Minamitani Takeharu, Ariga Hiroyoshi, Matsumoto Ken-Ichi
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Biol Pharm Bull. 2002 Nov;25(11):1472-5. doi: 10.1248/bpb.25.1472.
Extracellular matrix tenascin-X (TNX)-null mice, generated by disruption of the Tnx gene, display augmented invasion and metastasis of B16-BL6 melanoma tumor cells due to increased activities of matrix metalloproteinase (MMP)-2 and MMP-9. In this study, we investigated cell-matrix and cell-cell adhesions using TNX-null fibroblasts and wild-type fibroblasts. TNX-null fibroblasts exhibited a decreased attachment to fibronectin compared with that of wild-type fibroblasts. B16 melanoma cells were cocultured with wild-type or TNX-null fibroblasts, and the adhesion of B16 melanoma to the fibroblasts was assessed. B16 melanoma cells on wild-type fibroblasts proliferated and spread out in a horizontal direction, whereas those on TNX-null fibroblasts overlapped each other rather than migrating horizontally. These overlapping B16 melanoma cells on TNX-null fibroblasts peeled off faster than those on wild-type fibroblasts. To determine whether the decreased cell-matrix and cell-cell adhesions on TNX-null fibroblasts were due to increased MMP activity, the activities of MMPs in wild-type and TNX-null fibroblasts were compared by gelatinolytic assays. The analysis of MMPs from conditioned media demonstrated that almost the same levels of MMP activities were detected between wild-type and TNX-null fibroblasts. However, contrary to our expectations the activities of MMPs from conditioned media of B16 melanoma cells cocultured on TNX-null fibroblasts were rather reduced than those of B16 melanoma cells cocultured on wild-type. We concluded that the absence of TNX in the extracellular environment might play an important role in enhancement of the detachment of B16 melanoma cells.
通过破坏Tnx基因产生的细胞外基质肌腱蛋白X（TNX）基因敲除小鼠，由于基质金属蛋白酶（MMP）-2和MMP-9活性增加，B16-BL6黑色素瘤肿瘤细胞的侵袭和转移增强。在本研究中，我们使用TNX基因敲除的成纤维细胞和野生型成纤维细胞研究了细胞-基质和细胞-细胞黏附。与野生型成纤维细胞相比，TNX基因敲除的成纤维细胞对纤连蛋白的附着减少。将B16黑色素瘤细胞与野生型或TNX基因敲除的成纤维细胞共培养，并评估B16黑色素瘤细胞与成纤维细胞的黏附。野生型成纤维细胞上的B16黑色素瘤细胞呈水平方向增殖和扩散，而TNX基因敲除的成纤维细胞上的B16黑色素瘤细胞相互重叠，而不是水平迁移。TNX基因敲除的成纤维细胞上这些重叠的B16黑色素瘤细胞比野生型成纤维细胞上的细胞更快脱落。为了确定TNX基因敲除的成纤维细胞上细胞-基质和细胞-细胞黏附的减少是否是由于MMP活性增加，通过明胶酶分析比较了野生型和TNX基因敲除的成纤维细胞中MMP的活性。对条件培养基中MMP的分析表明，野生型和TNX基因敲除的成纤维细胞之间检测到的MMP活性水平几乎相同。然而，与我们的预期相反，在TNX基因敲除的成纤维细胞上共培养的B16黑色素瘤细胞条件培养基中MMP的活性比在野生型成纤维细胞上共培养的B16黑色素瘤细胞的活性降低。我们得出结论，细胞外环境中TNX的缺失可能在增强B16黑色素瘤细胞的脱离中起重要作用。
