Investigations of Mammalian Aminopropyltransferases

描述(改编自申请人的摘要)：多胺含量增加 在肿瘤促进和肿瘤生长过程中。有很好的证据表明 多胺生物合成途径的酶是有效的靶标 化学预防/化疗药物和多胺类似物的设计 N1，N11-双(乙基)去甲精胺(BE-3-3-3)目前正在接受临床治疗 作为抗肿瘤药物的试验。拟议的实验针对的是几个 多胺生理学和类似物作用的领域。其中包括 最大的多胺-精胺的生理作用；以及 精胺/精胺-N1-乙酰转移酶(SSAT)的调节 是将高级多胺转化为 腐胺。具体有三个目的：(一)考察(S)的作用 精胺。所有哺乳动物细胞都有精胺合成酶，并产生 精胺，但没有明确的迹象表明这种物质有任何独特的功能 多胺。这将使用缺乏精胺合成酶的细胞进行研究。 初步实验表明，这些细胞远远多于 对BCNU和其他破坏DNA的毒剂的杀戮敏感。的作用 因此，精胺在保护细胞免受DNA损伤方面将是主要的 这些研究的初步重点。(B)研究以下事项的规管及重要性 SSAT。(B-1)SSAT被多胺和某些多胺高度诱导 类似物，如BE-3-3-3。我们已经证明了这些诱导剂稳定了 抗细胞内降解的蛋白质，这种降解通过 泛素/蛋白酶体系统。将进行实验，以阐明 SSAT降解途径及多胺抑制机制 退化。(B-2)SSAT的结构/功能和底物专一性 将会被研究。这些研究将包括对突变体L156F的检查 存在于CHO55.7Res细胞中，如下所述。(b-3)有可能 SSAT形成的过量腐胺促进肿瘤生长将得到检验 使用我们培育的在皮肤中表达SSAT的转基因小鼠 K6启动子。(C)研究其抗增殖作用的机制 多胺的类似物。使用一种新的程序来避免运输突变，我们 已分离到对BE-3-3-3(CHO55.7Res)耐药的CHO细胞株。这些细胞可以 不表示SSAT是对模拟的反应。进一步的实验将测试 这是否导致了他们的抵抗，调查其他潜在因素 对照和CH055.7Res细胞之间的差异，并使用类似的但 适当改良的分离耐药人肿瘤细胞的方法 BE-3-3-3.对其他多胺类似物的交叉抗药性将使用 BE-3-3-3耐药细胞。我们已经发现缺乏交叉抗性 CHO55.7来自另一种类似物Chen-SPM，表明这种药物要么起作用 在不同的地点。与用于获得BE-3-3-3的协议类似 抗性细胞将被用来分离对CHE-SPM具有抗性的细胞，这将 然后用来研究其作用机理。

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.