Topoisomerase ll Beta in Myeloid Differentiation by Retionids

描述（由申请人提供）：本申请的总体目标是确定拓扑异构酶（topo） ii β在所有反式维甲酸（ATRA）诱导的急性髓性白血病（AML）细胞分化中的作用，在识别髓性分化涉及的信号机制的更广泛背景下。在一组AML细胞系中使用靶向si-RNA对topo IIbeta进行药理学抑制或下调的研究中，我们证实了atra分化细胞的存活需要topo IIbeta。对topo ii β缺乏促进atra诱导的细胞凋亡机制的初步研究发现，氧化还原调节剂过氧化物还蛋白2 （PRDX2）和atra诱导的活性氧（ROS）积累下调，g蛋白信号传导调节剂（RGS2）上调，参与髓细胞分化和应激反应。因此，我们的工作假设是ATRA诱导的APL细胞分化需要topo IIbeta和/或PRDX2作为生存信号，在缺乏这些信号的情况下，细胞死亡途径通过ROS积累和RGS2上调的机制被激活。为了验证这一假设，我们将使用不同表达topo IIbeta、PRDX2或RGS2的AML细胞模型来确定PRDX2下调和atra诱导的RGS2上调的功能意义。具体来说，我们将确定atra诱导的分化、ROS积累和凋亡：a)在表达topo IIbeta的AML细胞中PRDX2下调或RGS2过表达，或b)在topo IIbeta缺乏的AML细胞中PRDX2过表达或RGS2下调。接下来，我们将确定拓扑ii β和PRDX2的缺乏是否会导致atra诱导分化后外源性和/或内源性caspase途径的激活，以及这些途径之间是否涉及交叉对话。在这些研究中，我们将检测ATRA处理的拓扑ⅱβ -缺陷AML细胞中TRAIL、半胱天冬酶（9、8和3）和PARP的激活、BID的切割和细胞色素c的释放。我们将进一步研究灭活caspase 9或8对atra诱导的topo ibeta缺陷细胞凋亡的影响。在缺乏或存在topo ii β催化抑制剂的情况下，ATRA治疗对分化、生长停滞和细胞凋亡的影响将在患者源性髓性白血病中确定。这些结果将在AML细胞系中得到证实。拟议的研究将为atra诱导的AML细胞分化、生长停滞和凋亡的新靶点提供重要信息。从长远来看，确定这些靶点将有助于开发治疗骨髓增生异常综合征继发AML的新策略。

DESCRIPTION (provided by applicant): The overall goal of this application is to determine the role of topoisomerase (topo) IIbeta in all trans retinoic acid (ATRA)-induced differentiation of acute myeloid leukemia (AML) cells, within the broader context of identifying signaling mechanisms involved in myeloid differentiation. In studies employing pharmacologic inhibition or down- regulation with targeted si-RNA of topo IIbeta in a panel of AML cell lines we established that topo IIbeta is required for survival of ATRA-differentiated cells. Preliminary studies to identify the mechanisms by which deficiency in topo IIbeta promotes ATRA-induced apoptosis revealed down regulation of the redox regulator, peroxiredoxin 2 (PRDX2), and ATRA-induced accumulation of reactive oxygen species (ROS) and up regulation of regulator of G-protein signaling (RGS2), which is involved in myeloid differentiation and stress response. Thus, our working hypothesis is that ATRA- induced differentiation of APL cells requires topo IIbeta and/or PRDX2 as survival signals, in the absence of which the cell death pathway is activated via a mechanism involving accumulation of ROS and up-regulation of RGS2. To test this hypothesis we will use models of AML cells that differentially express topo IIbeta, PRDX2 or RGS2 to determine the functional significance of down regulation of PRDX2 and ATRA-induced up-regulation of RGS2. Specifically we will determine ATRA-induced differentiation, ROS accumulation and apoptosis following a) down regulation of PRDX2 or over expression of RGS2 in topo IIbeta expressing AML cells, or b) over expression of PRDX2 or down regulation of RGS2 in topo IIbeta deficient AML cells. We will next determine whether deficiency in topo IIbeta and PRDX2 leads to activation of the extrinsic and/or intrinsic caspase pathway following ATRA-induced differentiation and whether cross- talk between these pathways is involved. For these studies we will examine activation of TRAIL, caspases (9, 8 and 3) and PARP, cleavage of BID and release of cytochrome c in ATRA treated topo IIbeta -deficient AML cells. Further we will examine the effect of inactivating caspase 9 or 8 on ATRA-induced apoptosis in topo IIbeta -deficient cells. The effect of ATRA treatment on differentiation, growth arrest and apoptosis in the absence or presence of the topo IIbeta catalytic inhibitor will be determined in patient derived myeloid leukemias. These results will be corroborated with those in AML cell lines. The proposed studies should provide important information on novel targets regulating ATRA-induced differentiation, growth arrest and apoptosis of AML cells. In the long term, identification of these targets would assist in the development of novel strategies for treatment of AML secondary to myelodysplastic syndrome.