DECREASED EXPRESSION OF PROTOONCOGENES C-FOS, C-MYC, AND C-JUN FOLLOWING POLYAMINE DEPLETION IN IEC-6 CELLS

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence or absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% DFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days. Changes in c-myc and c-jun associated with polyamine depletion did not appear to result from a generalized decrease in gene expression, since junB and glyceraldehyde-3-phosphate dehydrogenase mRNA levels remained constant in DFMO-treated cells. DFMO also totally prevented the stimulated expression of c-fos when 5% dFBS was given after 24 h serum deprivation. These results indicate that 1) polyamine depletion induced by DFMO is associated with decreases in cell proliferation and in the expression of c-fos, c-myc and c-jun protooncogenes and 2) exogenous spermidine reverses inhibitory effects of DFMO. These findings suggest that polyamines may be required for GI mucosal growth in association with their ability to regulate protooncogene expression.