| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Translocation of macromolecules thorough the nuclear pore is involved in growth, differentiation and functions of eukaryotic cells. Growing evidence indicates that a nuclear exporter Crml plays a central role in the nuclear export system, since Leptomycin B was found to inhibit function of nuclear export signal (NES) in the cytoplasmic protein. The present study was designed to examine a hypothesis that endogenous substances inhibiting Crml function exist, because it is known that LMB binding domain of Crml protein is highly conserved in different species. Based on the fact that the a, β-unsaturated lactone ling of LMB is essential for binding with Crml, we focused on a toad steroid bufalin which possesses similar chemical structure with LMB and has been suggested to exist in human plasma. Bufalin halted THP-1 leukemia cell growth at G2/M boundary as similar to LMB. To examine the effect of bufain on NES, we established HepG2 cells stably express NES-tagged green fluorescent protein (GFP-NES). GFP-NES distributed exclusively in cytoplasm of untreated cells and LMB induced nuclear accumulation of the tagged protein in all cells observed, indicating that this system can properly be applied for searching compounds inhibiting NES. However, bufalin did not induce nuclear accumulation of the fluorescence at any time points or concentrations examined, indicating that this steroid compound does not directly inhibit Crml function. We next screened 82 natural compounds and 32 known kinase inhibitors or anticancer drugs using GFP-NES expressed HepG2 cells. Although there is no compound that inhibits NES as potent as LMB, kinase inhibitors staurosporine and K252a, progesterone, and a ginger component 8-shogaol induced nuclear accumulation of GFP-NES in parallel with cell death. It is, therefore, suggested that these compounds might inhibit the Crml-dependent nuclear export.
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