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Boron neutron capture therapy (BNCT) is based on nuclear reactions between thermal neutron and boron-10 preferentially distributed in the cancer cells. 10 B-boronophenylalanine (BPA) is the approved drug for treatment of oral cancers for BNCT. However, the predictive biomarkers to evaluate therapeutic efficacy and side-effects have not been clarified yet. Here we performed comprehensive analysis of mRNA expression using human oral squamous carcinoma SAS cells after BPA-BNCT. The expression of particular mRNAs including inflammatory and immune-related responses and transcription factors, namely CSF2, ATF3, MAFB, PTGS2 and TNFAIP3 was increased 24 h after neutron irradiation of therapeutic dose of BPA-BNCT. NF-κB pathway genes were also activated after BNCT. The early increase of the gene product of CSF2 gene, granulocyte-macrophage colony stimulating factor (GM-CSF), in culture supernatant of SAS cells was observed by ELISA analysis after BPA-BNCT at a setting dose of 24 Gy-eq. The GM-CSF level was also increased after equivalent dose of gamma-ray and carbon beam irradiation. GM-CSF may be involved in local and systemic early responses of BNCT for particular types of cancer., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MItsuko Masutani reports financial support was provided by Cancer Intelligence Care Systems, Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Accelerator-based boron neutron capture therapy (BNCT) systems employing a solid-state lithium target indicated the reduction of neutron flux over the lifetime of a target, and its reduction could represent the neutron flux model. This study proposes a novel compensatory approach for delivering the required neutron fluence and validates its clinical applicability. The proposed approach relies on the neutron flux model and the cumulative sum of real-time measurements of proton charges. The accuracy of delivering the required neutron fluence for BNCT using the proposed approach was examined in five Li targets. With the proposed approach, the required neutron fluence could be delivered within 3.0%, and within 1.0% in most cases. However, those without using the proposed approach exceeded 3.0% in some cases. The proposed approach can consider the neutron flux reduction adequately and decrease the effect of uncertainty in neutron measurements. Therefore, the proposed approach can improve the accuracy of delivering the required fluence for BNCT even if a neutron flux reduction is expected during treatment and over the lifetime of the Li target. Additionally, by adequately revising the approach, it may apply to other type of BNCT systems employing a Li target, furthering research in this direction., (© 2024. The Author(s).)

Developing strategies for the radiosensitization of cancer cells by the inhibition of genes, which harbor low toxicity to normal cells, will be useful for improving cancer radiotherapy. Here, we focused on a β-site of amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1; β-secretase, memapsin-2). By functional inhibition of this peptidase by siRNA, it has also recently been shown that the DNA strand break marker, γH2AX foci, increased, suggesting its involvement in DNA damage response. To investigate this possibility, we knocked down BACE1 with siRNA in cancer cell lines, and sensitization to γ-irradiation was examined by a colony formation assay, γH2AX foci and level analysis, and flow cytometry. BACE1 knockdown resulted in the sensitization of HeLa, MDA-MB-231, U2OS, and SAOS cells to γ-irradiation in a diverse range. BACE1 knockdown showed a weak radiosensitization effect in osteosarcoma U2OS cells, which has a normal p53 function. HeLa and SAOS cells, which harbor p53 dysfunction, exhibited a greater level of radiosensitization. These results suggest that BACE1 may be a potential target for the radiosensitization in particular cancer cells.

This study aimed to quantify the relative biological effectiveness (RBE) for epithermal neutron beam contaminated with fast neutrons in the accelerator-based boron neutron capture therapy (BNCT) system coupled to a solid-state lithium target. The experiments were performed in National Cancer Center Hospital (NCCH), Tokyo, Japan. Neutron irradiation with the system provided by Cancer Intelligence Care Systems (CICS), Inc. was performed. X-ray irradiation, which was assigned as the reference group, was also performed using a medical linear accelerator (LINAC) equipped in NCCH. The four cell lines (SAS, SCCVII, U87-MG and NB1RGB) were utilized to quantify RBE value for the neutron beam. Before both of those irradiations, all cells were collected and dispensed into vials. The doses of 10% cell surviving fraction (SF) (D10) were calculated by LQ model fitting. All cell experiments were conducted in triplicate at least. Because the system provides not only neutrons, but gamma-rays, the contribution from the gamma-rays to the survival fraction were subtracted in this study. D10 value of SAS, SCCVII, U87-MG and NB1RGB for the neutron beam was 4.26, 4.08, 5.81 and 2.72 Gy, respectively, while that acquired by the X-ray irradiation was 6.34, 7.21, 7.12 and 5.49 Gy, respectively. Comparison of both of the D10 values, RBE value of SAS, SCCVII, U87-MG and NB1RGB for the neutron beam was calculated as 1.7, 2.2, 1.3 and 2.5, respectively, and the average RBE value was 1.9. This study investigated RBE of the epithermal neutron beam contaminated with fast neutrons in the accelerator-based BNCT system coupled to a solid-state lithium target., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Boron neutron capture therapy (BNCT) is a selective radiotherapy based on nuclear reaction that occurs when 10 B atoms accumulated in cancer cells are irradiated by thermal neutrons, triggering a nuclear fission response leading to cell death. Despite its growing importance in cancer treatment, molecular characterization of its effects is still lacking. In this context, proteomics investigation can be useful to study BNCT effect and identify potential biomarkers. Hence, we performed proteomic analysis with nanoLC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) on extracellular vesicles (EVs) isolated from SAS cultures treated or not with 10 B-boronophenylalanine (BPA) and different doses of neutron irradiation, to study the cellular response related to both boron administration and neutrons action. Despite the interference of fetal bovine serum in the medium, we were able to stratify BPA- and BPA+ conditions and to identify EVs-derived proteins characterizing pathways potentially related to a BNCT effect such as apoptosis, DNA repair and inflammatory response. In particular, KLF11, SERPINA1 and SERPINF2 were up-regulated in BPA+, while POLE and SERPINC1 were up-regulated in BPA-. These results provide the first proteomic investigation of EVs treated with BNCT in different conditions and highlight the potentiality of proteomics for improving biomarkers identification and mechanisms understanding of BNCT.

This review discusses the strategies of preclinical studies intended for accelerator-based (AB)-boron neutron capture therapy (BNCT) clinical trials, which were presented at the National Cancer Institute (NCI) Workshop on Neutron Capture Therapy held from April 20 to 22, 2022. Clinical studies of BNCT have been conducted worldwide using reactor neutron sources, with most targeting malignant brain tumors, melanoma, or head and neck cancer. Recently, small accelerator-based neutron sources that can be installed in hospitals have been developed. AB-BNCT clinical trials for recurrent malignant glioma, head and neck cancers, high-grade meningioma, melanoma, and angiosarcoma have all been conducted in Japan. The necessary methods, equipment, and facilities for preclinical studies to evaluate the biological effects of AB-BNCT systems in terms of safety and efficacy are described, with reference to two examples from Japan. The first is the National Cancer Center, which is equipped with a vertical downward neutron beam, and the other is the University of Tsukuba, which has a horizontal neutron beam. The preclinical studies discussed include cell-based assays to evaluate cytotoxicity and genotoxicity, in vivo cytotoxicity and efficacy of BNCT, and radioactivation measurements.

Systems biology approach, carried out with high-throughput omics technologies, has become a fundamental aspect of the study of complex diseases like cancer. It can molecularly characterize subjects, physiopathological conditions, and interactions, allowing a precise description, to reach personalized medicine. In particular, proteomics, typically performed with liquid chromatography coupled to mass spectrometry, is a powerful tool for systems biology, giving the possibility to perform diagnosis, patient stratification, and prediction of therapy effects. Boron Neutron Capture Therapy (BNCT) is a selective antitumoral radiotherapy based on a nuclear reaction that occurs when Boron-10 ( 10 B) atoms are irradiated by low-energy thermal neutrons, leading to cell death, thanks to the production of high-energy α particles. Since BNCT is recently becoming an important therapy for the treatment of different types of solid tumors such as gliomas, head and neck cancers, and others, it can take advantage of molecular investigation to improve the understanding of effects and mechanisms and so help its clinical applications. In this context, proteomics can provide a better understanding of mechanisms related to BNCT effect, identify potential biomarkers, and individuate differential responses by specific patients, stratifying responders and nonresponders. Another key aspect of BNCT is the study of new potential 10 B carriers to improve the selectivity of Boron delivery to tumors and proteomics can be important in this application, studying the effectiveness of new boron delivery agents, including protein-based carriers, also using computational studies that can investigate new molecules, such as boronated monoclonal antibodies, for improving BNCT.

The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified apolipoprotein B mRNA editing enzyme catalytic subunit 3G ( APOBEC3G: A3G ) as a candidate target. APOBEC3G is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. APOBEC3G knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that APOBEC3G knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in APOBEC3G -knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and APOBEC3G knockdown was observed. These results suggest that the functional inhibition of APOBEC3G sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that APOBEC3G could be useful as a target for the radiosensitization of cancer therapy.

Boron neutron capture therapy (BNCT) is a non-invasive therapeutic technique for treating malignant tumors, however, methods to evaluate its therapeutic efficacy and adverse reactions are lacking. High mobility group box 1 (HMGB1) is an inflammatory molecule released during cell death. Therefore, we aimed to investigate HMGB1 as a biomarker for BNCT response, by examining the early responses of tumor cells to 10 B-boronophenylalanine (BPA)-based BNCT in the Kyoto University Nuclear Reactor. Extracellular HMGB1 release was significantly increased in human squamous carcinoma SAS and melanoma A375 cells 24 h after neutron irradiation but not after γ-irradiation. At 3 days post-BPA-based BNCT irradiation in a SAS xenograft mouse model, plasma HMGB1 levels were higher than those in the non-irradiation control, and HMGB1 was detected in both nuclei and cytoplasm in tumor cells. Additionally, increased plasma HMGB1 levels post-BNCT irradiation were detected even when tumors decreased in size. Collectively, these results indicate that the extracellular HMGB1 release occurs at an early stage and is persistent when tumors are reduced in size; therefore, it is a potential biomarker for evaluating the therapeutic response during BNCT.

Identifying patients resistant to cisplatin treatment is expected to improve cisplatin-based chemotherapy for various types of cancers. Excision repair cross-complementing group 1 (ERCC1) is involved in several repair processes of cisplatin-induced DNA crosslinks. ERCC1 overexpression is reported as a candidate prognostic factor and considered to cause cisplatin resistance in major solid cancers. However, anti-ERCC1 antibodies capable of evaluating expression levels of ERCC1 in clinical specimens were not fully optimized. A mouse monoclonal antibody against human ERCC1 was generated in this study. The developed antibody 9D11 specifically detected isoforms of 201, 202, 203 but not 204, which lacks the exon 3 coding region. To evaluate the diagnostic usefulness of this antibody, we have focused on gastric cancer because it is one of the major cancers in Japan. When ERCC1 expression was analyzed in seventeen kinds of human gastric cancer cell lines, all the cell lines were found to express either 201, 202, and/or 203 as major isoforms of ERCC1, but not 204 by Western blotting analysis. Immunohistochemical staining showed that ERCC1 protein was exclusively detected in nuclei of the cells and a moderate level of constant positivity was observed in nuclei of vascular endothelial cells. It showed a clear staining pattern in clinical specimens of gastric cancers. Antibody 9D11 may thus be useful for estimating expression levels of ERCC1 in clinical specimens., Competing Interests: Takuya Honda was endowed by Eli Lilly Japan K.K.. Other authors have no conflicts of interest to disclose., (2021, National Center for Global Health and Medicine.)

Background and Purpose: Dizziness may persist even after the causative vestibular imbalance subsides. Although the precise mechanism of chronic dizziness is unknown, various cerebral activity changes associated with it have been reported. To understand its mechanism in the absence of the causative vestibular imbalance, we compared cerebral changes in chronic dizziness with and without persistent vestibular imbalance., Methods: Between September 2014 and March 2020, we examined regional cerebral blood flow (rCBF) in 12 patients having chronic post-lateral medullary infarction dizziness with persistent brainstem vestibular imbalance and 23 patients having chronic dizziness without currently active vestibular imbalance using single-photon emission computed tomography (SPECT) with 99m Technetium-ethyl cysteinate dimer. Further, we analyzed the SPECT images using a voxel-based group comparison., Results: We observed a decreased rCBF in the occipital lobe and increased rCBF in the medial and inferior parts of the temporal lobe in patients having chronic dizziness with and without active vestibular imbalance compared to healthy controls. However, only patients having chronic dizziness without active vestibular imbalance exhibited increased rCBF in the frontal lobe, including the orbitofrontal cortex., Conclusion: This is the first study to highlight the difference in rCBF changes between patients having chronic dizziness with and without active vestibular imbalance. Decreased occipital lobe activity and increased medial and inferior temporal lobe activity may be related to keeping dizziness perception triggered regardless of the presence or absence of active vestibular imbalance, whereas increased frontal lobe activity may explain the dizziness background to persist after the disappearance of vestibular imbalance., Competing Interests: The authors have no conflicts of interest to disclose., (© 2021 The Authors.)

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes dendritic cell differentiation from precursors, and consequently, enhances the antigen presentation process and adaptive immune responses. With such functions, GM-CSF has been used as immunotherapy in combination with radiotherapy for cancer treatment to augment the survival and activity of immune cells. However, an immune-suppressive tumor microenvironment may cause anergy of T cells. It has also been reported that GM-CSF contributes to the development of myeloid-derived suppressor cells from the precursors. In this study, to analyze the combined effect of GM-CSF and released factors from cancer cells after gamma-ray irradiation on bone marrow cell differentiation and dynamics, we established an in vitro culture system using mouse bone marrow cells, GM-CSF, and conditioned medium from gamma ray irradiated mouse melanoma B16 cells at 24 Gy. We analyzed the gene expression changes of the bone marrow-derived cells on day 6. The results showed that GM-CSF dose-dependently enhanced the differentiation of macrophages from bone marrow cells, their antigen-presenting function and polarization to type I. The results implied the induced macrophages from the bone marrow may potentially contribute to tumor immune responses in a systemic manner when GM-CSF is boosted during photon-beam radiation therapy.

An accelerator-based boron neutron capture therapy (BNCT) system employing a solid-state Li target can achieve sufficient neutron flux for treatment although the neutron flux is reduced over the lifetime of its target. In this study, the reduction was examined in the five targets, and a model was then established to represent the neutron flux. In each target, a reduction in neutron flux was observed based on the integrated proton charge on the target, and its reduction reached 28% after the integrated proton charge of 2.52 × 10 6 mC was delivered to the target in the system. The calculated neutron flux acquired by the model was compared to the measured neutron flux based on an integrated proton charge, and the mean discrepancies were less than 0.1% in all the targets investigated. These discrepancies were comparable among the five targets examined. Thus, the reduction of the neutron flux can be represented by the model. Additionally, by adequately revising the model, it may be applicable to other BNCT systems employing a Li target, thus furthering research in this direction. Therefore, the established model will play an important role in the accelerator-based BNCT system with a solid-state Li target in controlling neutron delivery and understanding the neutron output characteristics.

PolyADP-ribosylation is a post-translational modification of proteins, and poly(ADP-ribose) (PAR) polymerase (PARP) family proteins synthesize PAR using NAD as a substrate. Poly(ADP-ribose) glycohydrolase (PARG) functions as the main enzyme for the degradation of PAR. In this study, we investigated the effects of Parg deficiency on tumorigenesis and therapeutic efficacy of DNA damaging agents, using mouse ES cell-derived tumor models. To examine the effects of Parg deficiency on tumorigenesis, Parg +/+ and Parg -/- ES cells were subcutaneously injected into nude mice. The results showed that Parg deficiency delays early onset of tumorigenesis from ES cells. All the tumors were phenotypically similar to teratocarcinoma and microscopic findings indicated that differentiation spectrum was similar between the Parg genotypes. The augmented anti-tumor therapeutic effects of X-irradiation were observed under Parg deficiency. These results suggest that Parg deficiency suppresses early stages of tumorigenesis and that Parg inhibition, in combination with DNA damaging agents, may efficiently control tumor growth in particular types of germ cell tumors.

An accelerator-based boron neutron capture therapy (BNCT) system that employs a solid-state Li target can achieve sufficient neutron flux derived from the 7Li(p,n) reaction. However, neutron production is complicated by the large thermal load expected on the target. The relationship between neutron production and thermal load was examined under various conditions. A target structure for neutron production consists of a Li target and a target basement. Four proton beam profiles were examined to vary the local thermal load on the target structure while maintaining a constant total thermal load. The efficiency of neutron production was evaluated with respect to the total number of protons delivered to the target structure. The target structure was also evaluated by observing its surface after certain numbers of protons were delivered. The yield of the sputtering effect was calculated via a Monte Carlo simulation to investigate whether it caused complications in neutron production. The efficiency of neutron production and the amount of damage done depended on the proton profile. A more focused proton profile resulted in greater damage. The efficiency decreased as the total number of protons delivered to the target structure increased, and the rate of decrease depended on the proton profile. The sputtering effect was not sufficiently large to be a main factor in the reduction in neutron production. The proton beam profile on the target structure was found to be important to the stable operation of the system with a solid-state Li target. The main factor in the rate of reduction in neutron production was found to be the local thermal load induced by proton irradiation of the target., Competing Interests: The authors have declared that no competing interests exist.

Poly(ADP-ribose) glycohydrolase (Parg) is a central enzyme for poly(ADP-ribose) degradation. We established a Parg +/- mice strain by deletion of a part of exon 1 and around 0.4-kb upstream of sequences of the Parg gene. Parg -/- embryos obtained by intercrossing the Parg +/- mice died in utero between 4.5 and 9.5 days postcoitum. We examined whether poly(ADP-ribose) polymerase-1 (Parp-1) deficiency could rescue embryonic lethality of Parg -/- mice. Parg -/- Parp-1 -/- mice were born viable at a reduced frequency from the expected mendelian ratio in the intercross progeny of Parg +/- Parp-1 -/- mice. The results suggest a possibility that the presence of Parp-1 is responsible for the lethality of Parg -/- embryos, and Parg molecules or Parg activity degrading poly(ADP-ribose) might be important for embryogenesis. In Parg -/- Parp-1 -/- mice, Parg protein was not detected in various tissues, and the protein level of Timm23, a 5'-upstream gene of Parg, was reduced compared with that in Parg +/+ Parp-1 -/- mice. Parg -/- Parp-1 -/- mice showed retarded growth compared with Parg +/+ Parp-1 -/- mice, and died within 3 months of age accompanied with severe renal failure. Glomerular sclerosis, tubular dilatation, and hyaline casts in the kidney were observed in Parg -/- Parp-1 -/- mice. An increase in blood urea nitrogen (p < 0.05), a marked increase of albumin level in urine (p < 0.01) and its concomitant decrease in serum (p < 0.05) were also detected in Parg -/- Parp-1 -/- mice compared with the Parg +/+ Parp-1 -/- counterpart. The results imply that the combined Parg and Parp-1 loss with a hypomorphic state of Timm23 leads to the development of severe renal failure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: An accelerator-based boron neutron capture therapy (BNCT) system with a solid-state Li target is reported to have degradation of the Li target. The degradation reduces the Li thickness, which may change spectra of the generated neutrons corresponding to the Li thickness. This study aims to examine the relationship between the Li thickness and the generated neutrons and to investigate the effects of the Li thickness on the absorbed dose in BNCT., Method: The neutron energy spectra were calculated via Monte Carlo simulation for Li thicknesses ranging from 20 to 150 μm. Using the system, the saturated radioactivity of gold induced by reactions between 197 Au and the generated neutrons was evaluated with the simulation and the measurement, and those were compared. Additionally, for each Li thickness, the saturated radioactivity was compared with the number of generated neutrons. The absorbed doses delivered by 10 B(n,α) 7 Li, 14 N(n,p) 14 C, 1 H(n, g) 2 H, and (n,n') reactions in water were also calculated for each Li thickness., Results: The measurement and simulation indicated a reduction in the number of neutrons due to the degradation of the Li target. However, the absorbed doses were comparable for each Li thickness when the requisite number of neutrons for BNCT was delivered. Additionally, the saturated radioactivity of 198 Au could be a surrogate for the number of neutrons even if the Li thickness was varied., Conclusions: No notable effect to the absorbed dose was observed when required neutron fluence was delivered in the BNCT even if the degradation of the Li was observed., (Copyright © 2019 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Glioblastoma is the most common and devastating type of malignant brain tumor. We recently found that eribulin suppresses glioma growth in vitro and in vivo and that eribulin is efficiently transferred into mouse brain tumors at a high concentration. Eribulin is a non-taxane microtubule inhibitor approved for breast cancer and liposarcoma. Cells arrested in M-phase by chemotherapeutic agents such as microtubule inhibitors are highly sensitive to radiation-induced DNA damage. Several recent case reports have demonstrated the clinical benefits of eribulin combined with radiation therapy for metastatic brain tumors. In this study, we investigated the efficacy of a combined eribulin and radiation treatment on human glioblastoma cells. The glioblastoma cell lines U87MG, U251MG and U118MG, and SJ28 cells, a patient-derived sphere culture cell line, were used to determine the radiosensitizing effect of eribulin using western blotting, flow cytometry and clonogenic assay. Subcutaneous and intracerebral glioma xenografts were generated in mice to assess the efficacy of the combined treatment. The combination of eribulin and radiation enhanced DNA damage in vitro. The clonogenic assay of U87MG demonstrated the radiosensitizing effect of eribulin. The concomitant eribulin and radiation treatment significantly prolonged the survival of mice harboring intracerebral glioma xenografts compared with eribulin or radiation alone (P < .0001). In addition, maintenance administration of eribulin after the concomitant treatment further controlled brain tumor growth. Aberrant microvasculature was decreased in these tumors. Concomitant treatment with eribulin and radiation followed by maintenance administration of eribulin may serve as a novel therapeutic strategy for glioblastomas., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

This study aimed to evaluate the residual radioactivity in mice induced by neutron irradiation with an accelerator-based boron neutron capture therapy (BNCT) system using a solid Li target. The radionuclides and their activities were evaluated using a high-purity germanium (HP-Ge) detector. The saturated radioactivity of the irradiated mouse was estimated to assess the radiation protection needs for using the accelerator-based BNCT system. 24 Na, 38 Cl, 80m Br, 82 Br, 56 Mn, and 42 K were identified, and their saturated radioactivities were (1.4 ± 0.1) × 10 2 , (2.2 ± 0.1) × 10 1 , (3.4 ± 0.4) × 10 2 , 2.8 ± 0.1, 8.0 ± 0.1, and (3.8 ± 0.1) × 10 1 Bq/g/mA, respectively. The 24 Na activation rate at a given neutron fluence was found to be consistent with the value reported from nuclear-reactor-based BNCT experiments. The induced activity of each nuclide can be estimated by entering the saturated activity of each nuclide, sample mass, irradiation time, and proton current into the derived activation equation in our accelerator-based BNCT system.

XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

To understand the mechanism of cell death induced by boron neutron capture reaction (BNCR), we performed proteome analyses of human squamous tumor SAS cells after BNCR. Cells were irradiated with thermal neutron beam at KUR after incubation under boronophenylalanine (BPA)(+) and BPA(-) conditions. BNCR mainly induced typical apoptosis in SAS cells 24h post-irradiation. Proteomic analysis in SAS cells suggested that proteins functioning in endoplasmic reticulum, DNA repair, and RNA processing showed dynamic changes at early phase after BNCR and could be involved in the regulation of cellular response to BNCR. We found that the BNCR induces fragments of endoplasmic reticulum-localized lymphoid-restricted protein (LRMP). The fragmentation of LRMP was also observed in the rat tumor graft model 20 hours after BNCT treatment carried out at the National Nuclear Center of the Republic of Kazakhstan. These data suggest that dynamic changes of LRMP could be involved during cellular response to BNCR., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4(K271R) mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4(K210R) mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3'-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4(K271R) was defective in the nuclear localization of itself and LIG4, whereas XRCC4(K210R) was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4(K271R), but not M10-XRCC4(K210R), showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4(WT). The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4(K271R) than in M10-XRCC4(WT), whereas it was only marginally increased in M10-XRCC4(K210R) as compared to M10-XRCC4(WT). The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4., (Copyright © 2015 Elsevier Inc. All rights reserved.)

XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4(N326L)) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4(N326L) in the nucleus but only partially rescued radiosensitivity of M10-XRCC4(N326L). These results collectively indicated that the functional defects of XRCC4(N326L) might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

XRCC4 (X-ray cross-complementation group 4) is a protein associated with DNA ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end-joining. It has been shown that, in response to irradiation or treatment with DNA damaging agents, XRCC4 undergoes phosphorylation, requiring DNA-PK. Here we explored possible role of ATM, which is structurally related to DNA-PK, in the regulation of XRCC4. The radiosensitizing effects of DNA-PK inhibitor and/or ATM inhibitor were dependent on XRCC4. DNA-PK inhibitor and ATM inhibitor did not affect the ionizing radiation-induced chromatin recruitment of XRCC4. Ionizing radiation-induced phosphorylation of XRCC4 in the chromatin-bound fraction was largely inhibited by DNA-PK inhibitor but further diminished by the combination with ATM inhibitor. The present results indicated that XRCC4 phosphorylation is mediated through ATM as well as DNA-PK, although DNA-PK plays the major role. We would propose a possible model that DNA-PK and ATM acts in parallel upstream of XRCC4, regulating through phosphorylation.

Two glycoproteins (LM-hCG and HM-hCG) with gonadotropic activity were purified from the chorionic tissue of patients with hydatidiform mole with low and high urinary human chorionic gonadotropin levels and were compared to each other. The immunologic, biologic, and physicochemical properties of the two preparations were very similar. Also, the quantities of LM-hCG and HM-hCG recovered from molar tissue from the two types of patients were similar. However, the molar tissue from patients who excreted high levels of human chorionic gonadotropin immunoreactivity also contained other molecular forms with apparent human chorionic gonadotropin immunoreactivity tissues from patients who excreted low levels did not. These latter molecular forms may account for the differences in the levels of human chorionic gonadotropin immunoreactivity excreted by the two types of patients.

Estrogen levels are a useful indicator to use in predicting of ovarian hyperstimulation syndrome (OHSS) which is one of the side effects of HMG-HCG therapy. However, the quantitative assay of estrogens entails cumbersome time-consuming procedures. The present study represents our attempt to establish criteria for predicting the occurrence of OHSS by the use of ultrasonography (USG), a diagnostic procedure that can be performed quickly and conveniently. The subjects were 40 anovulatory women (79 cycles) receiving HMG-HCG therapy. Each patient had USG performed at the time of switching to HCG in a regimen of sequentially administered gonadotropins and was measured for maximum follicular diameter (FD) and total of vertical follicular area (FA) to correlate measurements of these parameters with simultaneously determined serum estradiol (E2) levels. A study was also made of relationships of FD and FA with ovulation and OHSS. The results are summarized as follows: No distinct correlation was observed between FD and E2 (r = 0.3794). It should be noted, however, that the therapy was successful in inducing ovulation in those cases in which the patient was switched to HCG from HMG when FD was 18mm or above. There was a significant correlation between FA and E2 (r = 0.8113, p less than 0.001). FA was thus proven to well reflect E2 levels and hence to be a parameter of the predictive value for OHSS. All but one (with moderate OHSS) of 26 cases showing evidence of OHSS had FA values of more than 6.0cm2, while those developing severe OHSS invariably.(ABSTRACT TRUNCATED AT 250 WORDS)

Unlabelled: The ovulation-inducing effect of bromocriptine (CB-154) and the underlying mechanism in the management of anovulatory infertility with normoprolactinemia were investigated. Thirty-seven normoprolactinemic women (anovulatory cycle, 11 cases; 1st grade amenorrhea, 18; and 2nd grade amenorrhea 8) with anovulia showing plasma prolactin levels of less than 25 ng/ml were give CB-154 daily in doses of 2.5 mh from the second day of menstruation or withdrawal bleeding (1) to assess the ovulatory induction rate, (2) to determine the serum PRL, LH, FSH and E2 levels before and during the drug administration, (3) to measure the serum PRL concentration by TRH test prior to CB-154 medication and (4) to assess size heterogeneity of serum PRL with the use of lyophilization., Results: (1) Ovulation occurred in 21 of the 37 patients, of a total of 42 menstrual cycles studied, 33 were ovulatory, indicating a high rate of repeated success. (2) There was a significant decrease of serum PRL following administration of CB-154, and the finding was the same in the group of women with successful ovulatory induction. Serum LH, FSH and E2 levels did not show any significant change following CB-154 therapy. (3) Prl concentrations were suppressed more in cases of unsuccessful induction than those in successful induction. (4) PRL responses to TRH tended to be lower in cases of successful induction than those in unsuccessful cases. (5) The Little PRL content ratio decreased after administration of CB-154, and this data have suggested that not only the problem of immunologically measured serum PRL levels but the qualitative problems also bear relations to the effect of this hormone on the endocrinological environment.

A study was conducted to define biochemical characteristics of human chorionic gonadotropin (hCG) in total hydatidiform mole patients with low serum and urinary hCG levels and to inquire into the reason for its low immunological hCG level by comparing with the hCG of molar patients with a high hCG level. Both hCGs purified from chorionic tissue of molar patients with low and high hCG levels have essentially identical physicochemical, immunological and biological properties. They have low levels of biological activity and practically identical amino acid and carbohydrate compositions. It appears that moles of patients with high levels as well as those with low levels of immunoreactivity produce this purified form of hCG. Extracts of molar tissue from patients with high levels of hCG immunoreactivity also contained other substances with apparent immunoreactivity which can be found in small amounts in tissue extracts from patients who excreted low levels of hCG immunoreactivity. In contrast, extracts of molar tissue and urine from patients with low hCG levels contained hCG-like substances which cannot be detected with the ordinary commercial hCG immunoassay kits. These differences may account for the different levels excreted by these two types of patients.