1/Ms mice, an inbred strain derived from Japanese wild mice, carry a germline hypomorphic mutation in the endothelin receptor type B gene (Ednrb). We observed that the JF1/Ms mice develop various spontaneous tumors at a high incidence late in life. The aim of this study was to elucidate the mechanism responsible for spontaneous tumors in these mice. Possible relevance of milk-borne mammary tumor virus and gene alterations in Ednrb to tumorigenesis was explored. METHODS: Expression and methylation status of Ednrb were quantitatively analyzed in normal and cancer tissues of mammary gland, liver, submandibular gland as well as in a cultured cell line, MW1, established from a submandibular gland adenocarcinoma. The biological effects of EDNRB were examined in the MW1 cells transfected with wild-type Ednrb. RESULTS: Transcripts of Ednrb were barely detectable, and the promoter region of Ednrb was hypermethylated in tumor tissues and the MW1 cells. In contrast, normal counterpart tissues showed positive expression of Ednrb transcripts and had unmethylated promoter regions. Treatment of the MW1 cells with 5-Aza-dC restored transcription of Ednrb to normal levels. Transfection of the MW1 cells with Ednrb1 (MW1-Ednrb1) resulted in lower growth rates and morphological changes compared with the mock-transfected MW1 cells (MW1-mock1). Furthermore, the MW1-Ednrb1 cells transplanted in syngeneic mice showed a lower proliferation rate than the MW1-mock1 cells. CONCLUSIONS: Germline mutation and subsequent promoter methylation of Ednrb may be relevant to cancer susceptibility in the JF1/Ms mice. These data indicate that Ednrb acts as a tumor suppressor, as reported in human prostate, bladder, and clear cell renal carcinomas.

Individual radiation exposure dose due to support activities at safe shelters in Fukushima Prefecture.

Immediately after the accidents in the nuclear power stations in Fukushima on March 11, the Japanese Government ordered the evacuation of the residents within a 20-km radius from the station on March 12, and asked various institutions to monitor the contamination levels of the residents. Hirosaki University, which is located 355 km north of Fukushima City, decided to send support staff to Fukushima. This report summarizes the results of the exposure of 13 individual teams from March 15 to June 20. The support teams surveyed more than 5,000 people during this period. Almost all subjects had external contamination levels of less than 13 kcpm on Geiger-Müller (GM) survey meter, which is categorized as "no contamination level." The 1(st) team showed the highest external exposure dose, but the 4(th) team onward showed no significant change. Subsequently, the internal radiation exposure was measured using a whole body counter that indicated undetectable levels in all staff members. Although the measured external radiation exposure dose cannot have serious biological effects on the health of an individual, a follow-up study of the residents in Fukushima and other regions where the radioactive material has spread will be required for a long time.

Crucial role of c-Myc in the generation of induced pluripotent stem cells.

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.