Our Research 2018 (85) ～ (91)
(85) Effect of different dose rates of ionizing radiation of ciliogenesis in hTERT-RPE1 cells
Jan 9, 2018
Mikio SHIMADA
Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology
Ionizing radiation (IR) induces genome instability and chromosome aberration in mammalian cells. IR exposure generates DNA damage, which is repaired by several DNA repair pathways. Meanwhile, IR also induces ciliogenesis and centrosome overduplication associated with cell cycle checkpoint mechanism. Centrosome number is strictly regulated, since overduplicated centrosomes cause aneuploidy, leading to tumorigenesis. Primary cilia play a sensory role in several signaling pathways during development and cellular homeostasis. In this study, to address how IR affects ciliogenesis, we irradiated telomerase reverse transcriptase-immortalized retina pigmentation epithelial cell, hTERT-RPE1, with γ-ray at different dose rates, that is 2 mGy/s (low dose rate) and 100 mGy/s (high dose rate). Centrosome and primary cilia were detected by immunofluorescence using γ-tubulin and acetylated-α-tubulin antibodies, respectively. After IR exposure, we saw an increase in cells with primary cilia and the combined treatment of IR exposure with serum starvation stimulation showed an additive effect. This study provides a new insight into radiation effect on the extracellular response.
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日本語解説 「細胞内器官一次繊毛形成に対する線量依存的な放射線照射の影響」
一次繊毛は細胞内の中心体から伸長する細胞内器官のひとつで主に上皮細胞に存在し、細胞外の環境を感知するセンサーとして機能する。一次繊毛は成長した生物の上皮細胞だけでなく胎児期の生物の発生にも必須で一次繊毛に関連する遺伝子が欠損すると臓器の逆位や発育遅滞、小頭症など重篤な疾患の原因となる。上皮細胞では主に細胞周期G0期、いわゆる休止期に特異的に一次繊毛が形成されることが知られており、ヒト網膜色素上皮細胞hTERT-RPEは血清飢餓により一次繊毛を誘導することが可能であるために一次繊毛の研究に頻繁に用いられている。本研究では放射線照射が一次繊毛の形成にいかに影響を及ぼすかを調べるためにhTERT-RPE細胞に放射線を照射し、一次繊毛のマーカーであるアセチル化αチューブリン抗体を用いた免疫染色により顕微鏡観察を行った。その結果、1Gyと5Gyの放射線照射で有意に一次繊毛の形成が観察された。一方で線量の依存性は観察されなかった。さらに血清飢餓状態で放射線を照射すると血清飢餓による一次繊毛の形成に対して放射線照射では付加的に一次繊毛の形成が観察された。この結果は放射線照射に応答して特異的に一次繊毛が形成していることを示唆している。今後の展望として放射線照射依存的な一次繊毛の形成に対する生理的な意義を調べていきたい。
(86) Dose-dependent decrease in anti-oxidant capacity of whole blood after irradiation: A novel potential marker for biodosimetry
May 31, 2018
Lue SUN
Department of Radiation Biology, Faculty of Medicine, University of Tsukuba
Department of Radiological Health Science, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health
Many reports have demonstrated that radiation stimulates reactive oxygen species (ROS) production by mitochondria for a few hours to a few days after irradiation. However, these studies were performed using cell lines, and there is a lack of information about redox homeostasis in irradiated animals and humans. Blood redox homeostasis reflects the body condition well and can be used as a diagnostic marker. However, most redox homeostasis studies have focused on plasma or serum, and the anti-oxidant capacity of whole blood has scarcely been investigated. Here, we report changes in the anti-oxidant capacity of whole blood after X-ray irradiation using C57BL/6 J mice. Whole-blood anti-oxidant capacity was measured by electron spin resonance (ESR) spin trapping using a novel spin-trapping agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO). We found that whole-blood anti-oxidant capacity decreased in a dose-dependent manner (correlation factor, r > 0.9; P < 0.05) from 2 to 24 days after irradiation with 0.5-3 Gy. We further found that the red blood cell (RBC) glutathione level decreased and lipid peroxidation level increased in a dose-dependent manner from 2 to 6 days after irradiation. These findings suggest that blood redox state may be a useful biomarker for estimating exposure doses during nuclear and/or radiation accidents.
日本語解説
近年、放射線照射後数時間から数日にわたり、ミトコンドリアからのROS産生量が増加して細胞が酸化ストレス状態に陥ることが知られるようになった。この遅発性のROSは、アポトーシス、細胞生存率、突然変異の誘導等に関与していることが報告されているが、これらの報告は全て細胞株を使用した実験であり、「生体」でどのようなレドックスバランスの変化が現れるのかは、ほとんどわかっていない。本研究では、放射線被ばく後の生体(血液)の抗酸化能がどのように変化するか明らかにすることを目的とした。マウスの全身に0.5, 1, 2,3GyのX線を照射し、直後から50日後にかけて継時的に採血し、i-STrap法により血液(全血)抗酸化能を評価した。放射線照射2日後から24日後にかけて一過性の線量依存的な血液抗酸化能の低下が観察され、照射線量に比例して抗酸化能の低下が観察される期間が長くなった。血液抗酸化能は大規模放射線災害などにおける線量推定(バイオドシメトリ)の指標として利用できる可能性がある。
(87) Knowledge Discovery of Suppressive Effect of Disease and Increased Anti-oxidative Function by Low-dose Radiation Using Self-organizing Map
Oct 4, 2018
Norie KANZAKI
Graduate School of Health Sciences, Okayama University
Ningyo-toge Environmental Engineering Center of the Japan Atomic Energy Agency
We previously reported that low-dose radiation induces the anti-oxidative function in many organ systems of mice. This results in the suppression of several kinds of oxidative stress-induced damage. However, to date, a proven cure has not been established. This study was conducted with the objective of revealing the health effects of low-dose radiation obtained from our previous reports and searching for a new treatment based on low-dose radiation, such as radon therapy. We extracted the characteristics of the effects of low dose radiation suppressing diseases and enhancing the anti-oxidative function using fuzzy answer by self-organizing map (SOM) based on mutual knowledge. The relationship between the suppressive effect and increased antioxidative function was shown in our result, and the concentration dependence of the effect against pain was shown on the output map. Although the effect against other organs depending on concentration was unpredictable, our results indicate that low-dose radiation may also be suitable for treatment of liver disease and brain disease. The results presented could encourage development and application of treatment using low-dose radiation, such as radon therapy.
日本語解説
我々はこれまで，低線量放射線はマウス諸臓器中で抗酸化機能を亢進し，酸化ストレス関連疾患を抑制することを報告してきた。しかしながら，それらの結果は対象疾患も低線量放射線による処置の条件も様々で，有効性が立証された治療法は確立されていない。そこで，本研究では，それらの結果から低線量放射線の健康効果を明らかにすることを目的とし，ラドン療法のような低線量放射線を活用した治療法の新規適応症を探索した。データの解析には自己組織化マップ（SOM）を用い，不安定な抗酸化機能の変化を自己組織化マップの曖昧な表現で視覚的に直感的に捉えることにより，出力された疾患抑制効果と抗酸化機能亢進の関連性を検討した。その結果，ラドン療法の適応症である疼痛への効果には明らかな線量依存性があることがわかり，肝疾患や脳疾患においても，線量依存性はないもののその効果を期待できると予測できた。本研究は，ラドン療法のような低線量放射線を活用した治療法の応用に貢献できると考える。
(88) Effects of retinoic acid-inducible gene-I-like receptors activations and ionizing radiation cotreatment on cytotoxicity against human non-small cell lung cancer in vitro
Oct 4, 2018
Hironori YOSHINO
Hirosaki University Graduate School of Health Sciences
Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are pattern-recognition receptors that recognize pathogen-associated molecular patterns, and induce antiviral immune response. Recent studies have shown that RLR activation induces antitumor immunity and cytotoxicity against cancers, including lung cancer. On the other hand, our previous report has shown that ionizing radiation exerts a limited effect on RLR in human monocytic cell-derived macrophages, thus suggesting that RLR agonists may be used as effective immunostimulants during radiation therapy. However, it is unclear whether ionizing radiation affects the cytotoxicity of RLR agonists against cancer cells. Therefore, here, we investigated the effects of ionizing radiation and RLR agonists cotreatment on cytotoxicity against human non-small cell lung cancer A549 and H1299. Treatment with RLR agonist poly(I:C)/LyoVec™ (hereafter referred to as “poly[I:C]”) exerted cytotoxic effects against human non-small cell lung cancer. The cytotoxic effects of poly(I:C) were enhanced by cotreatment with ionizing radiation, and poly(I:C) pretreatment resulted in the radiosensitization of non-small cell lung cancer. Furthermore, cotreatment of A549 and H1299 cells with poly(I:C) and ionizing radiation effectively induced apoptosis in a caspase-dependent manner compared with treatment with poly(I:C) or ionizing radiation alone. These results indicate that RLR agonists and ionizing radiation cotreatment effectively exert cytotoxic effects against human non-small cell lung cancer through caspase-mediated apoptosis.
日本語解説
Retinoic acid-inducible gene-I (RIG-I)様受容体 (RLR：RIG-I-like receptors)は病原体関連分子を認識するパターン認識受容体の一種で，細胞質のウイルス核酸認識センサーとして機能する。RLRは抗ウイルス応答において重要な役割を果たすが，近年の研究によりRLRの活性化が肺癌を含む様々な癌に対して抗腫瘍免疫ならびにアポトーシスなどの細胞死誘導を介して抗癌効果を示すことが報告されている。我々はこれまでに，単球系細胞のパターン認識受容体に及ぼす放射線の影響を検討する中で，RLRが放射線の影響を受けにくいことを見出した。これら結果から，癌放射線治療における免疫活性を兼ね備えた抗癌効果誘導の標的としてRLRの有用性が期待されるが，RLR刺激因子の抗癌効果に及ぼすに放射線の影響については不明である。そこで，本研究ではRLR刺激因子とX線の併用によるヒト肺癌細胞に対する抗癌効果を検討した。
RLR刺激因子poly(I:C)/LyoVecTM (以後，poly(I:C))でヒト肺癌細胞（A549及びH1299細胞）を処理したところ，生細胞数の低下及びAnnexin V陽性の細胞死集団の増加が観察された。次にpoly(I:C)と放射線の併用効果を検討したところ，poly(I:C)は放射線増感作用を示した。さらに，poly(I:C)とX線の併用処理群では各単独処理群と比べてAnnexin V陽性の細胞死集団の割合およびcleaved caspase-3の発現が高く，併用による細胞死誘導はcaspase阻害剤により抑制された。以上の結果より，RLR刺激因子poly(I:C)と放射線の併用はヒト肺癌細胞に対して効果的に抗癌効果を示すこと，そしてその機構の一部としてcaspaseを介した細胞死増強が示唆された。
References:
Yoshino et al. Effects of retinoic acid-inducible gene-I-like receptors activations and ionizing radiation cotreatment on cytotoxicity against human non-small cell lung cancer in vitro.
Oncology Letters. 15: 4697-4705, 2018. DOI: 10.3892/ol.2018.7867
(89) Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas
Oct 5, 2018
Yoshitaka MATSUMOTO
Radiation Oncology, Clinical Medicine, Faculty of Medicine, University of Tsukuba
Proton Medical Research Center, University of Tsukuba Hospital
The goal of this work was to clarify the effect of carbon-ion beams on reduction of the metastatic potential of malignant melanoma using in vitro and in vivo techniques. We utilized a 290 MeV/u carbon beam with a 6-cm spread-out Bragg-peak (SOBP), 137 Cs c rays or 200 kVp X rays for irradiation, and in vitro murine melanoma B16/BL6 cells that were implanted into C57BL/6J mice. The metastatic abilities (migration, invasion and adhesion) were suppressed by carbon ion treatment at all doses that were tested, whereas invasion and migration tended to increase after X-ray irradiation at low dose. Biological effects of carbon ions increased with linear energy transfer (LET) for both cell killing and metastatic abilities, although the effects were more pronounced for migration and invasion. mRNA expression of E-cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expression of Mel-CAM and L1-CAM was upregulated after low-dose photon exposure, but decreased with dose, especially after carbon-ion treatment. Conversely, these molecules showed a reversal in expression changes, especially after low-dose photon exposure. Cell-cell adhesion may be an important contributor to the antimetastatic effect of carbon ion treatment. The number of lung metastases after local tumor irradiation significantly decreased with increased dose and LET, with carbon ions being more effective than c rays. Integrating dose-response curves to examine the relationship between cell killing and lung metastasis clearly showed that carbon ions inhibit lung metastasis more efficiently than photons at the iso-effective level of cell killing. Thus, carbon ions were more effective than photon beams, not only at killing tumor cells, but also at inhibiting metastatic spread caused by tumor cells that survived irradiation.
Reference:
Matsumoto et al. (2018) Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas. Radiat. Res. 190 (2018) 412-423.
http://www.rrjournal.org/doi/abs/10.1667/RR15075.1?code=rrs-site&journalCode=rare
(90) Radiation-induced nitric oxide production accelerates endothelial senescence
Oct 6, 2018
Masaki NAGANE
Azabu University
In radiation therapy for cancer treatment, the therapeutic efficiency represents an optimal balance between tumor control and normal tissue complications. To optimize the cancer radio-therapy, it is important to prevent late-effects of radiation. Recent studies shed light on the late-effect of ionizing radiation on cardiovascular disfunction. However, a comprehensive understanding of the underlying molecular and pathophysiological mechanism of radiation-induced cardiovascular disease (RICVD).
Endothelial nitric oxide synthase (eNOS), a constitutive enzyme expressed in vascular endothelial cells, is the main source of nitric oxide (NO), which plays key roles in diverse biological functions, including regulation of vascular tone and angiogenesis. In this study, we clarify the involvement of radiation-induced DNA damage response (DDR) on production of NO in endothelial cells. Radiation-induced NO inhibited cell death, as well as induced cellular senescence. This study established that radiation-induced NO production accelerates endothelial senescence, which may play a critical role in RICVD.
(91) Human Rad52 promotes XPG-mediated R-loop processing to initiate transcription-associated homologous recombination repair
Oct 6, 2018
Reona KATO
Laboratory of Molecular Radiology, Center for Disease Biology and Integrative Medicine,
Graduate School of Medicine, The University of Tokyo
It has been known that of our huge genome, only small regions are utilized. The genomic regions that is frequently transcribed are the most important part of our genome. DNA damage induced in these important genomic regions should be repaired accurately to protect the genomic information. Previous studies have reported that an accurate DNA repair pathway is activated upon DNA damage in transcribed genomic regions. However, it has remained unknown how cells recognize the importance of genomic information and activate the accurate repair pathway. In this study, we have revealed that R-loops, a structure consisting of DNA-RNA hybrid and single-stranded DNA, are formed when DNA double-strand breaks are induced in transcribed genomic regions. In addition, the recognition of R-loops by the protein Rad52 promotes the activation of the accurate repair pathway, i.e., homologous recombination repair. This mechanism was designated as Transcription-Associated Homologous Recombination Repair (TA-HRR). Furthermore, we demonstrated that if TA-HRR was inhibited, interchromatid fusions, a precursor of genomic abnormalities frequently observed in cancer, was significantly increased, suggesting that TA-HRR is critical for suppressing cancer development. Thus, these findings represent an intrinsic mechanism by which human cells suppress cancer development.
日本語解説
我々のゲノム情報は非常に大きいことが知られているが、実際にはそのうちのほんの一部分しか利用されていない。従って、膨大なゲノムの中でも、転写されて頻繁に読み出される情報が記録されている領域は重要な部位と考えられる。そのような重要なゲノム領域に起こったDNA損傷は、遺伝情報を守るために、正確に修復することが必要である。これまでの研究で、そのような現象は観察されていたが、どのようにしてその重要性を認識して、正確に修復する経路を誘導しているのかについては分かっていなかった。
今回の研究で我々は、転写が活性化している領域にDNA損傷、特にDNA二重鎖切断が生じた場合には、周辺にR-loop構造と呼ばれる特殊な構造が形成されることを見出した。さらに遺伝子Rad52がこの構造を認識することで、正確な修復経路、すなわち相同組換え修復を誘導することを発見し、このメカニズムを転写共役型相同組換え修復と名付けた。転写共役型相同組換え修復を阻害した場合にゲノムに起こる異常を解析したところ、不正確な修復の結果として、姉妹染色体間結合と呼ばれる染色体異常の頻度が顕著に上昇することが分かった。このような染色体異常は、がんなどで頻繁に見られるゲノム異常の前駆体となることから、転写共役型相同組換え修復は、がんの発生を防ぐために我々の細胞に備わる重要なメカニズムであることが示唆された。
Reference
Takaaki Yasuhara* † , Reona Kato † , Yoshihiko Hagiwara, Bunsyo Shiotani, Motohiro Yamauchi, Shinichiro Nakada, Atsushi Shibata*, Kiyoshi Miyagawa* ( † co-first author, *corresponding author) Human Rad52 promotes XPG-mediated R-loop processing to initiate transcription-associated homologous recombination repair Cell 175 558-570 (2018)
https://doi.org/10.1016/j.cell.2018.08.056