Arsenic Trioxide and Acute Myeloid Leukemia

描述（由申请人提供）：三氧化二砷（As 2 O3）诱导急性早幼粒细胞白血病（APL，AML-M3）患者完全缓解，这些患者在全反式维甲酸（tRA）和化疗治疗后复发。临床结果表明，As 2 O3对APL的治疗作用与t（15;17）易位产物PML-RAR α的表达有关，并通过诱导细胞凋亡和非终末分化而实现。我们已经发现，As 2 O3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，一方面PML-RAR α表达与另一方面As 2 O3诱导凋亡之间的联系尚不清楚。结果表明：（1）APL细胞内谷胱甘肽-S-转移酶（GSTpi）、谷胱甘肽过氧化物酶（GPx）、过氧化氢酶（CAT）含量较低，髓过氧化物酶（MPO）含量较高; 3）抗坏血酸选择性地增加As_2O_3诱导的HL-60细胞（高表达MPO）的凋亡，而不增加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As 2 O3抑制GPx。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As_2O_3诱导的凋亡敏感; 4）抗坏血酸通过产生H_2O_2和消耗还原型谷胱甘肽（GSH）（GSTpi和GPx的底物）选择性地协同As_2O_3诱导的MPO阳性AML细胞凋亡。本课题的初步目的是证实As 2 O3通过H2 O2介导的途径诱导细胞凋亡。这将通过比较As 2 O3处理的AML细胞中的H2 O2量和细胞凋亡诱导来测试。第二个目标将确定GST π的核心作用，以控制细胞的敏感性，以As 2 O3诱导的H2 O2的积累和第三个目标将检查MPO在敏化As 2 O3诱导的细胞凋亡的功能。这些将通过稳定转染正义或反义cDNA并使用特异性抑制剂进行检测。第四个目标将剖析PML-RAR α表达与GSTpi、GPx、过氧化氢酶和MPO水平之间的关系。PML-RAR α稳定转染的细胞将用于此目的。本研究的最后一个目的是探讨As_2O_3联合抗坏血酸对髓过氧化物酶（MPO）阳性和阴性AML细胞的选择性凋亡诱导作用及其机制。携带AML细胞的SCID模型将用于测试体内效应。成功完成拟议的研究不仅有助于阐明As 2 O3诱导APL缓解的机制，而且还可能提供As 2 O3在其他形式的AML中的创新用途。

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.