Hayward I P, Whitehead R H, Ward L, Gianello R, Dempsey P, Bates R, Burns G F
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Parkville, Australia.
Immunol Cell Biol. 1995 Jun;73(3):249-57. doi: 10.1038/icb.1995.41.
The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.
LIM 1863结肠癌细胞系在含血清培养基中以形态和功能上有组织的类器官形式悬浮生长。添加转化生长因子-β（TGF-β）会导致类器官黏附并抑制DNA合成。与TGF-β孵育20分钟就足以诱导黏附，且这种黏附可被放线菌酮抑制。已证明黏附和DNA合成抑制是独立的事件。我们未能检测到基质或细胞膜抗原的任何变化。同样，合成蛋白质也没有变化（通过二维凝胶电泳），蛋白聚糖也没有上调。当从组织培养塑料表面裂解已黏附的类器官时，未处理的类器官会黏附到该表面。这种“预处理”过的表面可被胰蛋白酶破坏，但不能被胶原酶或正常LIM 1863培养物的培养基破坏。然而，当类器官在正常LIM 1863培养物条件化的培养基而非新鲜培养基中用转化生长因子-β（TGF-β）处理时，黏附表型受到阻止。一种抗β1整合素抗体（QE2E5）抑制了黏附过程，尽管未观察到整合素的定量变化（通过免疫沉淀或RNA分析）。第二种抗β1整合素抗体（61.2C4）抑制了LIM 1863对胶原蛋白的黏附，但不抑制TGF-β诱导的黏附，这意味着TGF-β诱导了β1整合素的特定构象变化或相互作用。在这个形态结构系统中，TGF-β诱导了许多细微的效应，包括新细胞外基质的形成和β1整合素的构象变化，而不是先前报道的细胞/基质分子的主要定量变化。
