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author:("matsunaga, S")
1.  Difference in distribution profiles between CD163+ tumor-associated macrophages and S100+ dendritic cells in thymic epithelial tumors 
Diagnostic Pathology  2014;9(1):215.
Background
In a number of human malignancies, tumor-associated macrophages (TAMs) are closely involved in tumor progression. On the other hand, dendritic cells (DCs) that infiltrate tumor tissues are involved in tumor suppression. However, there have been very few reports on the distribution profiles of TAMs and DCs in thymic epithelial tumors. We examined the difference in the distribution profiles between TAMs and DCs in thymoma and thymic carcinoma.
Methods
We examined 69 samples of surgically resected thymic epithelial tumors, namely, 16 thymic carcinomas and 53 thymomas, in which we immunohistochemically evaluated the presence of TAMs using CD68 and CD163 as markers and DCs using S100 as the marker in tumor tissue samples in comparison with normal thymic tissues.
Results
The percentage of samples with a large number of CD68+ TAMs was not significantly different between thymic carcinoma and thymoma (7/16 versus 16/53, p = 0.904). However, the percentage of sample with a large number of CD163+ TAMs was significantly higher in thymic carcinoma than in thymoma (15/16 versus 34/53, p = 0.024). In contrast, the percentage of samples with a large number of S100+ DCs was significantly lower in thymic carcinoma than in thymoma (2/16 versus 23/53, p = 0.021).
Conclusions
To the best of our knowledge, we are the first to show a high percentage of CD163+ TAMs and a low percentage of S100+ DCs in thymic carcinoma samples, and our findings may provide an idea for future targeted therapeutic strategies for thymic carcinoma using antibodies that inhibit monocyte differentiation to TAMs, thereby skewing TAMs differentiation toward DCs.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_215
doi:10.1186/s13000-014-0215-7
PMCID: PMC4302590  PMID: 25499804
Thymic carcinoma; Thymoma; Tumor-associated macrophages; CD163; Dendritic cells; S100
2.  Assessment of Still and Moving Images in the Diagnosis of Gastric Lesions Using Magnifying Narrow-Band Imaging in a Prospective Multicenter Trial 
PLoS ONE  2014;9(7):e100857.
Objectives
Magnifying narrow-band imaging (M-NBI) is more accurate than white-light imaging for diagnosing small gastric cancers. However, it is uncertain whether moving M-NBI images have additional effects in the diagnosis of gastric cancers compared with still images.
Design
A prospective multicenter cohort study.
Methods
To identify the additional benefits of moving M-NBI images by comparing the diagnostic accuracy of still images only with that of both still and moving images. Still and moving M-NBI images of 40 gastric lesions were obtained by an expert endoscopist prior to this prospective multicenter cohort study. Thirty-four endoscopists from ten different Japanese institutions participated in the prospective multicenter cohort study. Each study participant was first tested using only still M-NBI images (still image test), then tested 1 month later using both still and moving M-NBI images (moving image test). The main outcome was a difference in the diagnostic accuracy of cancerous versus noncancerous lesions between the still image test and the moving image test.
Results
Thirty-four endoscopists were analysed. There were no significant difference of cancerous versus noncancerous lesions between still and moving image tests in the diagnostic accuracy (59.9% versus 61.5%), sensitivity (53.4% versus 55.9%), and specificity (67.0% versus 67.6%). And there were no significant difference in the diagnostic accuracy between still and moving image tests of demarcation line (65.4% versus 65.5%), microvascular pattern (56.7% versus 56.9%), and microsurface pattern (48.1% versus 50.9%). Diagnostic accuracy showed no significant difference between the still and moving image tests in the subgroups of endoscopic findings of the lesions.
Conclusions
The addition of moving M-NBI images to still M-NBI images does not improve the diagnostic accuracy for gastric lesions. It is reasonable to concentrate on taking sharp still M-NBI images during endoscopic observation and use them for diagnosis.
Trial registration
Umin.ac.jp UMIN-CTR000008048
doi:10.1371/journal.pone.0100857
PMCID: PMC4079511  PMID: 24988209
3.  Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines 
Genes & Cancer  2014;5(7-8):285-292.
Piperlongumine is a naturally-occurring small molecule with various biological activities. Recent studies demonstrate that piperlongumine selectively kills various types of transformed cells with minimal toxicity to non-transformed cells by inducing a high level of reactive oxygen species (ROS). ROS generates various types of DNA lesions, including base modifications and single strand breaks. In order to examine the contribution of ROS-induced DNA damage to the cytotoxicity by piperlongumine, various DNA repair-deficient chicken DT40 cell-lines with a single DNA repair gene deletion were tested for cellular sensitivity to piperlongumine. The results showed that cell lines defective in homologous recombination (HR) display hyper-sensitivity to piperlongumine, while other cell lines with a deficiency in non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), Fanconi anemia (FA) pathway, or translesion DNA synthesis (TLS) polymerases, show no sensitivity to piperlongumine. The results strongly implicate that double strand breaks (DSBs) generated by piperlongumine are major cytotoxic DNA lesions. Furthermore, a deletion of 53BP1 or Ku70 in the BRCA1-deficient cell line restored cellular resistance to piperlongumine. This strongly supports the idea that piperlongumine induces DSB- mediated cell death. Interestingly, piperlongumine makes the wild type DT40 cell line hypersensitive to a PARP-inhibitor, Olaparib. The results implicate that piperlongumine inhibits HR. Further analysis with cell-based HR assay and the kinetic study of Rad51 foci formation confirmed that piperlongumine suppresses HR activity. Altogether, we revealed novel mechanisms of piperlongumine-induced cytotoxicity.
PMCID: PMC4162141  PMID: 25221646
BRCA1; BRCA2; piperlongumine; oxidative stress; homologous recombination; chemotherapy
4.  Effect of bevacizumab combined with boron neutron capture therapy on local tumor response and lung metastasis 
The aim of the present study was to evaluate the effect of bevacizumab on local tumor response and lung metastatic potential during boron neutron capture therapy (BNCT) and in particular, the response of intratumor quiescent (Q) cells. B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously administered bromodeoxyuridine (BrdU) to label all proliferating (P) tumor cells. The tumors were irradiated with thermal neutron beams following the administration of a 10B-carrier [L-para-boronophenylalanine-10B (BPA) or sodium mercaptoundecahydrododecaborate-10B (BSH)], with or without the administration of bevacizumab. This was further combined with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH, 40°C for 60 min). Immediately following the irradiation, cells from certain tumors were isolated and incubated with a cytokinesis blocker. The responses of the Q cells and the total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumor-bearing mice, 17 days following irradiation, lung metastases were enumerated. Three days following bevacizumab administration, the sensitivity of the total tumor cell population following BPA-BNCT had increased more than that following BSH-BNCT. The combination with MTH, but not with nicotinamide, further enhanced total tumor cell population sensitivity. Regardless of the presence of a 10B-carrier, MTH enhanced the sensitivity of the Q cell population. Regardless of irradiation, the administration of bevacizumab, as well as nicotinamide treatment, demonstrated certain potential in reducing the number of lung metastases especially in BPA-BNCT compared with BSH-BNCT. Thus, the current study revealed that BNCT combined with bevacizumab has the potential to sensitize total tumor cells and cause a reduction in the number of lung metastases to a similar level as nicotinamide.
doi:10.3892/etm.2014.1704
PMCID: PMC4061189  PMID: 24944637
bevacizumab; boron neutron capture therapy; 10B-carrier; quiescent cell; nicotinamide; mild temperature hyperthermia
5.  The dependency of compound biological effectiveness factors on the type and the concentration of administered neutron capture agents in boron neutron capture therapy 
SpringerPlus  2014;3:128.
Purpose
To examine the effect of the type and the concentration of neutron capture agents on the values of compound biological effectiveness (CBE) in boron neutron capture therapy.
Methods and materials
After the subcutaneous administration of a 10B-carrier, boronophenylalanine-10B (BPA) or sodium mercaptododecaborate-10B (BSH), at 3 separate concentrations, the 10B concentrations in tumors were measured by γ-ray spectrometry. SCC VII tumor-bearing C3H/He mice received 5-bromo-2′-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells, then treated with BPA or BSH. Immediately after reactor neutron beam irradiation, during which intratumor 10B concentrations were kept at levels similar to each other, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of BrdU-unlabeled quiescent (Q) and total (= P + Q) tumor cells were assessed based on the frequencies of micronucleation using immunofluorescence staining for BrdU.
Results
The CBE values were higher in Q cells and in the use of BPA than total cells and BSH, respectively. In addition, the higher the administered concentrations were, the smaller the CBE values became, with a clearer tendency in the use of BPA than BSH. The values for neutron capture agents that deliver into solid tumors more dependently on uptake capacity of tumor cells became more changeable.
Conclusion
Tumor characteristics, such as micro-environmental heterogeneity, stochastic genetic or epigenetic changes, or hierarchical organization of tumor cells, are thought to partially influence on the value of CBE, meaning that the CBE value itself may be one of the indices showing the degree of tumor heterogeneity.
doi:10.1186/2193-1801-3-128
PMCID: PMC4320213
Boron neutron capture therapy; Neutron capture agent; Relative biological effectiveness; Compound biological effectiveness; Tumor heterogeneity; Quiescent cell
6.  Radiosensitivity of pimonidazole-unlabelled intratumour quiescent cell population to γ-rays, accelerated carbon ion beams and boron neutron capture reaction 
The British Journal of Radiology  2013;86(1021):20120302.
Objective
To detect the radiosensitivity of intratumour quiescent (Q) cells unlabelled with pimonidazole to accelerated carbon ion beams and the boron neutron capture reaction (BNCR).
Methods
EL4 tumour-bearing C57BL/J mice received 5-bromo-29-deoxyuridine (BrdU) continuously to label all intratumour proliferating (P) cells. After the administration of pimonidazole, tumours were irradiated with c-rays, accelerated carbon ion beams or reactor neutron beams with the prior administration of a 10B-carrier. Responses of intratumour Q and total (P+Q) cell populations were assessed based on frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of pimonidazole-unlabelled tumour cells was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole.
Results
Following c-ray irradiation, the pimonidazole-unlabelled tumour cell fraction showed significantly enhanced radiosensitivity compared with the whole tumour cell fraction, more remarkably in the Q than total cell populations. However, a significantly greater decrease in radiosensitivity in the pimonidazole-unlabelled cell fraction, evaluated using a delayed assay or a decrease in radiation dose rate, was more clearly observed among the Q than total cells. These changes in radiosensitivity were suppressed following carbon ion beam and neutron beam-only irradiaton. In the BNCR, the use of a 10B-carrier, especially L-para-boronophenylalanine-10B, enhanced the sensitivity of the pimonidazole-unlabelled cells more clearly in the Q than total cells.
Conclusion
The radiosensitivity of the pimonidazole-unlabelled cell fraction depends on the quality of radiation delivered and characteristics of the 10B-carrier used in the BNCR.
Advances in knowledge
The pimonidazole-unlabelled subfraction of Q tumour cells may be a critical target in tumour control.
doi:10.1259/bjr.20120302
PMCID: PMC3615400  PMID: 23255546
7.  Mental health status among younger generation around Chernobyl 
Archives of Medical Science : AMS  2013;9(6):1114-1116.
Introduction
In order to improve our understanding of how to approach the younger generation around Chernobyl, we screened mental health status among young adults born after the accident living in the Gomel region, Belarus.
Material and methods
We enrolled 697 medical students who were born after the accident. Participants were asked to answer self-administered questionnaires including the General Health Questionnaire (GHQ-12).
Results
GHQ-12 scores were 1.80 ±2.28 (mean ± SD) among all 697 subjects. When logistic regression analysis was performed with confounding factors, “economic situation” and “association of diseases and/or poor health condition with radiation exposure” were significantly associated with poor mental status.
Conclusions
Our findings suggest that anxiety about radiation exposure among highly educated medical students in the Gomel region, although they were born after the accident, affects their poor mental health status.
doi:10.5114/aoms.2013.39798
PMCID: PMC3902728  PMID: 24482659
Chernobyl; Fukushima; Mental health; General Health Questionnaire-12
8.  Sonoporation as an enhancing method for boron neutron capture therapy for squamous cell carcinomas 
Background
Boron neutron capture therapy (BNCT) is a selective radiotherapy that is dependent on the accumulation of 10B compound in tumors. Low-intensity ultrasound produces a transient pore on cell membranes, sonoporation, which enables extracellular materials to enter cells. The effect of sonoporation on BNCT was examined in oral squamous cell carcinoma (SCC) xenografts in nude mice.
Materials and methods
Tumor-bearing mice were administrated boronophenylalanine (BPA) or boronocaptate sodium (BSH) intraperitoneally. Two hours later, tumors were subjected to sonoporation using microbubbles followed by neutron irradiation.
Results
The 10B concentration was higher in tumors treated with sonoporation than in untreated tumors, although the difference was not significant in BPA. When tumors in mice that received BPA intraperitoneally were treated with sonoporation followed by exposure to thermal neutrons, tumor volume was markedly reduced and the survival rate was prolonged. Such enhancements by sonoporation were not observed in mice treated with BSH-mediated BNCT.
Conclusions
These results indicate that sonoporation enhances the efficiency of BPA-mediated BNCT for oral SCC. Sonoporation may modulate the microlocalization of BPA and BSH in tumors and increase their intracellular levels.
doi:10.1186/1748-717X-8-280
PMCID: PMC3904744  PMID: 24295213
Ultrasound; Microbubble; Boron neutron capture therapy; Oral squamous cell carcinoma
9.  Development of a simple and rapid method of precisely identifying the position of 10B atoms in tissue: an improvement in standard alpha autoradiography 
Journal of Radiation Research  2013;55(2):373-380.
Boron neutron capture therapy (BNCT) can be utilized to selectively kill cancer cells using a boron compound that accumulates only in cancer cells and not in normal cells. Tumor-bearing animals treated by BNCT are routinely used to evaluate long-term antitumor effects of new boron compounds. Alpha-autoradiography is one of the methods employed in the evaluation of antitumor effects. However, a standard alpha-autoradiography cannot detect the microdistribution of 10B because of the difficulty associated with the superposition of a tissue sample image and etched pits on a track detector with the etching process. In order to observe the microdistribution of 10B, some special methods of alpha-autoradiography have been developed that make use of a special track detector, or the atomic force microscope combined with X-ray and UV light irradiation. In contrast, we propose, herein, a simple and rapid method of precisely identifying the position of 10B using the imaging process and the shape of etched pits, such as their circularity, without the need to use special track detectors or a microscope. A brief description of this method and its verification test are presented in this article. We have established a method of detecting the microdistribution of 10B with submicron deviation between the position of etched pits and the position of reaction in a tissue sample, for a given circularity of etched pits.
doi:10.1093/jrr/rrt110
PMCID: PMC3951073  PMID: 24142968
boron neutron capture therapy; boron compound; alpha-autoradiography; microdistribution
10.  High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation 
Journal of Radiation Research  2013;55(1):75-83.
Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with 60Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM's resistance to X-rays in a clinical setting.
doi:10.1093/jrr/rrt095
PMCID: PMC3885128  PMID: 23955054
glioblastoma multiforme; glioma stem cells; linear energy transfer; neutron beams; gamma rays
11.  Boron neutron capture therapy outcomes for advanced or recurrent head and neck cancer 
Journal of Radiation Research  2013;55(1):146-153.
We retrospectively review outcomes of applying boron neutron capture therapy (BNCT) to unresectable advanced or recurrent head and neck cancers. Patients who were treated with BNCT for either local recurrent or newly diagnosed unresectable head or neck cancers between December 2001 and September 2007 were included. Clinicopathological characteristics and clinical outcomes were retrieved from hospital records. Either a combination of borocaptate sodium and boronophenylalanine (BPA) or BPA alone were used as boron compounds. In all the treatment cases, the dose constraint was set to deliver a dose <10–12 Gy-eq to the skin or oral mucosa. There was a patient cohort of 62, with a median follow-up of 18.7 months (range, 0.7–40.8). A total of 87 BNCT procedures were performed. The overall response rate was 58% within 6 months after BNCT. The median survival time was 10.1 months from the time of BNCT. The 1- and 2-year overall survival (OS) rates were 43.1% and 24.2%, respectively. The major acute Grade 3 or 4 toxicities were hyperamylasemia (38.6%), fatigue (6.5%), mucositis/stomatitis (9.7%) and pain (9.7%), all of which were manageable. Three patients died of treatment-related toxicity. Three patients experienced carotid artery hemorrhage, two of whom had coexistent infection of the carotid artery. This study confirmed the feasibility of our dose-estimation method and that controlled trials are warranted.
doi:10.1093/jrr/rrt098
PMCID: PMC3885131  PMID: 23955053
boron neutron capture therapy; head and neck tumors
12.  Effects of employing a 10B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy 
The British Journal of Radiology  2012;85(1011):249-258.
Objectives
To evaluate the effects of employing a 10B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy (BNCT) by measuring the response of intratumour quiescent (Q) cells.
Methods
B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumours received reactor thermal neutron beam irradiation following the administration of a 10B-carrier [L-para-boronophenylalanine-10B (BPA) or sodium mercaptoundecahydrododecaborate-10B (BSH)] in combination with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.
Results
BPA-BNCT increased the sensitivity of the total tumour cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without a 10B–carrier, MTH enhanced the sensitivity of the Q cell population. Without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with nicotinamide treatment, showed the potential to reduce the number of metastases more than BSH-BNCT.
Conclusion
BSH-BNCT in combination with MTH improves local tumour control, while BPA-BNCT in combination with nicotinamide may reduce the number of lung metastases.
doi:10.1259/bjr/20974899
PMCID: PMC3473983  PMID: 22391496
13.  Reducing intratumour acute hypoxia through bevacizumab treatment, referring to the response of quiescent tumour cells and metastatic potential 
The British Journal of Radiology  2011;84(1008):1131-1138.
Objectives
The aim was to evaluate the influence of bevacizumab on intratumour oxygenation status and lung metastasis following radiotherapy, with specific reference to the response of quiescent (Q) cell populations within irradiated tumours.
Methods
B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation following treatment with the acute hypoxia-releasing agent nicotinamide or local mild temperature hyperthermia (MTH) with or without the administration of bevacizumab under aerobic conditions or totally hypoxic conditions, achieved by clamping the proximal end of the tumours. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In the other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.
Results
3 days after bevacizumab administration, acute hypoxia-rich total cell population in the tumour showed a remarkably enhanced radiosensitivity to γ-rays, and the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without γ-ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment.
Conclusion
Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide.
doi:10.1259/bjr/38457938
PMCID: PMC3473837  PMID: 21586505
14.  Effect of bevacizumab treatment on p-boronophenylalanine distribution in murine tumor 
Journal of Radiation Research  2012;54(2):260-267.
Previous studies have demonstrated that angiogenesis inhibitors can enhance tumor inhibitory effects of chemo- and radiotherapy via their action on tumor vessels. Here, we studied the effect of the angiogenesis inhibitor, bevacizumab (Avastin), on boron distribution in a murine tumor model. The human head and neck squamous cell carcinoma cell line was used for inoculation into mice. Boron-10 concentrations in tissues were measured by prompt γ-ray spectrometry (PGA). Hoechst 33342 perfusion and p-boronophenylalanine (BPA) distribution were determined by immunofluorescence staining. Our results revealed enhanced tumor blood perfusion and BPA accumulation in tumors after Avastin treatment, suggesting that combination of angiogenesis inhibition with treatment with boron compound administration may improve the efficacy of boron neutron capture therapy (BNCT) by modifying tumor vessels. In addition, our results also demonstrated the usefulness of immunofluorescence staining for investigating boron compound distribution at the cellular level.
doi:10.1093/jrr/rrs102
PMCID: PMC3589940  PMID: 23135099
angiogenesis inhibitor; bevacizumab; boron compounds; BNCT
15.  Wortmannin efficiently suppresses the recovery from radiation-induced damage in pimonidazole-unlabeled quiescent tumor cell population 
Journal of Radiation Research  2012;54(2):221-229.
Labeling of proliferating (P) cells in mice bearing EL4 tumors was achieved by continuous administration of 5-bromo-2′-deoxyuridine (BrdU). Tumors were irradiated with γ-rays at 1 h after pimonidazole administration followed by caffeine or wortmannin treatment. Twenty-four hours later, assessment of the responses of quiescent (Q) and total (= P + Q) cell populations were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. The pimonidazole-unlabeled cell fraction showed significantly enhanced radio-sensitivity compared with the whole cell fraction more remarkably in Q cells than total cells. However, a significantly greater decrease in radio-sensitivity in the pimonidazole-unlabeled than the whole cell fraction, evaluated using an assay performed 24 hours after irradiation, was more clearly observed in Q cells than total cells. In both the pimonidazole-unlabeled and the whole cell fractions, wortmannin efficiently suppressed the reduction in sensitivity due to delayed assay. Wortmannin combined with γ-ray irradiation is useful for suppressing the recovery from radiation-induced damage especially in the pimonidazole-unlabeled cell fraction within the total and Q tumor cell populations.
doi:10.1093/jrr/rrs094
PMCID: PMC3589932  PMID: 23097299
Quiescent cell; recovery from radiation-induced damage; pimonidazole; wortmannin; caffeine
16.  DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction 
Background
Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He) particle and a recoiled lithium nucleus (7Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells.
Methods
Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity.
Results
Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity.
Conclusion
Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR.
doi:10.1186/1748-717X-6-106
PMCID: PMC3179943  PMID: 21888676
xrs-5; DNA-DSB; BNCR; gamma-H2AX; 53BP1
17.  Significance of manipulating tumour hypoxia and radiation dose rate in terms of local tumour response and lung metastatic potential, referring to the response of quiescent cell populations 
The British Journal of Radiology  2010;83(993):776-784.
The purpose of this study was to evaluate the influence of manipulating intratumour oxygenation status and radiation dose rate on local tumour response and lung metastases following radiotherapy, referring to the response of quiescent cell populations within irradiated tumours. B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation at high dose rate (HDR) or reduced dose rate (RDR) following treatment with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the quiescent (Q) and total (proliferating + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Following HDR irradiation, nicotinamide and MTH enhanced the sensitivity of the total and Q-cell populations, respectively. The decrease in sensitivity at RDR irradiation compared with HDR irradiation was slightly inhibited by MTH, especially in Q cells. Without γ-ray irradiation, nicotinamide treatment tended to reduce the number of lung metastases. With γ-rays, in combination with nicotinamide or MTH, especially the former, HDR irradiation decreased the number of metastases more remarkably than RDR irradiation. Manipulating both tumour hypoxia and irradiation dose rate have the potential to influence lung metastasis. The combination with the acute hypoxia-releasing agent nicotinamide may be more promising in HDR than RDR irradiation in terms of reducing the number of lung metastases.
doi:10.1259/bjr/57015642
PMCID: PMC3473411  PMID: 20739345
18.  Correction: FANCD1/BRCA2 Plays Predominant Role in the Repair of DNA Damage Induced by ACNU or TMZ 
PLoS ONE  2011;6(6):10.1371/annotation/c6be24d1-bc23-43b4-ae01-b86dad174069.
doi:10.1371/annotation/c6be24d1-bc23-43b4-ae01-b86dad174069
PMCID: PMC3112056
19.  FANCD1/BRCA2 Plays Predominant Role in the Repair of DNA Damage Induced by ACNU or TMZ 
PLoS ONE  2011;6(5):e19659.
Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA−/−, FANCC−/−, FANCA−/−C−/−, FANCD2−/− cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG−/− and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.
doi:10.1371/journal.pone.0019659
PMCID: PMC3090409  PMID: 21573016
20.  Adverse effect of mild temperature hyperthermia combined with hexamethylenetetramine compared to its effect combined with tirapazamine in the treatment of solid tumors 
This study aimed to assess the effect on solid tumors of mild temperature hyperthermia (MTH) combined with hexamethylenetetramine (HMTA) or tirapazamine (TPZ). Squamous cell carcinoma (SCC VII) tumor-bearing mice were continuously administered 5-bromo-2′-deoxyuridine (BrdU) to label intratumor proliferating (P) cells. Mice received HMTA or TPZ through intraperitoneal single or subcutaneous continuous administration, with or without MTH (40°C, 60 min), followed or not by γ-ray irradiation or cisplatin treatment. After HMTA or TPZ administration without γ-ray irradiation or cisplatin treatment, immediately after γ-ray irradiation, or 1 h after cisplatin treatment, the response of quiescent (Q) cells was assessed in terms of micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cells was determined based on a comparison with non-BrdU-treated tumors. Without MTH, HMTA and TPZ had a nearly equal radiosensitizing and cisplatin sensitivity-enhancing effect on both total and Q cells. With MTH, radio- and cisplatin-sensitizing effects by HMTA were reduced, particularly in the Q cells. In contrast, the enhancing effects of TPZ were increased, particularly in the Q cells. Continuous administration of HMTA and TPZ resulted in higher radio- and cisplatin-sensitizing effects than intraperitoneal single administration. In terms of tumor cytotoxicity as a whole, including Q cells, the administration of γ-ray irradiation or cisplatin treatment combined with continuous HMTA administration is promising, taking into account the clinical use of HMTA. However, MTH should not be combined with HMTA administration.
doi:10.3892/etm_00000027
PMCID: PMC3490386  PMID: 23136610
mild temperature hyperthermia; hexamethylenetetramine; tirapazamine; quiescent cell; hypoxia
21.  Ambient levels of volatile organic compounds in the vicinity of petrochemical industrial area of Yokohama, Japan 
Urban ambient air concentrations of 39 aromatic (including benzene, toluene, and xylenes) and aliphatic volatile organic compounds (VOCs) were measured in Yokohama city, Japan. Yokohama city was selected as a case study to assess the amount of VOC released from Industrial area to characterize the ambient air quality with respect to VOC as well as to know the impact of petrochemical storage facilities on local air quality. For this purpose, ambient air samples were collected (from June 2007 to November 2008) at six selected locations which are designated as industrial, residential, or commercial areas. To find out the diurnal variations of VOC, hourly nighttime sampling was carried out for three nights at one of the industrial locations (Shiohama). Samples were analyzed using gas chromatographic system (GC-FID). Results show strong variation between day and nighttime concentrations and among the seasons. Aliphatic fractions were most abundant, suggesting petrochemical storage facilities as the major source of atmospheric hydrocarbons. High concentrations of benzene, toluene, ethyl benzene, and xylene (BTEX) were observed at industrial locations. BTEX showed strong diurnal variation which is attributed to change in meteorology. During our campaign, low ambient VOC concentrations were observed at the residential site.
doi:10.1007/s11869-009-0052-0
PMCID: PMC2860102  PMID: 20495606
BTEX; Volatile organic compounds; Ozone formation potential; Interspecific ratio; Health risk
22.  Effects of Caloric Intake on Intestinal Mucosal Morphology and Immune Cells in Rats Treated with 5-Fluorouracil 
Anticancer drugs have been reported to damage the intestinal mucosa. We evaluated the effects of caloric intake on the mucosal morphology and immune cells in rats treated with 5-fluorouracil (5-FU). Rats were received a liquid diet plus 5-FU treatment for 8 days as follows: Low calorie group (25 kcal/day with 5-FU), Normal calorie group (50 kcal/day with 5-FU), and Control group (50 kcal/day with saline). The mucosal morphology, cell numbers and phenotypes of spleen and intraepithelial lymphocytes (IEL) were assessed. As compared with the control group, the villus heights were significantly lower in the Low calorie group, but not significantly lower in the Normal calorie group. The total cell yield from the spleen, CD4+ and CD8+ T cells decreased in the Low and Normal calorie group, but these changes were less pronounced in Normal calorie group. The total cell yield from the IEL also decreased in the Low calorie group, but not in the Normal calorie group. Our study demonstrated that sufficient caloric intake attenuated the damages in intestinal morphology and in the immune cell numbers. Clinically, nutritional support would be expected to be one approach to reducing the risk of bacterial translocation or infection induced by chemotherapy.
doi:10.3164/jcbn.08-264
PMCID: PMC2704328  PMID: 19590710
5-FU; mucosal atrophy; nutritional support
23.  Tissue oxygenation in a murine SCC VII tumor after X-ray irradiation as determined by EPR spectroscopy 
Purpose
The goal of this study was to clarify the dynamics of tumor oxygen (partial pressure of oxygen, pO2) in SCC VII murine tumors in mice after X-ray irradiation.
Materials and methods
Changes in pO2 in tumors were measured by 1.2-GHz electron paramagnetic resonance (EPR) spectroscopy after they were exposed to various doses of irradiation. The pO2 in tumors was followed for up to six days after irradiation at doses of 0, 5, 10, 15, and 20 Gy. Paramagnetic crystals were used as an oximetry probe and implanted into normal or tumor tissues in mice for prolonged periods.
Results
The pattern of tumor oxygen after a single dose of radiation with the 5-Gy dose was different from those with other doses (10, 15, and 20 Gy). After 5 Gy, pO2 increased rapidly (P < 0.01, Student’s t test) and then returned to the level observed before irradiation by 12 hours (P < 0.01). In contrast, after 10, 15, or 20 Gy, pO2 increased rapidly by 6 h after irradiation, continued to increase until at least 24 h (P < 0.01), and then gradually decreased.
Conclusion
In tumors that received 5 Gy, post-irradiation increases in pO2 at 4 h after irradiation were detected by EPR oximetry (P < 0.01) noninvasively.
doi:10.1016/j.radonc.2007.11.020
PMCID: PMC2362098  PMID: 18077029
EPR; Oximetry; Reoxygenation; Tumor; Radiation
24.  Changes in Lymphocyte Phenotypes and Cytokine Production by Surgical Stress in a Rat Small Intestinal Resection Model 
Small intestinal resection rats are used widely as a malabsorption model, but the immunological changes are unclear. We examined the changes in systemic and mucosal immune status after a small intestinal resection in rats with a controlled nutritional status. Rats had 60% of their small intestine removed. At 5 days after the surgery, spleen cells and intraepithelial lymphocytes (IEL) were isolated. The phenotypes of spleen cells and IEL, the production patterns of Th1 and Th2 cytokines, and the proinflammatory cytokine levels in the plasma were measured. CD4+ T cells in the blood and spleen were significantly decreased in the Resection group (p<0.05). In contrast, IEL subpopulations were not different between the two groups. Interferon-γ production from the spleen cells was significantly decreased in the Resection group (p<0.05). Interleukin (IL)-4 production was not different between the two groups. Plasma IL-6 concentrations were significantly elevated in the Resection group 6 h after surgery (p<0.05). In conclusions, small intestinal resection in rats suppressed systemic immunity, and this model is useful as a surgical stress model.
doi:10.3164/jcbn.40.216
PMCID: PMC2275767  PMID: 18398499
surgical stress; CD4; CD8; Th1/Th2; intraepithelial lymphocyte
25.  Tumour enhancement with newly developed Mn-metalloporphyrin (HOP-9P) in magnetic resonance imaging of mice 
British Journal of Cancer  2001;84(12):1681-1685.
The purpose of the study is to evaluate the tumour enhancing characteristics and biodistribution of a newly developed metalloporphyrin derivative, HOP-9P (13, 17-bis (1-carboxypropionyl) carbamoylethyl-3, 8-bis (1-phenylpropyloxyethyl)-2,7,12,18-tetra- methyl-porphynato manganese (III)). Seven mice bearing SCC VII tumours were imaged using T1-weighted conventional spin echo magnetic resonance images before and 5 min, 2 h and 24 h after intravenous injection of 0.1 mmol/kg of HOP-9P. For the acquired images, signal intensities of the tumour, muscle and oil-phantom were measured. Then, tumor/oil and tumor/muscle signal intensity ratios were calculated. Nineteen mice were sacrificed before or after the administration of HOP-9P (at 5 min, 2 h and 24 h), and the biodistribution of manganese in the tumour, muscle, liver, blood and kidneys was measured using optical emission spectrometers and was expressed as micrograms of manganese per gram of tissue. The tumour/muscle signal intensity ratio at 24 h (3.18 ± 0.34) was significantly higher than precontrast ratio (1.77 ± 0.20) (P < 0.05). The biodistribution assessment of manganese demonstrated that HOP-9P gradually and consistently accumulated in the tumour to reach the highest concentration at 24 h (3.49 ± 1.22 μ gMn/g). It is concluded that HOP-9P is a potential tumour-specific MR contrast agent. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1802
PMCID: PMC2363677  PMID: 11401324
animal; metalloporphyrins; manganese; contrast media; neoplasms; magnetic resonance imaging

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