EGF-stimulated polyamine accumulation in the colon carcinoma cell line, caco-2

Background: Polyamines (putrescine, spermidine and spermine) are ubiquitous molecules indispensable for cell proliferation. In the intestinal lumen th ey are present in high amounts. Polyamine accumulation in proliferating cel ls of the intestinal mucosa is high, and it occurs both by enhanced synthes is and by increased uptake from the lumen. Aims: To study mitogen-induced p olyamine accumulation in the gut, we treated proliferating Caco-2 cells wit h epidermal growth factor (EGF) and measured the activity of ornithine deca rboxylase (ODC) and putrescine uptake. Furthermore, we investigated whether EGF-induced changes in the apical membrane could be responsible for the ef fect of EGF on polyamine uptake in Caco-2 cells. Methods: Putrescine uptake , ODC activity and intracellular polyamine content were evaluated in the pr esence of 100 ng/ml EGF. To study the mechanisms of EGF-stimulated polyamin e uptake, apical membrane vesicles were isolated, and putrescine uptake int o the vesicles measured. Possible enrichment in brush border membrane cytos keleton proteins (ezrin and villin) was assessed by Western blot. Results: Treatment with EGF induced an increase in ODC activity, which occurred with in the first minutes of treatment and reached peak values after 3 h. In con trast, an increase in putrescine uptake was more sustained, with peak level s at 12 h. Both synthesis and uptake contributed to an over 60% increase in intracellular putrescine and spermidine after EGF treatment. There were no detectable changes in apical membrane cytoskeleton las concluded by the ab sence of ezrin and villin enrichment in EGF-treated Caco-2 cells). However, in apical membrane vesicles isolated from EGF-pretreated cells, putrescine uptake was enhanced twofold. Conclusions: EGF stimulates both synthesis an d uptake of polyamines in Caco-2 cells. Enhanced synthesis seems to ensure rapid supply with polyamines in the earliest stages of growth, while the up take is responsible for the maintenance of high polyamine intracellular lev els during late growth phases. EGF-stimulated polyamine uptake is apparentl y not a consequence of structural changes in the apical membrane, but is li kely to occur by a distinct EGF-induced alteration of the polyamine transpo rter itself. Copyright (C) 2000 S. Karger AG, Basel.