Polyamine Suppression By Antizyme inducing Analogs

描述（由申请人提供）： 这项工作的最终目标是发展一种药理学方法 提高已知的肿瘤抑制蛋白抗酶的水平， 减少人类的癌症促进和肿瘤形成。多胺， 亚精胺和精胺以及它们二胺前体腐胺， 脂肪族阳离子对哺乳动物细胞生长和生存力至关重要。癌 细胞需要高水平的多胺和抑制剂， 所需的多胺合成或细胞摄取可阻断促进的多胺合成， 肿瘤形成阶段。正常细胞中的多胺稳态主要是 依赖于调节蛋白抗酶的活性， 在亚精胺或精胺浓度升高时合成。在 反过来，这种小蛋白质介导了初始酶的快速降解， 在多胺生物合成中，鸟氨酸脱羧酶， 多胺吸收系统。通过改造癌基因转染的细胞， 表达抗酶，有可能抑制它们的转化表型 以及它们在动物体内形成肿瘤的能力。建议的具体目标 构建研究以检验多胺类似物可用于 以无偿地刺激抗酶的产生，从而阻断 肿瘤形成的促进阶段。(1)腐胺能模拟 将鉴定抗酶基因的刺激转录， 表征了(2)多胺类似物和寡胺，其特异性促进 完整哺乳动物细胞中抗酶信息的翻译将是 鉴定(3)选择的抗酶诱导类似物的潜力， 将评价哺乳动物细胞中增加和维持抗酶水平。（四） 所选类似物逆转转化细胞表型的能力将 使用H-ras转染的NIH 3 T3细胞进行评估。抗酶诱导研究 将使用大鼠肝脏（HTC）和仓鼠（CHO）细胞，并将涉及检测 抗酶mRNA的Northern印迹分析，抗酶活性的ODC酶 抑制测定和通过免疫印迹免疫检测抗酶蛋白。 这些研究将为进一步探索 抗酶的化学诱导作为动物化学预防的一种手段。

DESCRIPTION (provided by applicant): The eventual goal of this work is the development of a pharmacological method to elevate levels of a known, tumor-suppressing protein, antizyme, and thereby reduce cancer promotion and tumor formation in humans. The polyamines, spermidine and spermine, and their diamine precursor, putrescine, are aliphatic cations essential for mammalian cell growth and viability. Cancer cells require elevated levels of the polyamines, and inhibitors that prevent the requisite polyamine synthesis or cellular uptake can block the promotional stage of tumor formation. Polyamine homeostasis in normal cells is largely dependent upon the activity of a regulatory protein, antizyme, which is synthesized in response to elevations in either spermidine or spermine. In turn, this small protein mediates both rapid degradation of the initial enzyme in polyamine biosynthesis, ornithine decarboxylase, and down regulation of the polyamine uptake system. By engineering oncogene-transfected cells to over express antizyme it is possible to inhibit both their transformed phenotype and their tumor-forming ability in animals. The specific aims of the proposed study are structured to test the hypothesis that polyamine analogs can be used to gratuitously stimulate antizyme production, and thereby block the promotional phase of tumor formation. (1) Putrescine mimics that specifically stimulate transcription of the antizyme gene will be identified and characterized. (2) Polyamine analogs and oligoamines that specifically promote translation of the antizyme message in intact mammalian cells will be identified. (3) The potential of selected antizyme-inducing analogs to increase and sustain antizyme levels in mammalian cells will be evaluated. (4) The ability of selected analogs to reverse a transformed cell phenotype will be assessed using H-ras-transfected NIH 3T3 cells. Antizyme induction studies will use rat liver (HTC) and hamster (CHO) cells, and will involve detection of antizyme mRNA by northern-blot analysis, antizyme activity by ODC enzyme inhibition assays, and antizyme protein by immunodetection on western-blots. These studies will provide an essential foundation for further exploration of the chemical induction of antizyme as a means of chemoprevention in animals.