Expression of the transforming growth factor-β gene during growth inhibition following polyamine depletion

Polyamine depletion and cytokine transforming growth factor-β (TGF-β) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-β gene in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in DMEM in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not only depleted intracellular polyamines but also significantly increased the mRNA levels of TGF-β. Increased TGF-β mRNA in DFMO-treated cells was paralleled by an increase in TGF-β content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-β gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-β in normal cells was ~65 min and increased to >16 h in cells treated with DFMO for 6 or 12 days. Exogenous polyamine, when given together with DFMO, prevented the increased half-life of TGF-β mRNA in IEC-6 cells. TGF- β added to the culture medium significantly decreased the rate of DNA synthesis and final cell number in normal and polyamine-deficient cells. Furthermore, growth inhibition caused by polyamine depletion was partially but significantly blocked by addition of immunoneutralizing anti-TGF-β antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-β gene through posttranscriptional regulation and 2) increased expression of the TGF-β gene plays an important role in the process of growth inhibition following polyamine depletion.