Identification and characterization of inheritable structural variations induced by ion beam radiations in rice.

High energy ion beams are effective physical mutagens for mutation induction in plants. Due to their high linear energy transfer (LET) property, they are known to generate single nucleotide variations (SNVs) and insertion/deletions (InDels, <50 bp) as well as structural variations (SVs). However, due to the technical difficulties to identify SVs, studies on ion beam induced SVs by genome sequencing have so far been limited in numbers and inadequate in nature, and knowledge of SVs is scarce with regards to their characteristics. In the present study, we identified and validated SVs in six M4 plants (designated as Ar_50, Ar_100, C_150, C_200, Ne_50 and Ne_100 according to ion beam types and irradiation doses), two each induced by argon (40Ar18+), carbon (12C6+) and neon (20Ne10+) ion beams and performed in depth analyses of their characteristics. In total, 22 SVs were identified and validated, consisting of 11 deletions, 1 duplication, and 4 intra-chromosomal and 6 inter-chromosomal translocations. There were several SVs larger than 1 kbp. The SVs were distributed across the whole genome with an aggregation with SNVs and InDels only in the Ne_50 mutants. An enrichment of a 11-bp wide G-rich DNA motif 'GAAGGWGGRGG' was identified around the SV breakpoints. Three mechanisms might be involved in the SV formation, i.e., the expansion of tandem repeats, transposable element insertion, and non-allelic homologous recombination. Put together, the present study provides a preliminary view of SVs induced by Ar, C and Ne ion beam radiations, and as a pilot study, it contributes to our understanding of how SVs might form after ion beam irradiation in rice.

Lethal DNA Lesions Caused by Direct and Indirect Actions of X rays are Repaired via Different DSB Repair Pathways under Aerobic and Anoxic Conditions.

We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.

Scaling parameter of the lethal effect of mammalian cells based on radiation-induced OH radicals: effectiveness of direct action in radiation therapy.

We have been studying the effectiveness of direct action, which induces clustered DNA damage leading to cell killing, relative to indirect action. Here a new criterion Direct Ation-Based Biological Effectiveness (DABBLE) is proposed to understand the contribution of direct action for cell killing induced by C ions. DABBLE is defined as the ratio of direct action to indirect action. To derive this ratio, we describe survival curves of mammalian cells as a function of the number of OH radicals produced 1 ps and 100 ns after irradiation, instead of the absorbed dose. By comparing values on the vertical axis of the survival curves at a certain number of OH radicals produced, we successfully discriminate the contribution of direct action induced by C ions from that of indirect action. DABBLE increases monotonically with increasing linear energy transfer (LET) up to 140 keV/µm and then drops, when the survival curves are described by the number of OH radicals 1 ps after irradiation. The trend of DABBLE is in agreement with that of relative biological effectiveness (RBE) of indirect action. In comparison, the value of DABBLE increases monotonically with LET, when the survival curves are described by the number of OH radicals 100 ns after irradiation. This finding implies that the effectiveness of C ion therapy for cancer depends on the contribution of direct action and we can follow the contribution of direct action over time in the chemical phase.

Mutagenic Effect of Three Ion Beams on Rice and Identification of Heritable Mutations by Whole Genome Sequencing.

High-energy ion beams are known to be an effective and unique type of physical mutagen in plants. However, no study on the mutagenic effect of argon (Ar) ion beam radiation on rice has been reported. Genome-wide studies on induced mutations are important to comprehend their characteristics for establishing knowledge-based protocols for mutation induction and breeding, which are still very limited in rice. The present study aimed to investigate the mutagenic effect of three ion beams, i.e., Ar, carbon (C) and neon (Ne) on rice and identify and characterize heritable induced mutations by the whole genome sequencing of six M4 plants. Dose-dependent damage effects were observed on M1 plants, which were developed from ion beam irradiated dry seeds of two indica (LH15, T23) and two japonica (DS551, DS48) rice lines. High frequencies of chlorophyll-deficient seedlings and male-sterile plants were observed in all M2 populations (up to ~30% on M1 plant basis); plants from the seeds of different panicles of a common M1 plant appeared to have different mutations; the whole genome-sequencing demonstrated that there were 236-453 mutations in each of the six M4 plants, including single base substitutions (SBSs) and small insertion/deletions (InDels), with the number of SBSs ~ 4-8 times greater than that of InDels; SBS and InDel mutations were distributed across different genomic regions of all 12 chromosomes, however, only a small number of mutations (0-6) were present in exonic regions that might have an impact on gene function. In summary, the present study demonstrates that Ar, C and Ne ion beam radiation are all effective for mutation induction in rice and has revealed at the genome level the characteristics of the mutations induced by the three ion beams. The findings are of importance to the efficient use of ion beam radiation for the generation and utilization of mutants in rice.

Combination of agents modifying effects in hadrontherapy: modelization of the role of HO° free radicals.

Purpose: A study is presented of the irradiation of cancerous cervical cell line HeLa loaded with a platinum salt, betamethasone and deoxyglucose. The presence of the platinum increases the free-radical concentration and augments the cell death rate, whereas betamethasone or deoxyglucose induces radiosensitization by the alteration of metabolic pathways. Two by two combinations of these chemicals are made to investigate the possible benefit when two radiosensitizers are present. A model is proposed to understand the results of the presence of two modifying agents on the dose effects.Materials and methods: The cells were incubated for 6 h in the presence of the following molecules: dichloro terpyridine platinum, concentration C = 350 µM, betamethasone and deoxyglucose with concentrations of C = 0.2 µM and C = 6 mM, respectively. The cells were subsequently irradiated by carbon C6+ ion 290 MeV/amu up to a dose of 2.5 Gy, under atmospheric conditions.Results: The presence of the platinum salt or bethamethasone augments the cell death rate. The combination of betamethasone with the platinum salt also increases the cell death rate, but less than for the platinum salt alone. The explanation is that any radiosensitizer also behaves as a scavenger of free radicals. This dual behavior should be considered in any optimization of the design of radiosensitizers when different ionizing particles are used.

Overexpression of Ras-Related C3 Botulinum Toxin Substrate 2 Radiosensitizes Melanoma Cells In Vitro and In Vivo.

Radioresistance is the major obstacle in the radiotherapy of the malignant melanoma. Thus, it is of importance to increase the radiosensitivity of melanoma cells. In the present study, the radioresistant melanoma cell line OCM-1 with inducible overexpression of Ras-related C3 botulinum toxin substrate 2 was established based on a radiation-inducible early growth response gene (Egr-1) promoter. The effects of Ras-related C3 botulinum toxin substrate 2 overexpression on the radiosensitivity of melanoma cells exposed to either X-rays or carbon ion beams were evaluated in cultured cells as well as xenograft tumor models. In addition, both reactive oxygen species yield and the NADPH oxidase activity were measured in the irradiated melanoma cells. It was found that the radiation-inducible overexpression of Ras-related C3 botulinum toxin substrate 2 sensitized the melanoma cells to both X-rays and carbon ion irradiation by enhancing the NADPH oxidase activity and the subsequent reactive oxygen species production. Besides, the overexpression of Ras-related C3 botulinum toxin substrate 2 enhanced the tumor-killing effect of radiotherapy in xenograft tumors significantly. The results of this study indicate that Ras-related C3 botulinum toxin substrate 2 is promising in increasing the radiosensitivity of melanoma cells, which provides experimental evidence and theoretical basis for clinical radiosensitization of the malignant melanoma.

Both irradiated and bystander effects link with DNA repair capacity and the linear energy transfer.

AIMS: In comparison with a low linear energy transfer (LET) radiation, a high-LET radiation induces more complex DNA damage. This study wonders whether radiation-induced bystander effect (RIBE) is dependent of LET. MATERIALS AND METHODS: Chinese hamster ovary CHO-9 cells and its subline EM-C11 cells (SSB repair deficient) and XR-C1 cells (DSB repair deficient) were irradiated by γ-rays, α-particles, or carbon ions with different LETs of 13, 30 and 70 keV/µm. Cell proliferation, cell death, DNA damage, cell cycle distribution and some protein expressions were measured with the cell counting kit-8 (CCK-8), colony formation, micronuclei (MN), flow cytometry and western blot, respectively. KEY FINDINGS: A series of cell responses were induced by these radiations in a LET-dependent manner, including proliferation inhibition, cell death, MN induction, G2/M phase arrest and the expression of γH2AX protein. These cell injuries were also depended on DNA repair capacity, and XR-C1 cells were the most sensitive to each radiation. Furthermore, when the cells were treated with the conditioned medium (CM) collected from irradiated CHO-9 cells, the MN induction and cell death response in the bystander cells of EM-C11 or XR-C1 increased along with LET of irradiation, and the bystander damage was easier to be induced in EM-C11 and XR-C1 cells than that in CHO-9 cells. SIGNIFICANCE: Both cellular DNA repair capacity and the LET value of radiation could deeply influence damage extents of not only the irradiated cells but also the bystander cells.

ESTIMATION OF RBE VALUES FOR CARBON-ION BEAMS IN THE WIDE DOSE RANGE USING MULTICELLULAR SPHEROIDS.

Hypofractionated carbon-ion therapy has been applied to treatment of several tumours. In this case, relative biological effectiveness (RBE) at high dose region must be considered, however, the RBE calculated physically has been not verified biologically. In this study, spheroid technique was adopted to estimate RBE in wide dose range. Cells were irradiated with X-rays and heavy-ions with LET of 13, 35, 100 and 300 keV/µm with monolayer and spheroid condition. Surviving fractions in wide dose range (0-15 Gy) were obtained to combined monolayer with spheroid survival data. The linear-quadratic and multi-target single-hit equation fitted well in survival data at low dose, and high dose region, respectively. A multi-process equation showed best fitting for survival data in wide dose range. RBE values of heavy-ions could be estimated by combination of monolayer and spheroid data. The values converged at 1.1-1.4 and varied by LET values at high and low dose region, respectively.

Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas.

The goal of this work was to clarify the effect of carbon-ion beams on reduction of the metastatic potential of malignant melanoma using in vitro and in vivo techniques. We utilized a 290 MeV/u carbon beam with a 6-cm spread-out Bragg-peak (SOBP), 137Cs γ rays or 200 kVp X rays for irradiation, and in vitro murine melanoma B16/BL6 cells that were implanted into C57BL/6J mice. The metastatic abilities (migration, invasion and adhesion) were suppressed by carbon ion treatment at all doses that were tested, whereas invasion and migration tended to increase after X-ray irradiation at low dose. Biological effects of carbon ions increased with linear energy transfer (LET) for both cell killing and metastatic abilities, although the effects were more pronounced for migration and invasion. mRNA expression of E-cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expression of Mel-CAM and L1-CAM was upregulated after low-dose photon exposure, but decreased with dose, especially after carbon-ion treatment. Conversely, these molecules showed a reversal in expression changes, especially after low-dose photon exposure. Cell-cell adhesion may be an important contributor to the antimetastatic effect of carbon ion treatment. The number of lung metastases after local tumor irradiation significantly decreased with increased dose and LET, with carbon ions being more effective than γ rays. Integrating dose-response curves to examine the relationship between cell killing and lung metastasis clearly showed that carbon ions inhibit lung metastasis more efficiently than photons at the iso-effective level of cell killing. Thus, carbon ions were more effective than photon beams, not only at killing tumor cells, but also at inhibiting metastatic spread caused by tumor cells that survived irradiation.

The Potential Application of Heavy Ion Beams in the Treatment of Arrhythmia: The Role of Radiation-Induced Modulation of Connexin43 and the Sympathetic Nervous System.

It has been known that heart disease-such as myocardial infarction (MI), cardiac hypertrophy, or heart failure-alters the molecular structure and function of the gap junction, which can lead to an abnormal heart rhythm. Radiation has been shown to modulate intercellular communication in the skin and lungs by increasing connexin43 (Cx43) expression. Understanding how Cx43 upregulation is induced in a diseased heart can help provide a new perspective to radiation therapy for arrhythmias. In a recent study with rabbits after MI, carbon ions were accelerated to 290 MeV/u and extracted in the air; a biologically (cell kill) uniform 6-cm spread-out Bragg peak beam was generated, and beam tissue depth was set to 30 mm with energy degraders to the depth position. Targeted heavy ion irradiation (THIR) with 15 Gy to the left ventricle increased Cx43 expression, improved conductivity, decreased the spatial heterogeneity of repolarization, and reduced the vulnerability of rabbit hearts to ventricular arrhythmias after MI. In clinically normal rabbits, THIR > = 10 Gy caused a significant dose-dependent increase of Cx43 protein and messenger RNA 2 weeks after irradiation. The left (irradiated) and right (nonirradiated) ventricles exhibited circumferential upregulation of Cx43 lasting for at least 1 year. There were no significant changes in electrocardiograms and echocardiograms, indicating no apparent injury for 1 year. A single exposure of 135 MeV/u THIR with 15 Gy to a dog heart attenuated vulnerability to ventricular arrhythmia after the induction of MI for at least 1 year through the modulation of Cx43 expression. This long-lasting remodeling effect on gap junctions may lay the groundwork to novel therapies against life-threatening ventricular arrhythmias in structural heart disease. To date, there have been few investigations into the effects of carbon-ion irradiation on electrophysiological properties in the human heart. Patients with mediastinum cancer were investigated for 5 years after treatment that included irradiation to the heart, and investigators found that carbon-ion beam irradiation to the heart is not immediately cardiotoxic and demonstrates consistent signals of arrhythmia reduction. Its practical application in non-cancer treatment, such as in arrhythmia treatment, is highly anticipated.

Biological effects of ion beam irradiation on perennial gentian and apple.

The development of new varieties of perennial plants generally requires lengthy and laborious procedures. In this study, we used ion beam irradiation mutagenesis in an attempt to accelerate the breeding process for perennial plants. We evaluated the biological effects of five ion beam sources (carbon, neon, argon, silicon, and iron) and neutron irradiation on Japanese gentian and apple. These treatments were applied at the National Institute of Radiological Sciences (NIRS) using the Heavy Ion Medical Accelerator in Chiba (HIMAC) and the Neutron-exposure Accelerator System for Biological Effect Experiments (NASBEE). Biological effects were observed in in vitro gentian plants after irradiation with ion beams at <10 Gy, whereas apple trees were less sensitive to ion beam irradiation. The growth of gentians in vitro was repressed by 3 Gy neutron irradiation, while that of grafted apple trees was not affected by 4 Gy neutron irradiation. During in vitro proliferation, seven pink-flowered lines were obtained from originally blue-flowered gentian after C and Ne ion beam irradiation treatments. Genomic and reverse transcription-PCR analyses of these lines suggested that the mutations occurred in the genomic region containing F3'5'H (encoding flavonoid 3',5'-hydroxylase). These results provide useful information for the mutagenesis and breeding of gentian, apple, and other perennial plants.

Changes in arrhythmogenic properties and five-year prognosis after carbon-ion radiotherapy in patients with mediastinum cancer.

INTRODUCTION: Carbon-ion irradiation of rabbit hearts has improved left ventricular conduction abnormalities through upregulation of gap junctions. However, to date, there has been no investigation on the effect of carbon-ion irradiation on electrophysiological properties in human. We investigated this effect in patients with mediastinum extra-cardiac cancer treated with carbon-ion radiotherapy that included irradiating the heart. METHODS AND RESULTS: In April-December 2009, eight patients were prospectively enrolled (including two male, aged 72.5 ± 13.0 years). They were treated with 44-72 Gray equivalent (GyE), with their hearts exposed to 1.3-19.1 GyE. High-resolution ambulatory electrocardiography was performed before and after radiotherapy to investigate arrhythmic events, late potentials (LPs), and heart rate variability. Five patients had pre-existing premature ventricular contraction (PVC)/atrial contraction (PAC) or paroxysmal atrial fibrillation (PAF)/AF; after irradiation, this improved in four patients with PVC/PAF/AF and did not deteriorate in one patient with PAC. Ventricular LP findings did not deteriorate and improved in one patient. In eight cases with available atrial LP findings, there was no deterioration, and two patients showed improvements. The low frequency/high frequency ratio of heart rate variability improved or did not deteriorate in the six patients who received radiation exposure to the bilateral stellate ganglions. During the five-year follow-up for the prognosis, six of the eight patients died because of cancer; there was no history of hospitalization for cardiac events. CONCLUSION: Although this preliminary study has several limitations, carbon-ion beam irradiation to the heart is not immediately cardiotoxic and demonstrates consistent signals of arrhythmia reduction.

Biological effects of carbon ion beams with various LETs on budding yeast Saccharomyces cerevisiae.

It has been established that irradiation with higher linear energy transfer (LET) increases lethality and mutagenicity more than that with lower LET. However, the characteristics specific to carbon ion beam have not yet been elucidated. Yeast cells were irradiated with carbon ions with an LET of 13 or 50keV/µm, and cell survival and mutation frequency were analyzed. The results, combined with our previous findings for ions with an LET of 107keV/µm, demonstrated that, in conjunction with an increase in LET, cell survival decreased, while mutation frequency increased. This indicates that a carbon ion beam with a higher LET is more mutagenic than one with a lower LET.

Equivalency of the quality of sublethal lesions after photons and high-linear energy transfer ion beams.

The quality of the sublethal damage (SLD) after irradiation with high-linear energy transfer (LET) ion beams was investigated with low-LET photons. Chinese hamster V79 cells and human squamous carcinoma SAS cells were first exposed to a priming dose of different ion beams at different LETs at the Heavy Ion Medical Accelerator in the Chiba facility. The cells were kept at room temperature and then exposed to a secondary test dose of X-rays. Based on the repair kinetics study, the surviving fraction of cells quickly increased with the repair time, and reached a plateau in 2-3 h, even when cells had received priming monoenergetic high-LET beams or spread-out Bragg peak beams as well as X-ray irradiation. The shapes of the cell survival curves from the secondary test X-rays, after repair of the damage caused by the high-LET irradiation, were similar to those obtained from cells exposed to primary X-rays only. Complete SLD repairs were observed, even when the LET of the primary ion beams was very high. These results suggest that the SLD caused by high-LET irradiation was repaired well, and likewise, the damage caused by the X-rays. In cells where the ion beam had made a direct hit in the core region in an ion track, lethal damage to the domain was produced, resulting in cell death. On the other hand, in domains that had received a glancing hit in the low-LET penumbra region, the SLD produced was completely repaired.

Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling.

Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) "targeted"/irradiated A549-GFP cells; (2) "neighboring"/non-irradiated cells directly contacting the "targeted" cells; and (3) "distant"/non-irradiated cells, which were not in direct contact with the "targeted" cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi-directional signaling had extended a profound influence on cellular sensitivity to radiation as well as the sensitivity to RIBE.

Amino acid transport system - A substrate predicts the therapeutic effects of particle radiotherapy.

L-[methyl-11C]Methionine (11C-Met) is useful for estimating the therapeutic efficacy of particle radiotherapy at early stages of the treatment. Given the short half-life of 11C, the development of longer-lived 18F- and 123I-labeled probes that afford diagnostic information similar to 11C-Met, are being sought. Tumor uptake of 11C-Met is involved in many cellular functions such as amino acid transport System-L, protein synthesis, and transmethylation. Among these processes, since the energy-dependent intracellular functions involved with 11C-Met are more reflective of the radiotherapeutic effects, we evaluated the activity of the amino acid transport System-A as an another energy-dependent cellular function in order to estimate radiotherapeutic effects. In this study, using a carbon-ion beam as the radiation source, the activity of System-A was evaluated by a specific System-A substrate, alpha-[1-14C]-methyl-aminoisobutyric acid (14C-MeAIB). Cellular growth and the accumulation of 14C-MeAIB or 14C-Met were evaluated over time in vitro in cultured human salivary gland (HSG) tumor cells (3-Gy) or in vivo in murine xenografts of HSG tumors (6- or 25-Gy) before and after irradiation with the carbon-ion beam. Post 3-Gy irradiation, in vitro accumulation of 14C-Met and 14C-MeAIB decreased over a 5-day period. In xenografts of HSG tumors in mice, tumor re-growth was observed in vivo on day-10 after a 6-Gy irradiation dose, but no re-growth was detected after the 25-Gy irradiation dose. Consistent with the growth results, the in vivo tumor accumulation of 14C-MeAIB did not decrease after the 6-Gy irradiation dose, whereas a significant decrease was observed after the 25-Gy irradiation dose. These results indicate that the activity of energy dependent System-A transporter may reflect the therapeutic efficacy of carbon-ion radiotherapy and suggests that longer half-life radionuclide-labeled probes for System-A may also provide widely available probes to evaluate the effects of particle radiotherapy on tumors at early stage of the treatment.

Inducibility of Ventricular Arrhythmia 1 Year Following Treatment with Heavy Ion Irradiation in Dogs with Myocardial Infarction.

BACKGROUND: Targeted external heavy ion irradiation (THIR) of rabbit hearts 2 weeks after myocardial infarction (MI) reduced the vulnerability of fatal ventricular tachyarrhythmias (VT/VF) in association with the increased connexin43 (Cx43). Increased Cx43 was maintained for at least 1 year in normal rabbits, but the long-term antiarrhythmic effects in the MI model are unknown. We investigated the propensity for late potentials and VT/VF inducibility. METHODS: Intracoronary injection of microspheres was performed to induce nontransmural MI in anesthetized eight beagles. Four beagles were treated with THIR (12 C6+ , 15 Gy) 2 weeks later (MI + THIR group), and four without THIR served as controls (MI group). Signal-averaged electrocardiography, programmed electrical stimulation, immunohistochemical analysis, and echocardiograms were performed at 1 year. RESULTS: Filtered QRS duration was exacerbated after MI and remained unchanged for 1 year in the MI group (118 ± 1.4 ms), but significantly returned toward baseline in the MI + THIR group (109 ± 6.9 ms). Similarly, root mean square voltage of the last 40 ms was exacerbated after MI, but recovered after THIR. VT/VF inducibility decreased to 25% in the MI + THIR group compared with 100% in the MI group. Immunostaining Cx43 expression in cardiac tissues significantly increased by 24-45% in the MI + THIR group. Left ventricular ejection fractions remained within the normal range in both groups. CONCLUSION: A single exposure of the dog heart to 12 C irradiation attenuated vulnerability to ventricular arrhythmia after the induction of MI for at least 1 year through the modulation of Cx43 expression.

RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation.

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.

Metformin enhances the radiosensitivity of human liver cancer cells to γ-rays and carbon ion beams.

The purpose of this study was to investigate the effect of metformin on the responses of hepatocellular carcinoma (HCC) cells to γ-rays (low-linear energy transfer (LET) radiation) and carbon-ion beams (high-LET radiation). HCC cells were pretreated with metformin and exposed to a single dose of γ-rays or carbon ion beams. Metformin treatment increased radiation-induced clonogenic cell death, DNA damage, and apoptosis. Carbon ion beams combined with metformin were more effective than carbon ion beams or γ-rays alone at inducing subG1 and decreasing G2/M arrest, reducing the expression of vimentin, enhancing phospho-AMPK expression, and suppressing phospho-mTOR and phospho-Akt. Thus, metformin effectively enhanced the therapeutic effect of radiation with a wide range of LET, in particular carbon ion beams and it may be useful for increasing the clinical efficacy of carbon ion beams.

PU-H71, a novel Hsp90 inhibitor, as a potential cancer-specific sensitizer to carbon-ion beam therapy.

PU-H71, a heat shock protein 90 (Hsp90) inhibitor, has yielded therapeutic efficacy in many preclinical models and is currently in clinical trials. Carbon-ion radiotherapy (CIRT) has provided successful tumor control; however, there is still room for improvement, particularly in terms of tumor-specific radiosensitization. The Hsp90 inhibitor PU-H71 has been shown to sensitize tumor cells to X-ray radiation. A murine osteosarcoma cell line (LM8) and a normal human fibroblast cell line (AG01522) were treated with PU-H71 before X-ray, 14- or 50-keV/µm carbon-ion beam (C-ion) irradiation. Cell survival and protein expression were evaluated with colony formation and western blot, respectively. Treatment with PU-H71 alone was shown to be non-toxic to both cell lines; however, PU-H71 was shown to significantly sensitize LM8 cells to not only X-ray, but also to C-ion irradiation, while only a minimal sensitizing effect was observed in AG01522 cells. PU-H71 treatment was found to suppress the protein expression levels of Rad51 and Ku70, which are associated with the homologous recombination pathway and the non-homologous end-joining pathway of double-strand break repair. The findings reported here suggest that PU-H71 could be a promising radiosensitizer for CIRT.