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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.

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.

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.

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.

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.

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.

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.

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.

DNA mismatch repair (MMR) is an evolutionally conserved and major mechanism in cells that counteracts the occurrence of point mutations on the genome. Defective MMR confers the ‘mutator’ phenotype ...

In terms of biological dosimetry at the time of radiation exposure, the dicentric chromosome (Dic) assay (DCA) is the gold standard for assessing for the acute phase and chromosome translocation (Tr) analysis is the gold standard for assessing the chronic phase. It is desirable to have individual dose–response curves (DRCs) for each laboratory because the analysis criteria differ between laboratories. We constructed the DRCs for radiation dose estimation (with three methods) using peripheral blood (PB) samples from five healthy individuals. Aliquots were irradiated with one of eight gamma-ray doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy), then cultured for 48 h. The number of chromosome aberrations (CAs) was analyzed by DCA, using Giemsa staining and centromere-fluorescence in situ hybridization (centromere-FISH) and by chromosome painting (chromosome pairs 1, 2 and 4) for Tr analysis. In DCA, there was large variation between individuals in the frequency of Dics formed, and the slopes of the DRCs were different. In Tr analysis, although variation was observed in the frequency of Tr, the slopes of the DRCs were similar after adjusting the background for age. Good correlation between the irradiation dose and the frequency of CAs formed was observed with these three DRCs. However, performing three different biological dosimetry assays simultaneously on PB from five donors nonetheless results in variation in the frequency of CAs formed, especially at doses of 50 mGy or less, highlighting the difficulty of biological dosimetry using these methods. We conclude that it might be difficult to construct universal DRCs.

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.

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.

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.

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.

We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78–60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.

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.

Biallelic germline mutations in the DNA mismatch repair gene MLH1 lead to constitutional mismatch repair-deficiency syndrome and an increased risk for childhood hematopoietic malignancies, including lymphoma and leukemia. To examine how Mlh1 dysfunction promotes lymphoma as well as the influence of ionizing radiation (IR) exposure, we used an Mlh1-/- mouse model and whole-exome sequencing to assess genomic alterations in 23 T-cell lymphomas, including 8 spontaneous and 15 IR-associated lymphomas. Exposure to IR accelerated T-cell lymphoma induction in the Mlh1-/- mice, and whole-exome sequencing revealed that IR exposure neither increased the number of mutations nor altered the mutation spectrum of the lymphomas. Frequent mutations were evident in genes encoding transcription factors (e.g. Ikzf1, Trp53, Bcl11b), epigenetic regulators (e.g. Suv420h1, Ep300, Kmt2d), transporters (e.g. Rangap1, Kcnj16), extracellular matrix (e.g. Megf6, Lrig1), cell motility (e.g. Argef19, Dnah17), protein kinase cascade (e.g. Ptpro, Marcks) and in genes involved in NOTCH (e.g. Notch1), and PI3K/AKT (e.g. Pten, Akt2) signaling pathways in both spontaneous and IR-associated lymphomas. Frameshift mutations in mononucleotide repeat sequences within the genes Trp53, Ep300, Kmt2d, Notch1, Pten and Marcks were newly identified in the lymphomas. The lymphomas also exhibited a few chromosomal abnormalities. The results establish a landscape of genomic alterations in spontaneous and IR-associated lymphomas that occur in the context of mismatch repair dysfunction and suggest potential targets for cancer treatment.

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.

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 modified C-band technique was developed in order to analyze more accurately dicentric, tricentric, and ring chromosomes in irradiated human peripheral lymphocytes. Instead of the original method relying on treatment with barium hydroxide Ba(OH) 2 , C-bands were obtained using a modified form of heat treatment in formamide followed with DAPI staining. This method was tentatively applied to the analysis of dicentric chromosomes in irradiated human lymphocytes to examine its availability. The frequency of dicentric chromosome was almost the same with conventional Giemsa staining and the modified C-band technique. In the analysis using Giemsa staining, it is relatively difficult to identify the centromere on the elongated chromosomes, over-condensed chromosomes, fragment, and acentric ring. However, the modified C-band method used in this study makes it easier to identify the centromere on such chromosomes than with the use of Giemsa staining alone. Thus, the modified C-band method may give more information about the location of the centromere. Therefore, this method may be available and more useful for biological dose estimation due to the analysis of the dicentric chromosome in human lymphocytes exposed to the radiation. Furthermore, this method is simpler and faster than the original C-band protocol and fluorescence in situ hybridization (FISH) method with the centromeric DNA probe.

Biological dosimetry is an essential tool for estimating radiation doses received to personnel when physical dosimetry is not available or inadequate. The current preferred biodosimetry method is based on the measurement of radiation-specific dicentric chromosomes in exposed individuals' peripheral blood lymphocytes. However, this method is labour-, time- and expertise-demanding. Consequently, for mass casualty applications, strategies have been developed to increase its throughput. One such strategy is to develop validated cytogenetic biodosimetry laboratory networks, both national and international. In a previous study, the dicentric chromosome assay (DCA) was validated in our cytogenetic biodosimetry network involving five geographically dispersed laboratories. A complementary strategy to further enhance the throughput of the DCA among inter-laboratory networks is to use a triage DCA where dose assessments are made by truncating the labour-demanding and time-consuming metaphase-spread analysis to 20 to 50 metaphase spreads instead of routine 500 to 1000 metaphase spread analysis. Our laboratory network also validated this triage DCA, however, these dose estimates were made using calibration curves generated in each laboratory from the blood samples irradiated in a single laboratory. In an emergency situation, dose estimates made using pre-existing calibration curves which may vary according to radiation type and dose rate and therefore influence the assessed dose. Here, we analyze the effect of using a pre-existing calibration curve on assessed dose among our network laboratories. The dose estimates were made by analyzing 1000 metaphase spreads as well as triage quality scoring and compared to actual physical doses applied to the samples for validation. The dose estimates in the laboratory partners were in good agreement with the applied physical doses and determined to be adequate for guidance in the treatment of acute radiation syndrome.

This paper considers the dose–effect relationship for unstable chromosome aberration yields in human lymphocytes in very low-dose range. Data are presented for 60Co γ-ray doses of 0, 10, 20, 40 and 1000 mGy. More than 5,000 metaphases were scored for each data point at the very low doses, and each cell was double-checked using a semi-automated metaphase finding/relocation system. Aberration yields of dicentrics plus centric rings followed an excellent linear dose response down to zero dose; the yields were significantly above the control frequency from 20 mGy.

Purpose The relationship between radiation exposure by CT and the risk of leukemia and brain tumor has been reported in children, in which the risk of leukemia was tripled at a radiation dose of 60 mGy to bone marrow (Pearce MS, Lancet 2012). Therefore, there is a possibility that CT scans induce chromosome aberrations (CAs) causing malignant tumors. When chromosomes are cleaved by radiation, dicentric chromosomes (Dics) and chromosome translocations (Trs) are formed in the same ratio. We have reported that breakage of chromosomes was induced after a single CT scan in adults by Dic assay (DCA) (Scientific Rep 2015). On the other hand, the number of Trs formed also increased after a single CT scan, but, no significant change of the numbers was observed before and after the CT scan (J Rad Res 2016). We then investigated whether cumulative CAs would be observed when an annual CT scan was performed for 3 years. Methods Peripheral blood samples were collected from 5 patients (4 males and 1 female; age range 51-73 years; mean age, 64 years) 3 times before and after a CT scan at annual examinations. They included 4 patients with malignant lymphoma who had not been treated for more than 3 years after the end of treatment and 1 patient followed due to an abnormal lung shadow without treatment. We performed analysis of Dic and Tr formation by centromere-FISH and painting-FISH using isolated lymphocytes, respectively, and counted the number of CAs equivalent to 2,000 metaphases per sample. The effective radiation dose of a CT scan was calculated by computational dosimetry system (WAZA-ARI). Results Regardless of CT scans, the number of Trs formed was higher than that of Dics in all patients. The dose of radiation exposure in one CT scan was 22.0 to 73.5 mSv (mean, 42.3 mSv). Both DCA and Tr analyses showed an increase in Dic and Tr formation after all CT scans, except that their number decreased in one patient after the second CT scan. However, the increase in these CAs decreased after about one year, and no cumulative increase in the number of Dics and Trs formed due to three consecutive CT scans was observed. Conclusion A single CT scan tended to produce both Dic and Tr formation, but there was no cumulative increase in CAs after three consecutive CT scans. The reason that the number of Trs formed was basically higher than that of Dics is considered to be related to such factors as age, smoking status, and past medical radiation exposure especially in adults because Tr is a stable chromosome. Citation Format: Yu Abe, Hideyoshi Noji, Misaki Sugai, Yumiko Kurosu, Takashi Ohba, Aki Yanagi, Yukari Yanai, Naohiro Tsuyama, Tetsuo Ishikawa, Tomisato Miura, Kenji Kamiya, Mitsuaki A. Yoshida, Akira Sakai. Investigation of the cumulative number of chromosome aberrations induced by three consecutive CT scans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 853.

To investigate whether p16 inactivation is involved in the development of rat pulmonary tumors, we compared the p16 status and tumorigenicity of cell lines which indicated different p16 status. The tumor cell line (PuD2) was established from lung adenocarcinoma induced in plutonium dioxide-inhaled rat in this study. The virus-immortalized SV40T2 cells, benzo[a]pyrene-induced BP cells, BP-derived BP(P)Tu cells, and gamma ray-transformed RTiv3 cells were utilized as the respiratory epithelial cell lines. A tumorigenicity assay-inoculating cells into nude mice revealed that PuD2, BP, and BP(P)Tu cells were tumorigenic, but SV40T2 and RTiv3 cells were not. Methylation-specific PCR of the p16 promoter region revealed that SV40T2 cells were unmethylated, BP cells displayed heterogeneous methylation, and BP(P)Tu and RTiv3 cells were completely methylated. Methylation-specific PCR and PCR of genomic DNA in the p16 region did not amplify product in PuD2 cells, indicating deletion of p16. Banded karyotypes prepared from PuD2 cells exhibited trisomy of chromosome 4, inversion in chromosome 11, and partial deletion of chromosomes 4 and 5. The demethylating agent 5Aza2dC partially demethylated the p16 promoter region of BP(P)Tu, BP and RTiv3 cells, increasing expression of the p16 transcript and decreasing growth of the cells. These results indicate that hypermethylation of the p16 promoter region occurs early in neoplastic transformation before acquisition of tumorigenicity in rat respiratory epithelium. Loss of genes located on chromosomes 4 and 5 may be important for tumor progression and acquisition of high tumorigenic activity in the Pu-induced rat lung tumor.

Blakely, W. F., Carr, Z., Chu, M. C-M., Dayal-Drager, R., Fujimoto, K., Hopmeir, M., Kulka, U., Lillis-Hearne, P., Livingston, G. K., Lloyd, D. C., Maznyk, N., Perez, M. D. R., Romm, H., Takashima, Y., Voisin, P., Wilkins, R. C. and Yoshida, M. A. WHO 1st Consultation on the Development of a Global Biodosimetry Laboratories Network for Radiation Emergencies (BioDoseNet). Radiat. Res. 171, 127–139 (2009). The World Health Organization (WHO) held a consultation meeting at WHO Headquarters, Geneva, Switzerland, December 17–18, 2007, to develop the framework for a global biodosimetry network. The WHO network is envisioned to enable dose assessment using multiple methods [cytogenetics, electron paramagnetic resonance (EPR), radionuclide bioassays, etc.]; however, the initial discussion focused on the cytogenetic bioassay (i.e., metaphase-spread dicentric assay). Few regional cytogenetic biodosimetry networks have been established so far. The roles and resources available from United Nations (UN) agenc...

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.

Loss of heterozygosity (LOH) plays an important role in leukemogenesis via inactivation of tumor suppressor genes. Recent studies have demonstrated that mouse thymic lymphomas (TL), a suitable model for the mechanistic study of human acute lymphoblastic leukemia, show frequent LOH on chromosomes 4 ( p15/p16 ), 11 ( Ikaros ), 12 ( Bcl11b ), 19 ( Pten ), and X. To date, however, little data are available regarding the mechanism of LOH. In this study, we re-evaluated chromosomal abnormality and loss of heterozygosity in 26 TL-induced by x-rays in C57BL/6 (B6), C3H, and F1 (B6 × C3H) mice, using the quinacrine-Hoechst double-staining method and chromosome painting technique. Chromosomally abnormal cells were present in 25 TL examined (25/26, 96%). The most frequent abnormality was trisomy or partial trisomy of chromosome 15 (16/26, 62%), which is consistent with previous studies. Structural abnormalities of chromosome 11 with interstitial deletion of proximal region and chromosome 12 with translocation with deletion of distal region were newly identified in 7 (27%) and 12 (46%) cases, respectively. These results indicate that the distinct mechanism contributes to the LOH of each tumor suppressor gene on different chromosomal locations.

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes ofor =6 bp. Type B changes are more drastic and involve modifications ofor =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Ionizing radiation (IR) induces cellular stress responses, such as signal transduction, gene expression, protein modification, and metabolite change that affect cellular behavior. We analyzed X-irradiated human Epstein-Barr virus-transformed B lymphoblastoid cells and normal fibroblasts to search for metabolites that would be suitable IR-responsive markers by Liquid Chromotography–Mass spectrometry (LC–MS). Mass spectra, as analyzed with principal component analysis, showed that the proportion of peaks with IR-induced change was relatively small compared with the influence of culture time. Dozens of peaks that had either been upregulated or downregulated by IR were extracted as candidate IR markers. The IR-changed peaks were identified by comparing mock-treated groups to 100 mGy-irradiated groups that had recovered after 10 h, and the results indicated that the metabolites involved in nucleoside synthesis increased and that some acylcarnitine levels decreased in B lymphoblastoids. Some peaks changed by as much as 20 mGy, indicating the presence of an IR-sensitive signal transduction/metabolism control mechanism in these cells. On the other hand, we could not find common IR-changed peaks in fibroblasts of different origin. These data suggest that cell phenotype-specific pathways exist, even in low-dose responses, and could determine cell behavior.

BACKGROUND Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that expressed most abundantly in highly vascularized organs. The authors examined the effect of transfection of EPAS1 cDNA on the endogenous expression of vascular endothelial growth factor (VEGF) in the 293 Tet-Off cell line and the possible involvement of EPAS1 in the angiogenesis of renal cell carcinoma (RCC). METHODS Complete cDNA of EPAS1 was cloned and transfected to cells from the 293 Tet-Off fetal kidney cell line, in which the expression of EPAS1 can be inhibited by doxycycline. The subsequent changes in expression pattern of VEGF and transferrin receptor (TfR), a target gene of hypoxia-inducible factor 1α (HIF-1α), were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. In addition, expression of EPAS1, HIF-1α, and VEGF were analyzed by semiquantitative RT-PCR in five RCC cell lines and in 13 RCC tissue samples. In situ hybridization was performed on 7 of the 13 RCC tissue samples. RESULTS Endogenous VEGF was increased significantly by the introduction of EPAS1 cDNA at both the mRNA level and the protein level. With the inhibition of EPAS1 by doxycycline treatment, the expression of VEGF was significantly decreased accordingly, whereas the expression of TfR was not affected. EPAS1 was detected in all of the RCC cell lines examined. In RCC tissue samples, EPAS1 mRNA and VEGF mRNA were increased significantly in tumor tissues compared with normal adjacent kidney tissues. In situ hybridization showed that EPAS1 and VEGF were coexpressed topographically in tumor tissues. CONCLUSIONS These results suggest that endogenous VEGF can be up-regulated transcriptionally by EPAS1, and EPAS1 may be involved in the angiogenesis of RCC. Cancer 2001;91:1429–36. © 2001 American Cancer Society.

Cytogenetic and molecular studies have indicated that a putative tumor suppresor gene (s), which may play an important role in oral squamous cell carcinogenesis, is located on the short arm of chromosome 3 (3 p). We analyzed loss of heterozygosity (LOH) on 3 p, especially the 3 p25-pter region, in 17 oral squamous cell carcinomas (OSCCs) with the use of 5 microsatellite markers and constructed a deletion map for this chromosome arm. LOH at one or more loci was detected in 9 of 17 (53%) tumors. LOH on these region was more common in advanced OSCCs and may be associated with tumor progression. Our data support the notion that one of tumor suppressor gene (s) contributing to the progression of oral squamous cell carcinoma resides on 3 p25-pter.

To determine whether inactivation of the p16 gene mapped to the chromosome 9p21 region is associated with the development of oral squamous cell carcinoma (SCC), we investigated the mutational states of two forms of alternative transcripts (alpha and beta) from the p16 gene in 14 oral SCC cell lines by means of RT-PCR, PCR, direct sequencing and methylation analyses. Alterations of the alpha transcript were detected in all of the cell lines examined: homozygous deletions in three lines; subtle mutations in exons 1 alpha or 2 in four lines; skipping of exon 2 in two lines; hypermethylation of the 5' CpG island of the p16 gene in four lines; and an unknown mechanism in one line. On the other hand, abnormalities of the beta transcript were observed in seven of the 14 cell lines. Nonetheless, the mutations that essentially affect the function of the encoded protein were found only in five cell lines, including three lines with homozygous deletion. There was no cell line having only beta transcript alterations. Thus, alteration of the alpha transcript of the p16 gene was a highly frequent event in oral SCC. Since this type of alteration resulted in gene inactivation through multiple pathways, it may play a major role in the process of oral SCC development.

The Chromosome Network in Japan was established just after the critical Tokai-mura accident in 2001 with the purposes of (1) integrating work to be ready for a large-scale radiation accident, (2) establishing standard methods for cytogenetic dose estimation, (3) creating a standard dose response curve in Japan, (4) preparing a training program for cytogenetic dose estimation and (5) training successors for biodosimetry work. In order to perform the cytogenetic dose estimation according to the same criteria, an inter-laboratory comparison is being experimentally carried out among the laboratories in the Chromosome Network.

Response to a large-scale radiological incident could require timely medical interventions to minimize radiation casualties. Proper medical care requires knowing the victim's radiation dose. When physical dosimetry is absent, radiation-specific chromosome aberration analysis can serve to estimate the absorbed dose in order to assist physicians in the medical management of radiation injuries. A mock exercise scenario was presented to six participating biodosimetry laboratories as one individual acutely exposed to Co under conditions suggesting whole-body exposure. The individual was not wearing a dosimeter and within 2-3 h of the incident began vomiting. The individual also had other medical symptoms indicating likelihood of a significant dose. Physicians managing the patient requested a dose estimate in order to develop a treatment plan. Participating laboratories in North and South America, Europe, and Asia were asked to evaluate more than 800 electronic images of metaphase cells from the patient to determine the dicentric yield and calculate a dose estimate with 95% confidence limits. All participants were blind to the physical dose until after submitting their estimates based on the dicentric chromosome assay (DCA). The exercise was successful since the mean biological dose estimate was 1.89 Gy whereas the actual physical dose was 2 Gy. This is well within the requirements for guidance of medical management. The exercise demonstrated that the most labor-intensive step in the entire process (visual evaluation of images) can be accelerated by taking advantage of world-wide expertise available on the Internet.

Radionuclide concentrations in environmental samples such as surface soils, plants and water were evaluated by high purity germanium detector measurements. The contribution rate of short half-life radionuclides such as (132)I to the exposure dose to residents was discussed from the measured values. The highest values of the (131)I/(137)Cs activity ratio ranged from 49 to 70 in the environmental samples collected at Iwaki City which is located to the south of the F1-NPS. On the other hand, the (132)I/(131)I activity ratio in the same environmental samples had the lowest values, ranging from 0.01 to 0.02. By assuming that the (132)I/(131)I activity ratio in the atmosphere was equal to the ratio in the environmental samples, the percent contribution to the thyroid equivalent dose by (132)I was estimated to be less than 2%. Moreover, the contribution to the thyroid exposure by (132)I might be negligible if (132)I contamination was restricted to Iwaki City.

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.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.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.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.

Induced pluripotent stem cells (iPSCs) have been established from a variety of somatic cells not only for regenerative medicine but also for studies of the pathogenesis of inherited genetic diseases or neoplasms. In iPSCs established from T cells, the rearrangement of the T cell receptor (TCR) of the established T cell-derived iPSCs (TiPSCs) took over that of the original T cells. If B cell-derived iPSCs (BiPSCs) could be similarly established from mature B cells or plasma cells, the rearrangement of the B cell receptor (BCR) of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) together with ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) using a Sendai virus vector (Bueno C, et al. Leukemia 2016). We were able to also establish BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes using a method different from the above. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells, which were pre-stimulated with IL-21 and CD40L, using a retrovirus vector, and centrifugations of the cells after their activation by IL-4, IL-2, and CD40-ligand (CD40L) on a retronectin coated plate. We further established doxycycline-controlled (Tet-off system) activation-induced cytidine deaminase (AID)-induced BiPSCs (BiPSCs-A). AID is an enzyme that initiates somatic hypermutation (SHM) and class-switch recombination (CSR) in B cells. We hypothesized that the origin of a myeloma cell is a reprogrammed mature B cell, in which reciprocal chromosome translocation occurs by double stranded breakage (DSB) of DNA induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14. First, we did not detect an increase in dicentric chromosome (DIC) formation, which is evidence of DSB of DNA, in the BiPSCs-A. We next analyzed the ability of these BiPSCs to differentiate into hematopoietic stem cells (HSCs). Both the parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs as judged by confirmation of CD34 expression and colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45; we therefore think that these CD34+/CD38-/CD43-/CD45- cells might be hematoendothelial cells as Maxim proposed previously (Vodyanik MA, et al. Blood 2006). Furthermore, both BiPSCs with induced AID expression and BiPSCs without induced AID expression were capable of differentiating into HSCs. Based on the findings regarding the differentiation of BiPSCs-A into HSCs and their retention of the IgH gene rearrangement, there is a possibility that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HSCs and further into B cells in experiments using mouse. Thus these BiPSCs might be useful in elucidating the tumor origin of abnormal B cells in B cell tumor formation. These cells might be especially useful in understanding multiple myeloma, which is thought to originate from germinal center (GC) or post-GC B cells and has a productive (functional) allele with IgH gene rearrangement that produces M-protein, and another nonproductive (nonfunctional) allele of chromosome 14. Disclosures No relevant conflicts of interest to declare.

Telomeres, at the end of chromosomes, shorten with each cell division, resulting in cellular senescence. Tumor cells, unlike normal somatic cells, express a telomerase that maintains the telomere length. Deletion of a gene(s) on chromosome 3 is common in human renal cell carcinoma (RCC) and reintroduction of a normal chromosome 3 into an RCC immortal cell line restored the program of cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells with a normal chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to those of the parental RCC23. Thus, restoration of the cellular senescence program by chromosome 3 is associated with repression of telomerase function in RCC cells.

Cytogenetic analysis of a case of extraskeletal myxoid chondrosarcoma revealed a reciprocal translocation between 9q and 22q in almost all metaphases analyzed. Structural rearrangements involving 9q and 22q have been reported previously in three cases of extraskeletal myxoid chondrosarcoma. The breakpoints on chromosomes 9 and 22 in the present case were in regions 9q22-q31 and 22q11-q12.2, respectively. The same breakpoints were present in all three previously reported cases. Thus, this recently identified rearrangement of 9q and 22q may serve as a critical cytogenetic parameter for the diagnosis and classification of extraskeletal myxoid chondrosarcoma, as well as being a primary chromosomal event in the course of development of this rare malignancy.

Two different erythroleukemia cell lines have been established from the splenic lesions of transgenic mice possessing the Friend spleen focus-forming virus (F-SFFV) gp55 gene. One showed a near-diploid karyotype and a temperature-sensitive (ts) p53 mutation, and the other, a hyper-triploid karyotype with double p53 mutations found by single-strand conformation polymorphism (SSCP) analysis. The cell lines both retained No.11 chromosomes on which p53 genes are localized. Another p53 allele in the cell line with the ts-p53 mutation appeared intact in the SSCP analysis of the genomic exon 5. The cells with the ts-mutant p53 gene showed no apparent change with temperature shift in their growth or dimethylsulfoxide-induced differentiation, although the wild-type p53 gene on the other allele was not expressing. This ts-p53Val-135 gene made p53-deficient fibroblasts anchorage-independent at 37 degrees C but not at 32 degrees C. This non-virus-producing, mouse erythroleukemia cell line will be useful for the study of mutated p53 function during the induction of erythrodifferentiation or apoptotic change.

Major contaminants from venting and hydrogen explosions at the Fukushima Daiichi nuclear reactors between 12 and 15 March 2011 were transported northwestward and deposited on soil and plants via precipitation. Surface soils and plant leaves were sampled at 64 sites in the Fukushima Prefecture. The highest concentrations of (134)Cs (84.4 kBq kg(-1)) and (137)Cs (82.0 kBq kg(-1)) in surface soils were observed at Nagadoro in Iidate village located 32 km northwest from the Fukushima Daiichi nuclear power plant. Furthermore, (131)I, (129)Te, (129 m)Te, (110 m)Ag and (140)La were detected in the same samples. Outer surface of plant leaves, such as bamboo, cabbage and grasses were highly contaminated at the high-dose rate areas of Tsushima and Minami-Tsushima in Namie town. Mugwort leaves that grew after the pollution event had extremely low concentration of radionuclides; however, the plant/soil radiocaesium ratio was 0.023 ± 0.006. It is anticipated that decomposition of fallen leaves will promote recycling of radionuclides in the environment.

The analysis of dicentric chromosomes in human peripheral blood lymphocytes (PBLs) by Giemsa staining is the most established method for biological dosimetry. However, this method requires a well-trained person because of the difficulty in detecting aberrations rapidly and accurately. Here, we applied a fluorescence in situ hybridization (FISH) technique, using telomere and centromere peptide nucleic acid (PNA) probes, to solve the problem of biological dosimetry in radiation emergency medicine. A comparison by a well-trained observer found that FISH analysis of PBLs for the dose estimation was more accurate than the conventional Giemsa analysis, especially in samples irradiated at high doses. These results show that FISH analysis with centromeric/telomeric PNA probes could become the standard method for biological dosimetry in radiation emergency medicine.