Investigations of Mammalian Aminopropyltransferases

描述（改编自申请人摘要）：多胺含量增加 在肿瘤促进和肿瘤生长期间。有充分证据表明 多胺生物合成途径的酶是有效的靶标， 化学预防/化学治疗剂和多胺类似物的设计， N1，N11-双（乙基）去甲精胺（BE-3-3-3）目前正在临床上进行 作为抗肿瘤药物的试验。拟议的实验针对几个 多胺生理学和类似物作用领域。其中包括 生理作用的最大的多胺，精胺;和重要性， 调节酶亚精胺/精胺-N1-乙酰转移酶（SSAT）， 是将高级多胺转化为 腐胺有3个具体目标：（A）研究的功能（S） 精胺所有的哺乳动物细胞都有精胺合成酶， 但没有明确的迹象表明，任何独特的功能， 聚胺这将使用缺乏精胺合酶的细胞进行研究。 初步的实验表明，这些细胞是相当多的 对BCNU和其他破坏DNA的药物的杀伤敏感。的作用 因此，精胺在保护细胞免受DNA损伤方面将是主要的 这些研究的最初重点。(B)研究的规则和重要性， SSAT。(B-1)SSAT是非常高度诱导的多胺和一些多胺 类似物如BE-3-3-3。我们已经证明，这些诱导剂稳定了 蛋白质抵抗细胞内降解， 泛素/蛋白酶体系统。将进行实验以阐明 SSAT降解途径和多胺阻止SSAT降解的机制 降解(B-2)SSAT的结构/功能和底物特异性 将被研究。这些研究将包括检查突变体L156 F， 存在于下述CHO55.7Res细胞中。(B-3)的可能性 SSAT过量腐胺的形成会促进肿瘤生长 使用我们已经产生的在皮肤中表达SSAT的转基因小鼠， K6启动子(C)目的：探讨中药复方甘草酸苷抗肿瘤细胞增殖作用的机制 聚胺类似物。使用一种新的程序来避免运输突变体，我们 已分离出对BE-3-3-3具有抗性的CHO细胞系（CHO55.7Res）。这些细胞 不表达SSAT以响应模拟。进一步的实验将测试 这是否是它们抗药性的原因，调查其他潜在的 对照和CH 055.7Res细胞之间的差异，并使用类似但 适当修改的程序，以分离对以下耐药的人肿瘤细胞： BE-3 - 3对其它多胺类似物的交叉抗性将使用 BE-3-3-3抗性细胞。我们已经发现缺乏交叉耐药性， CHO55.7Res与另一种类似物CHEN-Spm相比，表明这种药物要么 在另一个地方与用于获得BE-3-3-3的方案相似的方案 抗性细胞将用于分离对CHE-Spm具有抗性的细胞， 然后用于研究其作用机制。

DESCRIPTION(adapted from applicant's abstract): Polyamine content is increased during tumor promotion and in neoplastic growth. There is good evidence that the enzymes of the polyamine biosynthetic pathway are valid targets for the design of chemopreventive/chemotherapeutic agents and polyamine analogs such as N1,N11-bis(ethyl)norspermine (BE-3-3-3) are currently undergoing clinical trials as anti-tumor agents. The proposed experiments are aimed at several areas of polyamine physiology and analog action. These include the physiological role of the largest polyamine, spermine; and the importance and regulation of the enzyme spermidine/spermine-N1-acetyltransferase (SSAT), which is the rate limiting step in the conversion of the higher polyamines into putrescine. There are 3 specific aims: (A) To investigate the function(s) of spermine. All mammalian cells have the enzyme spermine synthase and produce spermine but there are no clear indications of any unique function of this polyamine. This will be investigated using cells lacking spermine synthase. Preliminary experiments indicate that these cells are considerably more sensitive to killing by BCNU and other agents that damage DNA. The role of spermine in protection of cells from DNA damage will therefore be the major initial focus for these studies. (B) To study the regulation and importance of SSAT. (B-1) SSAT is very highly inducible by polyamines and by some polyamine analogs such as BE-3-3-3. We have shown that these inducers stabilize the protein against intracellular degradation which occurs via the ubiquitin/proteasome system. Experiments will be carried out to elucidate the pathway of SSAT degradation and the mechanism by which polyamines prevent degradation. (B-2) The structure/function and substrate specificity of SSAT will be studied. These studies will include examination of mutant L156F which is present in CHO55.7Res cells described below. (B-3) The possibility that excess putrescine formation by SSAT enhances neoplastic growth will be tested using transgenic mice that we have generated that express SSAT in the skin from the K6 promoter. (C) To study the mechanism of the antiproliferative action of the polyamine analogs. Using a novel procedure to avoid transport mutants, we have isolated CHO cell lines resistant to BE-3-3-3 (CHO55.7Res). These cells do not express SSAT in response to the analog. Further experiments will test whether this is responsible for their resistance, investigate other potential differences between the control and CH055.7Res cells and use a similar but appropriately modified procedure to isolate human tumor cells resistant to BE-3-3-3. Cross-resistance to other polyamine analogs will be tested using the BE-3-3-3 resistant cells. We have already found a lack of cross-resistance of CHO55.7Res to another analog, CHEN-Spm, suggesting that this drug either acts at a different site. A similar protocol to that used to obtain the BE-3-3-3 resistant cells will be used to isolate cells resistant to CHE-Spm which will then be used to study its mechanism of action.