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

我们在体外分离了具有独特形态的大鼠膀胱肿瘤NBT-II细胞系的自发变体。在获得的5个亚系中，有3个(NBT-L1、L2a和L2b)在体外表现出细长的形状和中等到高的侵袭活性。另外两个亚系(NBT-T1和T2)形成紧密的菌落，侵袭活性很低或可以忽略不计。编码细胞黏附分子(E-钙粘蛋白、α-连环蛋白和β-连环蛋白)的mRNAs含量与细胞的侵袭活性无关。然而，在侵袭性细胞(NBT-L1、L2a和L2b)中，E-钙粘蛋白的表达水平低于非侵袭性细胞(NBT-T1和T2)，而在连接素的表达水平则不是。此外，侵袭性细胞中上皮样前体蛋白和mRNA的表达上调。为了探讨上皮蛋白前体在膀胱癌细胞侵袭转移中的作用，我们在大肠杆菌中制备了重组上皮蛋白前体和截短型上皮蛋白前体蛋白-1和-2，并检测了重组蛋白对NBT-II亚系生长和运动能力的影响。Epithelin前体在培养的侵袭性而非侵袭性亚系上表现出明显的生长和运动刺激活性。Epithelin-1和Epithelin-2对所有亚系均无影响。然而，由于包涵体的形成，上皮蛋白前体制剂的比活性为10w，很难获得足够数量的纯重组蛋白。我们已经测试了几种细菌表达系统来生产上皮蛋白前体，但没有得到满意的结果。因此，可能需要利用真核细胞表达系统来生产高比活性的上皮蛋白前体。

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.