Taraboletti G, Roberts D D, Liotta L A
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892.
J Cell Biol. 1987 Nov;105(5):2409-15. doi: 10.1083/jcb.105.5.2409.
Thrombospondin induces the migration of human melanoma and carcinoma cells. Using a modified Boyden chamber assay, tumor cells migrated to a gradient of soluble thrombospondin (chemotaxis). Checkerboard analysis indicated that directional migration was induced 27-fold greater than stimulation of random motility. Tumor cells also migrated in a dose-dependent manner to a gradient of substratum-bound thrombospondin (haptotaxis). A series of human melanoma and carcinoma cells were compared for their relative motility stimulation by thrombospondin haptotaxis vs. chemotaxis. Some cell lines exhibited a stronger haptotactic response compared to their chemotactic response while other lines exhibited little or no migration response to thrombospondin. Human A2058 melanoma cells which exhibit a strong haptotactic and chemotactic response to thrombospondin were used to study the structural domains of thrombospondin required for the response. Monoclonal antibody C6.7, which binds to the COOH-terminal region of thrombospondin, inhibited haptotaxis in a dose-dependent optimal manner. C6.7 had no significant effect on thrombospondin chemotaxis. In contrast, monoclonal antibody A2.5, heparin, and fucoidan, which bind to the NH2-terminal heparin-binding domain of thrombospondin, inhibited thrombospondin chemotaxis but not haptotaxis. Monoclonal antibody A6.1 directed against the internal core region of thrombospondin had no significant effect on haptotaxis or chemotaxis. Synthetic peptides GRGDS (50 micrograms/ml), but not GRGES, blocked tumor cell haptotaxis on fibronectin, but had minimal effect on thrombospondin or laminin haptotaxis. The 140-kD fragment of thrombospondin lacking the heparin-binding amino-terminal region retained the property to fully mediate haptotaxis but not chemotaxis. When the COOH region of the 140-kD fragment, containing the C6.7-binding site, was cleaved off, the resulting 120-kD fragment (which retains the RGDA sequence) failed to induce haptotaxis. Separate structural domains of thrombospondin are therefore required for tumor cell haptotaxis vs. chemotaxis. This may have implications during hematogenous cancer metastases formation.
血小板反应蛋白可诱导人黑色素瘤细胞和癌细胞的迁移。利用改良的博伊登小室分析，肿瘤细胞向可溶性血小板反应蛋白梯度迁移（趋化作用）。棋盘分析表明，定向迁移的诱导作用比随机运动的刺激作用大27倍。肿瘤细胞也以剂量依赖的方式向基质结合的血小板反应蛋白梯度迁移（趋触性）。比较了一系列人黑色素瘤细胞和癌细胞对血小板反应蛋白趋触性与趋化性的相对运动刺激作用。一些细胞系表现出比趋化反应更强的趋触反应，而其他细胞系对血小板反应蛋白几乎没有或没有迁移反应。对血小板反应蛋白表现出强烈趋触性和趋化性反应的人A2058黑色素瘤细胞被用于研究该反应所需的血小板反应蛋白的结构域。与血小板反应蛋白COOH末端区域结合的单克隆抗体C6.7以剂量依赖的最佳方式抑制趋触性。C6.7对血小板反应蛋白趋化性没有显著影响。相反，与血小板反应蛋白NH2末端肝素结合结构域结合的单克隆抗体A2.5、肝素和岩藻依聚糖抑制血小板反应蛋白趋化性，但不抑制趋触性。针对血小板反应蛋白内部核心区域的单克隆抗体A6.1对趋触性或趋化性没有显著影响。合成肽GRGDS（50微克/毫升）可阻断肿瘤细胞在纤连蛋白上的趋触性，但对血小板反应蛋白或层粘连蛋白趋触性影响最小。缺乏肝素结合氨基末端区域的140-kD血小板反应蛋白片段保留了完全介导趋触性但不介导趋化性的特性。当包含C6.7结合位点的140-kD片段的COOH区域被切除时，产生的120-kD片段（保留RGDA序列）未能诱导趋触性。因此，肿瘤细胞趋触性与趋化性需要血小板反应蛋白的不同结构域。这可能对血源性癌症转移形成过程有影响。
