Polyamines are necessary for cell migration by a small intestinal crypt cell line

Abstract

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

Depolarization and decreased surface expression of K+ channels contribute to NSAID-inhibition of intestinal restitution

“…The determination of intracellular polyamine content yielded expected results for IEC-6 treated with DFMO with and without spermadine [29,31]. As shown in Figure 3A, exposure to DFMO significantly decreased the intracellular polyamine content of IEC-6 cells.…”

“…Previous studies have shown that depletion of intracellular polyamines by DFMO inhibits epithelial migration [22,25,29,31,37] and that NSAIDs accelerate polyamine catabolism in colonic epithelial cancer cells [26]. Based on these observations, we hypothesized that NSAIDs inhibit intestinal cell migration by depleting intracellular polyamines.…”

“…These cells, developed by Quaroni et al [27], are non-tumorigenic and retain the character of undifferentiated intestinal crypt cells. Cell culture conditions were similar to those described previously by others [28,29]. The basic medium consisted of DMEM supplemented with heat-inactivated fetal bovine serum (FBS, 5%), insulin (10 μg/ml) and gentamicin (50 μg/ ml).…”

MEK/ERK regulates adherens junctions and migration through Rac1

“…Since migration is involved in mucosal restitution, and polyamine depletion inhibits it, it is important to understand the mechanism by which polyamines affect this process. Previous reports from our laboratory have shown that polyamine-depletion inhibited migration by preventing the activation of Rho family GTPases and subsequent cytoskeletal remodeling [McCormack et al, 1993;Ray et al, 2002]. We have also shown that sustained activation of MEK1 restored migration of polyamine-depleted cells by activating Rac1 to cause the reorganization of cortical actin into stress fibers [Vaidya et al, 2005].…”

“…During the later stage, migration continues, and cell proliferation becomes a major factor [Silen and Ito, 1985]. Polyamine deficient cells fail to migrate normally during the early phase of restitution [McCormack et al, 1993;Rao et al, 1999] and throughout the process of repair. Normal intestinal epithelial cells are tightly bound into sheets, but during epithelial restitution, cell junctions must rapidly disassemble and reassemble.…”