TopoisomerasellBeta in Myeloid Differentiation by Retionids

描述（由申请人提供）：本申请的总体目标是在鉴定涉及髓样分化的信号传导机制的更广泛背景下，确定拓扑异构酶（topo）II β在急性髓样白血病（AML）细胞的全反式视黄酸（ATRA）诱导的分化中的作用。在一组AML细胞系中使用topo II β的靶向si-RNA进行药理学抑制或下调的研究中，我们确定了topo II β是ATRA分化细胞存活所需的。初步研究，以确定拓扑II β缺陷促进ATRA诱导的细胞凋亡的机制，揭示了下调的氧化还原调节剂，过氧化物酶2（PRDX 2），ATRA诱导的活性氧（ROS）的积累和上调的G蛋白信号转导（RGS 2），这是参与骨髓分化和应激反应的调节。因此，我们的工作假设是，ATRA诱导的APL细胞分化需要拓扑异构酶II β和/或PRDX 2作为存活信号，在没有它们的情况下，细胞死亡途径通过涉及ROS积累和RGS 2上调的机制被激活。为了检验这一假设，我们将使用不同表达拓扑异构酶II β、PRDX 2或RGS 2的AML细胞模型来确定PRDX 2下调和ATRA诱导的RGS 2上调的功能意义。具体地，我们将确定ATRA诱导的分化、ROS积累和凋亡，a）在表达topo II β的AML细胞中PRDX 2的下调或RGS 2的过表达，或B）在topo II β缺陷的AML细胞中PRDX 2的过表达或RGS 2的下调。我们接下来将确定拓扑II β和PRDX 2的缺乏是否导致ATRA诱导的分化后外源性和/或内源性胱天蛋白酶途径的激活，以及是否涉及这些途径之间的串扰。对于这些研究，我们将检查ATRA处理的topo II β缺陷型AML细胞中TRAIL、半胱天冬酶（9、8和3）和PARP的活化、BID的裂解和细胞色素c的释放。此外，我们将检查失活caspase 9或8对拓扑异构酶II β缺陷细胞中ATRA诱导的细胞凋亡的影响。将在患者来源的髓性白血病中测定在不存在或存在拓扑异构酶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。这些结果将与AML细胞系中的结果相证实。这些研究将为研究ATRA诱导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，这些目标的识别将有助于开发新的策略来治疗继发于骨髓增生异常综合征的AML。

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.