Agmatine is derived from arginine via arginine decarboxylase (ADC), and is produced principally and constitutively by the kidney. It is a novel endogenous inhibitor of cell proliferation whose effects are attributed, at least in part, to regulation of polyamines. Polyamines are required components of cell cycle progression. The rate-limiting enzyme of polyamine biosynthesis is ornithine decarboxylase (ODC), a proto-oncogene required for growth and significantly elevated in tumors. Intracellular polyamine levels are autoregulated by induction of antizyme, a protein that inhibits both ODC and cellular polyamine import. Agmatine lowers intracellular polyamine levels by inducing antizyme and SSAT, an enzyme involved in the metabolism of polyamines. In transformed NIH/3T3 cells agmatine inhibits proliferation via a G1 cell cycle arrest with induction of cyclin kinase inhibitors in a senescent-like manner, in effect, reverting a transformed to a senescent phenotype. Agmatine inhibits proliferation in all cell lines evaluated, even those deficient in cyclin kinase inhibitors, suggesting redundant modes of arrest. Finally, agmatine initiates a coordinated network of antiproliferative effects involving Akt pathways (linked with survival and growth), and angiogenic factors, which could also contribute to this arrest. Considering the agmatine system may provide a new therapeutic avenue we first have to understand its actions in more detail. OBJECTIVES: To define the mechanisms of agmatine's antiproliferative effects. Here we will develop tools vital for this and future work. We will combine siRNA and lentiviral vector technology to establish stable knock-down cell lines of candidate proteins induced by agmatine (cyclin kinase inhibitors, antizyme and SSAT) and delineate the mechanisms of agmatine-mediated senescence and growth arrest. The respective siRNA lentiviral vectors also provide tools for future assessment in animal models. Understanding the mechanisms involved in this network of antiproliferative responses elicited by agmatine would allow us to define, target and exploit critical pathways by molecular or pharmacologic approaches. These pathways will have particular application to diabetes and IRI in kidney, models we plan to pursue. ? ?
