Role of mutant E-cadherin and epithelin in invasion and metastasis of bladder tumor cells

我们已经分离出，在体外，自发变异的大鼠膀胱肿瘤NBT-II-细胞系具有独特的形态。在获得的五个亚系中，三个（NBT-L1、L2 a和L2 b）在体外表现出细长的形状和中度至高度的侵袭活性。另外两个亚系（NBT-T1和T2）形成紧密的集落，并表现出非常低或可忽略的侵袭活性。编码细胞粘附分子（E-cadherin、α-catenin和β-catenin）的mRNA含量与细胞的侵袭性无关。然而，E-cadherin蛋白的表达水平，而不是连环蛋白的表达水平，在侵袭性细胞（NBT-L1，L2 a和L2 b）中低于非侵袭性细胞（NBT-T1和T2）。此外，侵袭性细胞中上皮素前体蛋白和mRNA表达上调。为了研究上皮素前体在膀胱肿瘤细胞侵袭和转移中的作用，我们在大肠杆菌中表达了重组上皮素前体和上皮素-1和-2（上皮素前体的截短形式）。大肠杆菌中表达，并检测重组蛋白对NBT-II亚系生长和运动的影响。上皮素前体表现出明显的生长和运动刺激活动的侵袭性，但不noninvasve亚系在文化。Epithelin-1和Epithelin-2对所有亚系均无影响。然而，上皮素前体制剂的比活性为10 w，并且由于形成包涵体而难以获得足够量的纯重组蛋白。我们已经测试了几种细菌表达系统用于生产上皮素前体，但不能获得令人满意的结果。因此，可能需要利用真核表达系统来生产高比活性的上皮素前体。

We have isolated, in vitro, spontaneous variants of the rat bladder tumor NBT-II-cell line with a distinctive morphology. Of five sublines obtained, three (NBT-L1, L2a, and L2b) exhibited an elongated shape and moderate to high invasive activity in vitro. The other two sublines (NBT-T1 and T2) formed tight colonies and exhibited very low or negligible invasive activity. The contents of mRNAs coding for cell adhesion molecules (E-cadherin, α-catenin and β-catenin) were not correlated with the invasive activity of the cells. However, the expression level of the E-cadherin protein, but not those of catenins, was lower in invasive cells (NBT-L1, L2a and L2b) than in noninvasive cells (NBT-T1 and T2). In addition, the expression of epithelin precursor protein and mRNA was upregulated in invasive cells. To investigate the role of epithelin precursor in invasion and metastasis of bladder tumor cells, we have produced recombinant epithelin precursor and epithelin-1 and -2 (truncated forms of epithelin precursor) in E. coli and examined the effects of the recombinant proteins on growth and motility of NBT-II sublines. Epithelin precursor showed apparent growth- and motility-stimulating activities on Invasive but not noninvasve sublines in culture. Epithelin-1 and -2 had no effect on all the sublines. The specific activities of the epithelin precursor preparation, however, were 10w and it was difficult to obtain enough amount of pure recombinant proteins due to the formation of inclusion bodies. We have tested several bacterial expression systems for the production of epithelin precursor, but could not obtain satisfactory results. Thus it may be needed to use eukariotic expression system to produce epithelin precursor with high specific activities.