急性早幼粒细胞白血病对全反式维甲酸耐药的机制及其逆转

目的　揭示急性早幼粒细胞白血病 (APL)产生维甲酸耐药的机制 ,探讨耐药性逆转的方法。方法　MTT法测定细胞增殖 ,极限稀释法诱导APL细胞耐药性。RT PCR及流式细胞仪测定MDR1、消炎痛及PGE1增殖影响实验分别间接测定GST酶活性或cAMP作用。干扰素、高三尖杉酯碱(HHT)及三氧化二砷 (As2 O3 )进行体外耐药细胞逆转实验。结果　对全反式维甲酸 (ATRA)耐药的HL6 0细胞MDR1阴性 ,初发时MDR1为阴性的APL患者复发后仍为阴性。消炎痛对耐药的HL6 0增殖分化无影响 ,前列腺素E(PGE)可部分恢复ATRA对耐药HL6 0的作用。干扰素可明显逆转HL6 0的耐药性。As2 O3 与HHT对ATRA耐药的HL6 0细胞有明显作用。结论　APL细胞耐药并非多药耐药 ,消炎痛或干扰素明显逆转其耐药性 ,ATRA与As2 O3 化疗药无交叉耐药性。     

Relationship between G-CSF and hyperleukocytosis in patients with APL after treatment with all-trans retinoic acid

Thepatientswithacutepromyelocyticleukemia(APL)oftendiedfromearlyinfectionorcoagulationdisorders.lllCompleteremission(CR)ratesof50%-60%duringthechemotherapywerereportedinrecentyears.Studiesinvitrohaveshownthatall-transretinoicacid(ATRA)wascapableoflettingAPLcellstodifferentiateatsomelowconcentrations.ThispropertyhasbeenappliedinvivoinnewlydiagnosedandinrelapsingAPL.Otherwise,withlesscoagulationofdisordersandbonemarrowaplasia.DevelopmentofATRAtreatmenthasresultedinincreasingCRratestoabo…     

三氧化二砷诱导HL-60细胞凋亡效应及抑制细胞ERK的活性

目的:初步探讨三氧化二砷(arsenic trioxide,As2O3)促进HL-60细胞凋亡及其可能机制.方法:不同浓度的As2O3(0、2.5、5、10 μmol/L)作用于HL-60细胞24h后,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测Caspase-3活化、ERK活性和cyclinD1蛋白的表达,RT-PCR法检测cyclinD1 mRNA的表达.结果:As2O3可抑制HL-60细胞增殖,诱导HL-60细胞的凋亡,并呈浓度依赖性 (P<0.05);As2O3可使HL-60细胞Caspase-3激活,并抑制ERK活性,但不能下调cyclinD1的表达(P>0.05).结论:As2O3能抑制HL-60细胞增殖,并诱导其凋亡,其效应可能与As2O3抑制HL-60细胞ERK活性有关.     

Differentiating agents in pediatric malignancies: All-trans-retinoic acid and arsenic in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is the most potentially curable type of acute myeloid leukemia. It is characterized by the chromosomal translocation t(15;17), which results in the fusion gene PML-RAR-α. The introduction of all-trans- retinoic acid (ATRA) was a major advance in treatment of this disease. This agent induces terminal differentiation of malignant myeloid cells to mature neutrophils, and its side effects are usually well tolerated in children. ATRA does not eradicate the malignant myeloid clone in APL and, eventually, resistance develops. Arsenic trioxide induces nonterminal differentiation of malignant promyelocytes and promotes apoptosis. APL patients treated with ATRA or arsenic trioxide have rapid resolution of their coagulopathy. Because both of these drugs are well tolerated in children and their synergy has been shown in animal models, the possibility of combining ATRA and arsenic trioxide in frontline therapy for children with APL is being considered.     

Upregulation of Bfl-1/A1 in leukemia cells undergoing differentiation by all-trans retinoic acid treatment attenuates chemotherapeutic agent-induced apoptosis.

All-trans retinoic acid (ATRA) induces differentiation of NB4 and HL-60 leukemia cells, but not R4 and HL-60/Res cells. Three agents used in cancer therapy, doxorubicin (Dox), arsenic trioxide (As(2)O(3)) and paclitaxel, induce apoptosis, but not differentiation, in all of these cell lines. The induction of apoptosis by these agents is decreased in ATRA-pretreated NB4 and HL-60 cells, but not in ATRA-pretreated R4 and HL-60/Res cells. The level of Bcl-2 protein is decreased by ATRA treatment in NB4, HL-60 and HL-60/Res cells. The level of Mcl-1 protein is increased by ATRA treatment in NB4 and R4 cells, but not in HL-60 and HL-60/Res cells. Bfl-1/A1 mRNA is not expressed in these cell lines, however, its expression is markedly induced by ATRA treatment in NB4 and HL-60 cells, but not in R4 or HL-60/Res cells, which correlates with inhibition of apoptosis. Inhibiting Bfl-1/A1 mRNA upregulation in ATRA-pretreated NB4 cells using small interfering RNA (siRNA) partly recovers cell sensitivity to Dox-induced apoptosis. These data demonstrate that ATRA induction of Bfl-1/A1 in differentiated NB4 and HL-60 cells contributes to a loss of sensitivity to chemotherapy-induced apoptosis.     

三氧化二砷(As_2O_3)治疗2例儿童难治性急性早幼粒细胞白血病

为探讨三氧化二砷治疗儿童白血病的效果。对2例难治性儿童患者进行了治疗。1例达CR。使用过程无明显毒副反应，且与全反式维甲酸（ATRA）无交叉耐药。细胞形态学检查显示：AS2O3有诱导分化及促凋亡双重作用。     

三氧化二砷诱导HL-60细胞凋亡效应及抑制细胞ERK的活性

目的：初步探讨三氧化二砷（arsenic trioxide,As2O3）促进HL-60细胞凋亡及其可能机制。方法：不同浓度的As2O3（0、2.5、5、10μmol/L）作用于HL-60细胞24h后,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测Caspase-3活化、ERK活性和cyclinD1蛋白的表达,RT-PCR法检测cyclinD1mRNA的表达。结果：As2O3可抑制HL-60细胞增殖,诱导HL-60细胞的凋亡,并呈浓度依赖性（P〈0.05）;As2 O3可使HL-60细胞Caspase-3激活,并抑制ERK活性,但不能下调cyclinD1的表达（P〉0.05）。结论：As2 O3能抑制HL-60细胞增殖,并诱导其凋亡,其效应可能与As2 O3抑制HL-60细胞ERK活性有关。     

Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia.

Recent studies showed that arsenic trioxide (As2O3) could induce apoptosis and partial differentiation of leukemic promyelocytes. Here, we addressed the possible mechanisms underlying these two different effects. 1.0 microM As2O3-induced apoptosis was associated with condensation of the mitochondrial matrix, disruption of mitochondrial transmembrane potentials (DeltaPsim) and activation of caspase-3 in acute promyelocytic leukemia (APL) cells regardless of their sensitivity to all-trans retinoic acid (ATRA). All these effects were inhibited by dithiothreitol (DTT) and enhanced by buthionine sulfoximine (BSO). Furthermore, BSO could also render HL60 and U937 cells, which had the higher cellular catalase activity, sensitive to As2O3-induced apoptosis. Surprisingly, 1.0 microM As2O3 did not induce the DeltaPsim collapse and apoptosis, while 0.1 microM As2O3 induced partial differentiation of fresh BM cells from a de novo APL patient. In this study, we also showed that 0.2 mM DTT did not block low-dose As2O3-induced NB4 cell differentiation, and 0. 10.5 microM As2O3 did not induce differentiation of ATRA-resistant NB4-derived sublines, which were confirmed by cytomorphology, expression of CD11b, CD33 and CD14 as well as NBT reduction. Another interesting finding was that 0.10.5 microM As2O3 could also induce differentiation-related changes in ATRA-sensitive HL60 cells. However, the differentiation-inducing effect could not be seen in ATRA-resistant HL60 sublines with RARalpha mutation. Moreover, low-dose As2O3 and ATRA yielded similar gene expression profiles in APL cells. These results encouraged us to hypothesize that As2O3 induces APL cell differentiation through direct or indirect activation of retinoic acid receptor-related signaling pathway(s), while DeltaPsim collapse is the common mechanism of As2O3-induced apoptosis.     

Granulocyte colony-stimulating factor potentiates differentiation induction by all-trans retinoic acid and arsenic trioxide and enhances arsenic uptake in the acute promyelocytic leukemia cell line HT93A

The effects of arsenic trioxide (ATO), all-trans retinoic acid (ATRA) and granulocyte colony-stimulating factor (G-CSF), alone or in combination, were investigated by focusing on differentiation, growth inhibition and arsenic uptake in the acute promyelocytic leukemia (APL) cell line HT93A. ATO induced differentiation at low concentrations (0.125?μM) and apoptosis at high concentrations (1-2?μM). Furthermore, ATRA induced greater differentiation than ATO. No synergistic effect of ATRA and ATO was found on differentiation. G-CSF promoted differentiation-inducing activities of both ATO and ATRA. The combination of ATRA and G-CSF showed maximum differentiation and ATO addition was not beneficial. Addition of 1?μM ATRA and/or 50?ng/ml G-CSF to ATO did not affect apoptosis compared to ATO treatment alone. ATRA induced expression of aquaporin-9 (AQP9), a transmembrane transporter recognized as a major pathway of arsenic uptake, in a time- and dose-dependent manner. However, treatment with 1?μM ATRA decreased arsenic uptake by 43.7% compared to control subject. Although G-CSF addition did not enhance AQP9 expression in the cells, the reduced arsenic uptake was recovered to the same level as that in controls. ATRA decreased cell viability and addition of 50?ng/ml G-CSF to ATRA significantly increased the number of viable cells compared with that in ATRA alone treated cells. G-CSF not only promotes differentiation-inducing activities of both ATRA and ATO, but also makes APL cells vulnerable to increased arsenic uptake. These observations provide new insights into combination therapy using these three agents for the treatment of APL.     

亚砷酸联合阿米福汀对HL-60细胞中Survivin表达的影响

 目的　探讨亚砷酸（As2O3）联合阿米福汀（AMI）诱导人髓系白血病细胞HL-60凋亡的可能机制。方法　不同浓度As2O3单用和联合AMI对HL-60细胞进行不同时间干预,用MTT比色法检测细胞的生长抑制作用,用半定量RT-PCR法检测抑凋亡基因Survivin mRNA的表达水平。结果　As2O3组和联合组均可显著抑制HL-60细胞的增生,呈浓度依赖性,联合组对其抑制作用明显大于As2O3组。联合组降低Survivin的表达作用比As2O3组更明显。结论　As2O3通过下调抑凋亡基因Survivin的表达诱导HL-60凋亡;AMI可增强As2O3对Survivin的下调作用,增强HL-60细胞对As2O3的敏感性,发挥促凋亡效应。     

CYTOKINE SECRETION IN PATIENTS WITH ACUTE PROMYE—LOCYTIC LEUKEMIA AFTER TREATMENT WITH ALL—TRANS RETINOIC ACID

Acute promyelocytic leukemia (APL) is characterized by the clonal expansion of hematopoietic progenitor cells and by the presence of the specific chromosomal translocation t(15; 17) (q22; q21)[1], which results in a fusion gene PML/RARa by juxtaposing the PML gene on chromosome 15 and the RARa gene on chromosome 17[2]. PML/RARa plays a critical role in the high sensitivity of APL blasts to all-trans retinoic acid (ATRA). It was well known that all-trans retinoic acid (ATRA) was fi…     

三氧化二砷和曲古抑菌素A协同诱导HL-60细胞凋亡的作用及分子机制研究

目的 探讨三氧化二砷(As2O3)和曲古抑菌素A(TSA)在体外诱导急性早幼粒白血病细胞HL-60凋亡过程中的协同作用.方法 采用MTT法检测As2O3和(或)TSA对HL-60细胞增殖的影响,流式细胞术检测细胞周期和细胞凋亡,逆转录-聚合酶链反应(RT-PCR)检测凋亡相关基因Bax和Bcl-2的表达.结果 As2O3和TSA联用较单独用药能明显抑制细胞,引起细胞周期阻滞和凋亡,Bax基因表达升高,Bcl-2基因表达降低,Bax/Bcl-2比值升高.结论 As2O3和TSA均能够引起HL-60凋亡,二者具有协同效应,其机制是引起细胞周期阻滞,且上调Bax与Bcl-2比值.     

p27Kip1在As2O3诱导白血病细胞凋亡中的作用机制

目的:研究三氧化二砷(arsenic trioxide,As2O3)及转录生长因子(transforming growth factor-β1,TGF-β1)对人HL-60细胞增殖的影响,并探讨相关信号通路.方法:流式细胞术检测细胞周期及凋亡,RT-PCR检测p27Kip1、TGF-β1、Cyclin E和Bcl-2表达,免疫组织化学方法和Western blot方法检测p27Kip1、TGF-β1、Cyclin E和Bcl-2蛋白水平.结果:As2O3、TGF-β1单药或联合处理均可诱导细胞凋亡,以联合处理组凋亡最明显,并且As2O3单药组及联合处理组细胞周期阻滞于G1期.As2O3单药及联合处理组可见p27Kip1、内源性TGF-β1表达上调,Cyclin E和Bcl-2表达下调,外源性TGF-β1处理组可见p27Kip1 mRNA及蛋白表达上调,内源性TGF-β1 mRNA表达上调,内源性TGF-β1蛋白水平与对照组比较无明显变化,Cyclin E和Bcl-2表达下调,联合处理组p27Kip1及内源性TGF-β1表达上调及Cyclin E和Bcl-2表达下调程度强于单药处理组.结论:As2O3诱导细胞凋亡的机制可能是通过上调TGF-β1,从而上调p27Kip1,拮抗Cyclin E和Bcl-2的作用,抑制细胞增殖,使细胞周期阻滞于G1期,从而诱导细胞发生凋亡,外源性TGF-β1通过上调内源性TGF-β1,从而上调p27Kip1,增强As2O3诱导细胞凋亡的作用.     

Down-regulation of wt1 expression in leukemia cell lines as part of apoptotic effect in arsenic treatment using two compounds

Arsenic trioxide (As2O3) induces remission in patients with acute promyelocytic leukemia (APL). To better understand molecular mechanisms of arsenic actions, this study investigated the effect of two different arsenic compounds on gene expression of apoptosis and cellular proliferation related genes. The Wilms' tumor gene (wt1) is up-regulated in acute myeloid leukemia (AML) and a variety of leukemia cell lines. The expression of wt1 in these cells is proposed to have an anti-apoptotic effect. HL-60 and K562 were treated with arsenic trioxide (As2O3) and sodium arsenite (NaAsO2) at concentrations between 0 - 10 microM for up to 48 h. The induction of apoptosis was accompanied by down-regulation of hTERT and wt1 mRNA and protein expression but up-regulation of par-4. Low concentrations of 0.1 microM arsenic induced expression of the anti-apoptotic bcl-2 gene in both cell lines HL-60 and K562. There were no major differences encountered between compounds. After arsenic treatment of the leukemia cell lines HL-60 and K562 the up-regulation of par-4 may contribute to the induction of apoptosis rather than down-regulation of bcl-2. The therapeutic effect of arsenic is the induction of apoptosis by modulating the gene expression profile of pro- and anti-apoptotic genes including the wt1 gene.     

急性早幼粒细胞白血病耐药机制的研究进展

全反式维甲酸(ATRA)与三氧化二砷(As2O3)作为一线药物治疗急性早幼粒细胞白血病(APL)以来,APL患者的治愈率得到显著提高.但ATRA和(或)As2O3耐药的出现成为一个严重问题.现就近年来新提出的一些耐药机制如融合基因突变、细胞信号通路异常、染色质重构复合物异常、凋亡调控异常、骨髓微环境介导耐药等方面进行综述.     

Apoptosis induction by (+)α-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells

We analyzed the effect of (+)α-tocopheryl succinate (α-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). α-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58 μM, for NB4 and NB4-R2, respectively. α-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34⁺ hematopoietic progenitors in methylcellulose assays. α-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when α-TOS was added 24 h after ATO, an additive effect was observed. Our results support the concept of α-TOS as an anti-leukemic compound which spares normal hematopoiesis.     

Arsenic trioxide induces apoptosis in leukemia/lymphoma cell lines via the CD95/CD95L system

Although arsenic trioxide (As2O3) has been shown to be an effective anticancer agent for acute promyelocytic leukemia (APL), its mechanisms of action as well as its effect on other leukemias than APL remain unclear. We studied in vitro effects of As2O3 at low concentrations (1.0-2.0 microM) on two human leukemia/lymphoma cell lines, HL-60, an acute myeloid leukemia cell line, and RL, a B-cell lymphoma cell line. As2O3 inhibited proliferation of HL-60 cells and RL cells to the similar degree to the reported inhibition by an APL cell line, NB4. As2O3-treated cell lines exhibited typical morphologic changes of apoptosis such as nuclear condensation and apoptotic bodies, and a cell cycle arrest at the subG1 phase. As2O3-treated cell lines also showed upregulation of CD95/CD95L expression and activation of caspases 8 and 3. Treatment of these cells with anti-CD95 antibodies capable of blocking the CD95 signaling pathway ameliorated As2O3-induced apoptosis. These data suggest that As2O3 can inhibit growth of leukemia/lymphoma cells by inducing the cell cycle arrest and apoptosis that is partially mediated by the CD95/CD95L system.     

曲古抑菌素A促进As2O3诱导HL-60细胞分化与分子机制的研究

目的：研究曲古抑菌素A能否促进小剂量三氧化二砷（As2O3）诱导HL-60细胞分化并对其机制进行初步探讨.方法：用MTT实验测定药物对细胞增殖的变化;流式细胞仪检测细胞表面CD11b表达率观察药物对细胞的分化作用; RT-PCR方法检测p21和cyclin D3在mRNA水平的表达变化.结果：HL-60细胞经曲古抑菌素A和（或）小剂量As2O3作用24 h后,联合用药组较单用As2O3组能明显抑制细胞增殖,促进细胞分化,p21在mRNA水平表达增加,cyclin D3在mRNA水平表达降低.结论：曲古抑菌素A能促进小剂量As2O3诱导HL-60细胞的分化,其机制可能与细胞周期蛋白cyclin D3和细胞周期蛋白依赖性激酶抑制剂p21的表达变化有关.