Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (L?brich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.     

DNA double-strand break misrejoining after exposure of primary human fibroblasts to CK characteristic X rays, 29 kVp X rays and 60Co gamma rays

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co gamma rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co gamma rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co gamma rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.     

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.     

Comparative radiation tolerance based on the induction of DNA double-strand breaks in tobacco BY-2 cells and CHO-K1 cells irradiated with gamma rays

Higher plants are generally more tolerant to ionizing radiation than mammals. To explore the radiation tolerance of higher plants, the induction of DNA double-strand breaks (DSBs) by gamma rays was investigated in tobacco BY-2 cells and compared with that in Chinese hamster ovary (CHO)-K1 cells as a reference. This is the first examination of radiation-induced DSBs in a higher plant cell. The resulting DNA fragments were separated by pulsed-field gel electrophoresis and stained with SYBR Green I. The initial yield of DSBs was then quantified from the fraction of DNA fragments shorter than 1.6 Mbp based on the assumption of random distribution of DSBs. The DSB yield in tobacco BY-2 cells (2.0 +/- 0.1 DSBs Gbp(-1) Gy(-1)) was only one-third of that in CHO-K1 cells. Furthermore, the calculated number of DSBs per diploid cell irradiated with gamma rays at the mean lethal dose was five times greater in BY-2 cells (263 +/- 13) than in CHO-K1 cells. These results suggest that the radiation tolerance of BY-2 cells appears to be due not only to a lower induction of DNA damage but also to a more efficient repair of the induced DNA damage.     

Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.

Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.     

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

Induction and processing of oxidative clustered DNA lesions in 56Fe-ion-irradiated human monocytes

Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0-10 Gy of (56)Fe ions (1.046 GeV/ nucleon, LET = 148 keV/microm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The (56)Fe ions produced decreased yields of DSBs (10.9 DSB Gy(-1) Gbp(-1)) and clusters (1 DSB: approximately 0.8 Fpg clusters: approximately 0.7 Endo III clusters: approximately 0.5 Endo IV clusters) compared to previous results with (137)Cs gamma rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with gamma rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with gamma rays. The enhanced levels of apoptosis observed after exposure to (56)Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or "splitting" during DNA replication.     

Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells

A new method is described for detecting DNA double-strand breaks (DSBs) that utilizes asymmetric field inversion gel electrophoresis (AFIGE). DNA purified from cells in agarose plugs is subjected to AFIGE and DNA breakage quantitated by the fraction of DNA released from the plug. To test the specificity of the method for DNA DSBs, purified DNA in agarose plugs was treated for increasing times with restriction endonuclease, XhoI. After an initial time period, the fraction of DNA released increased in direct proportion to time. This correlates with the expected response for a randomly broken DNA molecule. In contrast, treatment with the single-strand breaking agent, hydrogen peroxide, over a 1000-fold range produced no release of DNA from the plug. Thus the assay appears to be specific for DNA DSBs and was used to measure DNA breaks induced by gamma radiation. Purified DNA, irradiated in agarose plugs, exhibited a log-linear dose response up to doses that release greater than 90% DNA from the plug. When live cells were irradiated in agarose, a similar linear dose response was observed up to 40 Gy and a significant signal as low as 2.5 Gy. Also in live cells, a threefold lower percentage of DNA was released from the plug over the same dose range. However, less DNA per gray is released at doses above 40 Gy and may reflect a crosslinking effect produced by the irradiation of DNA in live cells. DNA which was "pulse-labeled" was used to test the effect of DNA replication on the ability of AFIGE to detect DNA DSBs. Replicating DNA irradiated in the cell or after purification exhibited a reduced rate of release from the plug per dose of irradiation. Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.     

Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.     

Dose-dependent misrejoining of radiation-induced DNA double-strand breaks in human fibroblasts: experimental and theoretical study for high- and low-LET radiation

Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X rays and high-LET particles (helium, nitrogen and iron) in the dose range 10-80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2-Mbp restriction fragment was analyzed directly after exposure and after 16 h of repair incubation. It was found that the misrejoining frequency for X rays was nonlinearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high-LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X rays, particularly at the lower doses. These experimental results were simulated with a Monte Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that the data for both low- and high-LET radiation could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 microm. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al., Radiat. Res. 158, 568-580, 2002). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also be partly due to differences between fibroblasts and lymphocytes.     

肿瘤坏死因子α和β对电离辐射诱导细胞凋亡的效应

为探讨肿瘤坏死因子（tumor necrosis foctor)α和β（TNFα和β）对电离辐射诱发细胞凋亡的效应及其机理,采用DNA琼脂糖凝胶电泳和FACS分析等方法,观察了人肿瘤坏死因子α（hTNFα)和β（hTNFβ)对^60Co-γ射线诱发细胞凋亡的形态学,生化学变化。结果显示：hTNFα或hTNFβ均可明显抑制^60Co-γ射线诱发正常人胚肺二倍体细胞（2BS）的凋亡,而相同剂量的hTNFα能促进^60Co-γ射线诱发的人体肺腺癌细胞系A549细胞凋亡,而对另一株人体肺癌SPC细胞的效应比A549降低1倍;hTNFβ能分别增强A549和SPC的细胞凋亡频率。由此认为,hTNFα和hTNFβ均可通过调节细胞的生理生化反应来改变细胞对电离辐射的敏感性,可保护正常细胞免受辐射损伤,而增加某些肿瘤细胞对辐射的敏感性。     

Changes of chromatin structure state in thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation

The paper deals with changes in the structural state of chromatin in isolated thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation. Content of necrosis and apoptosis cells in the suspension of the isolated rat thymocites, during 3-hour incubation after X-ray irradiation in a dose of 4.5 Gy or with the presence of 0.1 microM of H2O2 by the method of double lifetime staining by fluorescent dye Hehst 33342 and propydium iodide has been estimated. Apoptogenic effect of the studied effects has been found out, the dynamics of condensation and internucleosomic chromatin fragmentation has been established. It has been shown that 100 microM alpha-tocopherol inhibited completely DNA fragmentation in the cells incubated with H2O2 and only partially in irradiated cells. Introduction of postmitochondrial supernatant, isolated from the incubated control or irradiated cells, into the cell-free system which included the ATP-regenerating system and nuclei of control thymocites did not affect the level of DNA fragmentation, while the increase of the level of fragmented DNA in nuclei was observed in the presence of the supernatant obtained by centrifugation of the cells treated by H2O2. Differences of mechanisms of thymocite apoptosis initiation, as affected by hydrogen peroxide and ionizing radiation, is discussed.     

Clustered DNA damages induced by x rays in human cells

Although DNA DSBs are known to be important in producing the damaging effects of ionizing radiation in cells, bistranded clustered DNA damages-two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns-are postulated to be difficult to repair and thus to be critical radiation-induced lesions. Gamma rays can induce clustered damages in DNA in solution, and high-energy iron ions produce DSBs and oxidized pyrimidine clusters in human cells, but it was not known whether sparsely ionizing radiation can produce clustered damages in mammalian cells. We show here that X rays induce abasic clusters, oxidized pyrimidine clusters, and oxidized purine clusters in DNA in human cells. Non-DSB clustered damages comprise about 70% of the complex lesions produced in cells. The relative levels of specific cluster classes depend on the environment of the DNA.     

HIV-1Tat蛋白抑制DNA修复和增强细胞辐射敏感性

近年来临床研究发现,艾滋病合并肿瘤患者放疗后产生的正常组织和皮肤毒性反应明显高于普通肿瘤患者.本研究将探讨HIV-1Tat蛋白是否影响细胞对电离辐射敏感性及机理. 两个表达Tat蛋白的细胞系TT2和TE671-Tat均来源于人的横纹肌肉瘤细胞（TE671）并已转染了不同来源的tat基因.使用细胞辐射后克隆形成率检测辐射敏感性,RT-PCR和Western 印迹检测基因表达,彗星电泳和γ-H2AX位点检测DNA双链断裂和修复. TT2和TE671-Tat细胞的辐射敏感性与转染空载体及对照细胞相比明显增加.彗星电泳和γ-H2AX位点检测表明,在表达Tat蛋白的细胞中,辐射诱导DNA双链断裂的修复水平明显降低.通过RT-PCR和Western 印迹检测进一步证实,表达Tat蛋白的细胞中DNA修复蛋白DNA-PKcs的表达被抑制. HIV-1Tat蛋白抑制DNA-PKcs的表达,降低DNA双链断裂的修复,使细胞的电离辐射敏感性增高.本研究为了解AIDS合并肿瘤患者对放射治疗敏感性变化提供了重要信息.     

Chromatin remodeling finds its place in the DNA double-strand break response

The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells.     

Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.     

Inhibition of repair of radiation-induced DNA double-strand breaks by nickel and arsenite

The effect of arsenite or nickel on the repair of DNA double-strand breaks (DSBs) was studied in gamma-irradiated Chinese hamster ovary cells using pulsed-field gel electrophoresis. After treatment with nickel chloride or arsenite for 2 h, cells were irradiated with gamma rays at a dose of 40 Gy, and the numbers of DNA DSBs were measured immediately after irradiation as well as at 30 min postirradiation. Both arsenite and nickel(II) inhibited repair of DNA DSBs in a concentration-dependent manner; 0.08 mM arsenite significantly inhibited the rejoining of DSBs, while 76 mM nickel was necessary to observe a clear inhibition. The mean lethal concentrations for the arsenite and nickel(II) treatments were approximately 0.12 and 13 mM, respectively. This indicates that the inhibition of repair by arsenite occurred at a concentration at which appreciable cell survival occurred, but that nickel(II) inhibited repair only at cytotoxic concentrations at which the cells lost their proliferative ability. These novel observations provide insight into the mechanisms underlying the effects of combined exposure to arsenite and ionizing radiation in our environment.     

ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks

Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU)—substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to radiation doses between 0.2–20 Gy, as determined by pulsed-field gel electrophoresis and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68) and p53 (ser15) phosphorylation. Interestingly, low levels (≤2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (>2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.Key words: bromodeoxyuridine, DNA repair, MAP kinase, p53, KU-55933, U87 glioma cells     

The Effects of Low-dose Ionizing Radiation in the Activated Rat Basophilic Leukemia (RBL-2H3) Mast Cells

Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, β-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca(2+)](i)). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcεRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcεRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.