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Bendamustine, a hybrid molecule of a purine analog and alkylator, induces cell death by the activation of apoptosis and the DNA damage response. The agent MK615 is produced from Japanese apricot and contains a number of cyclic triterpenes that exhibit antitumor activities. In the present study, the combined effects of bendamustine and MK615 on lymphoma cells were investigated. The combined compounds synergistically induced apoptosis in all lymphoid cell lines examined. MK615 inhibited the bendamustine-induced phosphorylation of checkpoint kinase 1 and 2. As ataxia telangiectasia mutated (ATM) and ataxia telangiectasia- and Rad3-related (ATR) kinases are key mediators of the DNA damage response, the effects of the combination of bendamustine and ATM/ATR inhibitors (KU-60019 and VE-821) on lymphoma cells were investigated. KU-60019 and/or VE-821 potentiated bendamustine activity in all cell lines tested, but did not affect MK615 activity, suggesting that these inhibitors have the same underlying mechanism of action as that of MK615. The results of the present study suggest that it may be feasible to use ATM/ATR inhibitors in combination with bendamustine for treating malignant lymphoma.

平成28年度衝撃波シンポジウム (2017年3月8日-10日. ヴェルクよこすか 横須賀市立勤労福祉会館）横須賀市, 神奈川県, Symposium on Shock Waves in Japan, FY2016 (March 8-10, 2017. Werk Yokosuka), Kanagawa, Japan, 資料番号: SA6000084049, レポート番号: 1D1-1【若】

Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions of these proteins in the regulation of apoptosis. This article discusses recent insights into this possible additional functions of p21 and p27.

総説, 細胞周期には,DNA複製を行うS期と細胞分裂を行うM期とがあり,その前にそれぞれG1期とG2期とが存在する.細胞周期が1周回るごとに細胞は倍加する.細胞が分裂して増加することは,成長の基本である.また,発生や再生の基本でもあり,これらのメカニズムを明らかにすることはがんを理解するうえでも非常に重要である.細胞周期の解析には数々の生物種が用いられてきたが,単細胞の真核生物である酵母も大きな貢献をした.細胞周期進行の機構は酵母からヒトに至るまで広く保存されているため,酵母で新しく得られた知見は高等真核生物にも応用でき,これにより細胞周期の研究が大きく進展した.本総説では,酵母にはどのような特徴があるのか,また,酵母を用いることによりどのような研究を行うことができるのかを紹介する.

Article, 信州医学雑誌 54(2): 59-68(2006)

総説, 細胞周期は単細胞生物から多細胞高等生物に至るまで,すべての生命体にとって欠くことのできない重要な過程である.細胞周期では単に1つの細胞が2つに分裂するだけではなく,遺伝情報の担い手である染色体DNAを正確に複製し,紡錘体を形成して染色体を正確に娘細胞へ分配する必要がある.そのため染色体DNAおよび紡錘体に損傷が生じた場合にはチェックポイントが発動して,損傷の修復が完了するまで細胞周期を停止する.また高等生物において細胞周期とチェックポイントは,発生や分化および個体の恒常性維持と深く関連しており,その破綻は発生異常や癌などの疾患につながる.本総説では真核生物の細胞周期とチェックポイントを制御する分子メカニズムを概説する.また細胞周期と概日リズムの機能的な関連に触れ,時計分子医療に対する展望を紹介する.

Survivin is one of the IAP (inhibitor of apoptosis proteins), which is expressed during embryonic development, but is not expressed in terminal differentiated adult tissue. Overexpression of survivin is observed in a variety of human tumors including thyroid cancers. We examined the role of survivin on the proliferation and differentiation of thyroid epithelial cells. Rat thyroid epithelial cell line FRTL-5 was stably transfected with human sense and antisense survivin genes. FRTL-5 cells transfected with a survivin sense gene (S cells) proliferated without TSH. In the presence of TSH, the proliferation of S cells was significantly faster than that of cells transfected with an empty vector (E cells) or cells transfected with a survivin antisense gene (AS cells). The proliferation of AS cells were retarded as compared with that of E cells. Western blot analysis demonstrated that phosphorylation of Rb protein at serine 795 was enhanced in S cells. Activation of E2F-1 was also enhanced in S cells as demonstrated by gel shift assay. Northern blot analysis showed that expression of Pax-8 mRNA was significantly enhanced in S cells, and was significantly decreased in AS cells. Expression of thyroglobulin (Tg) and TSH receptor (TSHR) mRNA was decreased in AS cells. There was no significant change in TTF-1 and TTF-2 mRNA expression. These observations suggest that survivin enhances the proliferation of thyroid epithelial cells through activation of E2F-1, and also enhance the expression of Pax-8, which then affect the expression of thyroid specific proteins. Survivin appears to play an important role in the proliferation as well as differentiation of thyroid epithelial cells.

Positron emission tomography using fluorine-18 fluoro-deoxyglucose (FDG PET) is useful for the detection of malignant tumor recurrences and for the evaluation of a therapeutic response to these;including ones found in the lung, colon, head and neck regions. The experimental study demonstrated FDG uptake was higher in faster-growing rather than in slower-growing tumors. These findings show FDG accumulation exhibits cell cycle dependency. However, the precise mechanism remains to be elucidated. In this study, the relationship between FDG uptake and the cell cycle phase in HeLa S3 cells, as well as how they compare to the conventional tracer ^67Ga citrate (Ga) with single photon emission tomography (SPECT) was assessed. Synchronization of HeLa S3 cells was accomplished via a double thymidine block. The uptake of FDG and Ga was determined after cell cycle synchronization. The glucose transporter was independently evaluated for the level of its Glut 1 glucose transporter membrane protein. FDG uptake in HeLa S3 cells was significantly higher in the early S phase and G_2/M phase compared to the G_1 phase. In addition, Ga uptake was higher in the G_2/M phase. Immunochemical assays for the Glut 1 transporter showed an increase in membrane expression within S phase cells. It has been concluded that cell cycle dependency is reflected in the uptake of FDG and Ga, seen during PET or SPECT imaging of tumor tissue. These results reveal tumor proliferative activity, and can assist in evaluating a therapeutic response. Furthermore, the increased FDG uptake, in part due to increased membrane expression of the Glut 1 glucose transporter, contributes to this phenomenon.

蛍光染色は,特異性に高くかつ高感度であることから,様々な組織細胞化学や分子生物学の研究に用いられてきた。蛍光特性の1 つでFluorescence Resonance Energy Transfer (FRET)は,適切な2種類の蛍光分子ペアが100Å以下の距離に近接した時に生じるエネルギー移動であり,その解析は,2つの分子の相対的位置関係,すなわち距離と方向性についての様々な情報を与えてくれる。近年,このFRETを顕微鏡下で視覚化するユニークな解析法,Microscopic FRET法が,細胞内の様々な生理活性物質の局在や相互作用をオングストローム単位で解析できる手法として注目を浴びている。しかし,顕微鏡下では,FRET の測定に様々な制約があるのみならず,細胞内は光学的に多様であるために,FRET の発生に際して,複雑な蛍光現象が起こっていると考えられる。本稿では,緩衝液に溶解されたDNA と細胞核内DNA 上で起こるFRET 現象を,蛍光強度と蛍光寿命の測定の二面から解析した結果を踏まえ,Microscopic FRET 法の原理および問題点を提示する。

1)シスプラチン投与により誘導されたAIは経日的及び濃度依存的に増加した. 2)腫瘍増殖の抑制とそれに伴うAIの増加は,アポトーシスによる腫瘍抑制効果による. 3)細胞周期ではG2+M期,G0+GI期の順に細胞集積を認め,アポトーシス細胞もG2+M期,G0+GI期の順に出現した. 4)各細胞周期において,AIの経日的な増加は認められなかった. 5)細胞周期調整機構とAIとの関連性が示唆された

Article, 信州医学雑誌 46(4): 239-248(1998)

In cells undergoing apoptosis, the characteristic "ladder" pattern of DNA fragments is observed on agarose gel electrophoresis. Such a pattern of DNA degradation generally serves as a criterion for the apoptosis. However, on agarose gel electrophoresis it is difficult to accurately quantify DNA fragmentation. Therefore, quantification of DNA fragmentation is determined by flow cytometry, DNA fragmentation assay etc. In this paper we improved the methods for the analysis of apoptosis by flow cytometry. Asa result, we can obtain much information (quantification of apoptosis and cell cycle analysis etc.) from a single sample (1～3×10 6 cells) with the improved methods. The sample is treated with phosphate-citrate buffer. These methods are very simple andconvenient.

ラット甲状腺由来のFRTL-5細胞においては細胞増殖時にコレステロール生合成系が活性化されることが知られている。このFRTL-5細胞の増殖時におけるコレステロール生合成経路の役割について, とくに細胞周期制御蛋白を中心に検討を加えた。FRTL-5細胞の増殖, とくにG1からS期への移行過程では, コレステロール生合成経路のメバロン酸代謝産物であるgeranylgeranylpyrophosphateが細胞周期制御蛋白であるp27の減少を介してcyclin dependent kinase 2を活性化し細胞周期の進行に不可欠であることがはじめて明らかになった。, Cyclin-dependent kinase (Cdk) enzymes are activated for entry into the S phase of the cell cycle. In growth-stimulated rat thyroid FRTL-5 cells, the elimination of Cdk inhibitor protein, p27^, and the activation of Cdk2 during the G1/S transition is required for the cell cycle progression. Pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, prevents the elimination of p27 and leads to G1 arrest. Mevalonate and its metabolite, geranylIgeranylpyrophosphate, but not farnesyl pyrophosphate, allow Cdk2 activation by causing the elimination of p27 through the acceleration of its degradation. These results indicate that among mevalonate metabolites, geranylgeranylpyrophosphate is absolutely required for the elimination of p27 followed by Cdk2 activation, and promote the degradation of p27 during G1/S transition in growth-stimulated FRTL-5 cells.

Retinoblastoma protein （pRb） interacts with transcriptional factors and, particulary, dephosphorylated pRb functions as a negative regulator of cell cycle. We have previously demonstrated that dephosphorylated pRb was associated with 73kDa heat shock cognate protein （HSC73） in certain tumor cell lines. In this experiment, we analyzed the interaction between these two proteins in vitro and determined the HSC73-binding region of pRb by using GST-deletion mutant pRb fusion proteins and synthetic peptides corresponding to the amino acid sequence of pRb. Our data showed that HSC73 interacted directly with a novel region which was located in N-terminal 328-340 amino acid residues outside the pocket region of pRb. Furthermore, we analyzed the function of HSC73 in its interaction with pRb in vitro. The analysis using native polyacrylamide gel electrophoresis indicated that the HSC73 could confer the conformational change on pRb, and might protect the aggregation. Dephosphorylated pRb, but not phosphorylated pRb was degraded by the liver-derived cytosolic extract when the interaction with HSC73 was blocked by the synthetic peptide. These data suggest that HSC73 acts as the molecular chaperone selectively for the dephosphorylated pRb, thereby potentiating the pRb function as the inhibitory regulator of cell cycle and, further, cell proliferation.

The Fas antigen is a cell surface protein that can mediate apoptosis in a variety of cell types, including the human leukemia T cells. We previously demonstrated that the Fas-resistant variants may exist in highly Fas-sensitive human leukemia T-cell lines. The surface expression of Fas antigen was unchanged in the variant cells, suggesting a defective intracytoplasmic mechanism leading to apoptosis. We found that the stimulation of Fas-sensitive cells （SUP-T13） with anti-Fas antibody 2D1, but not resistant variants （LAC2D1R）, induced repression of p34cdc2 and p33cdk2 along with apoptosis. There were no alterations in expression of bcl-2, HSP70, HSP90 and cyclin proteins examined. In the present study, we also examined the changes in other cell cycle-related proteins such as Rb and ubiquitin. We observed an increase of unphosphorylated Rb and of polymerization of ubiquitin after stimulation with 2D1 in a time-dependent fashion on SUP-T13. Similar results, that is, repression of p34cdc2 and p33cdk2, an increase of unphosphorylated Rb and an increase of polymerization of ubiquitin after stimulation with 2D1 were obtained from other Fassensitive cell lines such as Jurkat cells. Furthermore, we examined the relationship between cell cycle perturbation and cell killing. Interestingly, after stimulation with 2D1, Fas-sensitive cells （SUP-T13） incorporated as much bromodeoxyuridine as Fas-resistant cells （LAC2D1R） did, suggesting that SUP-T13 induced apoptosis after entering into the S phase of the cell cycle. Moreover, in histograms obtained from flow cytometric analysis of DNA with propidium iodide, there were no changes in the ratio of cells in the G1, S or G2/M phase in SUP-T13 stimulated with 2D1. In conclusion, our results suggest that Fas-mediated apoptosis may occur in any phase of the cell cycle and that depletion of p34cdc2 and p33cdk2 and an increase of unphosphorylated Rb are involved in the specific mechanism of Fas-mediated apoptosis, which is considered to be mediated by the ubiquitin dependent proteolytic system. In addition to investigating the expression of intracytoplasmic proteins in activated human lymphocytes, we confirmed that Fas-sensitivity was expressed when the ?cyclin kinases such as p34cdc2 and p33cdk2 were expressed sufficiently and the proliferative effect was enhanced. Furthermore, as an anti-CD3 mAb, EF6, reversed the antiproliferative effect of the antiT cell receptor mAb LC4, but not that of 2D1, Fas-mediated apoptosis seemed to be independent of TcR-mediated apoptosis.

微小重力は個体レベルでは骨･筋肉その他臓器に影響するが、細胞に対して直接的作用を及ぼすかどうか不明である。そこで培養細胞を用い、トリプシン処理により解離された細胞内骨格の再配列過程を観察することによって、細胞内骨格に及ぼす微小重力の直接作用を明らかにするとともに、細胞の形態、増殖、ウロキナーゼ産生、ブドウ糖消費に及ぼす微小重力の影響についても検索することを目的として実験を行った。この研究は、個体レベルで生ずる微小重力の影響のメカニズムを解明する上で重要であるばかりでなく、宇宙実験室における生物科学の基本技術の1つである細胞培養技法を確立する上で大きな意義を有する。飛行実験には、4個の培養容器で培養したサル腎由来培養細胞JTC-12が使用された。搭乗科学者は予定のスケジュール通りに実験を実施した。培養容器1および2の細胞について、位相差顕微鏡観察と写真撮影およびトリプシン処理と細胞固定を行い、冷蔵保存した。培養容器3の細胞については顕微鏡写真を撮影した。培養容器4の細胞については無血清培地に変換し、1日培養したのち、培地を採取し、冷蔵保存した。飛行延長にともない、顕微鏡写真撮影が1回追加実施された。軌道上撮影した顕微鏡写真観察から培養初期においては培養細胞の形態、増殖は地上対照群と差がないことが明らかとなった。しかし生きたまま帰還した細胞では細胞周期のS期頻度の減少とG2+M期頻度の増加が認められた。トリプシン処理により浮遊した細胞の基材への再付着は飛行群では地上対照群に比して少なかった。固定し帰還した細胞についての透過型および走査型電子顕微鏡観察から、トリプシン処理後の飛行群細胞は基材上でわずかに伸展するにすぎなかった。また、細胞辺縁におけるマイクロフィラメントの束状の再配列はわずかであり、地上対照群と著しく異なっていた。また、採取した培地中のブドウ糖量およびウロキナーゼ量はいずれも地上対照群と差が認められなかった。本研究の結果は、8日間の飛行では哺乳動物細胞の形態、増殖、生理活性物質の産生には宇宙実験室環境はほとんど影響しないが、トリプシンを用いた継代操作においては微小重力のもとでは沈降がない、あるいは膜機能の変化のため、浮遊した細胞の基材への付着が遅延し、細胞の継代が困難であることが示唆された。宇宙実験室における細胞培養技術の確立のための基礎的技術に関して情報が得られた。, Microgravity has effects on bones, muscles and other organs at an individual level. However, its direct effects on cells are unclear. The experiment is carried out to observe rearrangement of the cytoskeleton dissociated by trypsinization in cultured cells. The purpose is to understand the direct effects of microgravity on the cytoskeleton as well as on cell structure and proliferation, urokinase production, and glucose consumption. This study is important because it reveals the effect mechanism of microgravity at an individual level and because it establishes the cell culturing technique, one of the biologically basic techniques, in a space laboratory. This space flight experiment uses monkey-kidney-derived cultured cells, JTC-12, cultured in four cultures vessels No. 1 to 4. The scientist on board carried out the experiment as scheduled. The cells in No. 1 and 2 were observed and their photographs were taken with a phase contrast microscope. They were stored in a refrigerator after trypsinization and fixation. Microscopic photographs of the No. 3 cells were taken. The No. 4 cells were converted into a serum free medium, cultured for one day, and its medium was collected and stored in a refrigerator. Because of prolonged flight, one additional microscopic photographing was carried out. The microscopic photographs taken in orbit showed that, in the early culture stage, the cultured cells' structure and proliferation were not different from those of the ground control cells. However, the cells that returned alive showed decreased S stage and increased G2+M stages in cell cycle. Fewer trypsinized floating flight cells readhered to the medium than the ground control cells. Observing the cells that returned fixed with a transmission or scanning electron microscope showed slightly extended trypsinized flight cells on the medium. It also showed little bundle rearrangement of microfilaments at the cell margin, which was greatly different from that of the ground control cells. The glucose and urokinase amounts in the collected medium were not different between the flight and the ground control cells. These results suggest that the space laboratory environment has little effect on mammalian cells' structure and proliferation and bioactive substances' production. They also suggested the difficulty of cell passage in a microgravity state because, in passage technique using trypsin, floated cells takes much time to readhere to the medium due to microgravity or change in membrane function. This experiment provided information on the basic technique for establishing cell culturing technique in a space laboratory., 資料番号: AA0004115007, レポート番号: NASDA-TMR-940002 V.1

An improved method for staining DNA in phytoplankton nucleus is proposed. The method is for use in microfluorometry of fluorescence emitted from DNA stained with 4',6-diamidino-2-phenylindole (DAPl). Tris buffer composed of Tris (hydroxymethyl) aminomethane, 2-mercaptoethylamine hydrochloride, NaCl and EDTA-2Na yields low decay of the fluorescence under excitation light and stable intensity of fluorescence for 14 to 30 hours after staining. The low decay and the stability results in low variances in DAPI fluorescence. Suitable concentration of DAPI seems to be variable according to species. The method was applied to a natural population, and confirmed that cohorts were resolved well enough for cell cycle analysis of phytoplankton., DAPI染色による核内DNA量の測定を行うために，最適なDAPI染色法を確立することを目的として，緩衝液，染色時間，DAPI濃度に対する検討を行い，天然植物プランクトン群集への応用を試みた。主要な結果は以下の通りである。1．トリス緩衝液 (20mM Tris hydroxymethyl) aminomethane, 20mM 2- mercaptoethylamine hydrochloride, 100mM NaCl, 10mM EDTA-2Na, pH8.2) を用いることで，励起光下での蛍光の減衰が抑えられることが確認された。2．14時間から30時間の間に測定を行うことで，蛍光強度の経時変化は考慮する必要がなくなった。3．対象とする種によって，DAPI濃度を変える必要がある。4．本研究で得た染色条件によって天然植物プランクトン群集に対して，細胞周期解析を行うことが可能である。

Using CEA producing human ovarian cancer cell line （0MC-1）, changes in the secretion and the cellular content of CEA and its morphological changes were observed following the administration of various agents. Likewise, the relationship of these changes to the cell cycle was examined by flow cytometry （FCM）. CEA secretion of OMC-1 increased during the lag phase at 34.0ng/24hr/10? cells, and decreased during the logarithmic growth phase and the stationary phase at 2.4-3.5ng/24hr/10? cells. The cellu-lar content of CEA reached a temporary peak during the lag phase at 24.9ng/10? cells, increased to the highest level during the logarithmic growth phase at 10-18ng/10? cells and decreased to the lowest level during the stationary phase at about 5ng/10? cells. The temporary increase of the CEA secreted during the lag phase was considered to be regulated by the newly synthesized CEA released during the first 12 hours of culture. When investigation for manifestations of CEA in OMC-1 with the DNA/CEA double stain method using FCM was done, it was observed that CEA was manifested in all phases irrespective of cell cycle, for a CEA positive rate was 88.3%. Seventy-two hours after eight kinds of agents were administered, observations were made to determine possible increases in secretion of CEA, with due consideration being given to the inhibition rate of cellular proliferation. When the control level of CEA secretion-promoting NaBT 1mM, dbc-AMP 1mM and theo 2mM was increased 1.8 times of more, an increase in the cellular content of CEA of 1.7 times or more than that of control was observed for each agent. Using FCM, an increase in CEA positive fluorescence intensity was observed in each agent. It was considered that CEA positive cells occupied approximately 98.6% of the total cells when administering theo, which has the effect of transforming negative cells not manifesting CEA into positive ones. By administra-tion of NaBT, synchronous effects on the G? + G? phase of the cell cycle were recognized. When using Ki-67 antibody, which is considered to recognize cellular proliferation, analysis by the DNA/Ki-67 double stain method using FCM revealed an increase in Ki-67 negative G? phase cells with administration of NaBT and theo. By administration of dbc-AMP non-cycling cells, which were Ki-67 negative, of not only G?, but also S+G?/M phases, increased greatly. It was suggested that promotion of CEA production was closely related with the increase of G? cells or non-cycling cells.

Supervisor:小長谷 明彦, 知識科学研究科, 修士