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 MDC K cells with the inhibitor arrested their locomotion within half an hour, s uppressed their proliferation, and caused them to become round. The round c ells that were still attached to the culture plate were alive. Upon removal of the inhibitor these cells flattened out and resumed locomotion and prol iferation. 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-spe cific toxic mechanism. The inhibitor was both heat stable and resistant to several chemical treatments, including proteases. Its behavior upon ion exc hange chromatography suggested that it was positively charged at neutral pH , whilst its molecular mass was estimated to be 350 or larger by gel-filtra tion FPLC analysis. The inhibitory fraction reacted extensively with fluore scamine, suggesting that the inhibitory factor has amine groups, which are a possible candidate for its positive charges. Since spermine and spermidin e, unlike the inhibitor in the present study, irreversibly inhibited the gr owth of the MDCK cells, the inhibitory activity in the present study is thu s not due to contamination by these polyamines. Our experiments also suppor t that the inhibitor is not a peptide. (C) 2001 wiley-Liss, Inc.