Development of inhibitors for polyamine metabolism including cell death

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Aiming at elucidation of the mechanism of cell death accompanied by the polyamine accumulation in cells, the following research has been done to develop strong inhibitor for spermidine/spermine-N^1-acetyltransferase (SSAT): 1) Development of novel assay method for SSAT and screening assay method for compounds inhibiting SSAT activity, 2) Synthesis of polyamine analogues and examination of their effect on cell growth or apoptosis in cultured cell lines, 3) Changes in cell volume and polyamine distribution in cells accumulated with polyamine.It was found that N1-dansylnorspermine was very useful as a fluorescent substrate for non-radio isotopic SSAT assay method using recombinant human SSAT. The kinetic studies were performed for the recombinant SSAT protein and cellular SSAT activity in HTC cells. It was shown that the method was useful for measuring the activity in biological sample having low level of the enzyme. From the screening for the inhibitor, we found that bis(alkyl)norspermidine was a very potent one, whose IC50 to SSAT activity was much lower than bis(ethyl)spermine, and had inhibition activity for polyamine oxidase.It was also found that the compounds synthesized in this study were effective for cell growth inhibition or induction of apoptosis.The changes in cell volume and distribution of polyamine were examined by Coulter counter and ultrafiltration method, respectively, for the cells accumulated with spermidine. It was suggested that the cell volume was decreased and the distribution of polyamine was changed by the accumulation of cellular polyamine.