Tool to Define the Antiproliferative Effects of Agmatine

说明(申请人提供)：胍丁胺通过精氨酸脱羧酶(ADC)从精氨酸中提取，主要由肾脏产生。它是一种新的内源性细胞增殖抑制因子，其作用至少部分归因于多胺的调节。多胺是细胞周期进程所必需的成分。多胺生物合成的限速酶是鸟氨酸脱羧酶(ODC)，这是一种生长所需的原癌基因，在肿瘤中显著升高。细胞内的多胺水平是通过诱导抗酶来自动调节的，抗酶是一种同时抑制ODC和细胞多胺输入的蛋白质。胍丁胺通过诱导抗酶和SSAT(一种参与多胺新陈代谢的酶)来降低细胞内多胺水平。在转化的NIH/3T3细胞中，胍丁胺通过诱导细胞周期蛋白激酶抑制剂以类似衰老的方式阻止G1期细胞周期，从而抑制细胞增殖，实际上使转化的细胞恢复到衰老表型。Agmatine抑制所有被评估的细胞系的增殖，即使是那些缺乏周期蛋白激酶抑制剂的细胞系，这表明了多余的停滞模式。最后，胍丁胺启动了一个协调的抗增殖作用网络，涉及Akt通路(与生存和生长有关)和血管生成因子，这也可能有助于这种抑制。考虑到胍丁胺系统可能提供一种新的治疗途径，我们首先必须更详细地了解它的作用。目的：明确胍丁胺抗细胞增殖作用的机制。在这里，我们将开发对这项工作和未来的工作至关重要的工具。我们将结合siRNA和慢病毒载体技术，建立由胍丁胺(细胞周期蛋白激酶抑制剂、抗酶和SSAT)诱导的候选蛋白的稳定敲除细胞系，并阐明其介导的衰老和生长停滞的机制。各自的siRNA慢病毒载体也为未来动物模型的评估提供了工具。了解胍丁胺引起的这一抗增殖反应网络所涉及的机制，将使我们能够通过分子或药理学方法来定义、靶向和开发关键途径。这些途径将特别应用于糖尿病和肾脏IRI，这是我们计划追求的模式。

DESCRIPTION (provided by applicant): 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.