Nabeshima K, Inoue T, Shimao Y, Kataoka H, Koono M
Department of Pathology, Miyazaki Medical College, Kiyotake, Japan.
Virchows Arch. 1998 Sep;433(3):243-53. doi: 10.1007/s004280050243.
We have already presented a two-dimensional cell motility assay using a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1 as a motility model of tumour cells of epithelial origin. In this model, L-10 cells showed locomotion as a coherent sheet when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement "cohort migration". Electron and immunoelectron microscopic study of the migrating cell sheets demonstrated localized release from cell-cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex, including beta-catenin. Cell-extracellular matrix (ECM) interactions involved in this TPA-induced cohort migration and their effect on tyrosine phosphorylation of the E-cadherin-catenin complex have now been investigated. L-10 cell cohort migration was almost completely inhibited by addition of Arg-Gly-Asp (RGD) peptide into the medium, and thus RGD dependent. Cohort migration was stimulated on type I and IV collagens, fibronectin (FN)- and laminin-coated substratum, but was inhibited by RGD only on FN-coated surface. By using immunofluorescent techniques, FN was demonstrated preferentially around migrating cells, and a protein synthesis inhibitor, cycloheximide, inhibited the migration by about 75%. FN produced by L-10 cells were found to be mostly EDA+ FN when analysed by RT-PCR. Moreover, anti-FN antibody, but not anti-vitronectin antibody, inhibited the TPA-induced cohort migration almost completely. Thus, it was likely that L-10 cells produced FN themselves and moved on the FN substrate in an RGD-dependent manner. However, stimulation of migration by type I collagen coating and inhibition by RGD treatment did not affect the tyrosine phosphorylation of the E-cadherin-catenin complex induced by TPA, indicating that cell-cell interactions were adjusted to suit cell migration, irrespective of the condition of cell-ECM adhesion, during TPA-induced cohort migration.
我们已经提出了一种二维细胞运动分析方法，使用人直肠腺癌细胞系RCM-1的高转移变体（L-10）作为上皮来源肿瘤细胞的运动模型。在该模型中，当用12-O-十四烷酰佛波醇-13-乙酸酯（TPA）刺激时，L-10细胞作为一个连贯的细胞片层进行移动，我们将这种运动类型称为“群体迁移”。对迁移细胞片层的电子显微镜和免疫电子显微镜研究表明，仅在细胞下部细胞间黏附发生局部释放，同时E-钙黏蛋白免疫反应性丧失，并且这种变化与E-钙黏蛋白-连环蛋白复合物（包括β-连环蛋白）的酪氨酸磷酸化增加有关。现已对参与这种TPA诱导的群体迁移的细胞-细胞外基质（ECM）相互作用及其对E-钙黏蛋白-连环蛋白复合物酪氨酸磷酸化的影响进行了研究。向培养基中添加精氨酸-甘氨酸-天冬氨酸（RGD）肽几乎完全抑制了L-10细胞的群体迁移，因此其依赖于RGD。在I型和IV型胶原、纤连蛋白（FN）和层粘连蛋白包被的基质上群体迁移受到刺激，但仅在FN包被的表面上RGD可抑制迁移。通过使用免疫荧光技术，发现FN优先出现在迁移细胞周围，并且蛋白质合成抑制剂放线菌酮可将迁移抑制约75%。通过逆转录-聚合酶链反应（RT-PCR）分析发现，L-10细胞产生的FN大多为EDA+ FN。此外，抗FN抗体而非抗玻连蛋白抗体几乎完全抑制了TPA诱导的群体迁移。因此，L-10细胞很可能自身产生FN并以RGD依赖的方式在FN底物上移动。然而，I型胶原包被对迁移的刺激以及RGD处理对迁移的抑制并不影响TPA诱导的E-钙黏蛋白-连环蛋白复合物的酪氨酸磷酸化，这表明在TPA诱导的群体迁移过程中，无论细胞-ECM黏附情况如何，细胞间相互作用都会进行调整以适应细胞迁移。
