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.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/ß-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of ß-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of ß-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of ß-catenin pathway.

A mathematical model for cancer risk and accumulation of mutations caused by replication errors and external factors.

Replication errors influence mutations, and thus, lifetime cancer risk can be explained by the number of stem-cell divisions. Additionally, mutagens also affect cancer risk, for instance, high-dose radiation exposure increases lifetime cancer risk. However, the influence of low-dose radiation exposure is still unclear because this influence, if any, is very slight. We can assess the minimal influence of the mutagen by virtually comparing the states with and without mutagen using a mathematical model. Here, we constructed a mathematical model to assess the influence of replication errors and mutagens on cancer risk. In our model, replication errors occur with a certain probability during cell division. Mutagens cause mutations at a constant rate. Cell division is arrested when the number of cells reaches the capacity of the cell pool. When the number of cells decreases because of cell death or other reasons, cells resume division. It was assumed that the mutations of cancer driver genes occur stochastically with each mutation and that cancer occurs when the number of cancer driver gene mutations exceeds a certain threshold. We approximated the number of mutations caused by errors and mutagens. Then, we examined whether cancer registry data on cancer risk can be explained only through replication errors. Although the risk of leukemia was not fitted to the model, the risks of esophageal, liver, thyroid, pancreatic, colon, breast, and prostate cancers were explained only by replication errors. Even if the risk was explained by replication errors, the estimated parameters did not always agree with previously reported values. For example, the estimated number of cancer driver genes in lung cancer was larger than the previously reported values. This discrepancy can be partly resolved by assuming the influence of mutagen. First, the influence of mutagens was analyzed using various parameters. The model predicted that the influence of mutagens will appear earlier, when the turnover rate of the tissue is higher and fewer mutations of cancer driver genes were necessary for carcinogenesis. Next, the parameters of lung cancer were re-estimated assuming the influence of mutagens. The estimated parameters were closer to the previously reported values. than when considering only replication errors. Although it may be useful to explain cancer risk by replication errors, it would be biologically more plausible to consider mutagens in cancers in which the effects of mutagens are apparent.

Observation of Histone H2AX Phosphorylation by Radiation-Induced Bystander Response Using Titanium Characteristic X-ray Microbeam.

Radiation-induced bystander response (RIBR) is a response induced in non-irradiated cells that receive bystander signals from directly irradiated cells. X-ray microbeams are useful tools for elucidating the mechanisms underlying RIBR. However, previous X-ray microbeams used low-energy soft X-rays with higher biological effects, such as aluminum characteristic X-rays, and the difference from conventional X-rays and γ-rays has often been discussed. The microbeam X-ray cell irradiation system at the Central Research Institute of Electric Power Industry has been upgraded to generate higher energy titanium characteristic X-rays (TiK X-rays), which have a longer penetration distance sufficient to irradiate 3D cultured tissues. Using this system, we irradiated the nuclei of HeLa cells with high precision and found that the pan-nuclear induction of phosphorylated histone H2AX on serine 139 (γ-H2AX) in the non-irradiated cells increased 180 and 360 min after irradiation. We established a new method to quantitatively evaluate bystander cells, using the fluorescence intensity of γ-H2AX as an indicator. The percentage of bystander cells increased significantly to 23.2% ± 3.2% and 29.3% ± 3.5% at 180 and 360 min after irradiation, respectively. Our irradiation system and the obtained results may be useful for studies of cell competition as well as non-targeted effects.

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.

Ionizing radiation alters organoid forming potential and replenishment rate in a dose/dose-rate dependent manner.

Intestinal organoids are an in vitro cultured tissue model generated from intestinal stem cells, and they contain a mixture of epithelial cell types. We previously established an efficient 'one cell/well' sorting method, and defined organoid-forming potential (OFP) as a useful index to evaluate the stemness of individual cells. In this study, we assessed the response to radiation dose and dose-rate by measuring both OFP and the percentage of stem cells in the crypts. After high-dose-rate (HDR, 0.5 Gy/min) irradiation in vivo, the percentage of stem cells in the harvested crypt cells decreased, and the replenishment of cycling stem cells originating from dormant cells was enhanced, but OFP increased in cells irradiated with a total dose of >1 Gy. In contrast, at a total dose of 0.1 Gy the percentage of stem cells reduced slightly, but neither replenishment rate nor OFP changed. Furthermore, the response to 1 Gy of low-dose-rate (LDR) irradiation was similar to the response to 0.1 Gy HDR irradiation. These results suggest that 0.1 Gy HDR irradiation or 1 Gy LDR irradiation does not alter stemness. Additionally, the OFP increase in the colon in response to irradiation was smaller than that in the duodenum, similar to the percentage of stem cells. Understanding the differences in the response of stem cells between the colon and the duodenum to radiation is important to clarify the mechanisms underlying the development of radiation-associated intestinal cancers.

INTESTINAL ORGANOIDS FOR STUDYING THE EFFECTS OF LOW-DOSE/LOW-DOSE-RATE RADIATION.

Radiation response differs depending on the dose and dose rate in intestinal stem cells; however, the underlying mechanisms are not clear. To understand the effects of low-dose and low-dose-rate radiation, the authors established an organoid system that mimics the in vivo environment and sporadic low-dose-rate irradiation conditions in vitro. Organoid-forming potential and the number of stem cells in the organoids derived from 1 Gy-irradiated cells were lower than those from non-irradiated cells; however, the difference was not significant, although 1 Gy-irradiated stem cells exhibited significant growth disadvantage in the mixed-organoid with non-irradiated and irradiated stem cells. Furthermore, the authors irradiated a cell with X-ray microbeams and performed time-lapse observations and found that irradiated cells did not remain in the organoid. These results suggest that radiation-induced stem cell competition can occur in intestinal organoids and contribute to a low risk of cancers at low-dose-rate exposures.

Exposure of the cytoplasm to low-dose X-rays modifies ataxia telangiectasia mutated-mediated DNA damage responses.

We recently showed that when a low X-ray dose is used, cell death is enhanced in nucleus-irradiated compared with whole-cell-irradiated cells; however, the role of the cytoplasm remains unclear. Here, we show changes in the DNA damage responses with or without X-ray microbeam irradiation of the cytoplasm. Phosphorylated histone H2AX foci, a surrogate marker for DNA double-strand breaks, in V79 and WI-38 cells are not observed in nucleus irradiations at ≤ 2 Gy, whereas they are observed in whole-cell irradiations. Addition of an ataxia telangiectasia mutated (ATM) kinase inhibitor to whole-cell irradiations suppresses foci formation at ≤ 2 Gy. ABL1 and p73 expression is upregulated following nucleus irradiation, suggesting the induction of p73-dependent cell death. Furthermore, CDKN1A (p21) is upregulated following whole-cell irradiation, indicating the induction of cell cycle arrest. These data reveal that cytoplasmic radioresponses modify ATM-mediated DNA damage responses and determine the fate of cells irradiated at low doses.

A Mathematical Model for Stem Cell Competition to Maintain a Cell Pool Injured by Radiation.

The effect of low-dose-rate exposure to ionizing radiation on cancer risk is a major issue associated with radiation protection. Tissue stem cells are regarded as one of the targets of radiation-induced carcinogenesis. However, it is hypothesized that the effect of radiation may be reduced if damaged stem cells are eliminated via stem cell competition between damaged and intact stem cells. This would be particularly effective under very low-dose-rate conditions, in which only a few stem cells in a stem cell pool may be affected by radiation. Following this hypothesis, we constructed a simple mathematical model to discuss the influence of stem cell competition attenuating the accumulation of damaged cells under very low-dose-rate conditions. In this model, a constant number of cells were introduced into a cell pool, and the numbers of intact and damaged cells were calculated via transition and turnover events. A transition event emulates radiation dose, whereby an intact cell is changed into a damaged cell with a given probability. On the other hand, a turnover event expresses cell competition, where reproduction and elimination of cells occur depending on the properties of cells. Under very low-dose-rate conditions, this model showed that radiation damage to the stem cell pool was strongly suppressed when the damaged cells were less reproductive and tended to be eliminated compared to the intact cells. Furthermore, the size of the stem cell pool was positively correlated with reduction in radiation damage.

Ascochlorin activates p53 in a manner distinct from DNA damaging agents.

Ascochlorin, a prenylphenol antitumor antibiotic, profoundly increases the expression of endogenous p53 by increasing protein stability in the human osteosarcoma cells and human colon cancer cells. Ascochlorin also increases DNA binding activity to the p53 consensus sequence in nuclear extract and enhances transcription of p53 downstream targets. Ascochlorin specifically induces p53 phosphorylation at ser 392 without affecting ser 15 or 20, whereas DNA damaging agents typically phosphorylate these serines. Moreover, ascochlorin does not induce phosphorylation of ATM and CHK1, an established substrate of ATR that is activated by genotoxins, nor does it increase DNA strand break, as confirmed by comet assay. The structure-activity relationship suggests that p53 activation by ascochlorin is related to inhibition of mitochondrial respiration, which is further supported by the observation that respiratory inhibitors activate p53 in a manner similar to ascochlorin. These results suggest that ascochlorin, through the inhibition of mitochondrial respiration, activates p53 through a mechanism distinct from genotoxins.

Contributions of direct and indirect actions in cell killing by high-LET radiations.

The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/mum in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/mum. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/mum, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/mum, and decreased thereafter. When the RBE was estimated separately for direct action (RBE(D)) and indirect action (RBE(I)); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/mum. However, the peak value was much higher for RBE(D) (5.99) than RBE(I) (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.

A new paradigm in radioadaptive response developing from microbeam research.

A classic paradigm in radiation biology asserts that all radiation effects on cells, tissues and organisms are due to the direct action of radiation on living tissue. Using this model, possible risks from exposure to low dose ionizing radiation (below 100 mSv) are estimated by extrapolating from data obtained after exposure to higher doses of radiation, using a linear non-threshold model (LNT model). However, the validity of using this dose-response model is controversial because evidence accumulated over the past decade has indicated that living organisms, including humans, respond differently to low dose/low dose-rate radiation than they do to high dose/high dose-rate radiation. These important responses to low dose/low dose-rate radiation are the radiation-induced adaptive response, the bystander response, low-dose hypersensitivity, and genomic instability. The mechanisms underlying these responses often involve biochemical and molecular signals generated in response to targeted and non-targeted events. In order to define and understand the bystander response to provide a basis for the understanding of non-targeted events and to elucidate the mechanisms involved, recent sophisticated research has been conducted with X-ray microbeams and charged heavy particle microbeams, and these studies have produced many new observations. Based on these observations, associations have been suggested to exist between the radioadaptive and bystander responses. The present review focuses on these two phenomena, and summarizes observations supporting their existence, and discusses the linkage between them in light of recent results obtained from experiments utilizing microbeams.

Microbeam irradiation facilities for radiobiology in Japan and China.

In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.

An Efficient Intestinal Organoid System of Direct Sorting to Evaluate Stem Cell Competition in Vitro.

Stem cell competition could shed light on the tissue-based quality control mechanism that prevents carcinogenesis. To quantitatively evaluate stem cell competition in vitro, we developed a two-color intestinal organoid forming system. First, we improved a protocol of culturing organoids from intestinal leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5)- enhanced green fluorescent protein (EGFP)high stem cells directly sorted on Matrigel without embedding. The organoid-forming potential (OFP) was 25% of Lgr5-EGFPhigh cells sorted at one cell per well. Using this culture protocol with lineage tracing, we established a two-color organoid culture system by mixing stem cells expressing different fluorescent colors. To analyze stem cell competition, two-color organoids were formed by mixing X-ray-irradiated and non-irradiated intestinal stem cells. In the two-color organoids, irradiated stem cells exhibited a growth disadvantage, although the OFP of irradiated cells alone did not decrease significantly from that of non-irradiated cells. These results suggest that stem cell competition can be evaluated quantitively in vitro using our new system.

Rapid myeloid recovery as a possible mechanism of whole-body radioadaptive response.

We investigated the mechanism underlying the radioadaptive response that rescues mice from hematopoietic failure. C57BL/6 mice were irradiated with low-dose acute X rays (0.5 Gy) for priming 2 weeks prior to a high-dose (6 Gy) challenge irradiation. Bone marrow cells, erythrocytes and platelets in low-dose-preirradiated mice showed earlier recovery after the challenge irradiation than those in mice subjected only to the challenge irradiation. This suggests that hematopoiesis is enhanced after a challenge irradiation in preirradiated mice. The rapid recovery of bone marrow cells after the challenge irradiation was consistent with the proliferation of hematopoietic progenitors expressing the cell surface markers Lin-, Sca-1- and c-Kit+ in low-dose-preirradiated mice. A subpopulation of myeloid (Mac-1+/Gr-1+) cells, which were descendants of Lin-, Sca-1- and c-Kit+ cells, rapidly recovered in the bone marrow of low-dose-preirradiated mice, whereas the number of B-lymphoid (CD19+/B220+) cells did not show a statistically significant increase. Plasma cytokine profiles were analyzed using antibody arrays, and results indicated that the concentrations of several growth factors for myelopoiesis after the challenge irradiation were considerably increased by low-dose preirradiation. The rapid recovery of erythrocytes and platelets but not leukocytes was observed in the peripheral blood of preirradiated mice, suggesting that low-dose preirradiation triggered the differentiation to myelopoiesis. Thus the adaptive response induced by low-dose preirradiation in terms of the recovery kinetics of the number of hematopoietic cells may be due to the rapid recovery of the number of myeloid cells after high-dose irradiation.

Role of DNA double-strand break repair genes in cell proliferation under low dose-rate irradiation conditions.

Radiation-induced DNA double-stand breaks (DSBs) lead to numerous biological effects. To elucidate the molecular mechanisms involved in cellular responses to low dose and low dose-rate radiation, it is informative to clarify the roles of DSB repair related genes. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, it is shown that in chicken DT40 cells irradiated with gamma-rays at a low dose-rate (2.4 cGy/day), the growth delay in NHEJ-related KU70- and PRKDC (encoding DNA-PKcs)-defective cells were remarkably higher than in cells defective for the HR-related RAD51B and RAD54 genes. DNA-PKcs- defective human M059J cells also showed an obvious growth delay when compared to control M059K cells. RAD54(-/-)KU70(-/-) cells demonstrated their highest degree of growth delay after an X-irradiation with a high dose-rate of 0.9 Gy/min. However they showed a lower degree of growth delay than that seen in KU70(-/-) and PRKDC(-/-/-) cells exposed to low dose-rate irradiation. These findings indicate that cellular responses to low dose-rate radiation are remarkably different from those to high dose-rate radiation. The fact that both DT40 and mammalian NHEJ-defective cells were highly sensitive to low dose-rate radiation, provide a foundation for the concept that NHEJ-related factors may be useful as molecular markers to predict the sensitivity of humans to low dose-rate radiation.

Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector.

Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated "Focicles" harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn). We used CRISPR/Cas9-mediated genome editing to obtain Focicle knock-in cell lines in NIH3T3 cells, which were subject to X-ray irradiation that induced comparable numbers of Focicle and endogenous-53BP1 foci. In addition, the Focicle probes enabled the kinetic analysis of both DSB repair and cell-cycle arrest/progression after irradiation, demonstrating that the Focicle knock-in cells progressed to cell division after DNA damage elimination. These newly developed probes can help to gain a better understanding of the dynamics of DSB repair and cell-cycle control to in turn guide cancer treatment development and cancer-risk assessments.

Cellular responses and gene expression profiles of colonic Lgr5+ stem cells after low-dose/low-dose-rate radiation exposure.

We previously found that high-dose-rate radiation induced a replenishment of the colonic Lgr5+ stem cell pool, whereas low-dose-rate radiation did not. To identify key molecules that determine the dose-rate effects on this stem cell pool, we harvested colonic Lgr5+ stem cells by cell sorting at 2 weeks after exposure to 1 Gy of high-dose-rate (30 Gy/h) or low-dose-rate (0.003 Gy/h) radiation and analyzed their gene expression profiles using RNA-Seq. We found that pathways related to DNA damage response, cell growth, cell differentiation and cell death were upregulated in Lgr5+ stem cells irradiated with high dose rates, whereas pathways related to apical junctions and extracellular signaling were upregulated in low-dose-rate-irradiated colonic Lgr5+ stem cells. Interestingly, biological events involving apical junctions are known to play an important role in the exclusion of transformed cells that are surrounded by normal epithelial cells through 'cell competition'. We speculated that cell competition, through apical junctions and extracellular ligands, might contribute to the dose-rate effect on Lgr5+ cell replenishment. To understand this mechanism, we focused on 69 genes that were significantly upregulated in low-dose-rate-irradiated cells, which we named DREDGE (Dose-Rate Effect Determining GEnes). Based on these findings, we propose a possible mechanism underlying the dose-rate effect observed in the colonic stem cell pool.