Small molecular mass inhibitor of growth of MDCK cells derived from pig spinal cord

We have discovered cell growth inhibitory activity in a salt extract of pig spinal cords. The growth inhibitory factor was purified by gel-filtration, ion-exchange and high performance liquid chromatography. Incubation of MDCK cells with the inhibitor arrested their locomotion within half an hour, suppressed their proliferation, and caused them to become round. The round cells that were still attached to the culture plate were alive. Upon removal of the inhibitor these cells flattened out and resumed locomotion and proliferation. The inhibitor was 100 times less effective on CHO-K1 cells. The reversible effects of the inhibitor on MDCK cells and its little effects on CHO-K1 cells indicate that the inhibitory activity is not due to a non-specific toxic mechanism. The inhibitor was both heat-stable and resistant to several chemical treatments, including proteases. Its behavior upon ion exchange chromatography suggested that it was positively charged at neutral pH, whilst its molecular mass was estimated to be 350 or larger by gel-filtration FPLC analysis. The inhibitory fraction reacted extensively with fluorescamine, suggesting that the inhibitory factor has amine groups, which are a possible candidate for its positive charges. Since spermine and spermidine, unlike the inhibitor in the present study, irreversibly inhibited the growth of the MDCK cells, the inhibitory activity in the present study is thus not due to contamination by these polyamines. Our experiments also support that the inhibitor is not a peptide.