Abstract
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, AZI(Delta117-140), 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 AZI(DeltaY) 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.
Highlights
Levels of the enzyme ornithine decarboxylase (ODC) are tightly regulated during the cell cycle
Antizyme inhibits polyamine uptake and stimulates polyamine secretion (He et al, 1994; Mitchell et al, 1994; Mitchell et al, 1995; Sakata et al, 2000). These results demonstrate a role for antizyme in the negative regulation of cell proliferation and tumorigenesis, probably as a result of modulation of polyamine levels and facilitated degradation of growth regulatory molecules such as ODC and cyclin D1
Effect of antizyme inhibitor (AZI) overexpression on cell proliferation and transformation In order to study the effect of AZI on cell proliferation, AT2.1 rat prostate carcinoma cells were transfected with AZI or vector control and selected for stable expression
Summary
Levels of the enzyme ornithine decarboxylase (ODC) are tightly regulated during the cell cycle. ODC activity levels are activated in a biphasic manner during the cell cycle with a first activation phase in late G1, which is followed by a rapid decline in the half-life of ODC (Oredsson, 2003). This rapid degradation of ODC is mediated by the regulatory protein antizyme (Murakami et al, 1992). First thought to bind only to ODC, antizyme has been shown recently to bind and facilitate the ubiquitin-independent degradation of other small proteins, including Smad (Gruendler et al, 2001), Snip (Lin et al, 2002) and cyclin D1 (Newman et al, 2004)
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