Antizyme regulation of ornithine decarboxylase, polyamine metabolism and neoplastic growth

Ornithine decarboxylase (ODC) catalyzes formation of putrescine in the initial reaction of polyamine biosynthesis. Polyamines are essential for cell proliferation, therefore elevated ODC activity and polyamines are frequently detected within neoplastic tissues. Negative regulation of polyamine levels is mediated by antizyme, which inhibits ODC activity, stimulates ODC degradation and suppresses cellular uptake of exogenous polyamines.;Active ODC is a homodimer with a putative phosphorylation site at S303. To determine the importance of dimerization and phosphorylation to antizyme-stimulated ODC degradation, point mutations were introduced in the ODC protein. Substitutions at the conserved -FNGF- amino acids at positions 397--400 caused greater than 90% reduction in ODC activity, and the loss of activity for the F397, G399 and F400 mutants resulted from an inability to form an ODC dimer. Non-dimerizing F397D and F397E mutants were degraded more rapidly than wild type ODC in a reticulocyte lysate system supplemented with antizyme. The physiologic relevance of ODC phosphorylation was also investigated. The activity and dimerization of an S303A mutant was unchanged, but the half-life of the mutant in the presence of antizyme was also reduced. Therefore, ODC is protected from antizyme-stimulated degradation by association of the monomer into stable homodimers and by the phosphorylation of S303.;The effects of antizyme expression on polyamine metabolism in normal or neoplastic cells were also investigated. An inducible cell culture system demonstrated suppressed ODC activity and reduced polyamine levels following antizyme expression. To examine the significance of increased ODC activity and polyamines to chemical carcinogenesis, transgenic mice were generated with antizyme expression directed to the skin. ODC activity and polyamine levels in the skin of control mice increased dramatically following application of the tumor promoter TPA. However, TPA induction of ODC was significantly reduced or even prevented in the skin of mice with targeted antizyme expression. Furthermore, transgenic mice developed fewer tumors than normal littermates in response to a standard DMBA/TPA chemical carcinogenesis protocol. These studies demonstrate that increased ODC activity and polyamine content are essential for neoplastic growth induced by chemical carcinogenesis, and suggest that antizyme may act as a tumor suppressor in other experimental systems.