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: 6556228
• 负责人: David J Kroll
• 金额: $ 14.05万
• 依托单位: RESEARCH TRIANGLE INSTITUTE
• 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 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6556228
• 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的各种三级结构的相互转化。这 酶对细胞增殖是绝对必要的，因为它能分解 在有丝分裂之前物理上锁定的DNA。TOPO II也是一种临床上的 一类广泛应用的化疗药物的相关靶点 肺癌、乳腺癌和前列腺癌。TOPO II生物化学的研究 揭示了肿瘤细胞响应或逃避的几种机制 TOPO-IL导向抗肿瘤药物的细胞毒作用我们已经开发了 拓扑酶II活性可由蛋白质-蛋白质介导的假说 这些蛋白的相互作用和调节可能影响肿瘤细胞的反应 到TOPO II类毒药。因为我们最初确定了转录因子 CREB、ATF-2和c-jun作为拓扑结构相互作用蛋白(TIPS)刺激 Topo Li催化活性，其他人已经描述了来自酵母和 果蝇是忠实的染色体分离所必需的。一个人类 酵母尖端的同源基因SGSL很可能是当突变时 对Bloom综合征患者的高肿瘤发生率负责，大多数 最近，视网膜母细胞瘤抑癌基因产物Rb也被 最近发现与Topo II结合并抑制其催化活性。因此， Topo II蛋白质-蛋白质相互作用的研究之前已经揭示 这种酶在人类肿瘤中的作用未被认识到。这一续篇 建议扩展我们对Topo II蛋白质-蛋白质相互作用的研究 在TOPO-Il导向药物的细胞毒作用中。我们之前已经 将人类14-3-3蛋白的epsilon(E)亚型鉴定为来自 人HeLa细胞cDNA文库的蛋白质探针筛选 人拓扑酶II.14-3-3主要α亚型的C末端大片段 蛋白质，一种特殊基因的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品被认为与 原癌细胞信号通路，G2 DNA损伤检查点，以及 在细胞凋亡的调控中。然而，哺乳动物细胞保持了7个不同的 14-3-3基因产物，可能表明每种异构体都有独特的 功能。最近描述的一些14-3-3亚型的功能在 指导其他蛋白质的亚细胞区隔：要么是在 核出口(用于CDC25磷酸酶)或相反，用于核进口(用于 端粒酶和同源盒转录因子TLX-2)。使用倒数 亲和层析和免疫共沉淀方法，我们已经表明 14-3-3E，而不是G2停滞蛋白14-3-3E直接与人Topo 11a结合。 在功能上，这些与14-3-3e的相互作用，而不是14-3-3a，导致了 体外抑制Topo II DNA结合活性和适度减弱 依托泊苷对纯化酶和分离的核彗星DNA损伤的稳定作用 化验。我们建议检验一个由两部分组成的假设：1)不同的结构 Topo 11a和14-3-3蛋白中的决定因素导致这些异构体特异性 效应和相互作用可能受到特定细胞周期和DNA的影响 损伤依赖的磷酸化事件和，2)定点突变在 这些结构基序(在Topo II或14-3-3E上)或异位表达 特定的14-3-3亚型或它们的显性负对应物可以影响 TOPOLIA在体内的亚细胞区域化，并可能在 新近认识的酶和Topo Il-药物的细胞质积累 不同肿瘤细胞系平台期的耐药性。承认这一点 某些14-3-3亚型可能影响Topo II药物疗效，而不依赖于 与TOPO II的物理交互、适当的控制和替代 将使用范例来区分14-3-3对细胞的影响 相对于直接相关的周期分布或诱导细胞凋亡 与Topo IIa的蛋白质-蛋白质相互作用。因为目前的数据表明 14-3-3E可能长期保护细胞免受Topo Li导向的抗肿瘤药物的影响 这项工作的目标是确定TOPO II或TOPO II中的结构主题 14-3-3可能是小分子或多肽类药物或激酶的靶标 干扰其相互作用的抑制剂，以增强其抗肿瘤效果 TOPO II导向的药物和/或克服固有或获得性耐药性 这些特工。