軟骨性腫瘍におけるIX型コラーゲンの分布特性

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第982号, 学位授与年月日：平成3年3月25日,学位授与年：199

TNFαによるヒト骨肉腫細胞のα5β1インテグリン発現動態と浸潤転移における役割

金沢大学医薬保健研究域医学系低転移性であるヒト骨肉腫培養細胞OSTにおける各種インテグリンの発現をRT-PCR法を用いてさらに詳細に調べるために,ヒトで恒常的に発現しているといわれているβ2-microglobulinの発現量と比較することによってその増減を検討した.PCRによる増幅は,21サイクルから最初のスクリーニングで行った35サイクルの範囲で,β2-microglobulinの発現量がTNFα刺激前後で変化がないことを確認した.未処理OSTはα2,α3,α5,α6,αv,β1,β5,β6を発現しているが,TNFα刺激を受けるとα5ばかりでなくα3も激減した.α2,β1の発現量も有意に減少した.一方,α6は発現量が増加した.αv,β5,β6の発現量には差みられなかった.TNFα刺激によって新たにβ8の発現が確認された.このことから予想されることは,フィブロネクチンレセプターとしてのα5β1の減少,コラーゲンレセプターとしてのα2β1,α3β1の減少である.ビトロネクチンレセプターとしてのαvβ5の発現量に変化はみられなかった.しかし,細胞接着試験では,フィブロネクチンおよびコラーゲンに対する接着性にそれぞれ差はなく,ビトロネクチンに対する接着性はTNFα刺激で有意に低下した.このことから,蛋白レベルでのインテグリンの減少がTNFα刺激24時間ではまだ十分ではなく,分解・消失せずに残っており機能しているためではないかと考えている.実際,免疫沈降法では,ケミコン社のα5に対する抗体を用いると,TNFα刺激24時間ではα5β1の減少がみられなかった.さらに遊走試験でも,TNFα刺激を24時間したOSTは,フィブロネクチン,ビトロネクチンをコートした場合の両者ともにcontrolに対して遊走能が亢進した.研究課題/領域番号:08770151, 研究期間(年度):1996出典：研究課題「 TNFαによるヒト骨肉腫細胞のα5β1インテグリン発現動態と浸潤転移における役割」課題番号08770151（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-08770151/）を加工して作

in situ RT-PCR法を用いた接着分子およびプロテアーゼmRNAの局在観察

金沢大学医薬保健研究域医学系(1)RT-PCRによる検討。進行胃癌組織30例と非癌部胃粘膜組織20例について、胃癌組織と非癌部組織での各サブユニットの発現率をRT-PCR法で調べるとαv、β6、β8は癌組織で有意に発現率が高かった。β5は、癌部で高い傾向が見られたが、統計学的有意差は認められなかった(p=0.0582)。(2)競合RT-PCR法による検討。PCR競合鋳型合成キットを用いて、574塩基対からなる癌遺伝子v-erbBのDNA断片から、両端にβ5のプライマーの配列を含む240塩基対の競合鋳型を作成した。この競合鋳型をサンプルに等量混ぜることによってサンプル内のβ5の定量が可能であった。定量的検索が可能であった進行胃癌組織21例と非癌部胃粘膜組織10例について、β5は癌部で140倍多く発現していた。(3)in situ hybridizationによる検討。組織切片上でβ5がどの程度発現しているかを調べるためにin situ hybridizationを施行した。対象は、手術がなされた胃癌患者の術前の胃生検組織(10%中性緩衝ホルマリン固定パラフィン包埋切片)65例で、まずこれらの症例をmRNA全量を反映するd(T)20 oligonucleotideの発現を検討し、mRNAがよく保存されていることを確認した。プローブは、PCRでジゴキシゲニン標識したDNAプローブを用いた。陽性細胞の割合からβ5の陽性スコアを算出すると、癌部で有意に高いスコアが得られた。(4)まとめ。αvβ5はビトロネクチンをリガンドとするインテグリンである。これまでのところモノクローナル抗体を用いたPLP固定パラフィン切片での免疫組織化学的検討でスキラス型胃癌においてβ5が陽性になる傾向が示されている(Kawahara et al.Pathol.Int.45:493-500,1995)。本研究でも競合RT-PCR法とin situ hybridizationを用いた検討で、β5が胃癌に高発現していることが確かめられた。研究課題/領域番号:09770118, 研究期間(年度):1997 – 1998出典：「in situ RT-PCR法を用いた接着分子およびプロテアーゼmRNAの局在観察」研究成果報告書　課題番号09770118（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-09770118/)を加工して作

ヒト骨肉腫培養細胞におけるインテグリン分子の発現と浸潤能における役割

金沢大学がん研究所我々はこれまでに,低転移性であるヒト骨肉腫培養細胞OSTを用いて,細胞外マトリックスの分解に関わるMMP-9の発現が,浸潤転移にどのような役割を果たしているかを調べてきた.今回は,OSTにおける各種インテグリンの発現をRT-PCR法を用いてスクリーニングし,TNFα刺激によりどのインテグリンが増加あるいは減少するのか調べた.また,Fibronectin(FN),vitronectin(VN)をリガンドとする細胞接着試験を行った,さらに,細胞内伝達機構を調べる目的でPKC assayも行った.結果と考察 未処理OSTはα2,α3,α5,α6,αv,β1,β3,β5を発現しているが,TNFα刺激を受けるとα5の発現が激減した.免疫沈降法においても細胞表面のα5の減少が示された.αv,β1,β3,β5の発現には差は見られなかった.細胞接着試験では,FNに対する接着性に差はなかったが,VNに対する接着性はTNFα刺激で有意に低下した.またPKC活性はTNFαにより有意に低下した.FNは現在までに,FN分子中の細胞接着に関与する構造はRGD配列であることがわかっており,主としてα5β1(VLA-5)インテグリンがこの配列を認識する細胞膜レセプターであると言われている.OSTではα5の発現低下により正常に機能するフィブロネクチンレセプターの合成が障害され,基質に対する接着性の低下が運動能の増加をもたらし,さらにはMMP-9の分泌増加と相まって最終的にOSTの転移の増加につながったのではないかと推測している.PKC assayにおいて,protein kinaseの活性化剤として知られているPMA刺激でもPKCの活性が低下したので,OSTではインテグリンによって引き起こされる細胞内伝達機構にalternative pathwayがある可能性が示唆された.研究課題/領域番号:07770148, 研究期間(年度):1995出典：研究課題「ヒト骨肉腫培養細胞におけるインテグリン分子の発現と浸潤能における役割」課題番号07770148（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-07770148/）を加工して作

Androgen receptor and 5α-reductase immunohistochemical profiles in extramammary Paget disease

Background Extramammary Paget disease is an uncommon skin tumour occurring mostly in the genitoperineal region. Previous reports have shown frequent expression of androgen receptor, suggesting a tumour-proliferative effect of androgens on Paget cells. Androgen-converting enzymes such as 5α-reductase, which locally produces a bioactive androgen, have recently gained attention in studies of the intratumoral actions of androgens. Objectives We investigated correlations between the androgenic microenvironment and invasiveness in extramammary Paget disease, particularly in terms of sex differences. Methods We examined 58 cases of extramammary Paget disease (32 men, 26 women; 42 noninvasive, 16 invasive) using immunohistochemistry for androgen receptor and 5α-reductase. Results In all 58 cases, expression rates were 57% for androgen receptor and 55% for 5α-reductase, with 38% double-positivity for androgen receptor and 5α-reductase. Only 5α-reductase expression rate was significantly higher in invasive cases (81%) than in noninvasive cases (45%; P = 0·042). For invasive cases, numbers of double-positive results for androgen receptor and 5α-reductase were significantly higher in men (70%) than in women (17%; P = 0·039). Conclusions Double positivity for androgen receptor and 5α-reductase in Paget cells suggests autocrine synthesis of androgens in extramammary Paget disease. The different hormonal microenvironments in male and female cases and intratumoral androgen levels affect the invasiveness of extramammary Paget disease. © 2010 British Association of Dermatologists

Cooperative effect of radioimmunotherapy and antiangiogenic therapy with thalidomide in human cancer xenografts

金沢大学大学院医学系研究科Antiangiogenic therapy may prolong the dormancy of cancer lesions. Moreover, radioimmunotherapy (RIT) may eradicate this population of cells. This study dealt with determining the benefits associated with the combined usefulness of these 2 therapies with respect to inhibition of tumor growth. Methods: Antiangiogenic therapy using oral thalidomide (daily dose, 200 mg/kg) and RIT involving a single intravenous injection (4.63 MBq 131I-A7, an IgG1 murine monoclonal antibody) were conducted in mice bearing LS180 human colon cancer xenografts. RIT with an irrelevant IgG1, HPMS-1, was also performed as a control. Antiangiogenesis of thalidomide was investigated by immunohistochemical analysis of tumor sections. Results: Antiangiogenic therapy and RIT with 131I-A7 significantly suppressed the growth of xenografts. This combination produced more efficient tumor growth inhibition than did the monotherapy (P < 0.005). RIT using 131I-HPMS-1 was far less effective than 131I-A7, even when combined with thalidomide administration. Immunohistochemistry revealed a decrease in the microvessel number within tumors treated with thalidomide (P < 0.0001). Combined therapy further reduced the microvessel number (P < 0.01 vs. thalidomide monotherapy), Conclusion: The combination of RIT and thalidomide antiangiogenic therapy produces a better response of tumors than does monotherapy. Acting in concert, antiangiogenic therapy may prolong the dormancy of cancer lesions and RIT may eradicate this population of cells

Paracrine activation of MET promotes peritoneal carcinomatosis in scirrhous gastric cancer

がん進展制御研究所Scirrhous gastric cancer is associated with abundant stroma and frequently develops into peritoneal carcinomatosis with malignant ascites. Although malignant ascites is among the most deadly diseases worldwide, its molecular pathogenesis is poorly understood. We investigated the role of hepatocyte growth factor (HGF) in the production of peritoneal carcinomatosis with malignant ascites. We examined three scirrhous and three non-scirrhous human gastric cancer cell lines for the production of peritoneal carcinomatosis in vivo and responses to HGF in vitro. Furthermore, clinical scirrhous gastric cancer specimens were examined for HGF production. Among the six cell lines examined, only two scirrhous cell lines (NUGC4 and GCIY) produced peritoneal carcinomatosis with massive ascites after intraperitoneal injection in nude mice. Their proliferation was stimulated by exogenous HGF in vitro. On the other hand, a non-scirrhous cell line, MKN45, with MET amplification generated peritoneal tumors but not ascites. MET tyrosine kinase inhibitors, crizotinib and TAS-115, inhibited HGF-stimulated proliferation of NUGC4 and GCIY as well as constitutive proliferation of MKN45. Furthermore, crizotinib and TAS-115 prolonged the survival of mice bearing established tumors by NUGC4 or MKN45. In clinical specimens, HGF was markedly produced by stromal fibroblasts. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high levels of HGF. Our results strongly suggest that paracrine HGF-induced activation of MET-mediated signaling pathways plays an important role in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, MET signaling pathway may be a potential therapeutic target for peritoneal carcinomatosis of gastric cancer, even without MET amplification. © 2013 Japanese Cancer Association

Alteration of energy metabolism in the pathogenesis of bile duct lesions in primary biliary cirrhosis

Aim: Primary biliary cirrhosis (PBC) is characterised by antimitochondrial antibody against the pyruvate dehydrogenase complex (PDC) and chronic nonsuppurative destructive cholangitis (CNSDC). Pyruvate oxidation to acetyl-CoA by PDC is a key step in the glycolytic system. Oestrogen-related receptor-α (ERRa) is functionally activated by inducible coactivators such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and Bcl-3. Moreover, the PGC-1α-ERRa axis interrupts glycolytic metabolism through the upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4), which functionally inhibits PDC-E1α and stimulates fatty acid oxidation. In this study, we investigated the PGC-1α-ERRa axis to clarify PDC dysfunction in CNSDC of PBC. Methods: The expression of PGC-1α, Bcl-3, ERRa, PDK4 and PDC-E1α was examined by immunohistochemistry in liver sections from patients with PBC and controls. The expression of these molecules, the activity of mitochondrial dehydrogenase and PDC, and their alterations by starvation, a treatment used to induce PGC-1α expression, were examined in cultured human biliary epithelial cells (BECs). Results: The nuclear expression of PGC-1α, Bcl-3 and ERRa was exclusively observed in CNSDC of PBC. Moreover, the expression of PDK4 and PDC-E1α was enhanced in CNSDC of PBC. In cultured BECs, the amplification of Bcl-3 and PDK4 mRNAs by reversetranscription-PCR and mitochondrial dehydrogenase activity were markedly increased but PDC activity was decreased according to the upregulation of PGC-1α. Conclusions: In CNSDC of PBC, the activation of the ERRa-PGC-1α axis was exclusively observed, suggesting the interference of PDC-related glycolytic function and the induction of the fatty acid degradation system. The switching of the cellular energy system is possibly associated with the pathogenesis of CNSDC in PBC

Detection of cardiomyocyte death in a rat model of ischemia and reperfusion using 99mTc-labeled annexin V.

金沢大学大学院医学系研究科There is increasing evidence that cell death after myocardial ischemia and reperfusion may begin as apoptosis rather than necrosis. To determine the time course, location, and extent of this process, we studied groups of rats after a 20-min interval of coronary occlusion and reperfusion. METHODS: After thoracotomy, the left coronary artery was occluded for 20 min. After release and before study, groups of animals were allowed to recover for various intervals: 0.5 h (n = 6), 1.5 h (n = 7), 6 h (n = 7), 1 d (n = 8), 3 d (n = 8), or 2 wk (n = 5). At the time of study, the rats were injected with 99mTc-annexin V (80-150 MBq). One hour later, to verify the area at risk, 201Tl (0.74 MBq) was injected intravenously just after the left coronary artery reocclusion and the rats were sacrificed 1 min later. Dual-tracer autoradiography was performed to assess 99mTc-annexin V uptake and the area at risk. RESULTS: Extensive 99mTc-annexin V uptake was observed in the mid myocardium after 0.5-1.5 h of reperfusion. The area of annexin uptake had expanded in the subendocardial and subepicardial layers at 6 h after reperfusion and then gradually lessened over 3 d. At 0.5 and 1.5 h of reperfusion, 99mTc-annexin V uptake ratios were 7.36 +/- 2.95 and 6.34 +/- 2.24 (mean +/- SD), respectively. The uptake ratios gradually decreased at 6 h, 1 d, 3 d, and 2 wk after reperfusion (4.65 +/- 1.93, 3.27 +/- 0.92 [P < 0.01 vs. 0.5 h], 1.84 +/- 0.55 [P < 0.001 vs. 0.5 h, P < 0.005 vs. 1.5 h], and 1.65 +/- 0.31 [P < 0.001 vs. 0.5 h, P < 0.005 vs. 1.5 h], respectively). CONCLUSION: These data indicate that annexin binding commences soon after ischemia and reperfusion in the mid myocardium within the area at risk and expands to include the subendocardial and subepicardial layers at 6 h after reperfusion, followed by gradual reduction of activity over 3 d

Monocyte chemoattractant protein-1 derived from biliary innate immunity contributes to hepatic fibrogenesis

金沢大学医薬保健研究域医学系Aims: Monocyte chemoattractant protein-1 (MCP-1) is a major chemotactic factor for hepatic stellate cells (HSCs) associated with hepatic fibrosis. In this study, among several fibrogenetic factors derived from biliary epithelial cells (BECs), MCP-1 produced by the biliary innate immune system was found to be most critical in the histogenesis of hepatic fibrogenesis. Methods: Using cultured human BECs, the expression of five fibrogenetic factors including MCP-1 on stimulation with Toll-like receptor ligands, inflammatory cytokines or bile acids was examined. Moreover, in situ detection of MCP-1 and α-smooth muscle actin proteins was performed using sections from normal and diseased livers by immunohistochemistry. Results: All fibrogenetic factors were detected in BECs, but only MCP-1 expression was upregulated, by all the Toll-like receptor ligands, IL-1β, and tumour necrosis factor-alpha. Proliferating bile ductules in interface areas expressed MCP-1 in diseased livers accompanying α-smooth muscle actin-positive activated HSCs. Conclusions: Bile ductules proliferate in various hepatobiliary diseases, and its significance is still unknown. This study demonstrated that BECs in bile ductules could produce MCP-1, particularly, via biliary innate immunity, suggesting that MCP-1 derived from BECs plays an important role in the recruitment of HSCs to interface areas and the activation of HSCs resulting in the progression of periportal fibrosis. Copyright Article author (or their employer) 2011