High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

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A typical 2-cysteine peroxiredoxin (2-Cys Prx) PaPrx can act alternatively as thioredoxin (Trx)-dependent peroxidase and molecular chaperone in Pseudomonas aeruginosa PAO1. In addition, the functional switch of PaPrx is regulated by its structural change which is dependently induced by stress conditions. In the present study, we examined the effect of gamma ray on structural modification related to chaperone activity of PaPrx. The structural change of PaPrx occupied with gamma ray irradiation (2 kGy) based on polyacrylamide gel electrophoresis (PAGE) analysis and the functional change also began. The enhanced chaperone activity was increased about 3–4 folds at 30 kGy gamma irradiation compared with nonirradiated PaPrx, while the peroxidase activity was significantly decreased. We also investigated the influence of the gamma ray on protein hydrophobicity as related to chaperone function. The exposure of hydrophobic domains reached a peak at 30 kGy gamma ray and then decreased dependently with increasing gamma irradiation. Our results suggest that highly enhanced chaperone activity could be adapted for use in bio-engineering systems and industrial applications such as enzyme stabilization during industrial process (inactivation protection), improvement of useful protein productivity (refolding and secretion) and industrial animal cell cultivation (stress protection).

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The goal was to investigate the T helper (Th) response in splenocytes of mice exposed to low-dose/low-dose-rate (LDR) γ-rays, simulated solar particle event protons (sSPE), or combination of both. C57BL/6 mice were exposed to LDR γ-radiation (⁵⁷Co) to a total dose of 0.05 Gray (Gy) at 0.024 cGy/h, either with or without subsequent exposure to 2 Gy sSPE protons. Expression of genes related to Th cells was evaluated immediately after exposure (day 0). On day 21, intra- and extracellular cytokine production was assessed after activation with anti-CD3 monoclonal antibodies (mAb) or phorbol 12-myristate 13-acetate/ionophore (PMA/I). Five genes were significantly modulated on day 0 in one or more of the irradiated groups compared to controls (p < 0.05): Ccl11, Ccr5, Cd80, Inha, and Il9. On day 21, numbers of cells positive for interferon-γ were high in the LDR + sSPE group versus 0 Gy and LDR γ-rays (p < 0.05), but there was no difference in IL-2 and TNF-α. Levels of secreted cytokines after anti-CD3 mAb activation were high for 5 (MIP-1α, GM-CSF, IFN-γ, TNF-α, IL-13) and low for 2 (IL-7, IL-9) in all irradiated groups. Priming with LDR photons had a significant effect on IFN-γ and IL-17 compared to sSPE protons alone; IL-2 was low only in the LDR + sSPE group. The cytokine patterns after anti-PMA/I activation were different compared to anti-CD3 mAb and with fewer differences among groups. The data show that total-body exposure to space-relevant radiation has profound effects on Th cell status and that priming with LDR γ-rays can in some cases modulate the response to sSPE.

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p38 mitogen-activated protein kinases (p38) has been shown to be activated in hematopoietic stem and progenitors cells after exposure to ionizing radiation (IR) and its activation has been implicated in bone marrow (BM) suppression under various pathological conditions. Therefore, in the present study we investigated whether inhibition of p38 activity alone with SB203580 (SB, a specific p38 inhibitor) or in combination with granulocyte colony-stimulating factor (G-CSF) can mitigate total body irradiation (TBI)-induced BM damage and lethality. Our results showed that p38 inhibition with SB had no significant effect on the 30-day survival rates of the mice exposed to 7.2 Gy TBI when it was used alone but increased the survival of the mice when it was combined with G-CSF. This combined effect may be attributable to a better preservation or stimulation of hematopoietic stem and progenitor cells, because BM cells from SB and G-CSF-treated mice produced more colony forming units-granulocyte-macrophage (CFU-GM) and 4-week cobblestone area forming cells (CAFCs) than the cells from either SB or G-CSF-treated mice after TBI in a colony forming cell assay and a CAFC assay, respectively. These findings suggest that the combined therapy with SB and G-GSF is more effective in mitigating TBI-induced acute BM injury than either agent alone.

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The classical and alternative activation of macrophages has been proposed to play a role in radiation-induced pneumonitis and fibrosis, respectively. To test this hypothesis, the thoraces of C57BL/6 mice were irradiated with 12 Gy X-rays, and irradiated and control mice were euthanized at 1, 8, 12, 24 and 72 hours, and 2, 4, 8, 16 and 24 weeks after irradiation. The expression of inducible nitric oxide synthase (iNOS) and arginase type 1 (Arg-1) was evaluated at the mRNA and protein levels at different stages post-irradiation. We demonstrated that the enhanced mRNA and protein expression of iNOS occurred within the pneumonic stage, whereas the high levels of Arg-1 expression occurred within the fibrotic phase. Immunohistochemistry revealed that iNOS and Arg-1 were mainly expressed in macrophages. The expression of iNOS and Arg-1 may be associated with acute radiation pneumonitis and the development of radiation fibrosis, respectively. Although the function of macrophages cannot explain the whole process of radiation-induced pulmonary injury development, it may play an important regulatory role during this process.

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Understanding the effects of of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity.

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Rubrobacter radiotolerans is the most radio-resistant eubacterium without spore-formation in the life cycle, and its D₃₇ is 16,000 Gy against gamma-rays. To understand the molecular mechanism of the high radio-resistance, we purified and characterized superoxide dismutase (SOD) of this organism as enzymatic radical scavenger, and then analyzed its genetic information. The purified SOD protein formed homo-tetramerization of 24,000 Da-monomer, while maintaining its enzymatic activity against potassium cyanide and hydrogen peroxide. We obtained a partial amino acid sequence of the protein and cloned the gene from it. Sequence analysis of the cloned gene indicated that the protein showed a similarity to other bacterial manganese SODs (Mn-SODs). Sequencing for adjacent regions of the gene showed that the gene had promoter elements with an open reading frame for putative PAS/PAC sensor protein at the 5'-adjacent region. Introduction of the gene into Escherichia coli cells lacking intrinsic SOD genes restored the cellular enzymatic activity and resistance to methyl viologen, indicating the gene at work. A mutant cell harboring this gene also became resistant against gamma-rays. The present results suggest that the protein in question is the Mn-SOD of R. radiotolerans, a good candidate as a radio-protection factor for this bacterial radio-resistance.

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Radio-resistant or recurrent prostate cancer represents a serious health risk for approximately 20%–30% of patients treated with primary radiation therapy for clinically localized prostate cancer. In the present study, we investigated the expression profiles of 84 genes involved in various apoptosis pathways in two prostate cancer cell lines LNCaP (P53+ and AR+) and PC3 (P53– and AR–). We also studied the effect of monensin, an apoptosis inducing reagent, in X-ray-induced cell killing. Comparison of gene expressions between unirradiated LNCaP and PC3 cells revealed distinguished gene expression patterns. The data showed a significantly higher expression level of genes involved in the caspase/card family and the TNF ligand/receptor family in PC3 cells, whereas, LNCaP cells exhibited higher expressions in the p53 related genes. At 2 and 4 hrs post a 10 Gy X-ray exposure, changes of gene expressions were detected in a significant fraction of the genes in LNCaP cells, but no significant changes were found in PC3 cells. There was no significant apoptosis-inducing effect of X-rays (up to 10 Gy) in both cell lines; however, monensin was shown to be effective in inducing apoptosis in LNCaP, but not in PC3 cells. In addition, the effect of combined treatment of monensin and X-rays in LNCaP cells appeared to be synergistic. Our results suggest that monensin may be effective for both cancer cell killing and radiosensitizing, and the different expression profiles in apoptosis related genes in cancer cells may be correlated with their sensitivity to apoptosis inducing reagents.

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To investigate genes regulated by COX-2 or a COX-2 specific inhibitor, celecoxib, in irradiated cancer cells, we analyzed changes in gene expression using complementary DNA microarray following celecoxib or combined celecoxib and ionizing radiation (IR) treatment in a stable COX-2 knockdown A549 (AS) and a mock cell line (AN). Thirty-six genes were differentially expressed by COX-2 knockdown. Celecoxib changed the expressions of 40 and 69 genes in AN and AS cells, respectively. Twenty-seven genes were synchronously regulated by COX-2 and celecoxib. Among these, celecoxib regulated ras homolog gene family B and mitosin protein expression in a COX-2 dependent manner, especially in irradiated cells. In addition, we identified 11 genes that changed by more than 1.5 times the expected additive values after celecoxib and IR treatment. The current study may provide evidence that COX-2 or celecoxib regulates various intracellular functions in addition to their enzymatic activity regulation. We also identified candidate molecules that may be responsible for COX-2-dependent radiosensitization by celecoxib.

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Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm²). Cells were grown for up to 3 days, and phosphorylated ATM at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12–72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process.

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We examined dose–dependent or dose rate–dependent changes of superoxide dismutase (SOD) activity using a new large-scale facility for exposing small animals to radon. Mice were exposed to radon at a concentration of 250, 500, 1000, 2000, or 4000 Bq/m³ for 0.5, 1, 2, 4, or 8 days. When mice were exposed to radon at 2000 day•Bq/m³, activation of SOD activities in plasma, liver, pancreas, heart, thymus, and kidney showed dose–rate effects. Our results also suggested that continuous exposure to radon increased SOD activity, but SOD activity transiently returned to normal levels at around 2 days. Moreover, we classified the organs into four groups (1. plasma, brain, lung; 2. heart, liver, pancreas, small intestine; 3. kidney, thymus; 4. stomach) based on changes in SOD activity. Thymus had the highest responsiveness and stomach had lowest. These data provide useful baseline measurements for future studies on radon effects.

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We compared the prostate volumes and rectal doses calculated by CT and CT-MRI fusion, and verified the usefulness of CT-MRI fusion in three-dimensional (3D) radiotherapy planning for localized prostate cancer. Three observers contoured the prostate and rectum of 13 patients with CT and CT-MRI fusion. Prostate delineations were classified into three sub-parts, and the volumes and distances to the rectum (PR distance) were calculated. 3D radiotherapy plans were generated. A dose-volume histogram (DVH) was constructed for the rectum. The intermodality and interobserver variations were assessed. CT-MRI fusion yielded a significantly lower prostate volume by 31%. In the sub-part analysis, the greatest difference was seen for the apical side. The PR distance was significantly extended by 3.5-mm, and the greatest difference was seen for the basal side. The irradiated rectal volume was reduced in the CT-MRI fusion-based plan. The reduction rates were greater in the relatively high-dose regions. The decrease of the prostate volume and length alteration of the distance between the prostate and rectum were correlated with the decrease of the irradiated rectal volume. The prostate volume delineated by CT-MRI fusion was negatively correlated with the decrease of the irradiated rectal volume. CT showed a tendency towards overestimation of the prostate volume and underestimation of the PR distance as compared to CT-MRI fusion. The rectal dose was significantly reduced in CT-MRI fusion-based plan. Using CT-MRI fusion, especially in cases with a small prostate, the irradiated rectal volume can be reduced, with consequent reduction in rectal complications.

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Cerebral radionecrosis is a significant side effect in radiotherapy for brain cancer. The purpose of this study is to calculate the relative biological effectiveness (RBE) of carbon-ion beams on brain cells and to show RBE-weighted dose distributions for cerebral radionecrosis speculation in a carbon-ion treatment planning system. The RBE value of the radionecrosis for the carbon-ion beam is calculated by the modified microdosimetric kinetic model on the assumption of a typical clinical α/β ratio of 2 Gy for cerebral radionecrosis in X-rays. This calculation method for the RBE-weighted dose is built into the treatment planning system for the carbon-ion radiotherapy. The RBE-weighted dose distributions are calculated on computed tomography (CT) images of four patients who had been treated by carbon-ion radiotherapy for astrocytoma (WHO grade 2) and who suffered from necrosis around the target areas. The necrotic areas were detected by brain scans via magnetic resonance imaging (MRI) after the treatment irradiation. The detected necrotic areas are easily found near high RBE-weighted dose regions. The visual comparison between the RBE-weighted dose distribution and the necrosis region indicates that the RBE-weighted dose distribution will be helpful information for the prediction of radionecrosis areas after carbon-ion radiotherapy.

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One of the unusual patterns of local recurrence in breast cancer patient is an inflammatory skin recurrence (ISR) sharply demarcating the area of previous radiation fields. To clarify the characteristics of this recurrence, we conducted a nationwide survey. We sent a survey to radiation oncologists at 200 institutions in Japan and received answers from 92. Of these, 24 institutions had some experience with patients who developed ISR affecting the previously irradiated area. The case details of 16 patients from 11 institutions were available and analyzed in this study. Eight patients experienced ISR after breast conservative therapy (groupA) and 8 patients experienced ISR after post-mastectomy radiotherapy (groupB). The postoperative pathological examination of groups A and B showed positive axillary lymph-nodes in 7/8 and 8/8 patients, positive lymphatic invasion in 4/7 and 7/8 patients, and ER status negative in 7/8 and 6/7 patients respectively. Median survival period was 266 days in groupA and 1105 days in groupB (p = 0.0001). Patients who developed the ISR tended to have several characteristics, including positive lymph-node metastases, intensive lymphatic invasion, and ER status negative. Physicians should contemplate the diagnosis of ISR next to radiation recall or radiation dermatitis, especially when the aforementioned risk factors are present.

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The purpose of this study was to quantify the changes of tumor blood flow (BF) in cervical cancer after radiation therapy by using perfusion computed tomography (CT), and to examine the difference between maximum slope (MS) and single-input one-compartment model (SOCM) methods. Fourteen consecutive patients who received definitive radiation therapy for cervical cancer from October 2009 to February 2010 were enrolled in this study. Blood flow (BF) analyses were performed using both MS and SOCM methods. Quantitative BF maps were created using Body Perfusion (Toshiba Medical Systems, Co. Tokyo, Japan). Perfusion color maps were successfully created by the two analytical methods. BF of the tumors was clearly higher than that of normal cervix, making it possible to distinguish tumor tissue from normal cervical tissue. BF of the tumors after 20 Gy of radiation therapy calculated by the MS method was significantly larger than that before treatment (126.9 vs. 72.2 ml/min/100 ml, median; p < 0.05). Although BF calculated by the MS and SOCM methods showed a positive linear correlation (p < 0.001, r = 0.981), BF calculated by the MS method was lower than that obtained by the SOCM method (103.7 vs. 115.1 ml/min/100 ml, p < 0.01). The change of tumor BF in cervical cancer before and after radiation therapy can be monitored by conducting blood flow analysis using perfusion CT. BF by the MS method was lower than that by the SOCM method, but the two analytical methods correlated well. Perfusion CT may have potential in noninvasive monitoring of vascular and oxygenation status and for guiding adaptive therapy.

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The purpose of this study was to assess the toxicity and efficacy of multimodal approaches, including three-dimensional conformal re-irradiation, for patients with recurrent or persistent esophageal cancer after radiotherapy. Thirty-one patients with esophageal cancer treated with three-dimensional conformal re-irradiation were retrospectively analyzed. Of the 31 patients, 27 patients received concurrent chemotherapy, and 14 patients underwent regional hyperthermia during the re-irradiation. We divided the patients into two groups on the basis of their clinical condition: the curative group (n = 11) or the palliative group (n = 20). Severe toxicities were detected in one patient with Grade 3 esophageal perforation in the curative group, and 5 patients had a Grade 3 or higher toxicity of the esophagus in the palliative group. Advanced T stage at the time of re-irradiation was found to be significantly correlated with Grade 3 or higher toxicity in the esophagus. For the curative group, 10 (91%) of 11 patients had an objective response. For the palliative group, symptom relief was recognized in 8 (57%) of 14 patients with obvious swallowing difficulty. In conclusion, in the curative group with early-stage recurrent or persistent esophageal cancer, the multimodal approaches, including three-dimensional conformal re-irradiation, may be feasible, showing acceptable toxicity and a potential value of promising results, although further evaluations especially for the toxicities of the organs at risk are required. In the palliative group, the benefit of our therapy may be restrictive because severe esophageal toxicities were not uncommon in the patients with advanced T stage at the time of re-irradiation.

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Hypoxia-inducible factor 1α (HIF-1α) is an intrinsic marker of tumor hypoxia. It has been considered that hypoxic conditions reduce radiosensitivity, but the role of HIF-1α in patients treated with preoperative therapy for rectal cancer is still unclear. The aim of this study was to evaluate the predictive value of tumor response to preoperative hyperthermo-chemoradiotherapy (HCRT) and the prognostic significance of HIF-1α expression in patients with locally advanced rectal cancer. Between 2003 and 2006, 50 patients with histologically proven rectal adenocarcinoma who underwent HCRT followed by surgery were investigated. HIF-1α expression was immunohistochemically evaluated using pre-treatment biopsies. The total radiation dose was 40–50 Gy and chemotherapy consisted of 5-FU and LV administered by continuous infusion on Day 1–5, Day 15–19, and Day 29–33 during radiotherapy. Hyperthermia treatment was performed for once a week for 2–5 sessions. The surgical operation was performed 8 weeks after HCRT and each resected specimen was graded by histological criteria of the Japanese Classification of Colorectal Carcinoma. The effects of HIF-1α on clinical outcomes were analyzed by univariate and multivariate analysis. Positive HIF-1α expression was recognized in 42.0% of samples (21/50). Resected specimens that showed pathological grades 1, 2, and 3 numbered 17, 24, and 9 cases, respectively. There were no significant differences between the HIF-1α-positive group and HIF-1α-negative group for pathological grading and pCR. Overall survival (OS) rate at 3 years in the HIF-1α-negative group was 85.2%, which was significantly better than the 60.6% in the HIF-1α-positive group. Recurrence-free survival (RFS) rate at 3 years in the HIF-1α-negative group was 82.8%, being significantly better than 47.6% in the HIF-1α-positive group. In addition, elevated HIF-1α expression was significantly correlated with recurrence-free survival and metastasis-free survival rate in multivariate analysis. HIF-1α expression might be predictive of recurrence-free survival and metastasis-free survival rate for rectal cancer patients treated with HCRT.

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CpG-oligodeoxynucleotide (ODN), a synthetic analog of bacteria DNA, has attracted attention because it activates cells of an adaptive immune system and the innate immune system. In this study, we investigated whether CpG-ODN has radioprotective effects, when administered after total-body irradiation (TBI). Mice were treated with 50 μg CpG-ODN via intraperitoneal injection (i.p) within 30 min, 24 h and 48 h after TBI. Our results showed that the survival rate was enhanced at various levels of TBI. The calculated dose reduction factor (DRF) was 1.2. Bone marrow cell count and bone marrow histological examination indicated that CpG-ODN minimized the bone marrow damage induced by TBI. The data of the white blood cell (WBC) count, exogenous (CFU-S) and endogenous (endoCFU-S) colony forming unit-spleen count demonstrated that CpG-ODN reduced primitive hematopoietic stem cells damage and reconstituted hematopoiesis after TBI. Thus, we suggested that CpG-ODN had the potential to contribute to the improvement of the survival rate and limitation of myelosuppression induced by TBI.

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This study aimed to evaluate changes in the biodiversity of the oral microflora of patients with head and neck cancer treated with postoperative intensity-modulated radiotherapy (IMRT) or conventional radiotherapy (CRT). Pooled dental plaque samples were collected during the radiation treatment from patients receiving IMRT (n = 13) and CRT (n = 12). Denaturing gradient gel electrophoresis (DGGE) was used to analyze the temporal variation of these plaque samples. The stimulated and unstimulated salivary flow rates were also compared between IMRT and CRT patients. Reductions in the severity of hyposalivation were observed in IMRT patients compared with CRT patients. We also observed that the temporal stability of the oral ecosystem was significantly higher in the IMRT group (69.96 ± 7.82%) than in the CRT group (51.98 ± 10.45%) (P < 0.05). The findings of the present study suggest that IMRT is more conducive to maintaining the relative stability of the oral ecosystem than CRT.

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Paraaortic lymph nodal (PALN) recurrence is not a rare scenario of abdominal malignancies including pancreas cancer, even after radiotherapy. The role and indication of reirradiation is limited because it is often associated with significant late toxicity in the surrounding normal organs. We developed a new approach and technique of brachytherapy to overcome this difficult situation, with a paravertebral approach and hyaluronate gel injection (HGI). We encountered a patient with pancreatic cancer who developed PALN metastasis as in-field recurrence, 6 months after resection of pancreatic cancer with 50 Gy of preoperative radiotherapy. The applicator brachytherapy needles and gel injection needles were advanced by percutaneous paravertebral approach under local anesthesia, and by injection of a hyaluronate gel to separate the surrounding small intestines and the target. A single fraction of 18 Gy was delivered to the tumor (75.6 Gy equivalent in conventional schedule calculated with LQ model at α/β = 3) and total estimated D2cc (the minimum dose to the most irradiated volume of 2 cc, calculated at α/β = 2) in the small intestines was 61.6 GyE with HGI and 80.6 GyE without. No complications have been observed for six months. Three months later, FDG accumulation had disappeared, the tumor size was reduced, and serum CA-19-9 value decreased from 5150 U/mL to 36.6 U/mL (normal range < 37.5). Conclusively, the brachytherapy with the HGI procedure by paravertebral approach was thought safe and effective in reirradiation of PALN recurrence.

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