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Analysis of APL-Fusion Retinoic Acid Receptor Proteins

APL-融合视黄酸受体蛋白的分析

基本信息

批准号：
7098804
负责人：
SHUO DONG
金额：
$ 12.6万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2007-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7098804
关键词：
acute myelogenous leukemia cellular pathology chimeric proteins chromatin chromosome translocation confocal scanning microscopy fluorescence recovery after photobleaching genetic transcription intermolecular interaction lac operon molecular pathology nuclear receptors protein localization retinoate retinoid binding proteins

项目摘要

DESCRIPTION (provided by applicant): Acute promyelocytic leukemia (APL) is characterized by selective expansion of malignant cells blocked at the promyelocytic stage bearing the specific chromosomal translocation t(15;17)(q22;q21) and an effective clinical response to all-trans retinoic acid (ATRA) treatment. In addition to t(15;17), which produces the fusion protein PML-RARalpha, four other variant chromosomal translocations also have been described in APL patients: t(11;17), t(5;17), t(11;17) and del(17), which produce PLZF-RARalpha, NPM-RARalpha, NuMA-RARalpha and STAT5b-RARalpha fusion proteins, respectively. APL disease with PML-RARalpha, NPM-RARalpha, or NuMA-RARalpha fusion proteins is responsive to ATRA, while APL disease with PLZF-RARalpha or STATSb-RARalpha is ATRA-resistant. A considerable amount of evidence has suggested that these RARalpha fusion receptors cause leukemias by interfering with the function of retinoic acid receptors (RARs). Transgenic mouse studies demonstrate that XRARalpha (where X is one of the alternative RARalpha fusion partners: PML, PLZF, NPM, NuMA or STATSb) plays a pivotal role in the pathogenesis of APL. However, how X-RARalpha fusion proteins cause leukemia, and the molecular and cellular basis for the response to ATRA, are not completely known. The fact that the RARalpha gene is involved in each of the APL-translocations suggests that disruption of normal RARalpha function is critical for disease pathogenesis. We and others have demonstrated that each of the X-RARalpha fusion proteins bind RXRalpha, a heterodimerization partner of RARalpha essential for its transcriptional activity, and nuclear receptor (NR) coregulators, such as coactivator (CoA) and corepressor (CoR). Using CFP-tagged constructs and FRAP technique, we demonstrated that two RARalpha-containing fusion proteins, PML-RARalpha and NuMA-RARalpha, have nuclear localization patterns and nuclear mobility distinct from RARalpha that alter the nuclear localization and mobility of RXRalpha. These findings support our hypothesis that alterations in nuclear localization and mobility of APL fusion proteins as well as changes in nuclear localization and mobility of nuclear receptor coregulators by X-RARalpha, compared to wild-type RARalpha, contribute to the pathogenesis of APL. The real-time visualization of transcription dynamics will be explored in three specific aims: Aim I will further characterize differences in intranuclear organization, dynamics and interactions with other coregulators among unliganded or liganded X-RARalpha fusion proteins compared to wild-type RARalpha using confocal fluorescent microscopy and FRAP analysis. Aim II will determine the effects of unliganded or liganded XRARalpha proteins versus wild-type RARalpha protein on chromatin structure, as well as the different interactions between X-RARalpha proteins and chromatin-modifying coregulators at an integrated lac operator array. Aim III will determine the effects of key interactions identified in the lac operator system on real time gene transcription using cells containing a RARE-integrated array that drives an RFP-pts reporter. These experiments will give new insight into the molecular and cellular pathogenesis of APL and its response to ATRA and may prove useful as part of a rapid throughput drug screening assay for new APL treatments. The systems to be established in this proposal will also provide powerful tools and useful information that could be extended to other cancer investigations.

描述（由申请人提供）：急性早幼粒细胞白血病（APL）的特征是在携带特异性染色体易位t（15;17）（q22;q21）的早幼粒细胞阶段阻断的恶性细胞的选择性扩增，以及对全反式维甲酸（ATRA）治疗的有效临床应答。除了t（15; 17），其产生融合蛋白PML-RAR α，在APL患者中还描述了四种其他变体染色体易位：t（11;17）、t（5;17）、t（11;17）和del（17），其分别产生PLZF-RAR α、NPM-RAR α、NuMA-RAR α和STAT 5 b-RAR α融合蛋白。伴有PML-RAR α、NPM-RAR α或NuMA-RAR α融合蛋白的APL疾病对ATRA有反应，而伴有PLZF-RAR α或STATSb-RAR α的APL疾病对ATRA耐药。大量证据表明，这些RAR α融合受体通过干扰视黄酸受体（RAR）的功能而引起白血病。转基因小鼠研究表明，XRAR α（其中X是RAR α融合伴侣之一：PML，PLZF，NPM，NuMA或STATSb）在APL的发病机制中起着关键作用。然而，X-RAR α融合蛋白如何引起白血病，以及对ATRA反应的分子和细胞基础尚不完全清楚。 RAR α基因参与每种APL易位的事实表明，正常RAR α功能的破坏对于疾病的发病机制至关重要。我们和其他人已经证明，每一个X-RAR α融合蛋白结合RXR α，RAR α的异源二聚化伙伴，其转录活性，和核受体（NR）的辅调节因子，如辅激活因子（CoA）和辅阻遏物（CoR）。使用CFP标记的构建体和FRAP技术，我们证明了两种含有RAR α的融合蛋白PML-RAR α和NuMA-RAR α具有不同于RAR α的核定位模式和核移动性，其改变了RXR α的核定位和移动性。这些发现支持了我们的假设，即APL融合蛋白的核定位和移动性的改变以及X-RAR α核受体辅助调节因子的核定位和移动性的变化，与野生型RAR α相比，有助于APL的发病机制。实时可视化的转录动力学将探讨在三个具体的目标：目的我将进一步表征差异核内组织，动力学和与其他coregulators之间的unliganded或liganded X-RAR α融合蛋白相比，野生型RAR α使用共聚焦荧光显微镜和FRAP分析。目的II将确定unliganded或liganded XRAR α蛋白与野生型RAR α蛋白对染色质结构的影响，以及X-RAR α蛋白与染色质修饰辅助调节因子在一个集成的乳糖操作阵列之间的不同相互作用。目的III将确定关键的相互作用，确定在乳糖操纵子系统的真实的时间基因转录使用细胞含有一个罕见的集成阵列，驱动RFP-PTS报告的影响。这些实验将提供新的见解APL的分子和细胞的发病机制及其对ATRA的反应，并可能被证明是有用的一部分，快速通过药物筛选试验的新的APL治疗。本提案中建立的系统还将提供强大的工具和有用的信息，可扩展到其他癌症研究。