Direct observation of damage clustering in irradiated DNA with atomic force microscopy.

Ionizing radiation produces clustered DNA damage that contains two or more lesions in 10-20 bp. It is believed that the complexity of clustered damage (i.e., the number of lesions per damage site) is related to the biological severity of ionizing radiation. However, only simple clustered damage containing two vicinal lesions has been demonstrated experimentally. Here we developed a novel method to analyze the complexity of clustered DNA damage. Plasmid DNA was irradiated with densely and sparsely ionizing Fe-ion beams and X-rays, respectively. Then, the resulting DNA lesions were labeled with biotin/streptavidin and observed with atomic force microscopy. Fe-ion beams produced complex clustered damage containing 2-4 lesions. Furthermore, they generated two or three clustered damage sites in a single plasmid molecule that resulted from the hit of a single track of Fe-ion beams. Conversely, X-rays produced relatively simple clustered damage. The present results provide the first experimental evidence for complex cluster damage.

Detection of DNA-protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs.

Proteins are covalently trapped on DNA to form DNA-protein crosslinks (DPCs) when cells are exposed to DNA-damaging agents. DPCs interfere with many aspects of DNA transactions. The current DPC detection methods indirectly measure crosslinked proteins (CLPs) through DNA tethered to proteins. However, a major drawback of such methods is the non-linear relationship between the amounts of DNA and CLPs, which makes quantitative data interpretation difficult. Here we developed novel methods of DPC detection based on direct CLP measurement, whereby CLPs in DNA isolated from cells are labeled with fluorescein isothiocyanate (FITC) and quantified by fluorometry or western blotting using anti-FITC antibodies. Both formats successfully monitored the induction and elimination of DPCs in cultured cells exposed to aldehydes and mouse tumors exposed to ionizing radiation (carbon-ion beams). The fluorometric and western blotting formats require 30 and 0.3 µg of DNA, respectively. Analyses of the isolated genomic DPCs revealed that both aldehydes and ionizing radiation produce two types of DPC with distinct stabilities. The stable components of aldehyde-induced DPCs have half-lives of up to days. Interestingly, that of radiation-induced DPCs has an infinite half-life, suggesting that the stable DPC component exerts a profound effect on DNA transactions over many cell cycles.

Evaluation of SCCVII tumor cell survival in clamped and non-clamped solid tumors exposed to carbon-ion beams in comparison to X-rays.

The aim of this study was to measure the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for survival of cells within implanted solid tumors following exposure to 290MeV/nucleon carbon-ion beams or X-rays. Squamous cell carcinoma cells (SCCVII) were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (SOBP, at 46 and 80keV/µm) or X-rays was delivered to 5-mm or less diameter tumors. We defined three different oxygen statuses of the irradiated cells. Hypoxic and normoxic conditions in tumors were produced by clamping or not clamping the leg to avoid blood flow. Furthermore, single-cell suspensions were prepared from non-irradiated tumors and directly used to determine the radiation response of aerobic cells. Single-cell suspensions (aerobic condition) were fully air-saturated. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assays to obtain cell survival curves. The RBE values increased with increasing LET in SOBP beams. The maximum RBE values in three different oxygen conditions; hypoxic tumor, normoxic tumor and aerobic cells, were 2.16, 1.76 and 1.66 at an LET of 80keV/µm, respectively. After X-ray irradiation the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.87±0.13 and 2.52±0.11, respectively. The OER values of carbon-ion irradiated samples were small in comparison to those of X-ray irradiated samples. However, no significant changes of the OER at proximal and distal positions within the SOBP carbon-ion beams were observed. To conclude, we found that the RBE values for cell survival increased with increasing LET and that the OER values changed little with increasing LET within the SOBP carbon-ion beams.

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.

Carbon-Ion Beam Irradiation Alone or in Combination with Zoledronic acid Effectively Kills Osteosarcoma Cells.

Osteosarcoma (OSA) is the most common malignant bone tumor in children and adolescents. The overall five-year survival rate for all bone cancers is below 70%; however, when the cancer has spread beyond the bone, it is about 15-30%. Herein, we evaluated the effects of carbon-ion beam irradiation alone or in combination with zoledronic acid (ZOL) on OSA cells. Carbon-ion beam irradiation in combination with ZOL significantly inhibited OSA cell proliferation by arresting cell cycle progression and initiating KHOS and U2OS cell apoptosis, compared to treatments with carbon-ion beam irradiation, X-ray irradiation, and ZOL alone. Moreover, we observed that this combination greatly inhibited OSA cell motility and invasion, accompanied by the suppression of the Pi3K/Akt and MAPK signaling pathways, which are related to cell proliferation and survival, compared to individual treatments with carbon-ion beam or X-ray irradiation, or ZOL. Furthermore, ZOL treatment upregulated microRNA (miR)-29b expression; the combination with a miR-29b mimic further decreased OSA cell viability via activation of the caspase 3 pathway. Thus, ZOL-mediated enhancement of carbon-ion beam radiosensitivity may occur via miR-29b upregulation; co-treatment with the miR-29b mimic further decreased OSA cell survival. These findings suggest that the carbon-ion beam irradiation in combination with ZOL has high potential to increase OSA cell death.

Molecular mechanisms underlying the enhancement of carbon ion beam radiosensitivity of osteosarcoma cells by miR-29b.

Carbon ion radiotherapy (CIRT) is more effective than conventional photon beam radiotherapy in treating osteosarcoma (OSA); however, the outcomes of CIRT alone are still unsatisfactory. In this study, we aimed to investigate whether miR-29b acts as a radiosensitizer for CIRT. The OSA cell lines U2OS and KHOS were treated with carbon ion beam alone, γ-ray irradiation alone, or in combination with an miR-29b mimic. OSA cell death as well as invasive and migratory abilities were analyzed through viability, colony formation, Transwell, and apoptosis assays. miR-29 expression was downregulated in OSA tissues compared to that in normal tissues and was associated with metastasis and relapse in patients with OSA. Further, miR-29b was found to directly target the transcription factor Sp1 and suppress the activation of the phosphatase and tensin homolog (PTEN)-AKT pathway. Conversely, Sp1 was found to attenuate the inhibitory effects of miR-29b in OSA cells. When used in combination with miR-29b mimic, carbon ion beam markedly inhibited invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation in a Sp1/PTEN-mediated manner. Taken together, miR-29b mimic improved the radiosensitivity of OSA cells via the PTEN-AKT-Sp1 signaling pathway, presenting a novel strategy for the development of carbon ion beam combination therapy.

Transient infiltration of neutrophils into the thymus following whole-body X-ray irradiation in IL-10 knockout mice.

IL-10 is known to suppress the inflammatory responses in a variety of experimental models. Because we previously found that whole-body X-irradiation causes massive apoptosis in the thymus and transient infiltration of neutrophils, in this study, we examined whether or not IL-10 is involved in the regulation of neutrophil infiltration upon whole-body X-ray irradiation using IL-10 knockout mice. Although IL-10 was induced in the thymus on whole-body X-ray irradiation, apoptosis of thymocytes, neutrophil infiltration, and MIP-2 and KC production in the thymus were not affected by an IL-10 deficiency. Coculturing of bone marrow-derived macrophages with late apoptotic cells caused MIP-2 production, which was also not affected by an IL-10 deficiency. These results suggest the uniqueness of the inflammatory response induced by whole-body X-ray irradiation, which does not seem to be regulated by IL-10.

Radiobiologic significance of response of intratumor quiescent cells in vivo to accelerated carbon ion beams compared with gamma-rays and reactor neutron beams.

PURPOSE: To clarify the radiosensitivity of intratumor quiescent cells in vivo to accelerated carbon ion beams and reactor neutron beams. METHODS AND MATERIALS: Squamous cell carcinoma VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine to label all intratumor proliferating cells. Next, they received accelerated carbon ion or gamma-ray high-dose-rate (HDR) or reduced-dose-rate (RDR) irradiation. Other tumor-bearing mice received reactor thermal or epithermal neutrons with RDR irradiation. Immediately after HDR and RDR irradiation or 12 h after HDR irradiation, the response of quiescent cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for 5-bromo-2'-deoxyuridine. The response of the total (proliferating plus quiescent) tumor cells was determined from the 5-bromo-2'-deoxyuridine nontreated tumors. RESULTS: The difference in radiosensitivity between the total and quiescent cell populations after gamma-ray irradiation was markedly reduced with reactor neutron beams or accelerated carbon ion beams, especially with a greater linear energy transfer (LET) value. Clearer repair in quiescent cells than in total cells through delayed assay or a decrease in the dose rate with gamma-ray irradiation was efficiently inhibited with carbon ion beams, especially with a greater LET. With RDR irradiation, the radiosensitivity to accelerated carbon ion beams with a greater LET was almost similar to that to reactor thermal and epithermal neutron beams. CONCLUSION: In terms of tumor cell-killing effect as a whole, including quiescent cells, accelerated carbon ion beams, especially with greater LET values, are very useful for suppressing the dependency on the heterogeneity within solid tumors, as well as depositing the radiation dose precisely.

The radiosensitivity of total and quiescent cell populations in solid tumors to 290 MeV/u carbon ion beam irradiation in vivo.

PURPOSE: To clarify the radiosensitivity of intratumor total and quiescent (Q) cells in vivo to accelerated carbon ion beams compared with gamma-ray irradiation. MATERIALS AND METHODS: SCC VII tumor-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. Then they received 290 MeV/u carbon ions or gamma-rays. Immediately or 12 hours after the irradiation, the radiosensitivity of Q cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. That of the total (=P+Q) tumor cells was determined from the BrdU non-treated tumors based on the micronucleus frequency and clonogenic cell survival. RESULTS: The apparent difference in radiosensitivity between total and Q cell populations under gamma-ray irradiation was markedly reduced with carbon ion beam, especially with a higher linear energy transfer (LET) value. Clearer repair in Q cells than total cells through delayed assay under gamma-ray irradiation was efficiently inhibited with carbon ion beams, especially with a higher LET. CONCLUSION: In terms of tumor cell-killing effect as a whole, including intratumor Q cells, carbon ion beams, especially with higher LET values, were very useful for suppressing the dependency on the heterogeneity within solid tumors as well as depositing radiation dose precisely.

Responses of total and quiescent cell populations in solid tumors to carbon ion beam irradiation (290 MeV/u) in vivo.

PURPOSE: The aim of this study was to clarify the radiosensitivity of intratumor total cells and quiescent (Q) cells in vivo to accelerated carbon ion beams compared with gamma-ray irradiation. MATERIALS AND METHODS: Squamous cell carcinoma (SCC) VII tumor-bearing mice received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They then were exposed to carbon ions (290 MeV/u) or gamma-rays. Immediately after and 12 h after irradiation, immunofluorescence staining for BrdU was used to assess the response of Q cells in terms of micronucleus frequency. The response of the total (P + Q) tumor cells was determined from the tumors not treated with BrdU. RESULTS: The apparent difference in radiosensitivity between total and Q cell populations under gamma-ray irradiation was markedly reduced with carbon ion beams, especially with a higher linear energy transfer (LET) value. Clearer recovery in Q cells than in total cells through delayed assay under gamma-ray irradiation was efficiently inhibited by carbon ion beams, especially those with a higher LET. CONCLUSION: In terms of the tumor cell-killing effect as a whole, including intratumor Q cells, carbon ion beams, especially with higher LET values, were extremely useful for suppressing the dependence on the heterogeneity within solid tumors as well as depositing the radiation dose precisely.

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.

Repair of skin damage during fractionated irradiation with gamma rays and low-LET carbon ions.

In clinical use of carbon-ion beams, a deep-seated tumor is irradiated with a Spread-Out Bragg peak (SOBP) with a high-LET feature, whereas surface skin is irradiated with an entrance plateau, the LET of which is lower than that of the peak. The repair kinetics of murine skin damage caused by an entrance plateau of carbon ions was compared with that caused by photons using a scheme of daily fractionated doses followed by a top-up dose. Right hind legs received local irradiations with either 20 keV/microm carbon ions or gamma rays. The skin reaction of the irradiated legs was scored every other day up to Day 35 using a scoring scale that consisted of 10 steps, ranging from 0.5 to 5.0. An isoeffect dose to produce a skin reaction score of 3.0 was used to obtain a total dose and a top-up dose for each fractionation. Dependence on a preceding dose and on the time interval of a top-up dose was examined using gamma rays. For fractionated gamma rays, the total dose linearly increased while the top-up dose linearly decreased with an increase in the number of fractions. The magnitude of damage repair depended on the size of dose per fraction, and was larger for 5.2 Gy than 12.5 Gy. The total dose of carbon ions with 5.2 Gy per fraction did not change till 2 fractions, but abruptly increased at the 3rd fraction. Factors such as rapid repopulation, induced repair and cell cycle synchronization are possible explanations for the abrupt increase. As an abrupt increase/decrease of normal tissue damage could be caused by changing the number of fractions in carbon-ion radiotherapy, we conclude that, unlike photon therapy, skin damage should be carefully studied when the number of fractions is changed in new clinical trials.

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.

Protective effects of melatonin on gamma-ray induced intestinal damage.

PURPOSE: To examine the protective effects of melatonin on intestinal damage induced by gamma-rays. MATERIALS AND METHODS: Six-week-old Slc:ICR male mice were used. Mice were given whole-body irradiation at various exposure doses (7-21 Gy) with (137)Cs gamma-rays (0.98 Gy/min). The mice were orally administered 1 ml of either 1% carboxymethyl cellulose sodium salt (CMC) or melatonin (1, 5, 10 or 20 mg/ml) freshly prepared as a uniform suspension in CMC before or after irradiation. The concentrations of plasma melatonin were determined by the radioimmunoassay (RIA) method. The mice were killed at 3.5 days after the exposure. The jejunum was removed, fixed in formalin and then stained with hematoxylin and eosin. The numbers of crypts per transverse circumference were counted using a microscope for 10 histological sections of each mouse. RESULTS: The intestinal damage caused by gamma-ray irradiation was prevented by melatonin correlating to dosage. The D(0) (slope of the dose-survival curve) value significantly (p < 0.05) increased from 1.55 +/- 0.19 (mean +/- SD) Gy to 1.98 +/- 0.16 Gy by orally administering 20 mg melatonin 30 min before irradiation. The radioprotective effect of melatonin continued for 6 h after the administration. CONCLUSIONS: Melatonin is judged to be a potential protector against intestinal damage associated with radiotherapy. Further experimental and clinical studies on this subject are needed to allow its use for radiotherapy.

Glycine betaine, a beer component, protects radiation-induced injury.

Human whole-blood was exposed to 137Cs gamma-rays or 50 keV/microm carbon ions in the presence or absence of glycine betaine, a beer component in vitro. The dicentrics of chromosome aberrations in human lymphocytes were significantly (p < 0.05) reduced by glycine betaine after irradiation with 4 Gy of either gamma-rays or carbon ions. The maximum protection by glycine betaine for gamma-rays or carbon ions was 37% and 20%, respectively. C3H/He female mice, aged 14 weeks, received an i.p. injection of glycine betaine 15 min before whole-body irradiation with gamma-rays or 50 keV/microm carbon ions. Glycine betaine significantly (p < 0.05) increased the percent survival of irradiated mice with either gamma-rays or carbon ions. In conclusion, glycine betaine is a potent protector against damages caused by low- and high-LET radiation.

Biological gain of carbon-ion radiotherapy for the early response of tumor growth delay and against early response of skin reaction in mice.

The biological effectiveness of carbon ions relative to gamma rays (RBE) was compared between the tumor growth delay and an early skin reaction of syngeneic mice. The RBE was larger for a tumor than skin when irradiated with large doses of high-LET (linear energy transfer) carbon ions. The intra-track damage (a term of a linear quadratic model) of a tumor and skin increased equally with an increase of the LET, while the inter-track damage (beta term) of skin alone increased with the LET. These data provide evidence that high-LET radiotherapy could achieve therapeutic gain by minimizing the difference in response to fractionated irradiation between the tumor and normal tissue.

Designing a ridge filter based on a mouse foot skin reaction to spread out Bragg-peaks for carbon-ion radiotherapy.

BACKGROUND AND PURPOSE: Carbon-ion radiotherapy uses spread-out Bragg peaks (SOBP) to produce uniform biological effects within a target volume. The relative biological effectiveness is determined by the in vitro cell kill after a single dose is employed to design the SOBP. A question remains as to whether biological effects for in vivo tissues after fractionated doses are also uniform within the SOBP. MATERIAL AND METHODS: Mouse foot skin was irradiated with fractionated doses of carbon ions at various linear energy transfer (LET) values. A new ridge filter was designed based on alpha and beta values for each LET to cause moderate skin reaction, and was studied concerning its uniformity. RESULTS: The reciprocal total doses of intermediate-LET carbon ions and of reference gamma rays linearly increased with an increase of a dose per fraction in Fe-plots. As the single total dose of higher LET run off linearity, data obtained from 2 to 6 fractions were used to design a new ridge filter. The physical dose distribution of the new ridge filter was almost identical to, and indistinguishable from, the ridge filter designed based on the in vitro cell kill. CONCLUSIONS: The LET dependence of alpha is a principle of the biological factor to be used for designing spread-out Bragg peaks of carbon-ion radiotherapy.

Evaluation of therapeutic gain for fractionated carbon-ion radiotherapy using the tumor growth delay and crypt survival assays.

BACKGROUND AND PURPOSE: The aim of the study was to evaluate the therapeutic gain of carbon ion (C-ion) radiotherapy using a mouse model. MATERIALS AND METHODS: Transplanted fibrosarcoma (NFSa) growing in C3H/He mice and murine small intestine were irradiated with 290 MeV/nucleon C-ion beams (C-ions) in 1-12 fractions separated by 4h. The cell killing efficiencies of C-ions were measured using jejunum crypt survival and tumor growth delay (TGD) assays. RESULTS: The equieffect dose for crypt survival and TGD increased with increasing number of fractions after X-rays and 20 keV/µm C-ions, whereas TGD after 77 keV/µm C-ions rather decreased. Crypts showed stronger LET-dependent increase in α terms than the tumor while ß terms less depended on LET irrespective of tissues. Therapeutic gain factor, i.e., a ratio of tumor RBE over crypt RBE, of 77 keV/µm C-ions was more than unity at any doses while that of 20 keV/µm C-ions increased with an increase in dose per fraction. CONCLUSIONS: These specific data imply that use of large dose per fraction would be suitable for C-ion radiotherapy irrespective of LET from the point of view of therapeutic gain, though small dose per fraction by high-LET radiation decreases total dose for tumor.

The Effect of p53 Status of Tumor Cells on Radiosensitivity of Irradiated Tumors With Carbon-Ion Beams Compared With γ-Rays or Reactor Neutron Beams.

BACKGROUND: The aim of the study was to clarify the effect of p53 status of tumor cells on radiosensitivity of solid tumors following accelerated carbon-ion beam irradiation compared with γ-rays or reactor neutron beams, referring to the response of intratumor quiescent (Q) cells. METHODS: Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into hind legs of nude mice. Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received γ-rays or accelerated carbon-ion beams at a high or reduced dose-rate. Other tumor-bearing mice received reactor thermal or epithermal neutrons at a reduced dose-rate. Immediately or 9 hours after the high dose-rate irradiation (HDRI), or immediately after the reduced dose-rate irradiation (RDRI), the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. RESULTS: The difference in radiosensitivity between the total (P + Q) and Q cells after γ-ray irradiation was markedly reduced with reactor neutron beams or carbon-ion beams, especially with a higher linear energy transfer (LET) value. Following γ-ray irradiation, SAS/neo tumor cells, especially intratumor Q cells, showed a marked reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q cells within SAS/mp53 tumors that showed little repair capacity. In both total and Q cells within both SAS/neo and SAS/mp53 tumors, carbon-ion beam irradiation, especially with a higher LET, showed little recovery capacity through leaving an interval between HDRI and the assay or decreasing the dose-rate. The recovery from radiation-induced damage after γ-ray irradiation was a p53-dependent event, but little recovery was found after carbon-ion beam irradiation. With RDRI, the radiosensitivity to reactor thermal and epithermal neutron beams was slightly higher than that to carbon-ion beams. CONCLUSION: For tumor control, including intratumor Q-cell control, accelerated carbon-ion beams, especially with a higher LET, and reactor thermal and epithermal neutron beams were very useful for suppressing the recovery from radiation-induced damage irrespective of p53 status of tumor cells.

Induction of DNA-protein cross-links by ionizing radiation and their elimination from the genome.

Ionizing radiation produces various types of DNA lesions, such as base damage, single-strand breaks, double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, DSBs are the most critical lesions underlying the lethal effects of ionizing radiation. With DPCs, proteins covalently trapped in DNA constitute strong roadblocks to replication and transcription machineries, and hence can be lethal to cells. The formation of DPCs by ionizing radiation is promoted in the absence of oxygen, whereas that of DSBs is retarded. Accordingly, the contribution of DPCs to the lethal events in irradiated cells may not be negligible for hypoxic cells, such as those present in tumors. However, the role of DPCs in the lethal effects of ionizing radiation remains largely equivocal. In the present study, normoxic and hypoxic mouse tumors were irradiated with X-rays [low linear energy transfer (LET) radiation] and carbon (C)-ion beams (high LET radiation), and the resulting induction of DPCs and DSBs and their removal from the genome were analyzed. X-rays and C-ion beams produced more DPCs in hypoxic tumors than in normoxic tumors. Interestingly, the yield of DPCs was slightly but statistically significantly greater (1.3- to 1.5-fold) for C-ion beams than for X-rays. Both X-rays and C-ion beams generated two types of DPC that differed according to their rate of removal from the genome. This was also the case for DSBs. The half-lives of the rapidly removed components of DPCs and DSBs were similar (<1 h), but those of the slowly removed components of DPCs and DSBs were markedly different (3.9-5 h for DSBs versus 63-70 h for DPCs). The long half-life and abundance of the slowly removed DPCs render them persistent in DNA, which may impede DNA transactions and confer deleterious effects on cells in conjunction with DSBs.