| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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.
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