Arsenic Trioxide and Acute Myeloid Leukemia

描述（由申请人提供）：三氧化二砷（As2O3）诱导急性早幼粒细胞白血病（APL, AML-M3）患者在所有反式维甲酸（tRA）和化疗后复发的完全缓解。临床结果表明，As2O3对APL的治疗作用与t（15;17）易位产物pml - rar α的表达相关，并通过细胞凋亡和非终末分化诱导介导。我们发现As2O3降解pml - rar α，并允许rar α（来自野生型等位基因）驱动APL细胞部分分化。然而，pml - rar α表达与As2O3诱导细胞凋亡之间的关系尚不清楚。我们发现1)APL细胞含有少量谷胱甘肽s转移酶（GSTpi）、谷胱甘肽过氧化物酶（GPx）、过氧化氢酶和大量髓过氧化物酶（MPO）；2)过氧化氢（H2O2）介导的as2o3诱导APL细胞体外凋亡高度敏感；3)抗坏血酸选择性地增加as2o3诱导的HL-60细胞（表达大量MPO）的凋亡，而不是U937和正常骨祖细胞（不表达MPO）的凋亡。我们假设1)低水平的GSTpi允许As2O3抑制GPx。GPx抑制与过氧化氢酶低表达结合会导致H2O2积累；2)积累的H2O2经MPO转化为活性氧，触发细胞凋亡；3) pml - rar α通过上调MPO和/或下调GSTpi、过氧化氢酶和GPx使APL细胞对as2o3诱导的凋亡敏感；4)抗坏血酸通过产生H2O2和消耗GSTpi和GPx的底物还原型谷胱甘肽（GSH），选择性协同as2o3诱导的MPO阳性AML细胞凋亡。该项目的最初目的是确认As2O3通过h2o2介导的途径诱导细胞凋亡。这将通过比较H2O2量和As2O3处理的AML细胞的凋亡诱导来验证。第二个目的是确定GSTpi在控制细胞对as2o3诱导的H2O2积累的敏感性方面的核心作用，第三个目的是研究MPO在as2o3诱导的细胞凋亡中的增敏功能。这些将通过稳定转染正义或反义cDNA和使用特定抑制剂来测试。第四个目标将剖析pml - rar α表达与GSTpi、GPx、过氧化氢酶和MPO水平之间的联系。PML-RARalpha稳定转染的细胞将用于此目的。我们的最后一个目的是评估As2O3与抗坏血酸联合在体外表达MPO /不表达MPO的AML细胞中的选择性凋亡诱导及其机制。携带AML细胞的SCID模型将用于测试体内效果。这些研究的成功完成不仅有助于阐明As2O3诱导APL缓解的机制，而且可能为As2O3在其他形式的AML中的创新应用提供帮助。

DESCRIPTION (provided by applicant): Arsenic trioxide (As2O3) induced complete remission in acute promyelocytic leukemia (APL, AML-M3) patients that relapsed after all trans retinoic acid (tRA) and chemotherapy treatment. Clinical results indicated that the therapeutic effect of As2O3 in APL correlated with the expression of PML-RARalpha , the product of the t(15;17) translocation, and was mediated by apoptosis and non-terminal differentiation induction. We have found that As2O3 degraded PML-RARalpha and allowed RARalpha (from the wild-type allele) to drive APL cell partly differentiation. However, the connection between PML-RARalpha expression on one hand, and apoptosis induction by As2O3 on the other hand, is unclear. We have found that 1) APL cells contained low amounts of glutathione-s-transferase pi (GSTpi), glutathione peroxidase (GPx), catalase and high amounts of myeloperoxidase (MPO); 2) APL cells were highly sensitive to As2O3-induced apoptosis in vitro by a hydrogen peroxide (H2O2) mediated pathway; 3) Ascorbic acid selectively increased As2O3-induced apoptosis in HL-60 cells (which express high amounts of MPO) not in U937 and normal bone progenitors cells (which do not express MPO). We hypothesize that 1) low levels of GSTpi allow As2O3 to inhibit GPx. GPx inhibition in combination with low catalase expression will result in H2O2 accumulation; 2) accumulated H2O2 is converted into reactive oxygen species by MPO, and then trigger apoptosis; 3) PML-RARalpha sensitizes APL cells to As2O3-induced apoptosis by upregulating MPO and/or downregulating GSTpi, catalase and GPx; 4) Ascorbic acid selectively synergizes As2O3-induced apoptosis in MPO positive AML cells by producing H2O2 and depleting reduced form glutathione (GSH), the substrate of both GSTpi and GPx. The initial aim of the project is to confirm that As2O3 induces apoptosis through H2O2-mediated pathway. This will be tested by comparing H2O2 amount and apoptosis induction in As2O3 treated AML cells. The second aim will determine the central role of GSTpi to control the sensitivity of cells to As2O3-induced H2O2 accumulation and the third aim will examine the functions of MPO in sensitizing As2O3-induced apoptosis. These will be tested by stably transfecting sense or antisense cDNA and using specific inhibitors. The fourth aim will dissect the connection between PML-RARalpha expression and the levels of GSTpi, GPx, catalase and MPO. PML-RARalpha stably transfected cells will be used for this purpose. Our last aim will evaluate the selective apoptosis-induction and the mechanism of As2O3 in combination with ascorbic acid among AML cells with/without expressing MPO in vitro. SCID models bearing AML cells will be used to test the in vivo effect. Successful completion of the proposed studies will not only contribute to elucidation of the mechanism of As2O3-induced remission in APL, but may also provide innovative usage of As2O3 in other forms of AML.