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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Enhancement of DNA double-strand break induction and cell killing by K-shell absorption of phosphorus in human cell lines.

PURPOSE: To investigate an enhancement of DNA double-strand break (DSB) induction and cell killing effect by K-shell ionization of phosphorus atoms and Auger electrons on human cell lines. MATERIALS AND METHODS: Induction of DSB, DNA damage responses, cell cycle distributions, and cell killing effects were investigated after exposures of the cells with monochromatic synchrotron radiation soft X-rays of 2153 and 2147 eV, which were the resonance peak and off peak, respectively, of the K-shell photoabsorption of phosphorus. RESULTS: Higher biological effects in the cells irradiated with soft X-rays at 2153 eV than at 2147 eV were observed in (i) the efficiency of 53BP1/γ-H2AX co-localized foci formation per dose and residual number of foci, (ii) prolonged phosphorylation levels of DSB repair and/or cell cycle checkpoint related proteins and G2 arrest, (iii) the cell killing effects at the 10% survival level of normal human fibroblasts, HeLa cells, and human glioblastoma M059K cells (1.2-1.5 times higher) and that of human ataxia telangiectasia mutated (ATM)-defective cells and glioblastoma DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-defective cells (1.2 times). CONCLUSION: The yield of DSB and partly less-reparable complex DNA damage induction in human cells was enhanced by K-shell photoabsorption of phosphorus and low-energy Auger electrons.

In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage.

XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells.

Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation.

In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time after irradiation.

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.

Mechanisms and biological importance of photon-induced bystander responses: do they have an impact on low-dose radiation responses.

Elucidating the biological effect of low linear energy transfer (LET), low-dose and/or low-dose-rate ionizing radiation is essential in ensuring radiation safety. Over the past two decades, non-targeted effects, which are not only a direct consequence of radiation-induced initial lesions produced in cellular DNA but also of intra- and inter-cellular communications involving both targeted and non-targeted cells, have been reported and are currently defining a new paradigm in radiation biology. These effects include radiation-induced adaptive response, low-dose hypersensitivity, genomic instability, and radiation-induced bystander response (RIBR). RIBR is generally defined as a cellular response that is induced in non-irradiated cells that receive bystander signals from directly irradiated cells. RIBR could thus play an important biological role in low-dose irradiation conditions. However, this suggestion was mainly based on findings obtained using high-LET charged-particle radiations. The human population (especially the Japanese, who are exposed to lower doses of radon than the world average) is more frequently exposed to low-LET photons (X-rays or γ-rays) than to high-LET charged-particle radiation on a daily basis. There are currently a growing number of reports describing a distinguishing feature between photon-induced bystander response and high-LET RIBR. In particular, photon-induced bystander response is strongly influenced by irradiation dose, the irradiated region of the targeted cells, and p53 status. The present review focuses on the photon-induced bystander response, and discusses its impact on the low-dose radiation effect.

A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation.

The microcolony assay developed by Withers and Elkind has been a gold standard to assess the surviving fraction of small intestinal stem cells after exposure to high (≥8 Gy) doses of ionizing radiation (IR), but is not applicable in cases of exposure to lower doses. Here, we developed a novel in vitro assay that enables assessment of the surviving fraction of small intestinal stem cells after exposure to lower IR doses. The assay includes in vitro culture of small intestinal stem cells, which allows the stem cells to develop into epithelial organoids containing all four differentiated cell types of the small intestine. We used Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice to identify Lgr5(+) stem cells and their progeny. Enzymatically dissociated single crypt cells from the duodenum and jejunum of mice were irradiated with 7.25, 29, 101, 304, 1000, 2000 and 4000 mGy of X-rays immediately after plating, and the number of organoids was counted on Day 12. Organoid-forming efficiency of irradiated cells relative to that of unirradiated controls was defined as the surviving fraction of stem cells. We observed a significant decrease in the surviving fraction of stem cells at ≥1000 mGy. Moreover, fluorescence-activated cell sorting analyses and passage of the organoids revealed that proliferation of stem cells surviving IR is significantly potentiated. Together, the present study demonstrates that the in vitro assay is useful for quantitatively assessing the surviving fraction of small intestinal stem cells after exposure to lower doses of IR as compared with previous examinations using the microcolony assay.