三氧化二砷诱导鼻咽癌CNE-2Z细胞凋亡及其作用机制的初步研究

目的:研究三氧化二砷(As2O3)对人鼻咽癌CNE2Z细胞的生物学效应及其作用机制。方法:台盼蓝计数法计算As2O3的IC50;CNE-2Z细胞经As2O3处理后,通过流式细胞仪、荧光染色及DNA电泳检测细胞凋亡;流式细胞仪测定bcl2和bax阳性细胞百分率;罗丹明123染色后进行线粒体膜电位分析。结果:As2O3在一定浓度范围内以浓度依赖的方式抑制CNE2Z细胞生长,其IC50为(1.35±0.30)μmol/L;0.5～2.0μmol/LAs2O3处理CNE2Z细胞后,流式细胞仪检测出凋亡峰,荧光显微镜下可见明显的细胞凋亡形态特征,琼脂糖凝胶电泳出现DNA梯形条带;As2O3对bcl2的表达没有影响(P>0.05),但能显著增加bax的表达(P<0.01)及降低线粒体膜电位(P<0.01)。结论:As2O3在体外可诱导鼻咽癌CNE2Z细胞凋亡,其机制可能与上调bax表达和降低线粒体膜电位有关。     

抑制细胞外信号调节激酶对As2O3诱导胃癌细胞系MGC-803凋亡的作用

目的探讨细胞外信号调节激酶在三氧化二砷(As2O3)诱导胃癌细胞系MGC-803凋亡的信号转导途径中的作用.方法采用MTT法检测As2O3对胃癌细胞系MGC-803的增殖抑制作用,采用瑞氏-姬姆萨染色和流式细胞仪检测As2O3诱导MGC-803凋亡以及加入选择性ERK抑制剂PD98059后细胞凋亡率的变化.结果5 μmol/L与10 μmol/L的As2O3作用24 h后凋亡率分别为(4.74±0.03)%和(11.93±0.06)%,而与PD98059联合作用24 h后凋亡率分别增加至(11.65±0.09)%和(18.15±0.10)%,P＜0.05.As2O3抑制MGC-803细胞的增殖,并诱导凋亡.结论预先抑制ERK途径增加了As2O3诱导MGC-803细胞的凋亡效应.ERK途径可能参与了As2O3诱导胃癌细胞系MGC-803凋亡的信号转导途径.     

三氧化二砷诱导雌激素受体阴性乳腺癌细胞株MDA-MB-435s凋亡和bcl-2、bax表达关系的研究

目的 观察不同浓度三氧化二砷(As2O3)诱导人乳腺癌细胞系MDA-MB-435s凋亡的情况及对bcl-2和bax表达的影响,初步探讨了其诱导凋亡的途径.方法 不同浓度As2O3体外作用于MDA-MB-435s细胞24h后,采用TUNEL染色法和DNA梯状电泳法检测细胞凋亡情况,应用免疫组化法检测细胞中bcl-2和bax蛋白表达情况.结果 As2O3作用后,DNA琼脂糖凝胶电泳法呈现凋亡DNA梯状条带;As2O3作用组的凋亡指数明显高于阴性对照组(P＜0.05);bcl-2表达下调,bax表达上调.结论 As2O3能诱导MDA-MB-435s细胞凋亡,上调bcl-2蛋白表达、下调bax蛋白表达是其诱导凋亡的途径之一.     

三氧化二砷联合甲磺酸伊马替尼、咖啡因对K562白血病细胞的协同作用

 目的 探讨三氧化二砷（Arsenic trioxide,As2O3）与酪氨酸激酶抑制剂甲磺酸伊马替尼（实验药物代号STI571）以及细胞周期调节剂咖啡因联合诱导K562细胞凋亡的作用及机制,为寻找克服K562细胞对As2O3抵抗的有效手段提供实验依据。方法 以As2O3与STI571、咖啡因联合作用于K562细胞,采用MTT方法检测细胞增生活性,PI染色流式细胞仪检测细胞周期,PI和Annexin V双染色流式检测细胞凋亡,Western blot法检测细胞周期相关调节蛋白的表达。结果 5.0 μmol／L浓度的咖啡因对K562细胞增生无抑制作用,亦不能增强As2O3的抑制作用,单独以STI571 1.0 μmol／L处理,即可有效抑制K562细胞的生长,与As2O3联用能明显增加其抑制增生的效应;咖啡因与As2O3联合作用,不增加As2O3诱导的K562细胞凋亡率[（14.7±3.6）%vs（15.3±3.3）%,P＞0.05]; STI571具有轻度诱导K562细胞凋亡的作用[（18.3±4.5）%],与As2O3合用可显著增加诱导凋亡率[（14.7±3.6）%vs（42.8±4.2）%,P＜0.01],并明显降低As2O3诱导的G2／M期细胞比例。与单用As2O3比较,As2O3 + STI571明显抑制cdc2、cdc2-p及survivin蛋白表达,而As2O3与咖啡因合用不能诱导survivin蛋白表达下调,对cdc2、cdc2-p蛋白的表达无明显影响。结论 周期调节药物咖啡因对As2O3诱导K562细胞凋亡无增敏作用;酪氨酸激酶抑制剂STI571能协同As2O3诱导K562细胞凋亡,下调抗凋亡蛋白survivin的表达可能是其机制之一,值得深入研究其临床应用效果。     

三氧化二砷和胂酸基乙酸体外抗癌作用的比较

背景与目的:近来,有研究表明某些有机砷诱导细胞凋亡的能力强于无机砷,胂酸基乙酸(arsacetyl,ASAC)为有机砷的一种。本研究比较三氧化二砷(As2O3)和ASAC对肝癌细胞的抑制作用及诱导凋亡作用。方法:采用MTT(methythiazolyltetrazolium)法和细胞凋亡检测法观察As2O3和ASAC对SMMC-7721细胞增殖的影响及作用机制;用MTT法检测药物对正常血管内皮细胞(VEC)毒副作用。结果:As2O3和ASAC都明显抑制细胞增殖,抑制作用呈时间和浓度依赖性,且ASAC的抑制作用明显强于As2O3。如药物作用48h,0.1μmol/LAs2O3组抑制率为(14.2±1.5)%,0.1μmol/LASAC组为(27.1±3.0)%。1μmol/LAs2O3和0.1μmol/LASAC对VEC几乎无毒副作用,同样条件下作用于SMMC-7721细胞株,有凋亡小体生成,凝胶电泳出现DNA梯度图谱,流式细胞检查见明显的凋亡峰出现,并使细胞周期阻滞在S+G2/M期。结论:As2O3和ASAC都可以在体外诱导肝癌细胞凋亡,且ASAC诱导凋亡的能力强于As2O3,两者都作用于G0/G1期细胞。     

三氧化二砷联合miR-203对人白血病K562细胞的抑制作用及其机制

目的： 研究三氧化二砷（As 2O 3）联合过表达的微小RNA-203（microRNA-203,miR-203）对白血病K562细胞的抑制作用及其可能的分子机制。 方法： 将miR-203的真核表达载体pmiR-203转染K562细胞,Real-time PCR检测细胞内miR-203的表达。将K562细胞分为空白对照组、As 2O 3组、pmiR-203组、空质粒对照组、As 2O 3联合空质粒对照组和As 2O 3联合pmiR-203组,MTT法检测各组细胞增殖抑制率,流式细胞术仪检测细胞凋亡率,Western Blotting检测细胞内Bcr/abl蛋白的表达水平。构建Bcr/abl 3′UTR和Bcr/abl mut-3′UTR的双荧光素酶报告基因载体,将其与pmiR-203共转染K562细胞,通过荧光素酶活性分析判断miR-203是否与Bcr/abl基因的3′UTR结合。 结果： miR-203的真核表达载体pmiR-203转染K562细胞后,细胞内miR-203的表达水平明显增高( P <0.05)。高表达miR-203联合As 2O 3使K562细胞对As 2O 3的敏感性提高到单用As 2O 3的4.86倍,IC50 从3.4 μmol/L降低至0.7 μmol/L,两者联用表现为协同作用。As 2O 3联合pmiR-203组的细胞凋亡率显著高于As 2O 3联合空质粒对照组\[（29.97±3.19）% vs （10.77±1.71）%, P <0.05\]。过表达miR-203显著下调K562细胞内Bcr/abl蛋白的表达水平。Bcr/abl 3′UTR中带有明确的miR-203结合位点。 结论： miR-203可提高白血病K562细胞对As 2O 3的敏感性,miR-203和As 2O 3联用对K562细胞具有协同抑制作用,其作用机制可能与miR-203直接下调Bcr/abl融合基因的表达有关。     

紫杉醇诱导黑色素瘤A375细胞凋亡的分子机制

目的:探索紫杉醇对A375细胞的诱导凋亡作用及其机制。方法:采用四甲基偶氮唑蓝(MTT)法检测紫杉醇对细胞增殖能力及细胞生长的影响;流式细胞仪分析及细胞形态学观察检测细胞周期和细胞凋亡;Westernblot方法检测bcl2、Bax和Caspase3蛋白表达。结果:0.001～1.000μmol/L紫杉醇以时间和剂量依赖方式抑制A375细胞生长和诱导细胞凋亡,0.000～1.000μmol/L紫杉醇作用24h时,早期细胞凋亡率由(0.5±0.1)%增至(32.4±1.1)%,P<0.05,0.100μmol/L紫杉醇作用24和48h时,早期细胞凋亡率由(20.9±0.9)%增至(52.6±1.0)%,t=28.89,P=0.001;不同浓度紫杉醇诱导A375细胞凋亡的机制不同,0.001μmol/L时不影响细胞周期,0.010μmol/L时使G0/G1期细胞含量明显减少,0.100和1.000μmol/L时使细胞发生G2/M期阻滞。紫杉醇作用24h后,Caspase3被激活,同时bcl2和Bax蛋白表达分别被下调和上调。结论:紫杉醇可诱导A375细胞凋亡,bcl2/Bax蛋白比值的下降及Caspase3的激活参与了该细胞凋亡过程,但高浓度紫杉醇通过作用微管,低浓度紫杉醇则通过尚不清楚途径实现细胞凋亡过程。     

二烯丙基二硫诱导人鼻咽癌CNE2细胞凋亡及其可能的分子机制

背景与目的：二烯丙基二硫（DADS）是大蒜中的一种脂溶性单体化合物,对多种肿瘤细胞有促凋亡作用.本实验旨在探讨DADS抑制人鼻咽癌CNE2细胞增殖与诱导凋亡的生物学效应及可能的分子机制.方法：分别采用MTT、光学显微镜、流式细胞仪、DNA琼脂糖凝胶电泳与Western blot检测DADS抑制CNE2细胞增殖与诱导凋亡作用及Bcl-2、Bax和半胱氨酸天冬氨酸蛋白酶（Caspase 3）蛋白的表达.结果：MTT结果显示,50、100、200及400 μmol/L DADS作用CNE2细胞48 h,抑制率分别为3.66%、14.25%、29.66%及61.28%,呈浓度依赖性（P＜0.05）.形态学观察发现,DADS处理24 h后,部分CNE2细胞变圆,体积明显缩小,胞质强嗜酸性,核固缩、深染,核染色质积聚至核膜周边,呈现凋亡细胞形态学改变.流式细胞术分析显示,50、100、200和400 μmol/L DADS作用24 h,凋亡率分别为（10.8±1.3）%、（14.8±1.1）%、（21.7±2.0）%及（29.8±2.6）%,呈浓度依赖性诱导CNE2细胞凋亡（n=3,P＜0.05）.200及400 μmol/L DADS分别作用CNE2细胞24 h后,DNA凝胶电泳出现典型梯形条带.Western blot检测显示,50、100、200和400 μmol/L DADS处理CNE2细胞24 h后,Bcl-2蛋白与β-Actin的灰度值比分别是1.92±0.21、1.21±0.18、0.89±0.14及0.41±0.09,与对照组（2.24±0.26）比较,差异有显著性（P＜0.05）;Bax蛋白与β-Actin的灰度值比分别是0.78±0.14、1.12±0.19、1.54±0.22及2.08±0.27,与对照组（0.33±0.08）比较,差异有显著性（P＜0.05）;随浓度的增加,Bcl-2蛋白下调,Bax蛋白表达上调.200、400 μmol/L DADS处理CNE2细胞24 h后,与对照组相比,Caspase 3酶原蛋白条带变细并且可见到蛋白裂解产物,表明DADS可以促进Caspase 3的活化.结论：DADS具有抑制人鼻咽癌CNE2细胞增殖和诱导凋亡的作用,其机制可能与Bcl-2蛋白下调,Bax蛋白表达上调导致Bcl-2/Bax比值下降,促进Caspase 3的活化有关.     

三氧化二砷在诱导非小细胞肺癌NCI-H157细胞凋亡过程中对Survivin基因表达Caspase-3的影响

目的：研究三氧化二砷（As2O3）在诱导非小细胞肺癌NCI-H157凋亡过程中时Survivin基因表达及Cas-pase-3的影响．探讨As2O3，诱导肿瘤细胞凋亡的作用机理研究，方法：首先对NCI-H157细胞培养和增殖测定，然后采用RT-PCR法检测Surviv inⅡ1RNA表达；WestemBlot检测Survivin及Caspase-3蛋白，结果：RT-PCR结果显示，10txmol／LAs203作用于NCI-H157细胞12h，24h．48h同对照组相比，Survivin mNA表达分别降至85．7％，59．4％，22．5％，处理72h后几乎检测不到Survivin mNA的表达。，WesternBlot解析结果显示．Survivin蛋白表达在5txmol／LAs203处理后分别下调至75．2％（12h），54．3％（24h），37．5％（48h）及20．3％（72h）。分别用不同浓度的As2O3，5220moL／L作用NCI-H157细胞24h．Survivin蛋白表达随着A蜘0、浓度的增加逐渐下降．As2O3，作用12h之内只能检测到非激活状态32KD的proCaspase-3，处理24h后出现17KD的活性亚基，直至72h。结论：As2O33以时间、剂量依赖的方式抑制NCI-H157细胞增殖，诱导细胞凋亡过程中下调Survivin mNA及蛋白表达，并活化Caspases-3．提示As2O3、诱导的NCI-H157细胞凋亡机制可能与下调Survivin基因表达及激活Caspase-活性有关、     

三氧化二砷诱导胃癌细胞凋亡及对相关基因表达的影响

目的 研究三氧化二砷（As2O3)诱导胃癌细胞的凋亡作用及对凋亡相关基因p53、c-myc、bcl-2、bcl-xl和bcl-xs表达的影响,探讨其诱导胃 癌凋亡的作用机制。方法 研究As2O3对胃癌细胞SGC－07901和MKN－45的诱导凋亡作用和对凋亡相关基因表达的影响。结果 As2O3作用于细胞可见典型的细胞凋亡。出现典型的亚二倍体“凋亡峰”。10μmol／LAs2O3的诱导胃癌细胞SGC－7901和MKN－45的凋亡率为13．8％和22．5％。细胞凋亡指数在1．42％－9．5％之间。As2O3作用后能明显下调SGC－7901细胞的bcl-2蛋白和mRNA的表达;对SGC－7901细胞的p53、c-myc、bcl-xl和bcl-xs蛋白的表达无影响。As2O3能促进MKN－45细胞的p53蛋白和mRNA的表达,对MKN－45细胞的bcl-2、c-myc、bcl-xl和bcl-xs基因表达无影响。结论 As2O3可通过不同途径诱导胃癌细胞凋亡,通过下调SGC－7901细胞的bcl-2的蛋白表达诱导其凋亡,通过上调p53表达诱导胃癌MKN－45细胞凋亡。     

Indomethacin induces apoptosis and inhibits proliferation in chronic myeloid leukemia cells

A nonsteroidal anti-inflammatory drug (NSAID)--indomethacin (IN), was found to induce apoptosis and inhibit proliferation of K562 cells and primary culture bone marrow cells from six chronic myelogenous leukemia (CML) patients. IN induced cells apoptosis and inhibited cells proliferation in a dose-and time-dependent manner, the optimum IN concentration and incubation time for eliciting these effects were 400 micromol/l and 72 h, respectively. A synergic effect on Vp-16 (2.5 microg/ml) induced apoptosis was observed when combined with 100 micromol/l IN in K562 cells. RT-PCR results showed that IN down-regulated Bcl-2 mRNA expression, and did not change Bax mRNA expression; Western blot results confirmed that IN inhibited Bcl-2 protein expression, no influence was found on the translative level of bax protein. Our study indicate that IN induce apoptosis of CML cells by down-regulating Bcl-2 expression partially, and there is a potential significance in the treatment of CML.     

Apoptosis and erythroid differentiation triggered by Bcr-Abl inhibitors in CML cell lines are fully distinguishable processes that exhibit different sensitivity to caspase inhibition

Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.     

Imatinib (STI571)-mediated changes in glucose metabolism in human leukemia BCR-ABL-positive cells.

The therapeutic efficacy of imatinib mesylate (Gleevec) is based on its specific inhibition of the BCR-ABL oncogene protein, a widely expressed tyrosine kinase in chronic myelogenous leukemia (CML) cells. The goal of this study was to evaluate glucose metabolism in BCR-ABL-positive cells that are sensitive to imatinib exposure. Two human BCR-ABL-positive cell lines (CML-T1 and K562) and one BCR-ABL-negative cell line (HC-1) were incubated with different imatinib concentrations for 96 hours. Magnetic resonance spectroscopy on cell acid extracts was performed to evaluate [1-13C]glucose metabolism, energy state, and changes in endogenous metabolites after incubation with imatinib. Imatinib induced a concentration-dependent inhibition of cell proliferation in CML-T1 (IC50, 0.69 +/- 0.06 micromol/L) and K562 cells (IC50, 0.47 +/- 0.04 micromol/L), but not in HC-1 cells. There were no metabolic changes in imatinib-treated HC-1 cells. In BCR-ABL-positive cells, the relevant therapeutic concentrations of imatinib (0.1-1.0 micromol/L) decreased glucose uptake from the media by suppressing glycolytic cell activity (C3-lactate at 0.25 mmol/L, 65% for K562 and 77% for CML-T1 versus control). Additionally, the activity of the mitochondrial Krebs cycle was increased (C4-glutamate at 0.25 micromol/L, 147% for K562 and 170% for CML-T1). The improvement in mitochondrial glucose metabolism resulted in an increased energy state (nucleoside triphosphate/nucleoside diphosphate at 0.25 micromol/L, 130% for K562 and 125% for CML-T1). Apoptosis was observed at higher concentrations. Unlike standard chemotherapeutics, imatinib, without cytocidal activity, reverses the Warburg effect in BCR-ABL-positive cells by switching from glycolysis to mitochondrial glucose metabolism, resulting in decreased glucose uptake and higher energy state.     

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.     

Hu联合GM-CSF对K562细胞生长-凋亡相关基因表达的影响

探讨羟基脲（ｈｙｄｒｏｘｙｕｒｅａ,Ｈｕ）及Ｈｕ联合粒系－巨噬系集落刺激因子（ｇｒａｎｕｌｏｃｙｔｉｃ ｍａｃｒｏｐｈａｇｅ ｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇ ｆａｃｔｏｒ,ＧＭ－ＣＳＦ）对慢性粒细胞白血病（ｃｈｒｏｎｉｃ ｍｙｅｌｏｇｅｎｏｕｓ ｌｅｕｋｅｍｉａ,ＣＭＬ）细胞株Ｋ５６２细胞生长及凋亡相关基因表达的调节效应。方法：以Ｈｕ及Ｈｕ联合ＧＭ－ＣＳＦ培养Ｋ５６２细胞,采用逆转录－聚合酶     

Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in?vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In?vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.     

Cotreatment with vorinostat enhances activity of MK-0457 (VX-680) against acute and chronic myelogenous leukemia cells

PURPOSE: We determined the effects of vorinostat (suberoylanalide hydroxamic acid) and/or MK-0457 (VX-680), an Aurora kinase inhibitor on the cultured human (HL-60, OCI-AML3, and K562) and primary acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and mutant forms of Bcr-Abl. EXPERIMENTAL DESIGN: Following exposure to MK-0457 and/or vorinostat, apoptosis, loss of viability, as well as activity and levels of Aurora kinase and Bcr-Abl proteins were determined. RESULTS: Treatment with MK-0457 decreased the phosphorylation of Aurora kinase substrates including serine (S)10 on histone H3 and survivin, and led to aberrant mitosis, DNA endoreduplication as well as apoptosis of the cultured human acute leukemia HL-60, OCI-AML3, and K562 cells. Combined treatment with vorinostat and MK-0457 resulted in greater attenuation of Aurora and Bcr-Abl (in K562) kinase activity and levels as well as synergistically induced apoptosis of OCI-AML3, HL-60, and K562 cells. MK-0457 plus vorinostat also induced synergistic apoptosis of BaF3 cells with ectopic overexpression of wild-type or mutant Bcr-Abl. Finally, cotreatment with MK-0457 and vorinostat induced more loss of viability of primary AML and imatinib-refractory CML than treatment with either agent alone, but exhibited minimal toxicity to normal CD34+ progenitor cells. CONCLUSIONS: Combined in vitro treatment with MK-0457 and vorinostat is highly active against cultured and primary leukemia cells. These findings merit in vivo testing of the combination against human AML and CML cells, especially against imatinib mesylate-resistant Bcr-AblT315I-expressing CML Cells.     

In vitro effect of adenovirus-mediated human Gamma Interferon gene transfer into human mesenchymal stem cells for chronic myelogenous leukemia

For developing gene therapy for chronic myelogenous leukemia (CML), we evaluated the feasibility of using autologous bone marrow stromal cells (BMSCs) of one CML patient as a target cell population and studied the efficiency of recombinant adenovirus-mediated human Gamma Interferon (hIFN-gamma) gene transfer into BMSCs. BMSCs can be readily obtained, expanded, and successfully transduced with adenoviral vectors in vitro. We studied the in vitro expression of hIFN-gamma in human BMSCs following transduction with Ad/hIFN-gamma. On transduction of BMSCs at a MOI of 50, the expression and secretion of hIFN-gamma were achieved as high as 5492 +/- 660 approximately 50647 +/- 4049 ng/10(6) cells per 24 h over the course of 3 weeks. We further studied the effects of hIFN-gamma produced by transduced BMSCs on the proliferation of the human leukemia cell line K562 cells in vitro, proliferation of K562 cells was markedly inhibited in the experimental groups as compared with the other two control groups after 5 days of coculture. We also found that the percentage of K562 cells in the G(1) phase of cell cycle can be increased by treatment of hIFN-gamma produced by Ad/hIFN-gamma transduced BMSCs, but the percentage of K562 cells in the S phase of cell cycle can be decreased in the same time. Apoptosis rate of K562 cells in the experimental groups was 30.8 +/- 8.5%, as compared with the other two control groups (5.6 +/- 1.3% and 5.5 +/- 0.8%, respectively) (p < 0.01). Our results indicate that hIFN-gamma gene engineered BMSCs of CML donors could be successfully established and that local production of hIFN-gamma is sufficiently to inhibit the proliferation of K562 cells and induce apoptosis of K562 cells in vitro, suggesting an important potential use in the clinical gene therapy of CML.