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 polyamine s stimulates proliferation. This study tests the hypothesis that the p rotooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechan ism by which polyamines modulate mucosal growth. Studies were conducte d in the IEC-6 cell line, derived from rat small intestinal crypt cell s. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence or absence of al pha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthes is. 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, a nd 12 days after initial plating. The intracellular polyamines, spermi dine and spermine, and their precursor, putrescine, in DFMO-treated ce lls decreased significantly at 2 days and remained depleted thereafter . Although DFMO profoundly decreased growth and final cell number, bot h 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. Treatmen t 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 resul t from a generalized decrease in gene expression, since junB and glyce raldehyde-3-phosphate dehydrogenase mRNA levels remained constant in D FMO-treated cells. DFMO also totally prevented the stimulated expressi on of c-fos when 5% dFBS was given after 24 h serum deprivation. These results indicate that 1) polyamine depletion induced by DFMO is assoc iated with decreases in cell proliferation and in the expression of c- fos, c-myc and c-jun protooncogenes and 2) exogenous spermidine revers es inhibitory effects of DFMO. These findings suggest that polyamines may be required for GI mucosal growth in association with their abilit y to regulate protooncogene expression.