14 3 3 Implications in Topoisomerase II Pharmacology

14 3 3 拓扑异构酶 II 药理学的意义

• 批准号: 6333237
• 负责人: David J Kroll
• 金额: $ 12.57万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6333237
• 关键词: DNA damage; DNA gyrase; cell cycle; cell line; drug resistance; enzyme activity; etoposide; gene mutation; isozymes; mitochondria; multiple myeloma; neoplasm /cancer pharmacology; phosphoproteins; phosphorylation; plasmids; posttranslational modifications; protein protein interaction

DESCRIPTION: DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that catalyzes the interconversion of the various tertiary structures of DNA. This enzyme is absolutely essential to cellular proliferation since it decatenates physically interlocked DNA prior to mitosis. Topo II is also a clinically relevant target for a class of chemotherapeutic drugs used widely to treat cancers of the lung, breast, and prostate. The study of topo II biochemistry has revealed several mechanisms by which tumor cells respond to, or evade, the cytotoxic effects of topo Il-directed antitumor drugs. We have developed the hypothesis that topo II activity can be mediated by protein-protein interactions and modulation of these proteins may influence tumor cell response to topo II poisons. Since we originally identified the transcription factors CREB, ATF-2, and c-Jun as topo lI-interactive proteins (TIPs) that stimulate topo LI catalytic activity, others have described TIPs from yeast and Drosophila that are necessary for faithful chromosomal segregation. A human homolog of the yeast TIP, Sgsl, is likely to be the gene that when mutated is responsible for the high tumor frequency in Bloom's syndrome patients and, most recently, the retinoblastoma tumor suppressor gene product Rb has also been shown recently to bind topo II and inhibit its catalytic activity. Therefore, the study of topo II protein-protein interactions has revealed previously unappreciated roles for the enzyme in human neoplasia. This continuation proposal expands on our investigation of topo II protein-protein interactions in the cytotoxic action of topo Il-directed drugs. We have previously identified the epsilon (e) isoform of human 14-3-3 protein as a TIP from screening a human HeLa cell cDNA library with a protein probe comprising a large, C-terminal fragment of the major a isoform of human topo II. 14-3-3 proteins, an unusually highly conserved protein family of distinct gene products found across plants, fungi, and mammals, have been implicated in proto-oncogenic cellular signaling pathways, the G2 DNA damage checkpoint, and in apoptosis regulation. However, mammalian cells have maintained 7 distinct 14-3-3 gene products, perhaps indicating that each isoform possesses unique functions. A very recently described function of some 14-3-3 isoforms is in directing the subcellular compartmentalization of other proteins: either in nuclear export (for cdc25 phosphatase) or, conversely, in nuclear import (for telomerase and the homeobox transcription factor, TLX-2). Using reciprocal affinity chromatography and co-immunoprecipitation methods, we have shown that 14-3-3e, but NOT the G2 arrest protein 14-3-3E, directly binds human topo lla. Functionally, these interactions with 14-3-3e, but NOT 14-3-3a, lead to in vitro inhibition of topo II DNA binding activity and a modest attenuation of etoposide-stabilized DNA damage in purified enzyme and isolated nuclear comet assays. We propose to test a two-part hypothesis that 1) distinct structural determinants within topo lla and 14-3-3 proteins lead to these isoform specific effects and the interactions may be influenced by specific cell cycle and DNA damage dependent phosphorylation events and, 2) that site-directed mutations in these structural motifs (on either topo II or 14-3-3e) or ectopic expression of specific 14-3-3 isoforms or their dominant negative counterparts can influence subcellular compartmentalization of topo lIa in vivo and may play a role in the recently recognized cytoplasmic accumulation of the enzyme and topo Il-drug resistance of various tumor cell lines in plateau phase. Acknowledging that some 14-3-3 isoforms may influence topo II drug efficacy independently of physically interacting with topo II, appropriate controls and alternative paradigms will be employed to distinguish between effects of 14-3-3 on cell cycle distribution or apoptosis induction relative to those directly relating to protein-protein interactions with topo IIa. Since current data suggests that 14-3-3e may protect cells from topo lI-directed antitumor drugs, the long term goal of this work is to identify either structural motifs in either topo II or 14-3-3 that may be targeted by small molecules or peptidomimetics, or kinase inhibitors that disrupt their interaction, to enhance the antitumor efficacy of topo II-directed drugs and/or overcome intrinsic or acquired resistance to these agents.

描述：DNA 拓扑异构酶 II (topo II) 是一种普遍存在的核酶， 催化 DNA 各种三级结构的相互转化。这 酶对于细胞增殖绝对重要，因为它会连接 有丝分裂前 DNA 物理连锁。 Topo II 也是临床上 一类广泛用于治疗的化疗药物的相关靶点 肺癌、乳腺癌和前列腺癌。拓扑异构酶II的生物化学研究 揭示了肿瘤细胞响应或逃避的几种机制 Topo II 定向抗肿瘤药物的细胞毒作用。我们开发了 假设拓扑 II 活性可以由蛋白质-蛋白质介导 这些蛋白质的相互作用和调节可能会影响肿瘤细胞的反应 topo II 毒物。由于我们最初鉴定了转录因子 CREB、ATF-2 和 c-Jun 作为拓扑 I 相互作用蛋白 (TIP)，可刺激 topo LI 催化活性，其他人描述了来自酵母和 果蝇是染色体忠实分离所必需的。一个人类 酵母 TIP 的同源基因 Sgsl 很可能是当突变时 造成布卢姆氏综合征患者肿瘤发生率高的原因，并且大多数 最近，视网膜母细胞瘤抑癌基因产物Rb也被 最近显示可以结合拓扑 II 并抑制其催化活性。所以， 先前对拓扑 II 蛋白-蛋白相互作用的研究已揭示 该酶在人类肿瘤中的作用未被认识到。这个延续 该提案扩展了我们对拓扑 II 蛋白质-蛋白质相互作用的研究 拓扑异构酶 II 定向药物的细胞毒作用。我们之前有过 鉴定出人类 14-3-3 蛋白的 epsilon (e) 同工型作为 TIP 使用包含以下蛋白的蛋白质探针筛选人 HeLa 细胞 cDNA 文库 人类拓扑 II 的主要 a 亚型的大 C 端片段。 14-3-3 蛋白质，不同基因的异常高度保守的蛋白质家族 在植物、真菌和哺乳动物中发现的产物与 原癌细胞信号通路、G2 DNA 损伤检查点和 在细胞凋亡调控中。然而，哺乳动物细胞保留了 7 种不同的 14-3-3基因产物，或许表明每个异构体都具有独特的 功能。最近描述的一些 14-3-3 同工型的功能是 指导其他蛋白质的亚细胞区室化：要么在 核输出（对于 cdc25 磷酸酶），或相反，在核输入（对于 端粒酶和同源框转录因子 TLX-2）。使用倒数 亲和层析和免疫共沉淀方法，我们已经证明 14-3-3e（但不是 G2 阻滞蛋白 14-3-3E）直接结合人拓扑异构酶 IIa。 从功能上讲，这些与 14-3-3e（而不是 14-3-3a）的相互作用会导致 体外抑制拓扑 II DNA 结合活性并适度减弱 纯化酶和分离核彗星中依托泊苷稳定的 DNA 损伤 化验。我们建议测试一个由两部分组成的假设：1）不同的结构 topolla 和 14-3-3 蛋白内的决定簇导致这些亚型特异性 效应和相互作用可能受到特定细胞周期和 DNA 的影响 损伤依赖性磷酸化事件，2）定点突变 这些结构基序（在拓扑 II 或 14-3-3e 上）或异位表达 特定的 14-3-3 异构体或其显性负对应物可以影响 Topol Ila 在体内的亚细胞区室化可能在 最近认识到酶和拓扑II-药物在细胞质中的积累 多种肿瘤细胞系处于平台期的耐药性。承认这一点 一些 14-3-3 异构体可能独立影响拓扑 II 药物疗效 与拓扑 II 进行物理交互、适当的控制和替代方案 将采用范式来区分 14-3-3 对细胞的影响 相对于那些直接相关的周期分布或细胞凋亡诱导 与拓扑 IIa 的蛋白质-蛋白质相互作用。由于目前的数据表明 14-3-3e可以保护细胞免受拓扑异构酶II定向抗肿瘤药物的侵害，长期来看 这项工作的目标是识别拓扑 II 或拓扑结构基序 14-3-3 可能是小分子、肽模拟物或激酶的靶标 抑制剂破坏它们的相互作用，以增强抗肿瘤功效 拓扑II导向药物和/或克服内在或获得性耐药 这些代理。