Topoisomerase ll Beta in Myeloid Differentiation by Retionids

描述(申请人提供)：本申请的总体目标是确定拓扑异构酶(TOPO)IIbeta在全反式维甲酸(ATRA)诱导的急性髓系白血病(AML)细胞分化中的作用，在更广泛的背景下确定涉及髓系分化的信号机制。在一组AML细胞系中，我们采用药物抑制或下调Topo IIbeta的靶向si-RNA的研究，证实Topo IIbeta是ATRA分化细胞生存所必需的。初步研究发现Topo IIbeta缺陷促进ATRA诱导的细胞凋亡的机制显示，氧化还原调节因子过氧化还蛋白2(PRDX2)下调，ATRA诱导的活性氧(ROS)积累和参与髓系分化和应激反应的G蛋白信号转导调节因子(RGS2)上调。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，在没有Topo IIbeta和/或PRDX2的情况下，细胞死亡途径通过一种涉及ROS积累和RGS2上调的机制被激活。为了验证这一假设，我们将使用差异表达Topo IIbeta、PRDX2或RGS2的AML细胞模型来确定PRDX2下调和ATRA诱导RGS2上调的功能意义。具体地说，我们将在a)在表达Topo IIbeta的AML细胞中下调PRDX2或过度表达RGS2，或b)在Topo IIbeta缺陷的AML细胞中下调PRDX2或下调RGS2的情况下，确定ATRA诱导的分化、ROS积聚和细胞凋亡。接下来，我们将确定Topo IIbeta和PRDX2的缺失是否会导致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存在串扰。在这些研究中，我们将检测TRAIL、caspase(9、8和3)和PARP的激活，Bid的切割和细胞色素c的释放。此外，我们将研究失活caspase9或8在全反式维甲酸诱导Topo IIbeta缺陷细胞凋亡中的作用。在没有或存在Topo IIbeta催化抑制剂的情况下，ATRA治疗对患者来源的髓系白血病的分化、生长停滞和细胞凋亡的影响将被确定。这些结果将与AML细胞系中的结果相印证。这些研究将为调控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，这些靶点的确定将有助于开发治疗继发于骨髓增生异常综合征的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.