Linear Energy Transfer Radiation Research Articles

Interaction of low and high LET radiation in TK6 cells—mechanistic aspects and significance for radiation protection

Most environmental, occupational and medical exposures to ionising radiation are associated with a simultaneous action of different radiation types. An open question remains whether radiations of different qualities interact with each other to yield effects stronger than expected based on the assumption of additivity. It is possible that DNA damage induced by high linear energy transfer (LET) radiation will lead to an opening of the chromatin structure making the DNA more susceptible to attack by reactive oxygen species (ROS) generated by the low LET radiation. In such case, the effect of mixed beams should be strongly expressed in cells that are sensitive to ROS. The present investigation was carried out to test if cells with an impaired capacity to handle oxidative stress are particularly sensitive to the effect of mixed beams of alpha particles and x-rays. Clonogenic cell survival curves and mutant frequencies were analysed in TK6 wild type (wt) cells and in TK6 cells with a knocked down hMYH glycosylase. The results showed a synergistic effect of mixed beams on clonogenic cell survival of TK6wt but not TK6MYH- cells. The frequencies of mutants showed a high degree of interexperimental variability without any indications for synergistic effects of mixed beams. TK6MYH- cells were generally more tolerant to radiation exposure with respect to clonogenic cell survival but showed a strong increase in mutant frequency. The results demonstrate that exposure of wt cells to a mixed beam of alpha particles and x-rays leads to a detrimental effect which is stronger than expected based on the assumption of additivity. The role of oxidative stress in the reaction of cells to mixed beams remains unclear.

Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation

Background and purposeHigh linear energy transfer (LET) radiotherapy offers superior dose conformity and biological effectiveness compared with low-LET radiotherapy, representing a promising alternative for radioresistant tumours. A prevailing hypothesis is that energy deposition along the high-LET particle trajectories induces DNA lesions that are more complex and clustered and therefore more challenging to repair. The precise molecular mechanisms underlying the differences in radiobiological effects between high-LET and low-LET radiotherapies remain unclear. Material and MethodsHuman fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.5h and 5h post exposure, the DNA-damage pattern in the chromatin ultrastructure was visualised using gold-labelled DNA-repair factors. The induction and repair of single-strand breaks, double-strand breaks (DSBs), and clustered lesions were analysed in combination with terminal dUTP nick-end labelling of DNA breaks. ResultsHigh-LET irradiation induced clustered lesions with multiple DSBs along ion trajectories predominantly in heterochromatic regions. The cluster size increased over time, suggesting inefficient DSB repair. Low-LET irradiation induced many isolated DSBs throughout the nucleus, most of which were efficiently rejoined. ConclusionsThe clustering of DSBs in heterochromatin following high-LET irradiation perturbs efficient DNA repair, leading to greater biological effectiveness of high-LET irradiation versus that of low-LET irradiation.

O43. Estimating the effectiveness of high LET radiations to induce damage to DNA in human lymphocytes and modelling the repair thereof

Introduction Neutron and ion beam irradiations have an increased efficiency in cell killing, resulting from their high linear-energy-transfer (LET) which in turn induces clustered and complex DNA breaks. Similarly, Auger electron irradiations have been shown to a have an increased relative biological effectiveness (RBE) when compared to low-LET radiations. We report on our investigations into the RBE of the DNA incorporated Auger electron emitting 123 I and a model which may estimate the induction and repair of γ -H2AX foci (markers of DNA double strand breaks (DSBs)) in human lymphocytes after exposure to fast neutrons. Materials and methods The RBE of 123 I was determined by comparison of micronuclei inductions in T-lymphocytes using 5-[ 123 I]-iodo-2-deoxyuridine and similar inductions by graded doses of 60 Co γ -rays. Additionally, a theoretical model which describes the time-based repair pathways of DNA DSBs using a set of dependent partial differential equations, was investigated. The validity of the model was assessed by comparing the γ -H2AX foci kinetics predicted by the model to experimental curves observed for lymphocytes exposed to neutrons (20 keV/ μ m) and 60 Co γ -rays. Results The dose-limiting RBE of the DNA incorporated 123 I was found to range from 3 to 11. These observed RBE values relate well to those obtained in studies with 125 I incorporated into rodent cell lines. The higher micronuclei frequencies noted for Auger electrons in the study is indicative of the high-LET nature of these particles. Similar to experimental observations, the theoretical repair model predicted that a larger number of DSBs remain unrepaired after neutron irradiation compared to low-LET γ -irradiation. The model was shown to accurately describe and estimate the kinetics of γ -H2AX foci in human lymphocytes after exposure to fast neutrons and 60 Co γ -rays. Conclusion With respect to their role in radiation therapy and protection, there is a need for investigating and developing models which may predict the induction and repair of high-LET radiation induced DNA damage. Acknowledgement This work was supported by a University Development Cooperation VLIR Own Initiative Programme between Belgium and South Africa.

P8. The in vitro comparison of gold nanoparticles in different cell lines

Introduction Gold nanoparticles (AuNPs) have been recognized as effective radiosensitizers of low energy (e.g. 200–500 kV) X-rays, leading to the emission of Auger electrons that cause highly localized ionizing damage to cells. The aim of this study was to determine possible toxicity caused by AuNPs and to establish whether the AuNPs interacts with MV X-rays. Materials and methods The interaction between the 5 and 10 nm AuNPs with 6 MV X-rays were carried out in different cell lines, namely CHO-K1, BEnd5, MCF-7 and MCF-10A. The X-rays is a low linear energy transfer (LET) radiation type, which was scattered by using a Shonka chamber to obtain lower energies X-rays to interact with the AuNPs. The cells were incubated with 50 μg/ml of the respective AuNPs for 4 h and given a 2 Gy dose. The media was replaced and the cells were incubated for 24 h in the presence of cytochalasin B, then fixed and stained with acridine orange. Results In some of the cell types used in this study, higher MNi frequencies were induced by a combination treatment of radiation and AuNPs compared to the additive sum noted in samples exposed to AuNPs and radiation separately. The AuNPs alone reduced the growth rate of all types between 29% and 33%. Conclusion Interactions between the AuNPs and scattered 6 MV X-rays were noted for CHO-K1 and MCF-7 cells, but not for other cell types used in the study. The cellular uptake for AuNPs needs to be quantified to explain the results.

Radiation linear energy transfer and drop size dependence of the low frequency signal from tiny superheated droplets

The bubble nucleation in superheated tiny droplets of R-12 (CCl2F2, boiling point: −29.8°C) induced by neutrons and α-particles has been observed using condenser microphone as an acoustic sensor. The superheated droplets used in these experiments are of very small radii having distribution with main peak around 2–3µm expanding until about 40µm. The low frequency component of the acoustic shock wave released during bubble nucleation has been measured with condenser microphone and the analysis has been done using ROOT software. Pulses due to bubble nucleation have been recorded in the presence of 241Am–Be neutron source (few keV to about 10MeV) and 241Am alpha-source (5.48MeV with intensity 85.2%). The observables related to the power associated with bubble nucleation (P) and the area under the frequency spectrum of the signal (PFreq) have been estimated for the α-particle and neutron induced events. It shows that the α-particle induced bubble nucleation signals are of lower intensity than those obtained from the neutron induced bubble nucleation signals. This phenomenon as observed with tiny droplets is opposite to that observed so far for the larger droplets.

Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells

The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in National Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response.

Abstract 3713: Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles

Abstract Heavy ion therapy is a promising approach for cancer treatment, particularly for locally advanced solid tumors. High linear energy transfer (LET) radiation induces greater lesions density in close proximity (clusters) compared that in low-LET radiation. Such clustered DNA damage is thought to be more difficult to repair than isolated DNA damage produced by equivalent doses of low-LET radiation and persist longer in irradiated cells. It has been known that homologous combination contributes to the repair of clustered DNA damage other than classic non-homologous end-joining (NHEJ). DNA double-strand breaks are also repaired by an alternative NHEJ pathway with PARP1, DNA ligase III and Mre11. This alternative pathway is error prone and contributes to genomic instability, such as chromosomal translocation and telomere fusion. Genetic analysis of tumors in TCGA database revealed that these genes are amplified in human tumors and their overexpression is correlated with poor overall survival of cancer patients. However, influence of alternative NHEJ pathways on the relative biological effectiveness (RBE) of heavy ions remains unclear. Here we investigated the effect of PARP-1 and DNA ligase III on RBE of heavy ion particles on cell survival and chromosomal aberration in breast cancer cells and pancreatic cancer cells. Cells were irradiated with X-ray, Carbon (290 MeV/n, 13 KeV/μM) and Iron (1 GeV/n, 150 KeV/μM). We found that overexpression of PARP-1 and DNA ligase III increased RBE of carbon and iron. PARP inhibitor reduced chromosomal aberration induced by heavy ions. This study may provide predictor of radiation responsive of tumor cells from cancer patients that would lead to patient stratification for heavy ion therapy selection Citation Format: Huichen Wang, Premkuma Saganti. Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3713.

Novel Biological Approaches for Testing the Contributions of Single DSBs and DSB Clusters to the Biological Effects of High LET Radiation.

The adverse biological effects of ionizing radiation (IR) are commonly attributed to the generation of DNA double-strand breaks (DSBs). IR-induced DSBs are generated by clusters of ionizations, bear damaged terminal nucleotides, and frequently comprise base damages and single-strand breaks in the vicinity generating a unique DNA damage-clustering effect that increases DSB “complexity.” The number of ionizations in clusters of different radiation modalities increases with increasing linear energy transfer (LET), and is thought to determine the long-known LET-dependence of the relative biological effectiveness (RBE). Multiple ionizations may also lead to the formation of DSB clusters, comprising two or more DSBs that destabilize chromatin further and compromise overall processing. DSB complexity and DSB-cluster formation are increasingly considered in the development of mathematical models of radiation action, which are then “tested” by fitting available experimental data. Despite a plethora of such mathematical models the ultimate goal, i.e., the “a priori” prediction of the radiation effect, has not yet been achieved. The difficulty partly arises from unsurmountable difficulties in testing the fundamental assumptions of such mathematical models in defined biological model systems capable of providing conclusive answers. Recently, revolutionary advances in methods allowing the generation of enzymatic DSBs at random or in well-defined locations in the genome, generate unique testing opportunities for several key assumptions frequently fed into mathematical modeling – including the role of DSB clusters in the overall effect. Here, we review the problematic of DSB-cluster formation in radiation action and present novel biological technologies that promise to revolutionize the way we address the biological consequences of such lesions. We describe new ways of exploiting the I-SceI endonuclease to generate DSB-clusters at random locations in the genome and describe the possible utility of Zn-finger nucleases and of TALENs in generating DSBs at defined genomic locations. Finally, we describe ways to harness the revolution of CRISPR/Cas9 technology to advance our understanding of the biological effects of DSBs. Collectively, these approaches promise to improve the focus of mathematical modeling of radiation action by providing testing opportunities for key assumptions on the underlying biology. They are also likely to further strengthen interactions between experimental radiation biologists and mathematical modelers.

Transcription Factors in the Cellular Response to Charged Particle Exposure.

Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor’s p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles’ LET, with a maximal activation in the LET range of 90–300 keV/μm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also neighboring cells in the bystander effect.

Ionization density dependence of the curve shape and ratio of blue to UV emissions of Al2O3:C optically stimulated luminescence detectors exposed to 6-MV photon and therapeutic proton beams

In this work we characterized the dose and linear energy transfer (LET) (ionization density) dependence of commercial Al2O3:C optically stimulated luminescence (OSL) detectors (OSLDs) exposed to clinical photon and proton beams. We characterized the dose-dependence of the OSL signal, OSL curve shape, and the relative intensities of the blue and ultraviolet (UV) OSL emission bands using different readout protocols and beam qualities. We irradiated OSLDs with absorbed doses ranging from 0.1 Gy to 100 Gy in a 6-MV photon beam and from 0.1 Gy to 50 Gy in 140- and 250-MeV proton beams. Readouts were done with both continuous-wave (CWOSL) and pulsed (POSL) stimulation. The linearity of the OSLD dose–response depended on readout protocol and radiation type. Improved linearity was found for OSLDs irradiated with beams of increased LET, and for OSL signals containing only the blue emission band of Al2O3:C (which remained linear for doses up to 10 Gy for 140-MeV proton beam irradiations). The OSL curve shape did not vary with dose in the low-dose region (below 5 Gy depending on readout protocol), but beyond this, curves decayed more rapidly with increasing dose. Similarly, the ratio of blue to UV emission band intensities in the OSL signal did not vary with dose up to 5 Gy (depending on readout protocol), beyond which the ratio decreased with increasing dose. Because both the OSL curve shape and the ratio of blue to UV emission intensities have been investigated as potential parameters for measurements of LET, the constancy of these two quantities at doses relevant to radiotherapy is encouraging for the potential development of novel OSL methods to measure LET. Our findings are expected to contribute to the development of (i) improved readout protocols for commercially available Al2O3:C OSLDs and (ii) methods to measure radiation quality and LET.

고체비적검출기를 이용한 500 MeV/u 철 이온의 선에너지전이 교정

In this study, LET (Linear Energy Transfer) calibration of CR-39 SSNTD (Solid State Nuclear Track Detector) was performed using 500 MeV/u Fe heavy ions in HIMAC (Heavy Ion Medical Accelerator) for high LET radiation dosimetry. The irradiated CR-39 SSNDT were etched according JAXA (Japan Aerospace Exploration Agency) etching conditions. And the etched SSNTD were analyzed by using Image J. Determined dose-mean lineal energy () of 500 MeV/u Fe is about 283.3 keV/um by using the CR-39 SSNTD. This value is very similar result compare to the results calculated by GEANT4 Monte Carlo simulation and measured with TEPC active radiation detector. We confirmed that the CR-39 SSNTD was useful for high LET radiation dosimetry such as heavy iron ions.

A dose-response curve for biodosimetry from a 6 MV electron linear accelerator.

Biological dosimetry (biodosimetry) is based on the investigation of radiation-induced biological effects (biomarkers), mainly dicentric chromosomes, in order to correlate them with radiation dose. To interpret the dicentric score in terms of absorbed dose, a calibration curve is needed. Each curve should be constructed with respect to basic physical parameters, such as the type of ionizing radiation characterized by low or high linear energy transfer (LET) and dose rate. This study was designed to obtain dose calibration curves by scoring of dicentric chromosomes in peripheral blood lymphocytes irradiated in vitro with a 6 MV electron linear accelerator (Mevatron M, Siemens, USA). Two software programs, CABAS (Chromosomal Aberration Calculation Software) and Dose Estimate, were used to generate the curve. The two software programs are discussed; the results obtained were compared with each other and with other published low LET radiation curves. Both software programs resulted in identical linear and quadratic terms for the curve presented here, which was in good agreement with published curves for similar radiation quality and dose rates.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation.

BackgroundHigh linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions.FindingsHeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 μM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor.ConclusionsATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0464-y) contains supplementary material, which is available to authorized users.

Formation and repair of clustered damaged DNA sites in high LET irradiated cells

Purpose: Radiation with high linear energy transfer (LET) produces clustering of DNA double-strand breaks (DSB) as well as non-DSB lesions. Heat-labile sites (HLS) are non-DSB lesions in irradiated cells that may convert into DSB at elevated temperature during preparation of naked DNA for electrophoretic assays and here we studied the initial formation and repair of these clustered damaged sites after irradiation with high LET ions.Materials and methods: Induction and repair of DSB were studied in normal human skin fibroblast (GM5758) after irradiation with accelerated carbon and nitrogen ions at an LET of 125 eV/nm. DNA fragmentation was analyzed by pulsed-field gel electrophoresis (PFGE) and by varying the lysis condition we could differentiate between prompt DSB and heat-released DSB.Results: Before repair (t = 0 h), the 125 eV/nm ions produced a significant fraction of heat-released DSB, which appeared clustered on DNA fragments with sizes of 1 Mbp or less. These heat-released DSB increased the total number of DSB by 30–40%. This increase is similar to what has been found in low-LET irradiated cells, suggesting that the relative biological effectiveness (RBE) for DSB induction will not be largely affected by the lysis temperature. After 1–2 hours repair, a large fraction of DSB was still unrejoined but there was essentially no heat-released DSB present.Conclusions: These results suggest that high LET radiation, as low LET gamma radiation, induces a significant fraction of heat-labile sites which can be converted into DSB, and these heat-released DSB may affect both induction yields and estimates of repair.

Determination of the relative biological effectiveness and oxygen enhancement ratio for micronuclei formation using high-LET radiation in solid tumor cells: An in vitro and in vivo study

We determined the relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) of micronuclei (MN) formation in clamped (hypoxic) and non-clamped (normoxic) solid tumors in mice legs following exposure to X-rays and heavy ions. Single-cell suspensions (aerobic) of non-irradiated tumors were prepared in parallel and used directly to determine the radiation response for aerobic cells. Squamous cell carcinoma (SCCVII) cells were transplanted into the right hind legs of syngeneic C3H/He male mice. Irradiation doses with either X-rays or heavy ions at a dose-averaged LET (linear energy transfer) of 14–192keV/μm were delivered to 5-mm diameter tumors and aerobic single-cells in sample-tubes. After irradiation, the tumors were excised and trypsinized to observe MN in single-cells. The single-cell suspensions were used for MN formation assays. The RBE values increased with increasing LET. The maximum RBE values for the three different oxygen conditions; hypoxic tumor, normoxic tumor, and aerobic cells, were 8.18, 5.30, and 3.76 at an LET of 192keV/μm, respectively. After X-irradiation, the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.73 and 2.58, respectively. We found that the OER for the in vivo studies were smaller in comparison to that for the in vitro studies. Both of the OER values at 192keV/μm were small in comparison to those of the X-ray irradiated samples. The OERh/n and OERh/a values at 192keV/μm were 1.12 and 1.19, respectively. Our results suggest that high LET radiation has a large biological effect even if a solid tumor includes substantial numbers of hypoxic cells. To conclude, we found that the RBE values under each oxygen state for non-MN fraction increased with increasing LET and that the OER values for both tumors in vivo and cells in vitro decreased with increasing LET.

Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.

Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late-phase severe adverse effects independently of the TP53 status in vitro. Our findings indicated the feasibility of the combination of Ad-FIR with DNA damaging agents for future esophageal cancer treatment.

Abstract 1815: Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication

Abstract Purpose: Ionizing Radiation (IR)-induced bystander effects and genomic instability have important implication in radiotherapy and radioprotection. Their persistence in the progeny of normal cells may contribute to risk of long-term radiation-related health effect in human, including cancer. Hence, this study investigates the role of gap junction intercellular communication (GJIC) and the quality of radiation in the propagation of stressful effects in the unirradiated bystander cells and their progeny. Material and methods: Human skin fibroblasts in the confluent state were exposed to microbeam irradiations with different linear energy transfer (LET) at mean absorbed dose of 0.4 Gy, in the presence or absence of GJIC inhibitor (AGA) by which 0.036-0.4% of cells were directly targeted by IR. After 4 h irradiation or following 20 population doublings, the cells were harvested and assayed for micronucleus (MN) formation, gene mutation and protein oxidation. Results: Our results showed that high-LET carbon microbeams (LET ∼103 keV/μm) and high-LET proton microbeams (LET ∼11 keV/μm) were more effective than low-LET X ray microbeam (LET ∼6 keV/μm) in the induction of DNA damage (MN formation) in bystander cells. Interestingly, significant attenuation of MN formation occurred in bystander cells in the presence of AGA after proton and carbon microbeams. In contrast, incubation of the cells with AGA did not significantly affect the induction of MN formation in bystander cells during confluent holding after X irradiation. Further, the progeny of bystander cells exposed to X rays or protons showed persistent oxidative stress which correlated MN formation and mutation frequency. Such effects were not observed after irradiation by carbon ions. Importantly, the progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultured were exposed to X rays. Taken together, the overall results show the expression of stressful effects in the bystander cells and their progeny are dependent on the radiation quality or LET. Conclusions: Our findings suggest that the involvement of GJIC-dependent of radiation quality in the propagation of radiation induces stressful effects to bystander cells and their progeny. In addition, this work provides a strong support to the fact that carbon ions can significantly reduce the risk of cancer and have potential implications in the therapeutic outcome of radiotherapy compared to X rays or protons. Citation Format: Narongchai Autsavapromporn, Ianik Plante, Cuihua Liu, Teruaki Konishi, Noriko Usami, Tomoo Funayama, Yukio Uchihori, Tom K. Hei, Edouard I. Azzam, Sirikan Yamada, Takeshi Murakami, Masao Suzuki. Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2015-1815

ATR signaling cooperates with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam.

Little work has been done on the mechanism of low dose hyper-radiosensitivity (HRS) and later appeared radioresistance (termed induced radioresistance (IRR)) after irradiation with medium and high linear energy transfer (LET) particles. The aim of this study was to find out whether ATR pathway is involved in the mechanism of HRS induced by high LET radiation. GM0639 cells and two ATM deficient/mutant cells, AT5BIVA and AT2KY were irradiated by carbon ion beam. Thymidine block technique was developed to enrich the G2-phase population. Radiation induced early G2/M checkpoint was quantitatively assess with dual-parameter flow cytometry by detecting the cells positive for phospho-histone H3. The involvement of ATR pathway in HRS/IRR response was detected with pretreatment of specific inhibitors prior to carbon ion beam. The link between the early G2/M checkpoint and HRS/IRR under carbon ion beam was first confirmed in GM0639 cells, through the enrichment of cell population in G2-phase or with Aurora kinase inhibitor that attenuates the transition from G2 to M phase. Interestingly, the early G2/M arrest could still be observed in ATM deficient/mutant cells with an effect of ATR signaling, which was discovered to function in an LET-dependent manner, even as low as 0.2Gy for carbon ion radiation. The involvement of ATR pathway in heavy particles induced HRS/IRR was determined with the specific ATR inhibitor in GM0639 cells, which affected the HRS/IRR occurrence similarly as ATM inhibitor. These data demonstrate that ATR pathway may cooperate with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam.

Safe days in space with acceptable uncertainty from space radiation exposure

The prediction of the risks of cancer and other late effects from space radiation exposure carries large uncertainties mostly due to the lack of information on the risks from high charge and energy (HZE) particles and other high linear energy transfer (LET) radiation. In our recent work new methods were used to consider NASA's requirement to protect against the acceptable risk of no more than 3% probability of cancer fatality estimated at the 95% confidence level. Because it is not possible that a zero-level of uncertainty could be achieved, we suggest that an acceptable uncertainty level should be defined in relationship to a probability distribution function (PDF) that only suffers from modest skewness with higher uncertainty allowed for a normal PDF. In this paper, we evaluate PDFs and the number or “safe days” in space, which are defined as the mission length where risk limits are not exceeded, for several mission scenarios at different acceptable levels of uncertainty. In addition, we briefly discuss several important issues in risk assessment including non-cancer effects, the distinct tumor spectra and lethality found in animal experiments for HZE particles compared to background or low LET radiation associated tumors, and the possibility of non-targeted effects (NTE) modifying low dose responses and increasing relative biological effectiveness (RBE) factors for tumor induction. Each of these issues skew uncertainty distributions to higher fatality probabilities with the potential to increase central values of risk estimates in the future. Therefore they will require significant research efforts to support space exploration within acceptable levels of risk and uncertainty.

Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death.

The spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.