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

Polyamines (putrescine, spermidine and spermine) are ubiquitous molecules indispensable for cell proliferation. In the intestinal lumen they are present in high amounts. Polyamine accumulation in proliferating cells of the intestinal mucosa is high, and it occurs both by enhanced synthesis and by increased uptake from the lumen. To study mitogen-induced polyamine accumulation in the gut, we treated proliferating Caco-2 cells with epidermal growth factor (EGF) and measured the activity of ornithine decarboxylase (ODC) and putrescine uptake. Furthermore, we investigated whether EGF-induced changes in the apical membrane could be responsible for the effect of EGF on polyamine uptake in Caco-2 cells. Putrescine uptake, ODC activity and intracellular polyamine content were evaluated in the presence of 100 ng/ml EGF. To study the mechanisms of EGF-stimulated polyamine uptake, apical membrane vesicles were isolated, and putrescine uptake into the vesicles measured. Possible enrichment in brush border membrane cytoskeleton proteins (ezrin and villin) was assessed by Western blot. Treatment with EGF induced an increase in ODC activity, which occurred within the first minutes of treatment and reached peak values after 3 h. In contrast, an increase in putrescine uptake was more sustained, with peak levels 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 (as concluded by the absence 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. EGF stimulates both synthesis and uptake of polyamines in Caco-2 cells. Enhanced synthesis seems to ensure rapid supply with polyamines in the earliest stages of growth, while the uptake is responsible for the maintenance of high polyamine intracellular levels during late growth phases. EGF-stimulated polyamine uptake is apparently not a consequence of structural changes in the apical membrane, but is likely to occur by a distinct EGF-induced alteration of the polyamine transporter itself.