POLYAMINE DEPLETION INDUCES NPM MODULATING STABILITY AND TRANSCRIPTIONAL ACTIVITY OF p53 IN INTESTINAL EPITHELIAL CELLS

Our previous studies have shown that polyamines are required for normal intestinal mucosal growth and decreased levels of polyamines inhibit intestinal epithelial cell proliferation by stabilizing p53 and other growth-inhibiting proteins. Nucleophosmin (NPM) is a multifunctional protein and has been recently shown to regulate p53 activity. The current study sought to determine whether polyamine depletion increases NPM modulating the stability and transcriptional activity of p53 in normal intestinal epithelial cells (IEC-6 line). Depletion of cellular polyamines by α-difluoromethylornithine (DFMO), the specific inhibitor of polyamine biosynthesis, stimulated expression of the NPM gene and induced nuclear translocation of NPM protein. Polyamine depletion stimulated NPM expression primarily by increasing NPM gene transcription and its mRNA stability, and induced NPM nuclear translocation through activation of phosphorylation of mitogen-activated protein kinase kinase (MEK). Increased NPM interacted with p53 and formed a NPM/p53 complex in polyamine-deficient cells. Inhibition of NPM expression by small interfering RNA targeting NPM (siNPM) not only destabilized p53 as indicated by a decrease in its protein half-life but also prevented the increased p53-dependent transactivation as shown by a suppression of the p21-promoter activity. Decreased expression of NPM by siNPM also promoted cell growth in polyamine-deficient cells. These results indicate that 1) polyamine depletion increases expression of the NPM gene and enhances NPM nuclear translocation and 2) increased NPM interacts with and stabilizes p53 leading to inhibition of IEC-6 cell proliferation.