Human-mouse comparison of the multistage nature of radiation carcinogenesis in a mathematical model.

Mouse models are vital for assessing risk from environmental carcinogens, including ionizing radiation, yet the interspecies difference in the dose response precludes direct application of experimental evidence to humans. Herein, we take a mathematical approach to delineate the mechanism underlying the human-mouse difference in radiation-related cancer risk. We used a multistage carcinogenesis model assuming a mutational action of radiation to analyze previous data on cancer mortality in the Japanese atomic bomb survivors and in lifespan mouse experiments. Theoretically, the model predicted that exposure will chronologically shift the age-related increase in cancer risk forward by a period corresponding to the time in which the spontaneous mutational process generates the same mutational burden as that the exposure generates. This model appropriately fitted both human and mouse data and suggested a linear dose response for the time shift. The effect per dose decreased with increasing age at exposure similarly between humans and mice on a per-lifespan basis (0.72- and 0.71-fold, respectively, for every tenth lifetime). The time shift per dose was larger by two orders of magnitude in humans (7.8 and 0.046 years per Gy for humans and mice, respectively, when exposed at ~35% of their lifetime). The difference was mostly explained by the two orders of magnitude difference in spontaneous somatic mutation rates between the species plus the species-independent radiation-induced mutation rate. Thus, the findings delineate the mechanism underlying the interspecies difference in radiation-associated cancer mortality and may lead to the use of experimental evidence for risk prediction in humans.

Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Morphology dynamics in intestinal crypt during postnatal development affect age-dependent susceptibility to radiation-induced intestinal tumorigenesis in ApcMin/+ mice: possible mechanisms of radiation tumorigenesis.

Age at exposure is a major modifier of radiation-induced carcinogenesis. We used mouse models to elucidate the mechanism underlying age-related susceptibility to radiation-induced tumorigenesis. Radiation exposure in infants was effective at inducing tumors in B6/B6-Chr18MSM-F1 ApcMin/+ mice. Loss of heterozygosity analysis revealed that interstitial deletion may be considered a radiation signature in this model and tumor number containing a deletion correlated with the susceptibility to radiation-induced tumorigenesis as a function of age. Furthermore, in Lgr5-eGFP-ires-CreERT2; Apcflox/flox mice, deletions of both floxed Apc alleles in Lgr5-positive stem cells in infants resulted in the formation of more tumors than in adults. These results suggest that tumorigenicity of Apc-deficient stem cells varies with age and is higher in infant mice. Three-dimensional immunostaining analyses indicated that the crypt architecture in the intestine of infants was immature and different from that in adults concerning crypt size and the number of stem cells and Paneth cells per crypt. Interestingly, the frequency of crypt fission correlated with the susceptibility to radiation-induced tumorigenesis as a function of age. During crypt fission, the percentage of crypts with lysozyme-positive mature Paneth cells was lower in infants than that in adults, whereas no difference in the behavior of stem cells or Paneth cells was observed regardless of age. These data suggest that morphological dynamics in intestinal crypts affect age-dependent susceptibility to radiation-induced tumorigenesis; oncogenic mutations in infant stem cells resulting from radiation exposure may acquire an increased proliferative potential for tumor induction compared with that in adults.

Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level.

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.

Nuclear DNA damage-triggered ATM-dependent AMPK activation regulates the mitochondrial radiation response.

PURPOSE: AMP-activated protein kinase (AMPK) acts as a cellular energy sensor and is essential for controlling mitochondrial homeostasis. Here, we investigated the regulatory mechanisms involved in AMPK activation to elucidate how networks of intracellular signaling pathways respond to stress conditions. MATERIALS AND METHODS: Inhibitors of ATM, DNA-PK, and AKT were tested in normal TIG-3 and MRC-5 human fibroblasts to determine which upstream kinases are responsible for AMPK activation. SV40 transformed-human ATM-deficient fibroblasts (AT5BIVA) and their ATM-complemented cells (i.e., AT5BIVA/ATMwt) were also used. Protein expression associated with AMPK signaling was examined by immunostaining and/or Western blotting. RESULTS: Radiation-induced nuclear DNA damage activates ATM-dependent AMPK signaling pathways that regulate mitochondrial quality control. In contrast, hypoxia and glucose starvation caused ATP depletion and activated AMPK via a pathway independent of ATM. DNA-PK and AKT are not involved in AMPK-mediated mitochondrial signaling pathways. CONCLUSION: Activation of the AMPK signaling pathway differs depending on the stimulus. Radiation activates AMPK through two pathways: depletion of ATP-mediated LKB1 signaling and nuclear DNA damage-induced ATM signaling. Nuclear DNA damage signaling to mitochondria therefore plays a pivotal role in determining the cell fates of irradiated cells.

Establishment and activity of the planning and acting network for low dose radiation research in Japan (PLANET): 2016-2023.

The Planning and Acting Network for Low Dose Radiation Research in Japan (PLANET) was established in 2017 in response to the need for an all-Japan network of experts. It serves as an academic platform to propose strategies and facilitate collaboration to improve quantitative estimation of health risks from ionizing radiation at low-doses and low-dose-rates. PLANET established Working Group 1 (Dose-Rate Effects in Animal Experiments) to consolidate findings from animal experiments on dose-rate effects in carcinogenesis. Considering international trends in this field as well as the situation in Japan, PLANET updated its priority research areas for Japanese low-dose radiation research in 2023 to include (i) characterization of low-dose and low-dose-rate radiation risk, (ii) factors to be considered for individualization of radiation risk, (iii) biological mechanisms of low-dose and low-dose-rate radiation effects and (iv) integration of epidemiology and biology. In this context, PLANET established Working Group 2 (Dose and Dose-Rate Mapping for Radiation Risk Studies) to identify the range of doses and dose rates at which observable effects on different endpoints have been reported; Working Group 3 (Species- and Organ-Specific Dose-Rate Effects) to consider the relevance of stem cell dynamics in radiation carcinogenesis of different species and organs; and Working Group 4 (Research Mapping for Radiation-Related Carcinogenesis) to sort out relevant studies, including those on non-mutagenic effects, and to identify priority research areas. These PLANET activities will be used to improve the risk assessment and to contribute to the revision of the next main recommendations of the International Commission on Radiological Protection.

Newly discovered genomic mutation patterns in radiation-induced small intestinal tumors of ApcMin/+ mice.

Among the small intestinal tumors that occur in irradiated mice of the established mouse model B6/B6-Chr18MSM-F1 ApcMin/+, loss of heterozygosity analysis can be utilized to estimate whether a deletion in the wild-type allele containing the Adenomatous polyposis coli (Apc) region (hereafter referred to as Deletion), a duplication in the mutant allele with a nonsense mutation at codon 850 of Apc (Duplication), or no aberration (Unidentified) has occurred. Previous research has revealed that the number of Unidentified tumors tends to increase with the radiation dose. In the present study, we investigated the molecular mechanisms underlying the development of an Unidentified tumor type in response to radiation exposure. The mRNA expression levels of Apc were significantly lower in Unidentified tumors than in normal tissues. We focused on epigenetic suppression as the mechanism underlying this decreased expression; however, hypermethylation of the Apc promoter region was not observed. To investigate whether deletions occur that cannot be captured by loss of heterozygosity analysis, we analyzed chromosome 18 using a customized array comparative genomic hybridization approach designed to detect copy-number changes in chromosome 18. However, the copy number of the Apc region was not altered in Unidentified tumors. Finally, gene mutation analysis of the Apc region using next-generation sequencing suggested the existence of a small deletion (approximately 3.5 kbp) in an Unidentified tumor from a mouse in the irradiated group. Furthermore, nonsense and frameshift mutations in Apc were found in approximately 30% of the Unidentified tumors analyzed. These results suggest that radiation-induced Unidentified tumors arise mainly due to decreased Apc expression of an unknown regulatory mechanism that does not depend on promoter hypermethylation, and that some tumors may result from nonsense mutations which are as-yet undefined point mutations.

Effects of oxygen on the response of mitochondria to X-irradiation and reactive oxygen species-mediated fibroblast activation.

PURPOSE: In living organisms, sensitivity to radiation increases in the presence of oxygen (O2) compared with that under anoxic or hypoxic conditions. Here, we investigated whether O2 concentration affected the response of mitochondria to X-rays radiation, which is associated with tumor microenvironment formation via fibroblast activation in radiation-related tumors. MATERIALS AND METHODS: O2 concentrations were controlled at <5% (internal environmental oxygen condition) or anoxic levels during culture of normal human diploid lung fibroblasts TIG-3 and MRC-5. Protein expression associated with the response of mitochondria to radiation was assessed using immunostaining or western blotting. RESULTS: Induction of DNA damage (marker: γ-H2A histone family member X) and mitochondrial signaling (AMP-activated protein kinase), suppression of mitochondrial metabolic activity, and generation of reactive oxygen species occurred with radiation in cells cultured under 5% and 20% O2 conditions. However, reducing O2 concentration mitigated the effects of radiation on cell growth, mitochondrial damage (parkin), induction of antioxidant responses (nuclear factor E2-related factor 2), and fibroblast activation (α-smooth muscle actin). Radiation did not affect the markers used in this study in the absence of O2. CONCLUSION: O2 concentration affected the response of mitochondria to radiation and reactive oxygen species-mediated fibroblast activation. Higher O2 concentrations enhanced the effects of radiation on mitochondria in human fibroblasts. In vitro studies may overestimate in vivo radiation effects due to high O2 concentrations.

Molecular and cellular basis of the dose-rate-dependent adverse effects of radiation exposure in animal models. Part I: Mammary gland and digestive tract.

While epidemiological data are available for the dose and dose-rate effectiveness factor (DDREF) for human populations, animal models have contributed significantly to providing quantitative data with mechanistic insights. The aim of the current review is to compile both the in vitro experiments with reference to the dose-rate effects of DNA damage and repair, and the animal studies, specific to rodents, with reference to the dose-rate effects of cancer development. In particular, the review focuses especially on the results pertaining to underlying biological mechanisms and discusses their possible involvement in the process of radiation-induced carcinogenesis. Because the concept of adverse outcome pathway (AOP) together with the key events has been considered as a clue to estimate radiation risks at low doses and low dose-rates, the review scrutinized the dose-rate dependency of the key events related to carcinogenesis, which enables us to unify the underlying critical mechanisms to establish a connection between animal experimental studies with human epidemiological studies.

Molecular and cellular basis of the dose-rate-dependent adverse effects of radiation exposure in animal models. Part II: Hematopoietic system, lung and liver.

While epidemiological data have greatly contributed to the estimation of the dose and dose-rate effectiveness factor (DDREF) for human populations, studies using animal models have made significant contributions to provide quantitative data with mechanistic insights. The current article aims at compiling the animal studies, specific to rodents, with reference to the dose-rate effects of cancer development. This review focuses specifically on the results that explain the biological mechanisms underlying dose-rate effects and their potential involvement in radiation-induced carcinogenic processes. Since the adverse outcome pathway (AOP) concept together with the key events holds promise for improving the estimation of radiation risk at low doses and low dose-rates, the review intends to scrutinize dose-rate dependency of the key events in animal models and to consider novel key events involved in the dose-rate effects, which enables identification of important underlying mechanisms for linking animal experimental and human epidemiological studies in a unified manner.

Mutation and apoptosis are well-coordinated for protecting against DNA damage-inducing toxicity in Drosophila.

BACKGROUND: Apoptotic cell death is an important survival system for multicellular organisms because it removes damaged cells. Mutation is also a survival method for dealing with damaged cells in multicellular and also unicellular organisms, when DNA lesions are not removed. However, to the best of our knowledge, no reports have comprehensively explored the direct relationship between apoptosis and somatic cell mutations induced by various mutagenic factors. RESULTS: Mutation was examined by the wing-spot test, which is used to detect somatic cell mutations, including chromosomal recombination. Apoptosis was observed in the wing discs by acridine orange staining in situ. After treatment with chemical mutagens, ultraviolet light (UV), and X-ray, both the apoptotic frequency and mutagenic activity increased in a dose-dependent manner at non-toxic doses. When we used DNA repair-deficient Drosophila strains, the correlation coefficient of the relationship between apoptosis and mutagenicity, differed from that of the wild-type. To explore how apoptosis affects the behavior of mutated cells, we determined the spot size, i.e., the number of mutated cells in a spot. In parallel with an increase in apoptosis, the spot size increased with MNU or X-ray treatment dose-dependently; however, this increase was not seen with UV irradiation. In addition, BrdU incorporation, an indicator of cell proliferation, in the wing discs was suppressed at 6 h, with peak at 12 h post-treatment with X-ray, and that it started to increase again at 24 h; however, this was not seen with UV irradiation. CONCLUSION: Damage-induced apoptosis and mutation might be coordinated with each other, and the frequency of apoptosis and mutagenicity are balanced depending on the type of DNA damage. From the data of the spot size and BrdU incorporation, it is possible that mutated cells replace apoptotic cells due to their high frequency of cell division, resulting in enlargement of the spot size after MNU or X-ray treatment. We consider that the induction of mutation, apoptosis, and/or cell growth varies in multi-cellular organisms depending on the type of the mutagens, and that their balance and coordination have an important function to counter DNA damage for the survival of the organism.

[Effects of radiation exposure on human body].

There are two types of radiation health effect; acute disorder and late on-set disorder. Acute disorder is a deterministic effect that the symptoms appear by exposure above a threshold. Tissues and cells that compose the human body have different radiation sensitivity respectively, and the symptoms appear in order, from highly radiosensitive tissues. The clinical symptoms of acute disorder begin with a decrease in lymphocytes, and then the symptoms appear such as alopecia, skin erythema, hematopoietic damage, gastrointestinal damage, central nervous system damage with increasing radiation dose. Regarding the late on-set disorder, a predominant health effect is the cancer among the symptoms of such as cancer, non-cancer disease and genetic effect. Cancer and genetic effect are recognized as stochastic effects without the threshold. When radiation dose is equal to or more than 100 mSv, it is observed that the cancer risk by radiation exposure increases linearly with an increase in dose. On the other hand, the risk of developing cancer through low-dose radiation exposure, less 100 mSv, has not yet been clarified scientifically. Although uncertainty still remains regarding low level risk estimation, ICRP propound LNT model and conduct radiation protection in accordance with LNT model in the low-dose and low-dose rate radiation from a position of radiation protection. Meanwhile, the mechanism of radiation damage has been gradually clarified. The initial event of radiation-induced diseases is thought to be the damage to genome such as radiation-induced DNA double-strand breaks. Recently, it is clarified that our cells could recognize genome damage and induce the diverse cell response to maintain genome integrity. This phenomenon is called DNA damage response which induces the cell cycle arrest, DNA repair, apoptosis, cell senescence and so on. These responses act in the direction to maintain genome integrity against genome damage, however, the death of large number of cells results in acute disorder, and then DNA mis-repair and mutation is speculated to cause cancer. The extent to which this kind of cellular response could reduce the low-dose radiation risk is a major challenge for future research.

Kinetics of DNA Repair Under Chronic Irradiation at Low and Medium Dose Rates in Repair Proficient and Repair Compromised Normal Fibroblasts.

We present time and dose dependencies for the formation of 53BP1 and γH2AX DNA damage repair foci after chronic radiation exposure at dose rates of 140, 250 and 450 mGy/day from 3 to 96 h, in human and mouse repair proficient and ATM or DNA-PK deficient repair compromised cell models. We describe the time/dose-response curves using a mathematical equation which contains a linear component for the induction of DNA damage repair foci after irradiation, and an exponential component for their resolution. We show that under conditions of chronic irradiation at low and medium dose rates, the processes of DNA double-strand breaks (DSBs) induction and repair establish an equilibrium, which in repair proficient cells manifests as a plateau-shaped dose-response where the plateau is reached within the first 24 h postirradiation, and its height is proportionate to the radiation dose rate. In contrast, in repair compromised cells, where the rate of repair may be exceeded by the DSB induction rate, DNA damage accumulates with time of exposure and total absorbed dose. In addition, we discuss the biological meaning of the observed dependencies by presenting the frequency of micronuclei formation under the same irradiation conditions as a marker of radiation-induced genomic instability. We believe that the data and analysis presented here shed light on the kinetics of DNA repair under chronic radiation and are useful for future studies in the low-to-medium dose rate range.

Melatonin and MitoEbselen-2 Are Radioprotective Agents to Mitochondria.

Mitochondria are responsible for controlling cell death during the early stages of radiation exposure, but their perturbations are associated with late effects of radiation-related carcinogenesis. Therefore, it is important to protect mitochondria to mitigate the harmful effects of radiation throughout life. The glutathione peroxidase (GPx) enzyme is essential for the maintenance of mitochondrial-derived reactive oxygen species (ROS) levels. However, radiation inactivates the GPx, resulting in metabolic oxidative stress and prolonged cell injury in irradiated normal human fibroblasts. Here, we used the GPx activator N-acetyl-5-methoxy-tryptamine (melatonin) and a mitochondria-targeted mimic of GPx MitoEbselen-2 to stimulate the GPx. A commercial GPx activity assay kit was used to measure the GPx activity. ROS levels were determined by using some ROS indicators. Protein expression associated with the response of mitochondria to radiation was assessed using immunostaining. Concurrent pre-administration or post-administration of melatonin or MitoEbselen-2 with radiation maintained GPx activity and ROS levels and suppressed mitochondrial radiation responses associated with cellular damage and radiation-related carcinogenesis. In conclusion, melatonin and MitoEbselen-2 prevented radiation-induced mitochondrial injury and metabolic oxidative stress by targeting mitochondria. These drugs have the potential to protect against acute radiation injury and late effects of carcinogenesis in a variety of radiation scenarios assuming pre-administration or post-administration.

Radiation affects glutathione redox reaction by reduced glutathione peroxidase activity in human fibroblasts.

The glutathione (GSH) redox control is critical to maintain redox balance in the body's internal environment, and its perturbation leads to a dramatic increase in reactive oxygen species (ROS) levels and oxidative stress which have negative impacts on human health. Although ionizing radiation increases mitochondrial ROS generation, the mechanisms underlying radiation-induced late ROS accumulation are not fully understood. Here we investigated the radiation effect on GSH redox reactions in normal human diploid lung fibroblasts TIG-3 and MRC-5. Superoxide anion probe MitoSOX-red staining and measurement of GSH peroxidase (GPx) activity revealed that high dose single-radiation (SR) exposure (10 Gy) increased mitochondrial ROS generation and overall oxidative stress in parallel with decrease in GSH peroxidase (GPx) activity, while GSH redox control was effective after exposure to moderate doses under standard serum conditions. We used different serum conditions to elucidate the role of serum on GSH redox reaction. Serum starvation, serum deprivation and DNA damage response (DDR) inhibitors-treatment reduced the GPx activity and increased mitochondrial ROS generation regardless of radiation exposure. Fractionated-radiation was used to evaluate the radiation effect on GSH reactions. Repeated fractionated-radiation induced prolonged oxidative stress by down-regulation of GPx activity. In conclusion, radiation affects GSH usage according to radiation dose, irradiation methods and serum concentration. Radiation affected the GPx activity to disrupt fibroblast redox homeostasis.

Formation of DNA Damage Foci in Human and Mouse Primary Fibroblasts Chronically Exposed to Gamma Radiation at 0.1 mGy/min.

Low-dose-rate radiation exposures and their associated cancer risk are an important concern for radiation protection today. Nevertheless, there is almost no data concerning DNA damage at dose rates below 0.1 mGy/min. In this study, we investigated the formation of DNA damage repair foci under chronic low-dose-rate irradiation relative to acute high-dose-rate irradiation and assessed the magnitude of the dose-rate effect. Four human and four mouse normal fibroblast cell models from different organs were subjected to gamma irradiation at 0.096 mGy/min or 0.81 Gy/min, and dose-response curves were established for the dose range from 0.1 to 0.8 Gy. The results indicate that prolonged low-dose-rate exposures cause modestly increased levels of DNA repair foci, with a strongly supralinear dose-response relationship, where 40-70% of the radiation effect at 1 Gy was already present at the total dose of 0.1 Gy. Thus, compared to acute irradiation, low-dose-rate exposure was 6-9 times less efficient at a total dose of 0.1 Gy, and 10-20 times less efficient at 1 Gy. Comparison between cell models revealed a certain correlation between the presence of persistent, above-background foci at 48 h after irradiation and the sensitivity to low-dose-rate radiation, suggesting that repair capacity plays an important role in the cellular response to chronic irradiation. Given the findings reported here, we propose that establishing detailed dose-response curves and accounting for the repair rates of different cell models are essential steps in elucidating dose-rate effects.

Possible relationship between mitochondrial changes and oxidative stress under low dose-rate irradiation.

Objectives: High dose-rate ionizing radiation (IR) causes severe DSB damage, as well as reactive oxygen species (ROS) accumulation and oxidative stress. However, it is unknown what biological processes are affected by low dose-rate IR; therefore, the molecular relationships between mitochondria changes and oxidative stress in human normal cells was investigated after low dose-rate IR.Methods: We compared several cellular response between high and low dose-rate irradiation using cell survival assay, ROS/RNS assay, immunofluorescence and western blot analysis.Results: Reduced DSB damage and increased levels of ROS, with subsequent oxidative stress responses, were observed in normal cells after low dose-rate IR. Low dose-rate IR caused several mitochondrial changes, including morphology mass, and mitochondrial membrane potential, suggesting that mitochondrial damage was caused. Although damaged mitochondria were removed by mitophagy to stop ROS leakage, the mitophagy-regulatory factor, PINK1, was reduced following low dose-rate IR. Although mitochondrial dynamics (fission/fusion events) are important for the proper mitophagy process, some mitochondrial fusion factors decreased following low dose-rate IR.Discussion: The dysfunction of mitophagy pathway under low dose-rate IR increased ROS and the subsequent activation of the oxidative stress response.

Evaluation of sodium orthovanadate as a radioprotective agent under total-body irradiation and partial-body irradiation conditions in mice.

PURPOSE: Our previous study indicated that sodium orthovanadate (vanadate), a strong inhibitor of p53, effectively suppressed the lethality from the hematopoietic (HP) and gastrointestinal (GI) syndromes after 12 Gy total-body irradiation (TBI) in mice. This conclusion, however, was inconsistent with the fact that p53 plays a radioprotective role in the intestinal epithelium. The death after TBI of around 12 Gy was attributed to a combined effect of HP and GI syndromes. To verify the effect from prophylactic administration of p53 inhibitor on protection of HP and GI syndromes, in this study, the radioprotective effects from vanadate were investigated in TBI and lower half-body irradiation (partial-body irradiation: PBI) mouse models. METHODS: Female ICR mice were given a single injection of vanadate or vehicle, followed by a lethal dose of TBI or PBI. Radioprotective effects of vanadate against the irradiations were evaluated by analyzing survival rate, body weight, hematopoietic parameters, and histological changes in the bone marrow and intestinal epithelium. RESULTS: TBI-induced HP syndrome was effectively suppressed by vanadate treatment. After TBI, the vanadate-treated mice retained better bone marrow cellularity and showed markedly higher survival rate compared to the vehicle-treated animals. In contrast, vanadate did not relieve loss of intestinal crypts and failed to rescue mice from GI death after PBI. CONCLUSION: Vanadate is a p53 inhibitor that has been shown to be beneficial as a radiation protective agent against HP but was not effective in protecting against acute GI radiation injury.

Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells.

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain potential to transform by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we have already established normal B cell-derived induced pluripotent stem cells (BiPSCs). Here we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eµ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5'-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14). These BiPSCs differentiated into hematopoietic progenitor cells (HPCs). However, unlike cord blood, those HPCs did not differentiated into B lymphocytes by co-culture with BM stromal cell. Therefore, further ingenuity is required to differentiate those BiPSCs-derived HPCs into B lymphocytes.

The in vivo role of Rev1 in mutagenesis and carcinogenesis.

Translesion synthesis (TLS) is an error-prone pathway required to overcome replication blockage by DNA damage. Aberrant activation of TLS has been suggested to play a role in tumorigenesis by promoting genetic mutations. However, the precise molecular mechanisms underlying TLS-mediated tumorigenesis in vivo remain unclear. Rev1 is a member of the Y family polymerases and plays a key role in the TLS pathway. Here we introduce the existing to date Rev1-mutated mouse models, including the Rev1 transgenic (Tg) mouse model generated in our laboratory. We give an overview of the current knowledge on how different disruptions in Rev1 functions impact mutagenesis and the suggested molecular mechanisms underlying these effects. We summarize the available data from ours and others' in vivo studies on the role of Rev1 in the initiation and promotion of cancer, emphasizing how Rev1-mutated mouse models can be used as complementary tools for future research.