To accurately describe the radiation response over a wide dose and ionization density range Binomial and Poisson statistics have been combined with the recently developed potentially Repairable-Conditionally-Repairable (RCR) damage response model and the combination is shown to have several advantages for the accurate description of the cell survival at both low and very high doses and LET's, especially when compared with the classical Linear and Linear Quadratic cell survival models. Interestingly, the potentially and conditionally repairable damage types of the RCR model may also be linked to the two major radiation damage repair pathways of eukaryotic cells namely Non Homologous End Joining (NHEJ) and Homologous Recombination (HR) respectively. In addition it describes the damage interaction of low and high LET damage in different dose fractions more accurately than any other model (cf. (6) and Fig. 7d). This is of considerable importance when describing the response of tumors and normal tissues during pencil beam scanning with light ion beams where low and high LET dose fractions from the plateau and Bragg peak can interact synergistically when being delivered quasi simultaneously. In conclusion, considering the unique biological properties of light ion beams such as their increased effect on hypoxic tumors, their microdosimetric energy deposition heterogeneity and their pencil beam energy deposition kernels the largest clinical advantages are obtained with medium LET beams (≈ 20–50 eV/nm). This applies even for radiation resistant tumors, at least when the goal is to maximize tumor cure with minimal adverse reactions in normal tissues.

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Understanding the mechanisms that underlay the biological effects of particulate radiations is essential for space exploration and for radiotherapy. Here, we investigated the role of gap junction intercellular communication (GJIC) in modulating harmful effects induced in confluent cultures wherein most cells are traversed by one or more radiation tracks. We focused on the effect of radiation quality (linear energy transfer; LET) on junctional propagation of DNA damage and cell death among the irradiated cells. Confluent normal human fibroblasts were exposed to graded doses of 1 GeV protons (LET ~0.2 keV/μm) or 1 GeV/u iron ions (LET ~151 keV/μm) and were assayed for clonogenic survival and for micronucleus formation, a reflection of DNA damage, shortly after irradiation and following longer incubation periods. Iron ions were ~2.7 fold more effective than protons at killing 90% of the cells in the exposed cultures when assayed within 5–10 minutes after irradiation. When cells were held in the confluent state for several hours after irradiation, substantial potentially lethal damage repair (PLDR), coupled with a reduction in micronucleus formation, occurred in cells exposed to protons, but not in those exposed to iron ions. In fact, such confluent holding after exposure to a similarly toxic dose of iron ions enhanced the induced toxic effect. However, following iron ion irradiation, inhibition of GJIC by 18-α-glycyrrhetinic acid eliminated the enhanced toxicity and reduced micronucleus formation to levels below those detected in cells assayed shortly after irradiation. The data show that low-LET radiation induces strong PLDR within hours, but that high-LET radiation with similar immediate toxicity does not induce PLDR and its toxicity increases with time following irradiation. The results also show that GJIC among irradiated cells amplifies stressful effects following exposure to high-, but not low-LET radiation, and that GJIC has only minimal effect on cellular recovery following low-LET irradiation.

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Non-homologous end-joining (NHEJ) is the predominant pathway for the repair of DNA double-strand breaks (DSBs) in human cells. XRCC4 is indispensable to NHEJ and functions together with DNA ligase IV in the rejoining of broken DNA ends. Artemis is a nuclease required for trimming of some, but not all, types of broken DNA ends prior to rejoining by the DNA ligase IV/XRCC4 complex. To better understand the roles of these factors, we generated XRCC4- and Artemis-deficient cells from the human colon adenocarcinoma cell line HCT116 by gene targeting and examined their cellular responses to several DNA-damaging agents including X-rays. As anticipated, kinetic analyses of γ-H2AX foci and chromosomal aberrations after ionizing radiation (IR) demonstrated a serious incompetence of DSB repair in the XRCC4-deficient cells, and relatively moderate impairment in the Artemis-deficient cells. The XRCC4-deficient cells were highly sensitive to etoposide and 5-fluoro-2'-deoxyuridine as well as IR, and moderately sensitive to camptothecin, methyl methanesulfonate, cisplatin, mitomycin C, aphidicolin and hydroxyurea, compared to the parental HCT116 cells. The Artemis-deficient cells were not as sensitive as the XRCC4-deficient cells, except to cisplatin and mitomycin C. By contrast, the Artemis-deficient cells were significantly more resistant to hydroxyurea than the parental cells. These observations suggest that Artemis also functions in some DNA damage response pathways other than NHEJ in human cells.

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Up to now, more than 1048 human miRNAs have been identified. However, the recognition of new human miRNAs is becoming more and more difficult. Based on the hypothesis that the expression of some miRNAs can be induced by ionizing radiation, total RNAs of HeLa cells were isolated 1 h after exposure to 2 Gy of X-rays, and total small RNAs were enriched and sequenced by PAGE and Solexa technology, respectively. As a result, 421 kinds of known miRNAs and 337 kinds of unknown sequences were identified, among which 10 novel miRNAs were characterized by bioinformatic approach and verified by qRT-PCR. Finally, putative targets of these miRNAs were predicted by TargetScan software and compared with known proteins down-regulated by radiation. It was confirmed that some of the targets of these novel miRNAs were radiation-related proteins. These results imply that these 10 novel miRNAs are radiation-related miRNAs. This study reveals a new way to find novel miRNAs.

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African green monkey kidney cells, CV-1, were irradiated with Carbon ions (LET: 735 keV/μm Argon ions (LET: 3,000 keV/μm) to visualize ion tracks through the cell nucleus by labeling the 3'-OH termini result of DNA strand breaks. The 3'-OH termini of DNA were labeled with BrdU-triphosphate catalyzed by TdT. This method of TUNEL (TdT-mediated dUTP Nick End labeling) is based on the specific binding of TdT to 3'-OH termini of DNA. Subsequent immuno-fluorescent staining with the primary monoclonal antibody against BrdU, followed by a secondary antibody of Alexa Fluor 488, was performed to visualize the BrdU labeled DNA termini. Images of the cell nuclei were acquired by confocal laser microscopy. When cell monolayers were irradiated perpendicularly with argon ions, induced DSBs in cell nuclei were identifiable as fluorescent spots. In another irradiation setup, when cells were irradiated at a small angle with incident argon ions, DNA strand breaks were detected as fluorescent stripes across the cell nucleus. These results demonstrate the induction of 3'-OH termini at sites of DNA strand breaks along Argon ion tracks.

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The developmental stage has an influence on the overall responses of plants under biotic or abiotic stress conditions. However, there is a lack of data about the effects of ionizing radiation in plants at different developmental stages. We examined radiation sensitivity of Arabidopsis plants in terms of photosynthetic ability and oxidative stress resistance at two distinct vegetative and reproductive stages, which correspond to 23 and 43 d after seeding (DAS), respectively. When plants were exposed to γ rays at a dose rate 50 Gy h^–1 for 4 h, they were characterized as various common or differential cellular responses depending on the developmental stage. Radial expansion of leaves, inhibition of non-photochemical quenching, and production of •O₂^– and H₂O₂ under methyl viologen-induced photooxidative stress were commonly more conspicuous in the irradiated leaves of both plants than in the respective control. In contrast, the 23 and 43-DAS plants were explicitly discriminated in growth, chloroplast number & ultrastructure, photosynthetic pigment content & activity, and protein damage after γ irradiation. Natural leaf senescence was thereby enhanced in the irradiated leaves of the 23-DAS plants, while it was reversely alleviated in those of the 43-DAS ones. These results suggest that photosynthetic machineries of Arabidopsis plants at the reproductive stage can be relatively tolerant to γ rays of 200 Gy.

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Nasal administration of thallium-201 (²⁰¹Tl) has previously been shown to be useful for the assessment of olfactory nerve connectivity in vivo. We assessed the biological effects of nasal ²⁰¹Tl administration in mice to determine its safety before conducting clinical trials on humans. ²⁰¹Tl uptake was evaluated in normal mice (n = 5) in vivo by using a high-resolution gamma camera and radiography 15 min, 1, 2 and 9 d after administration of ²⁰¹TlCl to the right side of the nasal cavity (10 μl ²⁰¹TlCl per nostril, 74 MBq/ml). Murine olfactory epithelial thickness (n = 5) was measured 9 d following nasal administration of ²⁰¹TlCl. We assessed the odor detection ability of normal mice (n = 8) following nasal administration of ²⁰¹TlCl to both sides of the nasal cavity, by observing cycloheximide solution avoidance behavior. We subsequently administrated ²⁰¹TlCl (n = 4) or saline (n = 4) to both nostrils to assess the odor detection ability of mice following bilateral olfactory nerve transection. ²⁰¹Tl uptake by the nasal cavity decreased immediately following nasal administration of ²⁰¹Tl in normal mice. Nasal administration of ²⁰¹Tl did not affect the olfactory epithelial thickness or the odor detection ability of normal mice. Recovery of odor detection ability following olfactory nerve transection was not significantly different between mice nasally administered with ²⁰¹Tl, and mice administered with saline. Thus, nasal administration of ²⁰¹Tl for the diagnosis of traumatic olfactory impairment did not produce harmful biological effects in vivo.

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The combination of a chemotherapeutic agent and radiation is widely applied to enhance cell death in solid tumor cells in cancer treatment. The purine analogue 8-aminoadenosine (8-NH₂-Ado) is known to be a transcription inhibitor that has proved very effective in multiple myeloma cell lines and primary indolent leukemia cells. In this report, to examine whether 8-NH₂-Ado had the ability to enhance the radiation-induced cell killing in solid tumor cells, human lung adenocarcinoma A549 cells were irradiated in the presence and absence of 8-NH₂-Ado. 8-NH₂-Ado significantly increased reproductive cell death and apoptosis in A549 cells exposed to X-rays. When peptide inhibitors against caspase-3, -8, and -9 were utilized to evaluate the involvement of caspases, all inhibitors suppressed the enhancement of radiation-induced apoptosis, suggesting that not only mitochondria-mediated apoptotic signal transduction pathways but also death receptor-mediated pathways were involved in this enhancement of apoptosis. In addition, in the cells exposed to the treatment combining X-irradiation and 8-NH₂-Ado, reduction of the intracellular ATP concentration was essential for survival, and down-regulation of the expression of antiapoptotic proteins such as survivin and XIAP was observed. These results indicate that 8-NH₂-Ado has potential not only as an anti-tumor drug for leukemia and lymphoma but also as a radiosensitizing agent for solid tumors.

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In order to elucidate the mechanism of concerted expression of various proteins related to DNA-PK activity, we examined the relationship of the expression of these proteins with that of a transcription factor Sp1. We also explored whether the expression of genes related to DNA-PK activity and Sp1 changed after hypoxia and reoxygenation. RT-PCR was employed to measure expression of various proteins related to DNA-PK activity in peripheral blood cells (PBLs) of normal healthy volunteers and cancer patients. M059K and DLD1 cells were made hypoxic with the hypoxia chamber, and the expression of various proteins in the cells was examined. DNA-PK activity was measured using synthetic peptide substrate mimicking the sequence around Ser15 of human p53. Expression of examined genes whose expression was related to DNA-PK activity was significantly correlated with expression of Sp1. The expression of most genes lost its relationship with the expression of Sp1 after hypoxic exposure and reoxygenation. DNA-PK activity tended to decrease after hypoxic exposure and to recover after reoxygenation. Sp1 may control expression of genes related to DNA-PK activity. Expression of those genes deviated from the control Sp1 under hypoxia and reoxygenation. The control mechanism for expression of genes related to DNA-PK activity under hypoxia and reoxygenation may be different from that under aerobic conditions.

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Fission-neutron radiation damage is hard to treat due to its critical injuries to hematopoietic and gastrointestinal systems, and so far few data are available on the therapeutic measures for neutron-radiation syndrome. This study was designed to test the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in dogs which had received 2.3 Gy mixed fission-neutron-γ irradiation with a high ratio of neutrons (~90%). Following irradiation, rhG-CSF treatment induced 100% survival versus 60% in controls. Only two of five rhG-CSF-treated dogs experienced leukopenia (white blood cells [WBC] count < 1.0 × 10⁹/L) and neutropenia (neutrophil [ANC] count < 0.5 × 10⁹/L), whereas all irradiated controls displayed a profound period of leukopenia and neutropenia. Furthermore, administration of rhG-CSF significantly delayed the onset of leukopenia and reduced the duration of leucopenia as compared with controls. In addition, individual dogs in the rhG-CSF-treated group exhibited evident differences in rhG-CSF responsiveness after neutron-irradiation. Finally, histopathological evaluation of the surviving dogs revealed that the incidence and severity of bone marrow, thymus and spleen damage decreased in rhG-CSF-treated dogs as compared with surviving controls. Thus, these results demonstrated that rhG-CSF administration enhanced recovery of myelopoiesis and survival after neutron-irradiation.

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To understand how human tumor cells respond to the combined treatment with nocodazole and high LET radiation, alterations in cell cycle, mitotic disturbances and cell death were investigated in the present study. Human cervix carcinoma HeLa cells were exposed to nocodazole for 18 h immediately followed by high LET iron ion irradiation and displayed a sequence of events leading to DNA damages, mitotic aberrations, interphase restitution and endocycle as well as cell death. A prolonged mitotic arrest more than 10 h was observed following nocodazole exposure, no matter the irradiation was present or not. The occurrence of mitotic slippage following the mitotic arrest was only drug-dependent and the irradiation did not accelerate it. The amount of polyploidy cells was increased following mitotic slippage. No detectable G₂ or G₁ arrest was observed in cells upon the combined treatment and the cells reentered the cell cycle still harboring unrepaired cellular damages. This premature entry caused an increase of multipolar mitotic spindles and amplification of centrosomes, which gave rise to lagging chromosomal material, failure of cytokinesis and polyploidization. These mitotic disturbances and their outcomes confirmed the incidence of mitotic catastrophe and delayed apoptotic features displayed by TUNEL method after the combined treatment. These results suggest that the addition of high-LET iron ion irradiation to nocodazole enhanced mitotic catastrophe and delayed apoptosis in HeLa cells. These might be important cell death mechanisms involved in tumor cells in response to the treatment of antimitotic drug combined with high LET radiation.

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The aims of this study were to carry out a dose volume analysis of high-dose-rate interstitial brachytherapy with computed tomography-based treatment planning and to investigate the treatment outcome of patients with locally advanced bulky and/or irregularly shaped uterine cervical carcinoma. Between July 2003 and December 2007, 15 patients were treated with external beam radiation therapy and high-dose-rate interstitial brachytherapy with or without intracavitary brachytherapy. Seven patients were treated with interstitial brachytherapy alone, and 8 were treated with combined use of intracavitary and interstitial brachytherapy. A comparison of the volume and dose parameters with intracavitary and interstitial brachytherapy in patients who received both treatments showed that the median D90 of the high-risk clinical target volume per fraction was 4.4 Gy with intracavitary brachytherapy and 5.6 Gy with interstitial brachytherapy, and the median V100 was 66% with intracavitary brachytherapy and 85% with interstitial brachytherapy. The median D2cc of the bladder with intracavitary and interstitial brachytherapy per fraction was 5.5 Gy and 4.7 Gy, respectively, and the median D2cc of the rectum with intracavitary and interstitial brachytherapy was 5.9 Gy and 4.1 Gy, respectively. The median follow-up time was 37 months, and the overall and progression-free survival rates for all patients at 3 years were 78% and 51%, respectively. The actuarial 2-year and 3-year locoregional control rates were 80% and 71%, respectively. Dose distribution was improved with image-based interstitial brachytherapy, and satisfactory local control was achieved for patients with locally advanced uterine cervical carcinoma in which intracavitary brachytherapy may result in a suboptimal dose distribution.

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The purpose of this study was to evaluate the significance of skin phototype (suntan or sunburn type) in association with radiation dermatitis in patients with breast cancer who underwent postoperative radiotherapy after breast-conserving surgery because phototype could predict sunlight reaction. We divided patients into two phototypes (58 suntan/darkening and 28 sunburn/reddening types) according to self-reports before radiotherapy. We examined skin color changes in 86 patients who underwent breast-conserving surgery followed by 50 Gy/25 fractions (median) of radiotherapy with or without boost radiation (10 Gy/5 fractions). Color change was assessed according to CIE L*a*b* space, which is defined by the Commission Internationale de l'Éclairage (CIE) in 1976 for quantitative color assessment. The patients were also assessed by individual typology angle (ITA°; indicator of skin color calculated by L*a*b* space) and Common Terminology Criteria for Adverse Event v3.0 (CTCAE v3). Radiation therapy changed all values except the b* value, and the suntan type showed a greater darkening response associated with radiation dermatitis than did the sunburn type in terms of ITA° value change (p = 0.04), whereas the sunburn type did not show higher a* value (reddening). By CTCAE v3 classifications, a Grade 2 reaction appeared in 14% sunburn patients and in 31% of the suntan group, respectively (p = 0.16). Suntan type predicted higher pigmentation associated with radiation dermatitis. Self-reported phototype has the potential to be a good predictor of skin sensitivity to radiation exposure for clinical screening.

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Total body irradiation (TBI) is an important component of conditioning regimens for Allogeneic bone marrow transplantation (BMT). Interstitial pneumonitis (IP) and other pulmonary disorders are known regimen-related complications. The incidence of IP is related to the dose rate and dose fractionation; however, there is a paucity of clinical data regarding the optimal dose fractionation. This retrospective study evaluated patients to determine the influence of dose fractionation during TBI in preparation for allogeneic BMT on the subsequent development of IP and other pulmonary complications. Fifty-six patients were treated with TBI followed by BMT at our institute. All patients received a total TBI dose of 12 Gy given in 6 fractions over 3 days or in 4 fractions over 2 days. The prevalence of unrelated donors in the 4-fraction group was higher than that in the 6-fraction group. The overall and freedom from progression rates for patients in the 4-fraction group were better than those for patients in the 6-fraction group, but the difference did not reach significance. Clinically significant lung complications occurred in 19 (10: infectious and 9: non-infectious diseases) of 33 patients in the 6-fraction group and 12 (7: infectious and 5: non-infectious diseases) of 23 in the 4-fraction group. There was no significant difference between the two groups. There was no significant difference in pulmonary complications between patients treated with a TBI dose of 12 Gy in 6 fractions over 3 days and patients treated with a TBI dose of 12 Gy in 4 fractions over 2 days.

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Brain metastasis from esophageal carcinoma has been considered rare and survival following esophageal carcinoma with distant metastasis is poor. The purpose of this report was to clarify cumulative incidence and risk factors for brain metastasis after chemoradiotherapy for esophageal carcinoma, and to consider recommended treatments for brain metastasis from esophageal carcinoma. We reviewed 391 patients treated with chemoradiotherapy. Median age was 65 years. Clinical stages were I, II, III, and IV in 32, 47, 150, and 162 patients, respectively. Brain imaging was performed usually when patients revealed neurological symptoms. The 3-year cumulative incidence of brain metastasis after chemoradiotherapy was 6.6%. There were 4 patients with single metastasis and 8 with multiple metastases. Initial clinical stages were II, III, and IV in 1, 2, and 9 patients, respectively. Histology included squamous cell carcinoma in 10 patients and others in 2 patients. Univariate analysis demonstrated M factor, distant lymph node relapse, and recurrent lung and liver metastasis as significant risk factors of brain metastasis (P < 0.05). Median survival time after diagnosis of brain metastasis was 2.1 months. Brain metastasis was not directly related to cause of mortality. The causes were extracranial tumor deterioration in 8 patients and infection in 4 patients. Brain metastasis may increase in the future with improving survival from esophageal carcinoma. However, considering the poor survival after diagnosis of brain metastasis, short-term palliative therapy for brain metastasis appears preferable to vigorous long-term therapy.

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Patients with implanted cardiac pacemakers (ICPs) or implantable cardioverter defibrillators (ICDs) are increasing in number, and the incidence of treating these patients with radiation therapy also is increasing. Thus, a prospective survey was conducted of patients with these devices receiving radiation therapy. A prospective survey of patients with ICPs or ICDs treated with radiation therapy was conducted on methods of radiation therapy, status of ICP/ICD, and management of patients before, during, and after radiation therapy. After completion of radiation therapy, study participants were registered via mail, fax, or e-mail. Sixty-two patients from 29 institutions were registered from September 2006 to December 2008. Sixty patients had an ICP and 2 had an ICD. The total dose was estimated before radiation therapy by dose-volume histogram in 26 patients (42%) and by measurement of actual doses in 9 (15%). In one patient, the maximum total dose was 2069 cGy; however, in the other patients, the ICP/ICD dose did not exceed 478 cGy. Function of ICPs and ICDs was checked before radiation therapy in 38 patients (61%), after radiation therapy in 32 (52%), and both before and after radiation therapy in 29 (47%). ICP malfunction occurred in a patient with prostate cancer treated by intensity-modulated radiation therapy to the prostate. Even when an ICP or ICD is not within the field of radiation, malfunction of the device may still occur. To minimize the risk to patients, precautions must be taken during the planning and administration of radiation therapy.

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Purpose: The purpose of this study was to investigate the clinical factors for predicting local failure after definitive radiotherapy in oropharyngeal or hypopharyngeal squamous cell carcinoma. Materials and Methods: Between July 2006 and December 2008, 64 consecutive patients with squamous cell carcinoma of the hypopharynx or the oropharynx treated with definitive radiotherapy were included in this study. Clinical factors, such as pretreatment hemoglobin (Hb) level, T-stage, gross tumor volume of primary tumors (pGTV), and maximum standardized uptake value (SUV_max) on FDG-PET, were evaluated for the correlation with local failure. A subset analysis of 32 patients with MR images including diffusion-weighted images (DWI) as a pretreatment evaluation was also performed. The Kaplan-Meier curves, the log-rank test, and the Cox proportional hazards model were used to evaluate these clinical factors. Results: Eleven of 64 patients experienced local recurrence, with a median follow-up time of 15 months. In the univariate analysis, Hb level (p = 0.0261), T-stage (p = 0.012), pGTV (p = 0.0025), and SUV_max (p = 0.024) were significantly associated with local failure. In the multivariate analysis, pGTV (p = 0.0070) remained an adverse factor for local control. In the subset analysis of 32 patients with DWI, the median apparent diffusion coefficient (ADC) value of primary tumors on DWI was 0.79 × 10^–3 mm²/s (range, 0.40–1.60 × 10^–3 mm²/s). Patients with a high ADC value (> 0.79 × 10^–3 mm²/s) had a significantly lower local control rate than patients with a low ADC value (100% vs. 44%, p = 0.0019). The rate of local failure among patients with a large pGTV and a high ADC value was 55% (6/11), whereas no local failures occurred (0%, 0/21) among patients with a small pGTV or a low ADC. Conclusions: These results suggest that a combination of a large tumor volume and a high ADC value could be predictive of local recurrence after definitive radiotherapy in hypopharyngeal or oropharyngeal squamous cell carcinoma.

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Calibration curves for fission spectrum neutrons and other high LET radiations are scarce in cytogenetic dosimetry and particularly for Prematurely Condensed Chromosome Rings (PCC-ring). Here we analyzed the behavior of the PCC-ring frequency and PCC index after neutron irradiation in a broad dose interval from 1 to 26 Gy. PCC-rings were induced in lymphocytes with Calyculin A. 6455 PCC cells in G1, G2/M and M/A stages were analyzed. The best fitting between the frequency of PCC ring (Y) and the Dose (D) was obtained with the equation Y = (0.059 ± 0.003) D. The saturation of the PCC-ring was observed after around 4 Gy, but it was still possible to analyze cells exposed up to 26 Gy. The distribution of rings by cell follows Poisson or Neyman type distribution for all doses. This PCC-ring dose effect curve can be used in case of accidental overexposure to neutron radiation, allowing a dose assessment in a reliable way. Additionally, the PCC index seems to be well correlated with radiation dose and decrease in a dose dependent manner from 13% in non exposed sample down to 0.2%. This observation allows the possibility to perform a quick classification of victims exposed to high doses of both gamma and neutron radiations. The PCC assay can then be used for both neutron dose estimation up to 4 Gy and for the rapid classification of victims exposed to higher doses. This assay could be included in the multiparametric approach developed to optimize the medical treatment of radiation victims.

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