Pouliot N, Burgess A W
The Ludwig Institute for Cancer Research, Melbourne Branch, Australia.
Growth Factors. 2000;18(1):31-49. doi: 10.3109/08977190009003232.
The human colon carcinoma cell line LIM1215 proliferates and changes morphology (spread) in a cell density-dependent manner in response to epidermal growth factor (EGF). At high density, production of autocrine transforming growth factor-alpha enables the cells to proliferate and spread in the absence of exogenous EGF or serum. At low cell density (< 1 x 10(4)/cm2) EGF alone fails to elicit a mitogenic or morphological response and requires the presence of conditioned medium (derived from high cell density serum-free culture of the same cells) to exert its effects. This synergy between EGF and LIM1215 conditioned medium was investigated further. Using a low cell density assay and fractionated LIM1215 conditioned medium, we show that EGF-mediated mitogenic and morphological responses are separable. These responses are dependent on the synergistic action of a low molecular weight autocrine survival factor and an extracellular matrix-like spreading factor(s) secreted into the culture medium respectively. We find that under low cell density, serum-free conditions, EGF alone is insufficient to rescue LIM1215 from rapid apoptotic death. Catalase or LIM1215 autocrine survival factor prevent the death of LIM1215 cells and restore their proliferative (but not morphological) response to EGF, suggesting that cell death under these conditions may be the result of oxidative stress. Combination of EGF, partially purified autocrine survival and spreading factors induced proliferation and spreading of low density LIM1215 cells similar to that observed with EGF and unfractionated conditioned medium. GRGDS peptides strongly inhibited the spreading of LIM1215 cells in the presence of EGF and the partially purified autocrine spreading factor, demonstrating that integrin receptors are involved in the spreading process. Comparison of the spreading response of LIM1215 and Colo 526 cells on ASF and various adhesion proteins indicate that ASF is not collagen-I, collagen-IV, fibronectin or vitronectin. Taken together, these results support the concept that the autonomous growth of colon carcinoma cells in vitro is dependent on the synergistic interaction between several autocrine systems.
人结肠癌细胞系LIM1215在表皮生长因子（EGF）作用下，以细胞密度依赖性方式增殖并发生形态改变（铺展）。在高密度时，自分泌转化生长因子-α的产生使细胞能够在无外源性EGF或血清的情况下增殖和铺展。在低细胞密度（<1×10⁴/cm²）时，单独的EGF无法引发有丝分裂或形态学反应，需要有条件培养基（源自相同细胞的高密度无血清培养）存在才能发挥其作用。进一步研究了EGF与LIM1215条件培养基之间的这种协同作用。使用低细胞密度测定法和分级分离的LIM1215条件培养基，我们表明EGF介导的有丝分裂和形态学反应是可分离的。这些反应分别依赖于低分子量自分泌存活因子和分泌到培养基中的细胞外基质样铺展因子的协同作用。我们发现，在低细胞密度、无血清条件下，单独的EGF不足以使LIM1215免于快速凋亡死亡。过氧化氢酶或LIM1215自分泌存活因子可防止LIM1215细胞死亡，并恢复它们对EGF的增殖（但不是形态学）反应，这表明在这些条件下细胞死亡可能是氧化应激的结果。EGF、部分纯化的自分泌存活因子和铺展因子的组合诱导低密度LIM121细胞增殖和铺展，类似于用EGF和未分级的条件培养基观察到的情况。GRGDS肽在EGF和部分纯化的自分泌铺展因子存在下强烈抑制LIM1215细胞的铺展，表明整合素受体参与铺展过程。比较LIM1215和Colo 526细胞在自分泌铺展因子（ASF）和各种粘附蛋白上的铺展反应表明，ASF不是I型胶原、IV型胶原、纤连蛋白或玻连蛋白。综上所述，这些结果支持这样的概念，即结肠癌细胞在体外的自主生长依赖于几种自分泌系统之间的协同相互作用。
