14 3 3 Implications in Topoisomerase II Pharmacology

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 酶对细胞增殖是绝对必要的，因为它 在有丝分裂之前物理上互锁的DNA。Topo II也是一种临床 广泛用于治疗的一类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 已经揭示了肿瘤细胞响应或逃避免疫应答的几种机制， Topo II导向的抗肿瘤药物的细胞毒性作用。我们开发了 蛋白质-蛋白质介导Topo II活性假说 这些蛋白的相互作用和调节可能影响肿瘤细胞的反应 拓扑二型毒药。因为我们最初鉴定了转录因子 CREB、ATF-2和c-Jun作为拓扑异构酶I相互作用蛋白（TIP），刺激 除了topo L1催化活性外，其他人已经描述了来自酵母的TIP， 是果蝇染色体分离所必需的。人类 酵母TIP的同源物Sgsl很可能是突变时 负责布卢姆综合征患者的高肿瘤发生率， 最近，视网膜母细胞瘤肿瘤抑制基因产物Rb也被 最近显示结合拓扑异构酶II并抑制其催化活性。因此，我们认为， Topo II蛋白质-蛋白质相互作用的研究先前已经揭示了 酶在人类肿瘤形成中未被重视的作用。这一延续 该提案扩展了我们对topo II蛋白质-蛋白质相互作用的研究 topo II导向药物的细胞毒性作用。我们先前已经 鉴定了人14-3-3蛋白的β（e）同种型为TIP， 用蛋白质探针筛选人HeLa细胞cDNA文库，所述蛋白质探针包含 人拓扑异构酶II的主要α同种型的大的C末端片段。14-3-3 蛋白质，一个由不同基因组成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， 原癌细胞信号通路，G2 DNA损伤检查点，以及 在细胞凋亡调控中的作用然而，哺乳动物细胞保持了7种不同的 14-3-3基因产物，这可能表明每种亚型具有独特的 功能协调发展的最近描述的一些14-3-3同种型的功能是在 指导其他蛋白质的亚细胞区室化： 核输出（对于cdc 25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）。使用互易 亲和层析和免疫共沉淀方法，我们已经表明， 14-3-3e，但不是G2阻滞蛋白14-3-3E，直接结合人拓扑异构酶。 在功能上，这些与14-3-3e的相互作用，而不是14-3-3a，导致在 Topo II DNA结合活性的体外抑制和 纯化酶和分离核彗星中依托泊苷稳定的DNA损伤 测定。我们提出了一个两部分的假设，1）不同的结构， Topolla和14-3-3蛋白内的决定簇导致这些同种型特异性 作用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，以及，2） 这些结构基序（在拓扑异构酶II或14-3-3e上）或异位表达 特定的14-3-3同种型或其显性阴性对应物可以影响 在体内Topo IIa的亚细胞区室化，并可能在 最近认识到的酶和拓扑II药物的细胞质积累 各种肿瘤细胞系在平台期的耐药性。承认 一些14-3-3同种型可能独立于 与topo II物理交互，适当的控制和替代 将采用范例来区分14-3-3对细胞的影响， 周期分布或凋亡诱导相对于那些直接相关 蛋白质间的相互作用。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I导向的抗肿瘤药物的影响， 这项工作的目标是确定拓扑结构II或 14-3-3，其可被小分子或肽模拟物或激酶靶向 抑制剂，破坏它们的相互作用，以提高抗肿瘤功效 Topo II导向的药物和/或克服固有的或获得性的耐药性， 这些代理人。

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.