Klominek J, Robért K H, Sundqvist K G
Department of Lung Medicine, Karolinska Institute, Huddinge University Hospital, Sweden.
Cancer Res. 1993 Sep 15;53(18):4376-82.
A human malignant pleural mesothelioma cell line (STAV) was studied with respect to production of the extracellular matrix components laminin, type IV collagen, and fibronectin, and interactions with these proteins in vitro. We also analyzed STAV cell serum-free conditioned medium with respect to the possible presence of "autocrine motility factor-like" substance. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of biosynthetically labeled STAV serum-free conditioned medium showed that STAV cells released several proteins into the medium, including components with molecular weights of 850,000, 540,000 and 440,000. Using Western blotting we identified these proteins as laminin, type IV collagen, and fibronectin, respectively. By immunocytochemistry laminin, type IV collagen, and fibronectin were detected as a matrix surrounding the cells. Plastic culture dishes coated with microgram quantities of laminin, type IV collagen, and fibronectin induced attachment and spreading of STAV cells. Laminin, type IV collagen, and fibronectin stimulated directional (chemotactic) migration of STAV cells in Boyden chambers fitted with 8 microns filters. The same cells also migrated to insoluble step gradients of filter-bound extracellular matrix components (haptotaxis). When STAV serum-free conditioned medium was separated by using fast protein liquid chromatography Superose 6 gel filtration, two motility-inducing protein peaks were detected. The first peak contained proteins with molecular weight > 220,000 that had both chemotactic and haptotactic properties, while the second peak contained material with apparent molecular weights of approximately 67,000 that had chemotactic and chemokinetic (random motility) but not haptotactic properties. Analysis of the M(r) 67,000 material indicated that it was a heat-sensitive and trypsin-digestible protein. The production of both soluble and insoluble extracellular matrix components by human mesothelioma cells and the motile response to these molecules as well as the production of a M(r) 67,000 autocrine motility factor-like substance may be important for the highly invasive motile behavior of this tumor.
对一种人类恶性胸膜间皮瘤细胞系（STAV）进行了研究，内容涉及细胞外基质成分层粘连蛋白、IV型胶原和纤连蛋白的产生，以及在体外与这些蛋白质的相互作用。我们还分析了STAV细胞无血清条件培养基中是否可能存在“自分泌运动因子样”物质。对生物合成标记的STAV无血清条件培养基进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，STAV细胞向培养基中释放了几种蛋白质，包括分子量为850,000、540,000和440,000的成分。通过蛋白质印迹法，我们分别将这些蛋白质鉴定为层粘连蛋白、IV型胶原和纤连蛋白。通过免疫细胞化学检测到层粘连蛋白、IV型胶原和纤连蛋白是围绕细胞的基质。涂有微克量层粘连蛋白、IV型胶原和纤连蛋白的塑料培养皿可诱导STAV细胞附着和铺展。层粘连蛋白、IV型胶原和纤连蛋白在装有8微米滤膜的博伊登小室中刺激STAV细胞的定向（趋化性）迁移。同样的细胞也向滤膜结合的细胞外基质成分的不溶性阶梯梯度迁移（趋触性）。当使用快速蛋白质液相色谱Superose 6凝胶过滤法分离STAV无血清条件培养基时，检测到两个诱导运动的蛋白峰。第一个峰包含分子量>220,000的蛋白质，具有趋化性和趋触性，而第二个峰包含表观分子量约为67,000的物质，具有趋化性和化学动力学（随机运动）但不具有趋触性。对分子量为67,000的物质分析表明，它是一种对热敏感且可被胰蛋白酶消化的蛋白质。人间皮瘤细胞产生可溶性和不溶性细胞外基质成分以及对这些分子的运动反应，以及产生分子量为67,000的自分泌运动因子样物质可能对该肿瘤的高度侵袭性运动行为很重要。
