EFFECTORS OF POLYAMINE POOL MAINTENANCE

我们最近提出，细胞内多胺池是 通过体内平衡复合体小心地维持在一个狭窄的范围内 由三个效应器系统组成，所有这些系统都是快速响应的， 对其特定活性的速率限制，特定于多胺和 受细胞内多胺池的敏感调节。效应器 包括鸟氨酸脱羧酶和S- 控制多胺的腺苷蛋氨酸脱羧酶(SAMDC) 生物合成；一种控制摄取的假定转运蛋白；以及 乙酰化酶精胺/精胺N1乙酰转移酶(SSAT) 它具有控制多胺回归的多功能潜力- 转化、结合、输出和分解代谢。这个概念已经被证明 有助于解释多胺类似物的细胞效应和 抑制剂及其在抗癌应用中的有效性预测 战略。在此，我们建议利用最近开发的探测器和 研究未解决的和治疗相关的生物系统 每个效应器的各个方面，试图更好地了解其功能 和/或监管，并更好地界定其与多胺的关系 动态平衡。该计划的具体目标是：(1)亲和力 细胞质膜蛋白(S)的标记和鉴定 多胺结合和转运；(2)克隆人多胺结合和转运基因(S) 转运蛋白(S)用于分子机制研究 多胺对多胺运输和生长的潜在调控 刺激；(3)开发一组过度生产的细胞系变异体 用于进一步定义调节的多胺生物合成酶 酶和其他动态平衡效应器之间的相互关系； (4)研究SSAT和多胺SSAT诱导的意义 新陈代谢、多胺稳态与细胞生理 用SSAT mRNA导向的反义寡核苷酸阻断表达 (D)开发2-氟鸟氨酸作为高效液相标记物 监测通过多胺池的代谢通量并使用这一点 研究多胺动态平衡的方法学。预计 这些研究将提供与多胺池相关的新信息 动态平衡；它作为一个概念的有效性；它的影响至少有两个 多胺导向的药物(起源于本实验室)，现在 接近临床试验和适应症，可能会更多 在未来的药物设计中有效地利用和/或瞄准。

We have recently proposed that intracellular polyamine pools are carefully maintained within a narrow range via a homeostatic complex comprised of three effector systems all of which are rapidly responsive, rate limiting for their specified activity, specific for polyamines and sensitively regulated by intracellular polyamine pools. The effectors include the enzymes ornithine decarboxylase (ODC) and S- adenosylmethionine decarboxylase (SAMDC) which control polyamine biosynthesis; a putative transporter protein which controls uptake; and the acetylating enzyme spermidine/spermine N1 acetyltransferase (SSAT) which has the multifunctional potential to control polyamine back- conversion, binding, export and catabolism. The concept has proven useful in interpreting cellular effects of polyamine analogs and inhibitors and in predicting the effectiveness of their use in anticancer strategies. Herein, we proposed to utilize recently developed probes and biological systems to study unresolved and therapeutically relevant aspects of each effector in an attempt to better understand its function and/or regulation and to better define its relationship to polyamine homeostasis. The Specific Aims of the program are (1) photoaffinity labelling, and identification of plasma membrane protein(s) involved in polyamine binding and transport; (2) cloning the cDNA(s) for the transporter protein(s) for use in studying the molecular mechanisms underlying regulation of the polyamine transport by polyamines and growth stimuli; (3) to develop a panel of cell line variants which overproduce polyamine biosynthetic enzymes for use in further defining the regulatory interrelationships between the enzymes and other homeostatic effectors; (4) to study the significance of SSAT and SSAT induction in polyamine metabolism polyamine homeostasis and cellular physiology by transfecting and by blocking expression with SSAT mRNA-directed antisense oligonucleotide; (d) to develop 2-fluoroornithine as a HPLC marker for monitoring metabolic flux through the polyamine pools and to use this methodology to studying polyamine homeostasis. It is anticipated that these studies will provide novel information relevant to polyamine pool homeostasis; its validity as a concept; its implications to at least two polyamine-directed agents (with origins in this laboratory) which are now approaching clinical trial and indications by which it might be more effectively exploited and/or targeted in future drug design.