Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation

The p53 nuclear phosphoprotein plays a critical role in transcriptional reg ulation of target genes involved in growth arrest and apoptosis. The natura l polyamines, including spermidine, spermine, and their precursor putrescin e, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we in vestigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the proce ss of growth inhibition after polyamine depletion. Studies were conducted b oth in vivo and in vitro using rats and the IEC-6 cell line, derived from r at small intestinal crypt cells. Levels for p53 mRNA and protein, transcrip tion and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with a-difluoromethylornithin e (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA ha lf-life but had no effect on p53 gene transcription in IEC-6 cells. Inducti on of p53 mRNA levels in DFMO-treated cells was paralleled by an increase i n the rate of newly synthesized p53 protein. The stability of p53 protein w as also increased after polyamine depletion, which was associated with a de crease in Mdm2 expression. When polyamine-deficient cells were exposed to e xogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregula te p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least par tially, through the activation of p53 gene.