Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

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.

descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

Insights into radiation carcinogenesis based on dose-rate effects in tissue stem cells.

PURPOSE: Increasing epidemiological and biological evidence suggests that radiation exposure enhances cancer risk in a dose-dependent manner. This can be attributed to the 'dose-rate effect,' where the biological effect of low dose-rate radiation is lower than that of the same dose at a high dose-rate. This effect has been reported in epidemiological studies and experimental biology, although the underlying biological mechanisms are not completely understood. In this review, we aim to propose a suitable model for radiation carcinogenesis based on the dose-rate effect in tissue stem cells. METHODS: We surveyed and summarized the latest studies on the mechanisms of carcinogenesis. Next, we summarized the radiosensitivity of intestinal stem cells and the role of dose-rate in the modulation of stem-cell dynamics after irradiation. RESULTS: Consistently, driver mutations can be detected in most cancers from past to present, supporting the hypothesis that cancer progression is initiated by the accumulation of driver mutations. Recent reports demonstrated that driver mutations can be observed even in normal tissues, which suggests that the accumulation of mutations is a necessary condition for cancer progression. In addition, driver mutations in tissue stem cells can cause tumors, whereas they are not sufficient when they occur in non-stem cells. For non-stem cells, tissue remodeling induced by marked inflammation after the loss of tissue cells is important in addition to the accumulation of mutations. Therefore, the mechanism of carcinogenesis differs according to the cell type and magnitude of stress. In addition, our results indicated that non-irradiated stem cells tend to be eliminated from three-dimensional cultures of intestinal stem cells (organoids) composed of irradiated and non-irradiated stem cells, supporting the stem-cell competition. CONCLUSIONS: We propose a unique scheme in which the dose-rate dependent response of intestinal stem cells incorporates the concept of the threshold of stem-cell competition and context-dependent target shift from stem cells to whole tissue. The concept highlights four key issues that should be considered in radiation carcinogenesis: i.e. accumulation of mutations; tissue reconstitution; stem-cell competition; and environmental factors like epigenetic modifications.

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.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli.

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E. coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E. coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50 µmol m-2 s-1 at 37 °C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E. coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.

Minimization of Elements for Isothermal DNA Replication by an Evolutionary Approach.

DNA replication is one of the central functions of the cell. The complexity of modern DNA replication systems raises a question: is it possible to achieve a simpler continuous isothermal DNA replication using fewer proteins? Here, we searched such replication using an evolutionary approach. Through a long-term serial dilution experiment with phi29 DNA polymerase, we found that large repetitive DNAs spontaneously appear and continuously replicate. The repetitive sequence is critical for replication. Arbitrary sequences can replicate if they contain many repeats. We also demonstrated continuous DNA replication using expressed polymerase from the DNA for 10 rounds. This study revealed that continuous isothermal DNA replication can be achieved in a scheme simpler than that employed by modern organisms, providing an alternative strategy for simpler artificial cell synthesis and a clue to possible primitive forms of DNA replication.

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.

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.

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.

Activation of antioxidative enzymes induced by low-dose-rate whole-body gamma irradiation: adaptive response in terms of initial DNA damage.

An adaptive response induced by long-term low-dose-rate irradiation in mice was evaluated in terms of the amount of DNA damage in the spleen analyzed by a comet assay. C57BL/ 6N female mice were irradiated with 0.5 Gy of (137)Cs gamma rays at 1.2 mGy/h; thereafter, a challenge dose (0.4, 0.8 or 1.6 Gy) at a high dose rate was given. Less DNA damage was observed in the spleen cells of preirradiated mice than in those of mice that received the challenge dose only; an adaptive response in terms of DNA damage was induced by long-term low-dose-rate irradiation in mice. The gene expression of catalase and Mn-SOD was significantly increased in the spleen after 23 days of the low-dose-rate radiation (0.5 Gy). In addition, the enzymatic activity of catalase corresponded to the gene expression level; the increase in the activity was observed at day 23 (0.5 Gy). These results suggested that an enhancement of the antioxidative capacities played an important role in the reduction of initial DNA damage by low-dose-rate radiation.

Differences in Radiation Dose Response between Small and Large Intestinal Crypts.

The protection of intestinal epithelial cells from the lethal effects induced by high-dose radiation is an important issue in radiotherapy and in the treatment of acute radiation syndrome. However, the effects of middle- and low-dose radiation on intestinal epithelial cells remain unclear. Because the accumulation of DNA damage in intestinal stem cells may be crucial for the development of cancer-initiating cells, it is important to understand the kinetics of DNA repair and tissue response (which are involved in the elimination of damaged cells and tissue injury repair) to middle- to low-dose irradiation. In this study, mice were X-ray irradiated with 0.1, 1 or 4 Gy, after which the small intestine (duodenum and ileum) and colon were harvested from the animals. DNA damage repair and the elimination of damaged cells were quantified by measuring the number of foci of 53BP1, a surrogate marker for DNA double-strand breaks. Tissue-proliferative response was evaluated by determining the number of Ki-67(+) and mitotic cells. Intra-crypt response differed considerably between the small intestine and the colon. In the small intestine, 53BP1 foci were detected immediately after irradiation, but rapidly disappeared thereafter, especially noticeable in Lgr5(+) stem cells. Cellular growth was temporally arrested; however, cell numbers and mitotic cell numbers in the crypt did not change. The kinetics of DNA damage repair in Lgr5(+) stem cells were similar to those in the small intestines, while the colon was more susceptible to radiation-induced damage. Preferential cell loss in the lower crypt was clearly observed in the colon; and after low-dose X-ray irradiation, only the colon exhibited considerably reduced cell numbers and dramatic induction of mitosis. These results suggest that differences in radiation dose response between the small and the large intestine may depend on the growth activity of stem cells after DNA repair.

The role of gene mutations and gene products in intestinal tissue reactions from ionising radiation.

The response of the intestine to (low linear-energy-transfer) ionising radiation is reviewed regarding the cellular basis to the reactions, the regenerative processes which restore the tissue, and external agents which aid its recovery. In the steady-state, it is generally considered that the crypt cell lineages in both small and large intestine are maintained by a small number of stem cells, but there are differences for example in the composition of their niche residence and in the numbers of transit cell generations. Various cell surface markers are now available to indentify particular lineage cell types. Radiation doses up to 1Gy cause apoptotic stem-cell death in particular locations, at higher doses to >6Gy Lgr5+ stem cells are required for normal intestinal recovery, and at >8Gy some crypts are sterilised and the probability of animal death from intestinal injury increases with higher doses. Mutations in repair genes, tumour suppressor genes, and survival genes cause various degrees of stem cell and clonogenic cell radiosensitisation. Recent evidence is suggesting much plasticity in the crypt cell lineage, potentially contributing to flexibility in the hierarchical lineage, clonogen number variations and the sensitisation differences. Knockout mice for many different genes have been used to detect their role in both steady state and in irradiated conditions, expected to lead to further insight to the damage and restorative processes. Many different external agents have been used to ameliorate intestinal reactions, including prostaglandins, interleukins, angiogenic and epithelial growth factors, other cytokines, and intraluminal factors.

Effects of dose rates on radiation-induced replenishment of intestinal stem cells determined by Lgr5 lineage tracing.

An understanding of the dynamics of intestinal Lgr5(+) stem cells is important for elucidating the mechanism of colonic cancer development. We previously established a method for evaluating Lgr5(+) stem cells by tamoxifen-dependent Lgr5-lineage tracing and showed that high-dose-rate radiation stimulated replenishment of colonic stem cells. In this study, we evaluated the effects of low-dose-rate radiation on stem cell maintenance. Tamoxifen (4OHT)-injected Lgr5-EGFP-IRES-Cre(ERT2) × ROSA-LSL-LacZ mice were used, LacZ-labeled colonic crypts were enumerated, and the loss of LacZ(+) crypts under low-dose-rate radiation was estimated. After 4OHT treatment, the number of LacZ-labeled Lgr5(+) stem cells was higher in the colon of infant mice than in adult mice. The percentage of LacZ-labeled crypts in infant mice rapidly decreased after 4OHT treatment. However, the percentage of labeled crypts plateaued at ∼2% at 4 weeks post-treatment and remained unchanged for up to 7 months. Thus, it will be advantageous to evaluate the long-term effects of low-dose-rate radiation. Next, we determined the percentages of LacZ-labeled crypts irradiated with 1 Gy administered at different dose rates. As reported in our previous study, mice exposed to high-dose-rate radiation (30 Gy/h) showed a marked replenishment (P = 0.04). However, mice exposed to low-dose-rate radiation (0.003 Gy/h) did not exhibit accelerated stem-cell replenishment (P = 0.47). These findings suggest the percentage of labeled crypts can serve as a useful indicator of the effects of dose rate on the stem cell pool.

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.