NOVEL TRANSLOCATION PRODUCT IN APL

粒细胞成熟阻滞的分子机制， 急性早幼粒细胞白血病（APL）的特征仍不清楚。 最 APL患者存在t（15;17）染色体易位， 导致PML-RAR融合蛋白的表达。 竞争车型 提示PML-RAR作为PML的显性阴性， 野生型视黄酸受体α（RARa）阴性，或 转录激活因子 为了更深入地了解 APL的潜在机制，我们研究了一个APL患者与t（5;17） 染色体易位 我们表明，这种易位连接5'， 核仁蛋白质核磷蛋白（NPM）的3'元件， 拉拉。 在我们的初步数据中，我们鉴定并测序了嵌合cDNA， 并证明它编码一个52 kD的蛋白。 我们假设这 融合通过一种新的表达途径影响保留的RAR结构域的功能。 改变RAR结构域的核区室的机制 隔离。 我们假设NPM基序将RAR结构域重定向到一个 异常的核区室，融合结合并隔离RAR 异源二聚化伴侣。 通过这种新的重定向机制， 融合伙伴进入核的不习惯的结构区域， NPM将连接的RAR结构域转化为RAR α的显性阴性，并且 从而阻断骨髓成熟。 为了验证这个假设，我们将 完成NPM-RAR的完全开放阅读框的测序，和 确定融合蛋白确实在t中表达（5;17） 白血病细胞 我们将确定NPM-RAR是否再现APL 通过研究NPM-RAR的强制表达对表型的影响， HL-60细胞的分化潜能。 我们将决定 NPM-RAR表达调节野生型RAR α功能，以及NPM-RAR是否 与RAR α异二聚化配偶体的复合物。 我们将决定， 免疫荧光检测NPM-RAR在细胞中的分布，以及是否 它的表达影响了RARa的建筑隔间， 二聚化伴侣定位。 最后，我们将确定一个 一种人工构建体，将RAR定向到一个不习惯的核 结构域类似地阻断髓样分化。 这些 研究将测试破坏视黄酸的假设， 信号传导是APL中成熟阻滞的基础。 此外，他们将 作为未来工作的开创性研究， 隔间

The molecular mechanism underlying the granulocytic maturation arrest that characterizes Acute Promyelocytic Leukemia (APL) remains obscure. Most patients with APL manifest the t(15;17) chromosomal translocation, which results in expression of the PML-RAR fusion protein. Competing models suggest that PML-RAR acts as a dominant negative for PML, a dominant negative for wild type retinoic acid receptor alpha (RARa), or a rogue transcriptional activator. In an effort to gain greater insight into the mechanism underlying APL we have studies an APL patient with a t(5;17) chromosomal translocation. We show that this translocation links 5' elements of the nucleolar protein nucleophosmin (NPM) to 3' elements of RARa. In our preliminary data we identify and sequence the chimeric cDNA, and demonstrate that it encodes a 52 kd protein. We hypothesize that this fusion affects the function of the retained RAR domains through a novel mechanism of changing the nuclear compartment in which the RAR domains segregate. We hypothesize that NPM motifs redirect RAR domains into an aberrant nuclear compartment, where the fusion binds to and sequesters RAR heterodimerization partners. By this novel mechanism of redirecting its fusion partner into an unaccustomed architectural region of the nucleus, NPM converts the linked RAR domain into a dominant negative for RARa, and thereby blocks myeloid maturation. To test this hypothesis we will complete the sequencing of the full open reading frame for NPM-RAR, and establish that the fusion protein is indeed expressed in the t(5;17) leukemic cells. We will determine whether NPM-RAR reproduces the APL phenotype by investigating the effects of forced expression of NPM-RAR on the differentiation potential of HL-60 cells. We will determine whether NPM-RAR expression modulates wild type RARa function, and whether NPM-RAR complexes with RARa heterodimerization partners. We will determine by immunofluorescence the distribution of NPM-RAR in the cell, and whether its expression affects the architectural compartment in which RARa dimerization partners localize. Finally, we will determine whether an artificial construct that directs RAR to an unaccustomed nuclear architectural domain similarly blocks myeloid differentiation. These investigations will test the hypothesis that disrupted retinoic acid signaling underlies the maturational block in APL. In addition, they will serve as seminal studies for future work mapping nuclear functional compartments.