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.

G1 Premature Chromosome Condensation (PCC) Assay.

Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge the gap between radiation-induced DNA damage (mainly double strand breaks) and chromosome aberrations observable at metaphase stage.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Strategies to Enhance Radiosensitivity to Heavy Ion Radiation Therapy.

Heavy ion radiation therapy has been increasingly used due to several advantages over low linear energy transfer (LET) photon therapy, but further improvement of its therapeutic efficacy would be necessary. In this review, we summarize effective radiosensitizers for heavy ion radiation therapy and mechanisms associated with the radiosensitization. High LET heavy ions induce more complex and clustered DNA damage than low LET radiation. Inhibition of homologous recombimation repair or nonhomologous end rejoining and dysfunctional cell cycle checkpoint have been reported to sensitize cancer cells to heavy ions. Radiosenstizing agents, including DNA damage response inhibitors, Hsp90 inhibitors, histone acetylase inhibitors, and nanomaterials have been found to enhance cell killing by heavy ion irradiation through disrupted DNA damage response, cell cycle arrest, or other cellular processes. The use of these radiosensitizers could be a promising strategy to enhance the efficacy of heavy ion radiation therapy.

Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells.

High-linear energy transfer (LET) heavy ions cause higher therapeutic effects than low-LET radiation due to lower dependency on oxygen concentration in tumor cell killing. The lethality after irradiation largely depends on DNA double-strand breaks (DSBs), however the detailed LET dependency for DSB induction under oxic and hypoxic conditions has not been reported. Therefore, we evaluated the oxygen enhancement ratio (OER) of heavy ion-induced DSB induction using a highly-optimized flow cytometry-based method of γ-H2AX detection. Non-small cell lung cancer (NSCLC) A549 cells were exposed to X-ray, carbon-ion and iron-ion radiations under oxic or hypoxic condition. As a DSB marker, the γ-H2AX signal was measured 1 h postirradiation and analyzed by flow cytometry. DSB slope values were calculated as DSB induction per Gy. Our method was able to detect high-LET radiation-induced DSBs even from clustered DNA damage sites. We also showed a decrease in OER value in an LET-dependent manner regardless of radiation type. In summary, we demonstrated a simple, quick and highly-optimized flow cytometry-based method of DSB analysis that detects DSBs induced by heavy-ion radiation for hypoxic and nonhypoxic cancer cells. Our study may provide a useful biological basis for heavy-ion radiotherapy.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

STARLIFE-An International Campaign to Study the Role of Galactic Cosmic Radiation in Astrobiological Model Systems.

In-depth knowledge regarding the biological effects of the radiation field in space is required for assessing the radiation risks in space. To obtain this knowledge, a set of different astrobiological model systems has been studied within the STARLIFE radiation campaign during six irradiation campaigns (2013-2015). The STARLIFE group is an international consortium with the aim to investigate the responses of different astrobiological model systems to the different types of ionizing radiation (X-rays, γ rays, heavy ions) representing major parts of the galactic cosmic radiation spectrum. Low- and high-energy charged particle radiation experiments have been conducted at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. X-rays or γ rays were used as reference radiation at the German Aerospace Center (DLR, Cologne, Germany) or Beta-Gamma-Service GmbH (BGS, Wiehl, Germany) to derive the biological efficiency of different radiation qualities. All samples were exposed under identical conditions to the same dose and qualities of ionizing radiation (i) allowing a direct comparison between the tested specimens and (ii) providing information on the impact of the space radiation environment on currently used astrobiological model organisms. Key Words: Space radiation environment-Sparsely ionizing radiation-Densely ionizing radiation-Heavy ions-Gamma radiation-Astrobiological model systems. Astrobiology 17, 101-109.

TAS-116, a Novel Hsp90 Inhibitor, Selectively Enhances Radiosensitivity of Human Cancer Cells to X-rays and Carbon Ion Radiation.

Hsp90 inhibitors have been investigated as cancer therapeutics in monotherapy and to augment radiotherapy; however, serious adverse effects of early-generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here, we investigated the radiosensitizing effects of TAS-116 in low linear energy transfer (LET) X-ray and high LET carbon ion-irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of noncancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci and delayed the repair of DNA double-strand breaks (DSB). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and nonhomologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell-cycle progression marker, markedly increasing G2-M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared with either individual treatment. These results demonstrate that TAS-116 radiosensitizes human cancer cells to both X-rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell-cycle arrest. The promising results of combination TAS-116 + carbon ion radiotherapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. Mol Cancer Ther; 16(1); 16-24. ©2016 AACR.

The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.

BACKGROUND AND PURPOSE: PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. MATERIALS AND METHODS: The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by γ-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. RESULTS: PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of γ-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. CONCLUSION: Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy.

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.

Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair.

High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/µm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 µM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions.

A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.

A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1ß-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1ß in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1ß complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1ß foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses.

DNA Damage Response Proteins and Oxygen Modulate Prostaglandin E2 Growth Factor Release in Response to Low and High LET Ionizing Radiation.

Common cancer therapies employ chemicals or radiation that damage DNA. Cancer and normal cells respond to DNA damage by activating complex networks of DNA damage sensor, signal transducer, and effector proteins that arrest cell cycle progression, and repair damaged DNA. If damage is severe enough, the DNA damage response (DDR) triggers programed cell death by apoptosis or other pathways. Caspase 3 is a protease that is activated upon damage and triggers apoptosis, and production of prostaglandin E2 (PGE2), a potent growth factor that can enhance growth of surviving cancer cells leading to accelerated tumor repopulation. Thus, dying tumor cells can promote growth of surviving tumor cells, a pathway aptly named Phoenix Rising. In the present study, we surveyed Phoenix Rising responses in a variety of normal and established cancer cell lines, and in cancer cell lines freshly derived from patients. We demonstrate that IR induces a Phoenix Rising response in many, but not all cell lines, and that PGE2 production generally correlates with enhanced growth of cells that survive irradiation, and of unirradiated cells co-cultured with irradiated cells. We show that PGE2 production is stimulated by low and high LET ionizing radiation, and can be enhanced or suppressed by inhibitors of key DDR proteins. PGE2 is produced downstream of caspase 3 and the cyclooxygenases COX1 and COX2, and we show that the pan COX1-2 inhibitor indomethacin blocks IR-induced PGE2 production in the presence or absence of DDR inhibitors. COX1-2 require oxygen for catalytic activity, and we further show that PGE2 production is markedly suppressed in cells cultured under low (1%) oxygen concentration. Thus, Phoenix Rising is most likely to cause repopulation of tumors with relatively high oxygen, but not in hypoxic tumors. This survey lays a foundation for future studies to further define tumor responses to radiation and inhibitors of the DDR and Phoenix Rising to enhance the efficacy of radiotherapy with the ultimate goal of precision medicine informed by deep understanding of specific tumor responses to radiation and adjunct chemotherapy targeting key factors in the DDR and Phoenix Rising pathways.

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

BACKGROUND: High 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. FINDINGS: HeLa, 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. CONCLUSIONS: ATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.

Pre-exposure to ionizing radiation stimulates DNA double strand break end resection, promoting the use of homologous recombination repair.

The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.

Novel characteristics of CtIP at damage-induced foci following the initiation of DNA end resection.

Homologous recombination (HR) is a major repair pathway for DNA double strand breaks (DSBs), and end resection, which generates a 3'-single strand DNA tail at the DSB, is an early step in the process. Resection is initiated by the Mre11 nuclease together with CtIP. Here, we describe novel characteristics of CtIP at DSBs. At early times following exposure of human cells to ionizing radiation, CtIP localized to the DSB, became hyperphosphorylated and formed foci in an ATM-dependent manner. At later times, when the initiation of resection had occurred, CtIP foci persist but CtIP is maintained in a hypophosphorylated state, which is dependent on ATM and ATR. Exposure to cycloheximide revealed that CtIP turns over at DSB sites downstream of resection. Our findings provide strong evidence that CtIP is continuously recruited to DSBs downstream of both the initiation and extension step of resection, strongly suggesting that CtIP has functions in addition to promoting the initiation of resection during HR.

The combination of Hsp90 inhibitor 17AAG and heavy-ion irradiation provides effective tumor control in human lung cancer cells.

Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors.

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function.

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.

Heterochromatin domain number correlates with X-ray and carbon-ion radiation resistance in cancer cells.

Although it is known that cancer cells can develop radiation resistance after repeated exposures to X rays, the underlying mechanisms and characteristics of this radiation-induced resistance of cancer cells are not well understood. Additionally, it is not known whether cells that develop X-ray resistance also would develop resistance to other types of radiation such as heavy-ions including carbon ions (C-ion). In this study, we established X-ray resistant cancer cell lines by delivering repeated exposures to X rays, and then assessed whether the cells were resistant to carbon ions. The mouse squamous cell carcinoma cell line, NR-S1, was X irradiated six times with 10 Gy, and the X-ray resistant cancer cells named X60 and ten subclones were established. Significant X-ray resistance was induced in four of the subclones (X60, X60-H2, X60-A3 and X60-B12). The X60 cells and all of the subclones were resistant to carbon ions. The correlation analysis between radioresistance and morphological characteristics of these cells showed that X-ray (R=0.74) and C-ion (R=0.79) resistance correlated strongly with the number of heterochromatin domains. Moreover, the numbers of γ-H2AX foci remaining in irradiated X60 cells and radioresistant subclones X60-A3 and X60-H2 were lower than in the NR-S1 cells after X-ray or C-ion irradiation, indicating that X60 cells and the radioresistant subclones rapidly repaired the DNA double-strand breaks compared with NR-S1 cells. Our findings suggest that the underlying causal mechanisms of X-ray and C-ion radiation resistance may overlap, and that an increase in heterochromatin domain number may be an indicator of X-ray and C-ion resistance.

Carbon ion beam is more effective to induce cell death in sphere-type A172 human glioblastoma cells compared with X-rays.

PURPOSE: To obtain human glioblastoma cells A172 expressing stem cell-related protein and comparison of radiosensitivity in these cells with X-rays and carbon beam. METHODS: Human monolayer-type A172 glioblastoma cells were maintained in normal medium with 10% bovine serum. In order to obtain sphere-type A172 cells the medium was replaced with serum-free medium supplemented with growth factors. Both types of A172 cells were irradiated with either X-rays or carbon ion beams and their radiosensitivity was evaluated. RESULTS: Serum-free medium induced expression of stem cell-related proteins in A172 cells along with the neurosphere-like appearance. These sphere-type cells were found resistant to both X-rays and carbon ion beams. Phosphorylation of histone H2A family member X persisted for a longer period in the cells exposed to carbon ion beams than in those exposed to X-rays and it disappeared quicker in the sphere type than in the monolayer type. Relative radioresistance of the sphere type cells was smaller for carbon ion beams than for X-rays. CONCLUSIONS: We demonstrated that glioblastoma A172 cells with induced stem cell-related proteins turned resistant to irradiation. Accelerated heavy ion particles may have advantage over X-rays in overcoming the tumor resistance due to cell stemness.