Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation-and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation. The regulatory protein antizyme (AZ)1 has been studied primarily in the context of its role in facilitating degradation of the enzyme ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine synthesis (reviewed in Refs. 1-3). AZ is therefore thought to be dedicated principally to the feedback regulation of polyamine levels. AZ synthesis is controlled via an unusual mechanism of translational frameshifting. The ribosomal frameshift required for the translation of full-length AZ is directly induced by polyamines when their levels rise (4, 5). Polyamines can thus inhibit their own synthesis via AZ-mediated down-regulation of ODC. This mechanism serves to prevent extreme fluctuations in polyamine levels, which are thought to be toxic (6 -8). Despite this homeostatic mechanism, the levels of polyamines and ODC vary markedly during the cell cycle, indicating that additional factors control polyamine levels and suggesting a role for this pathway in the regulation of cell proliferation (9).Overproduction of AZ in a variety of cell types, including malignant oral keratinocytes, hepatoma cell lines, and prostate cancer cells, coincides with growth inhibition (10 -12) and cell cycle arrest in the G 1 phase (13,14). Furthermore, overexpression of antizyme in both mouse skin cancer and gastric epithelia models has been shown to result in tumor suppression (15,16). These and other observations of antiproliferative effects of antizyme prompted us to test whether antizyme may have a specific role in cell cycle regulation, thereby accounting for its potential role as a tumor suppressor.The cell cycle arrest previously seen in prostate carcinoma ...

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCrosl, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia cofi recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 ag/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE1OH) and nerol-10-hydroxylase (NE1OH). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE1OH is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE1OH failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus. P-4502 are the terminal oxidases of a large number of biotransformations. The enzymic reactions include metabo-1 This work was supported by the Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft (SFB206).2Abbreviations: P-450, cytochrome(s) P-450; CA4H, cinnamicacid-4-hydroxylase; 14DM, lanosterol 14a-demethylase; FL3'H, flavonoid-3'-hydroxylase; FL3'5'H, flavonoid-3',5'-hydroxylase; GE1OH, geraniol-10-hydroxylase; LAH, lauric-acid-hydroxylase (in chain); NE1OH, nerol-10-hydroxylase; EROD, 7-ethoxyresorufin-0-deethylase; MX medium, growth medium; IM2 medium, alkaloid induction medium.lism of steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, pheromones, drugs, plant metabolites, and numerous other substances, including mutagens (19). The importance of these enzymes led in the last 10 years to a dramatic increase of molecular research in animal systems (20).The importance of P-450 enzymes in plants has been recognized (5), but the molecular analysis is lagging. The approach from purified protein to molecular biology has been difficult. In plants, the concentrations of these proteins are usually lower than in animals, the activities are unstable, the reconstitution of solubilized components to enzymically active complexes is difficult, and antisera against purified plant proteins or against P-450 enzymes from other sources most often recognize several...

SummaryThe family of antizymes functions as regulators of polyamine homeostasis. They are a class of small, inhibitory proteins, whose expression is regulated by a unique ribosomal frameshift mechanism. They have been shown to inhibit cell proliferation and possess anti-tumor activity. Antizymes bind ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. They inhibit its enzymatic activity and promote the ubiquitin-independent degradation of ODC by the 26S proteasome. In addition, they also negatively regulate polyamine transport. Antizyme-mediated, ubiquitin-independent degradation of ODC is conserved from yeast to man. But recent data suggest that this degradation pathway might not be restricted to ODC alone and could involve newly discovered antizyme binding partners. Interestingly, antizyme proteins have been strictly preserved over a vast evolutionary timeframe. Antizymes consequently represent an important class of proteins that regulate cell growth and metabolism by a diverse set of mechanisms that include protein degradation, inhibition of enzyme activity, small molecule transport and other, potentially not yet discovered properties. IUBMB Life, 57: 671 -676, 2005

The antizyme inhibitor was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Blocking antizyme activity subsequently leads to increased intracellular levels of ODC and increased ODC enzymatic activity. We now report that antizyme inhibitor is a positive modulator of cell growth. Overexpression of antizyme inhibitor in NIH-3T3 mouse fibroblasts or in AT2.1 Dunning rat prostate carcinoma cells resulted in an increased rate of cell proliferation and an increase in saturation density of the cultured cells. This was accompanied by an increase in intracellular levels of the polyamine putrescine. In AT2.1 cells, antizyme inhibitor overexpression also increased the ability of the cells to form foci when grown under anchorage-independent conditions. In order to determine the role of antizyme on antizyme inhibitor activity we created an antizyme inhibitor mutant, AZIΔ117-140, which lacks the putative antizyme-binding domain. We show that this mutant fails to bind to antizyme, but remains capable of inducing increased rates of cell proliferation, suggesting that antizyme inhibitor has antizyme-independent functions. Silencing antizyme inhibitor expression leads to diminished levels of cyclin D1 and to reduced cell proliferation. Antizyme inhibitor is capable of preventing cyclin D1 degradation, and this effect is at least partially independent of antizyme. We show that wild-type antizyme inhibitor and the AZIΔY mutant are capable of direct interaction with cyclin D1 suggesting a potential mechanism for the antizyme-independent effects. Together, our data suggest a novel function for antizyme inhibitor in cellular growth control.

In contrast to the considerable interest in the oncogene ornithine decarboxylase (ODC) and in the family of antizymes with regard to cell proliferation and tumorigenesis, the endogenous antizyme inhibitor (AZI) has been less well studied. AZI is highly homologous to the enzyme ODC but does not possess any decarboxylase activity. Elevated ODC activity is associated with most forms of human malignancies. Antizymes bind ODC, inhibit ODC activity and promote the ubiquitin-independent degradation of ODC. Consequently they are proposed as tumor suppressors. In particular, the most studied member of the antizyme family, antizyme 1, has been demonstrated to play a role in tumor suppression. AZI inactivates all members of the antizyme family, reactivates ODC and prevents the proteolytic degradation of ODC, which may suggest a role for AZI in tumor progression.