口腔扁平上皮癌の転移形成に及ぼすマトリックスメタロプロテナーゼの影響

金沢大学医学部・附属病院口腔扁平上皮癌の転移形成におけるマトリックスメタロプロテナーゼ(MMPs)の果たす役割を、われわれが最近開発したin vivo転移モデルを用いて検索するとともに、臨床材料を用いて実際の口腔扁平上皮癌組織の産生するMMPsについても検討し、これらの酵素と転移との関係について検討を行った。まず、in vivo転移モデルを用いて転移能の異なるヒト口腔扁平上皮癌細胞株のOSC-19細胞とOSC-20細胞をヌードマウスの舌に移植し、舌腫瘍組織におけるMMPsの発現について免疫組織化学的および生化学的に検討した。その結果、頚部リンパ節に高率に転移するOSC-19細胞では、低転移性のOSC-20細胞と比較してゼラチナーゼ活性が高く、免疫染色およびゼラチンザイモグラムにおいてMMP-9(92kDa IV型コラゲナーゼ、ゼラチナーゼB)の発現が顕著に認められた。次に、口腔扁平上皮癌38例の初診時生検材料を用いてMMP-1,-2,-3,-9およびTIMP-1に対する免疫染色を行ったところ、癌細胞に陽性所見を示したのは、主としてMMP-1(70.0%)とMMP-9(45.7%)であり、MMP-2,MMP-3およびTIMP-1の発現率はそれぞれ21.1%、10.5%、22.2%と低かった。また、これらMMPsの陽性癌細胞率と頚部リンパ節転移との関係について検討したところ、MMP-QとMMP-9の陽性細胞率は転移を認めた群では非転移群に比較して有意に高値を示した。以上の結果より、口腔扁平上皮癌の頚部リンパ節転移にはMMP-1とMMP-9が深く関与し、特にMMP-9が重要な役割を果たしている可能性が示唆された。今後は、これらの結果をふまえて、さらに、これらの酵素のインヒビターを用いた転移抑制の可能性について研究を行っていく予定である。In order to study the roles of matrix metalloproteinases (MMPs) in lymph node metastasis of oral squamous cell carcinoma (SCC), the expression of MMP-1 (tissue collagenase), MMP-2 (72kDa gelatinase/type IV collagenase), MMP-3 (stromelysin-1), MMP-9 (92kDa gelatinase/type IV collagenase) and TIMP-1 (tissue inhibitor of metalloproteinase-1) in oral SCC tissues obtained from biopsy specimens of 46 cases and from in vivo metastasis model was examined immunohistochemically or biochemistrically. Among these MMPs, MMP-1 and MMP-9 were immunolocalized in the carcinoma cells in 70.0% and 45.7% of oral SCC,respectively. On the other hand, MMP-2 and MMP-3 were positively stained only in 21.1% and 10.5%, respectively. TIMP-1 was immunolocalized in 22.2% of the cases, but the ratio of immunoreactive cells to the total carcinoma cells was only 0.6(]SY.+-.])0.2%. The ratio of positive cells for MMP-1 or MMP-9 in carcinomas with lymph node metastasis was significantly higher than that in carcinomas without metastasis. In in vivo metastasis model, gelatinolytic activity was higher in the culture media of OSC-19 cells (high metastatic phenotype) than that of OSC-20 cells (low metastatic phenotype). Gelatin-substrate gel electrophoresis also showed that the gelatin-degrading activity with molecular weight of 92.000 was higher in the culture media of OSC-19 cells than that of OSC-20 cells. These results suggest that MMP-1 and MMP-9 play pivotal roles in the degradation of extracellular matrix macromolecules during oral SCC metastasis.研究課題/領域番号:06671997, 研究期間(年度):1994 – 1995出典：研究課題「口腔扁平上皮癌の転移形成に及ぼすマトリックスメタロプロテナーゼの影響」課題番号06671997（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/report/KAKENHI-PROJECT-06671997/066719971995kenkyu_seika_hokoku_gaiyo/）を加工して作

口腔扁平上皮癌の浸潤・転移に関与する運動因子の解析

金沢大学医学部附属病院口腔扁平上皮癌における癌細胞の運動能と浸潤様式との関連を明らかにする目的で浸潤様式の異なる3種類の口腔扁平上皮癌細胞株(OSC20,3型;OSC19,4C型;HOC313,4D型)を用いて細胞の運動能を比較検討するとともに,それらの運動能に関与する運動促進因子の発現と機能について検討した.金コロイド法を用いて細胞の運動性を測定したところ,血清存在下での運動能はHOC313細胞が最も高く、次いでOSC19細胞,OSC20細胞の順であったが,無血清培地ではHOC313細胞のみが大きな運動能を示した.この結果からHOC313細胞が運動促進因子を自ら産生していることが推測された.これらの細胞における自己分泌型運動促進因子(autocrine motility factor.AMF)遺伝子の発現を検討した結果,いずれの細胞でもmRNAの存在が認められたがHOC313細胞で特に強く発現していた.またAMFに対する免疫組織化学染色ではHOC313細胞のみにAMF蛋白の存在が認められ,また抗AMF抗体により運動性が抑制されたことから,HOC313細胞が自己運動促進因子としてAMFを産生していることが明らかになった.扁平上皮癌細胞がAMFを産生・分泌しているのを証明したのは本研究が初めてである.AMFレセプターのmRNAの発現はいずれの細胞にも認められたが,レセプター蛋白の存在量はHOC313細胞>OSC20細胞の順に高く、OSC19細胞では認められなかった.AMFと同一アミノ酸配列を有するグルコース6-リン酸イソメラーゼ(glucose 6-phosphate isomerase,G6PI)を作用させると,HOC313細胞とOSC20細胞は濃度依存的に運動性が亢進し,これはAMFレセプター蛋白の発現量と相応していた.分散因子/肝細胞増殖因子(scatter factor/hepatocyte growth factor,SF/HGF)に対しては,HOC313細胞とOSC20細胞で濃度依存的な遊走性の亢進が認められた.フィブロネクチン(fibronectin,Fn)およびビトロネクチン(vitoronectin,Vn)に対する遊走性はHOC313細胞で著しく,OSC20細胞では認めなかった.以上より,口腔扁平上皮癌細胞の運動能と各種運動促進因子に対する感受性は浸潤様式によって異なり,特に4D型の癌細胞は自己分泌型の運動促進因子を産生・分泌して自らの運動能を高め,周囲組織へび漫性に浸潤していくものと考えられた.The present study was conducted to clarify the mechanism that determines the mode of invasion of human oral squamous cell carcinomas. Three cell lines derived from human oral squamous cell carcinomas with different modes of invasion were assayed for their ability to migrate or invade in vitro. The cell lines employed were HOC3 13, OSO 19 and OSC2O cells, which respectively derive from oral tumors of the highest (grade 4D), high (grade 40) and moderate (grade 3) invasiveness, A phagokinesis assay with gold particles demonstrated that, among the three cell lines, the rate of migration in serum-containing media was ranked in the following order : H0C313 * OSCI9 > OSC2O.In serum free media, H0C313 cells also exhibited a high motility rate, whereas OSCl9 and OSC2O cells migrated to much lesser extents, suggesting that the HOC313 cells produce motility factors that act on themselves in an autocrine manner. Next, the nature of this activity was investigated. Northern blot analysis revealed that the gene coding for autocrine motility factor (AMF) was extremely strongly expressed in HOC313 cells ; two prominent hybridizing bands were detected at 2.0 and 4.0 kb. On the other hand, OSC19 cells expressed the 2.0 kb mRNA species at a low level, and OSC19 cells had barely detectable amounts of AMF mRNA.AMF proteins were clearly marked by immunohistochemical staining with anti-AMF antibodies in H0C313 cells, but not in OSC19 and OSC2O cells ; the immunoreactivity was localized in pseudopodia in a granular pattern. mRNA encoding AMF receptor was detected at a high level in H0C313 and OSC20 cells, and at a low level in OSCl9 cells. In Western blotting, AMF receptor proteins (gp78) were detected at a high level in H0C313 cells, at a low level in OSC2O cells, and not at all in OSCl9 cells. Relative amounts of the AMF and AMF receptor proteins in three cell lines were well correlated with their motility rates. The migration of HOC313 cells in serum-free media was markedly blocked by anti-AMF antibodies. Further, exogenously added glucose 6-phosphate isomerase, which is known to have the same amino acid sequence as AMF, stimulated HOC313 cell motility in a dose-dependent manner. These results indicate that AMF would at least partly account for the high motility of HOC313 cells, and this is the first demonstration that squamous carcinoma cells produce an autocrine factor that induces their own motility. The present study also established the effects of hepatocyte growth factor (HGF)/scatter factor (SF) and cxtracellular matrix components such as fibronectin and vitronectin on the chemotaxis of the oral squamous carcinoma cells. HGF/SF stimulated in a dose dependent manner the H0C313 cell migration through 8 mum pore sized filter to the highest extent. OSC2O cells exhibited a moderate migration toward HGF/SF, while OSCl9 cells did not respond to it. Fibronectin and vitronectin were also found to stimulate the migration of H0C313 cells. The migration of OSC19 cells was研究課題/領域番号:09672041, 研究期間(年度):1997 – 1998出典：研究課題「口腔扁平上皮癌の浸潤・転移に関与する運動因子の解析 」課題番号09672041（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/report/KAKENHI-PROJECT-09672041/096720411998kenkyu_seika_hokoku_gaiyo/）を加工して作

Thin observation module by bound optics (TOMBO) : concept and experimental verification

This paper was published in Optics Express and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://dx.doi.org/10.1364/AO.40.001806 Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law

Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of αv integrins

金沢大学大学院医学系研究科保健学専攻We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3, poorly differentiated type; OSC-19, well-differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-αv antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-β1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-β5 and anti-αv antibodies, and only HSC-β3 cells were positive for antiβ-3 antibody. α5β1 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects αvβ3-and/or αvβ5-mediated carcinoma cell invasion

Explosive Nucleosynthesis in Axisymmetrically Deformed Type II Supernovae

Explosive nucleosynthesis under the axisymmetric explosion in Type II supernova has been performed by means of two dimensional hydrodynamical calculations. We have compared the results with the observations of SN 1987A. Our chief findings are as follows: (1) $^{44}Ti$ is synthesized so much as to explain the tail of the bolometric light curve of SN 1987A. We think this is because the alpha-rich freezeout takes place more actively under the axisymmetric explosion. (2) $^{57}Ni$ and $^{58}Ni$ tend to be overproduced compared with the observations. However, this tendency relies strongly on the progenitor's model. We have also compared the abundance of each element in the mass number range $A= 16-73$ with the solar values. We have found three outstanding features. (1) For the nuclei in the range $A=16-40$, their abundances are insensitive to the initial form of the shock wave. This insensitivity is favored since the spherical calculations thus far can explain the solar system abundances in this mass range. (2) There is an enhancement around A=45 in the axisymmetric explosion compared with the spherical explosion fairly well. In particular, $^{44}Ca$, which is underproduced in the present spherical calculations, is enhanced significantly. (3) In addition, there is an enhancement around A=65. This tendency does not rely on the form of the mass cut but of the initial shock wave. This enhancement may be the problem of the overproduction in this mass range, although this effect would be relatively small since Type I supernovae are chiefly responsible for this mass number range.Comment: 32 pages, 12 figures, LaTe