三氧化二砷对HL-60细胞hTERT基因表达和端粒酶活性的影响

目的：探讨三氧化二砷（As2O3)处理HL－60细胞前后人类端粒酶逆转灵酶（hTERT)基因表达和端粒酶活性的改变。方法：采用姬姆萨染色及碘化丙锭染色置流式细胞仪检测来观察细胞的凋亡，以RT－PCR分析hTERT 基因的mRNA表达水平；应用间接免疫荧光标记法通过流式细胞仪检测hTERT蛋白含量；利用多聚酶链反应－酶联免疫反应（PCR－ELISA）方法检测As2O3处理HL－60前后端粒酶活性的改变。结果：2umol/L As2O3诱导HL－60细胞凋亡的过程中，hTERT mRNA表达水平显著下降，细胞内hTERT蛋白含量也相应降低，其端粒酶活性明显受到抑制，结论：As2O3可通过下调hTERT基因的表达而抑制HL－6细胞的端粒酶活性。     

Arsenic trioxide induces oxidative stress,DNA damage,and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells

Background

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of all acute myloid leukemia cases. It is a blood cancer that is formed by chromosomal mutation. Each year in the United States, APL affects about 1,500 patients of all age groups and causes approximately 1.2% of cancer deaths. Arsenic trioxide (ATO) has been used successfully for treatment of APL patients, and both induction and consolidated therapy have resulted in complete remission. Recently published studies from our laboratory have demonstrated that ATO pharmacology as an anti-leukemic drug is associated with cytotoxic and genotoxic effects in leukemia cells.

Methods

In the present study, we further investigated the detailed molecular mechanism of ATO-mediated intrinsic pathway of apoptosis; using HL-60 cells as a test model. Oxidative stress was assessed by spectrophotometric measurements of MDA and GSH levels while genotoxicity was determined by single cell gel electrophoresis (Comet assay). Apoptosis pathway was analyzed by Western blot analysis of Bax, Bcl2 and caspase 3 expression, as well as immunocytochemistry and confocal imaging of Bax and Cyt c translocation and mitochondrial membrane potential depolarization.

Results

ATO significantly (p < 0.05) induces oxidative stress, DNA damage, and caspase 3 activityin HL-60 cells in a dose-dependent manner. It also activated the intrinsic pathway of apoptosis by significantly modulating (p < 0.05) the expression and translocation of apoptotic molecules and decreasing the mitochondrial membrane potential in leukemia cells.

Conclusion

Taken together, our research demonstrated that ATO induces mitochondrial pathway of apoptosis in HL-60 cells. This apoptotic signaling is modulated via oxidative stress, DNA damage, and change in mitochondrial membrane potential, translocation and upregulation of apoptotic proteins leading programmed cell death.     

三氧化二砷对HL-60细胞凋亡及Survivin和Caspase-3表达的影响

目的:探讨三氧化二砷(As2O3)诱导HL-60细胞凋亡过程中Survivin基因表达以及Caspase-3蛋白变化情况。方法:采用7.5μmol/L As2O3和20μmol/LAc-devd-cho单独或联合作用于HL-60细胞,半定量逆转录聚合酶链反应(RT-PCR)检测Survivin mRNA、免疫组织化学法检测Caspase-3蛋白的变化,流式细胞术检测HL-60细胞的凋亡率。结果:7.5μmol/LAs2O3作用HL-60细胞24h和48h后,其凋亡率分别为(31.93±0.34)%和(55.91±0.52)%,Survivin mRNA比值分别为51.42%和22.37%,Caspase-3蛋白激活逐渐增加;20μmol/L Ac-devd-cho对HL-60细胞凋亡和Survivin mRNA表达均无影响;20μmol/L Ac-devd-cho和7.5μmol/L As2O3共同作用HL-60细胞24h和48h,细胞凋亡率分别为(9.61±0.21)%和(13.05±0.35)%,分别与As2O3单独作用组相比有显著性差异(P<0.05),Survivin mRNA比值分别为52.14%和21.08%,分别与As2O3单独作用组相比无显著性差异(P>0.05),Caspase-3蛋白激活被阻断。结论:As2O3诱导HL-60细胞凋亡与抑制Survivin基因表达、激活Caspase-3蛋白的活性有关。     

Z-ajoene引起HL-60细胞G2/M期阻滞及端粒酶表达活性的降低

目的 探讨Z-ajoene诱导细胞周期阻断于G2／M期的分子机制以及对端粒酶活性的抑制作用。方法 采用MTT法测定Z-ajoene对HL-60细胞的增殖抑制率;以流式细胞仪测定Z-ajoene对HL-60细胞的周期阻断作用,并采用Western blot法测定相关蛋白p34^cdc2、cyclinB1、cyclinA的表达水平;TRAP-银染法测定端粒酶活性的变化,同时采用RT-PCR方法检测端粒酶hTRT和TP1mRNA的表达。结果 10μmol／L Z-ajoene作用后能明显将HL-60细胞阻滞于G2／M期。在药物作用10h后,G2／M期细胞的比例达到顶峰,与对照组比较增加了1．95倍。另外,10μmol／L Z-ajoene作用后出现cyclinB1蛋白的聚集及p34^cdc2蛋白表达的降低,但cyclinA蛋白的水平并无显著性变化。经Z-ajoene作用24h后,HL-60细胞端粒酶活性显著降低。而不同浓度的药物作用12h,其端粒酶活性均无显著性变化。在10μmol／L Z-ajoene作用24h后,能够降低端粒酶hTRT和TP1 mRNA的水平。结论 Z-ajoene能够在作用较短时问内明显将细胞周期阻断于G2／M期,同时伴有cyclinB1聚集及p34^cdc2的抑制;而HL-60细胞的端粒酶活性仅在药物作用较长时间后才有一定降低,并且端粒酶hTRT和TP1mRNA的表达也同时降低。结果表明,Z-ajoene的细胞周期阻断作用可能是造成端粒酶缺失及HL-60细胞生长抑制的重要原因。     

hTERT反义寡核苷酸对HL-60细胞凋亡及细胞周期的影响

目的:研究hTERT基因反义寡核苷酸(ASODN)对HL-60细胞诱导凋亡作用及细胞周期的影响。方法:应用hTERT ASODN封闭HL-60细胞hTERT基因,RT-PCR方法检测目的基因的表达;流式细胞仪检测细胞凋亡及细胞周期分析,TUNEL方法检测细胞凋亡,琼脂糖凝胶电泳检测凋亡细胞DNA梯带。结果:hTERT ASODN作用细胞72h后,hTERT基因表达明显受到抑制(P<0.01)。流式细胞仪检测显示:ASODN组细胞阻滞于G₀/G₁期,S、G₂/M期细胞减少(P<0.05);细胞增殖指数(PI)明显降低(P<0.05);检测出早期凋亡峰。Annexin V/PI检测及TUNEL检测均显示:ASODN组凋亡细胞阳性率明显高于SODN组(P<0.01)。琼脂糖凝胶电泳显示:ASODN组出现凋亡DNA梯带。结论:hTERT ASODN能够封闭目的基因表达,阻滞HL-60细胞于G₀/G₁期,并诱导细胞凋亡,对治疗白血病具有潜在应用价值。     

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.     

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。     

黄芩苷作用HL-60细胞前后c-myc基因表达的变化

 目的 研究中药黄芩苷（baicalin）对人髓系白血病细胞株HL-60细胞增生、凋亡的影响及探讨c-myc基因在其中的作用。方法 培养人髓系白血病细胞株HL-60细胞,应用MTT法绘制细胞生长曲线观察黄芩苷对HL-60细胞增生的影响;DNA凝胶电泳观察细胞凋亡;RT-PCR检测黄芩苷作用前后c-myc基因mRNA表达水平的变化;Western blot检测黄芩苷作用前后c-myc蛋白表达水平的变化。结果　细胞生长曲线结果示黄芩苷能明显抑制HL-60细胞增生,半数抑制浓度（IC50）约为20μmol／L,DNA 片段化的检出表明黄芩苷能有效诱导HL-60细胞凋亡;黄芩苷作用后HL-60细胞c-myc基因和蛋白的表达水平均下降,并且随作用时间延长,表达下降更明显。结论 黄芩苷能有效抑制HL-60细胞增生,诱导其凋亡,c-myc基因可能参与了黄芩苷抑制HL-60细胞增生和诱导凋亡的过程。     

苦参碱诱导HL-60细胞株凋亡作用的实验研究

 目的　研究苦参碱（MAT）诱导HL-60细胞株凋亡的作用及其机制。方法　采用 CCK-8法检测不同浓度MAT对HL-60细胞的增殖抑制作用;流式细胞术检测细胞凋亡以及线粒体跨膜电位的变化;分光光度法检测Caspase-9活性。结果　0.25～2.0 mg／ml MAT对HL-60细胞有生长抑制作用,与对照组相比差异有统计学意义（F＝67.83,P＜0.05）;MAT处理48 h后,细胞凋亡率明显增高,并呈剂量依赖性（t值分别为4.685、6.300、9.641、6.786,均P＜0.05）;1.0 mg／ml MAT处理后48 h内,细胞线粒体跨膜电位逐渐降低（F＝54.83,P＜0.05）,Caspase-9活性逐渐升高,呈时间依赖性（F＝72.31,P＜0.05）。结论　MAT可能通过降低细胞线粒体跨膜电位、激活Caspase-9而诱导HL-60细胞凋亡。     

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.     

全反式维甲酸对HL-60细胞hTERT基因表达和端粒酶活性的影响

目的 探讨全反式维甲(ATRA)诱导HL-60细胞分化过程中人类端粒酶逆转录酶(hTERT)蛋白表达和端粒酶活性的改变。 方法 应用间接免疫荧光标记法通过流式细胞仪检测hTERT蛋白含量的变化;采用多聚酶链反应-酶联免疫反应(PCR-ELISA)方法检测ATRA处理HL-60细胞前后端粒酶活性的改变,用碘化丙锭染色经流式细胞仪检测细胞周期变化。 结果 1μmol/L ATRA作用HL-60细胞24、48、72h,hTERT蛋白平均荧光强度分别为61.87±4.36、37.47±2.85、33.45±2.37,与空白对照组相比,具有显著性差异,P<0.05。1μmol/L ATRA作用48h后,HL-60细胞的端粒酶活性即出现下降,作用72h,端粒酶的活性明显受到抑制。 结论 在HL-60细胞分化过程中,ATRA能抑制HL60细胞的hTERT基因的表达和端粒酶活性。     

Alkylphosphocholines induce apoptosis in HL-60 and U-937 leukemic cells

Alkylphosphocholines (APC) represent a new group of ether-lipid-related compounds with remarkable activity against transformed cells in vitro and good tolerability in vivo. Their mechanism of action remains unknown. The aim of the present study was to investigate the effects of a series of APC on three human leukemic cell lines: K-562, HL-60, and U-937. The tetrazolium dye-reduction (MTT) assay and cell counting were used to determine the cytotoxicity of the APC used. DNA gel electrophoresis and enzyme-linked immunosorbent assay (ELISA) detection of oligonucleosomes were performed to identify and quantify DNA fragmentation. Electron and phase-contrast microscopy were used to detect morphologic changes specific for programmed cell death. HL-60 and U-937 cells were found to be sensitive, but K-562 cells were relatively resistant to APC exposure. APC with long alkyl chains exerted stronger cytotoxicity than did those with short alkyl chains. DNA fragmentation was found after treatment with APC in HL-60 and U-937 cells but not in K-562 cells. In HL-60 cells the increase in mono- and oligonucleosome formation as measured by ELISA was correlated with the length of the alkyl chains at 14 h of exposure to APC but plateaued at 20 h. The morphologic alterations in HL-60 and U-937 cell lines, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies, confirmed the induction of apoptosis after APC exposure. It is concluded that programmed cell death plays an important role in the cytotoxicity of APC against certain human leukemic cell lines. The antineoplastic profiles of APC with long alkyl chains render them attractive for further therapeutic application. Received: 1 September 1996 / Accepted 4 July 1997     

hTERT基因反义核酸对K562细胞端粒酶活性的影响

探讨人类端粒酶逆转录酶（ｈｕｍａｎ ｔｅｌｏｍｅｒａｓｅ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔａｓｅ,ｈＴＥＲＴ）基因的反义寡核苷酸（ａｎｔｉｓｅｎｓ ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,ＡＳＯＮＤ）对Ｋ５６２细胞端粒酶活性的影响。方法：采用多聚酶链反应－酶联免疫测定（ＰＣＲ－ＥＬＩＳＡ）方法检测人工合成的反义脱氧寡核苷酸处理Ｋ５６２细胞前后端粒酶活性的改变,以逆转录ＰＣＲ（ＲＴ－ＰＣＲ）分     

三氧化二砷对肝癌细胞survivin基因和端粒酶活性的影响

目的:探讨三氧化二砷(As2O3)诱导肝癌细胞凋亡时survivin基因表达和端粒酶活性的变化。方法:采用倒置相差显微镜观察细胞形态,流式细胞仪分析细胞亚二倍体百分率,逆转录-多聚酶链式反应检测细胞survivin mRNA表达,免疫组织化学染色检测细胞survivin蛋白的表达,端粒酶检测试剂盒检测端粒酶活性。结果:0.25~2.00μmol/L As2O3明显抑制肝癌Bel-7402细胞生长,诱导细胞凋亡,survivin mRNA和蛋白表达上调,端粒酶活性下降,呈时间、剂量依赖关系。结论:As2O3抑制肝癌Bel-7402细胞增殖,诱导细胞凋亡,可能与其降低端粒酶活性有关;而survivin基因表达上调,可能是抵抗As2O3诱导凋亡的途径之一。     

阿糖胞苷诱导HL-60细胞端粒酶各组分基因mRNA水平变化的研究

背景与目的阿糖胞苷(Ara-C)是治疗急性非淋巴细胞白血病常用而有效的药物之一。端粒酶是一种特殊的核糖核蛋白复合物,在肿瘤的发生、发展中起着重要作用。本研究以Ara-C为诱导剂,检测了不同浓度、不同时间Ara-C作用HL-60细胞端粒酶各组分基因mRNA水平的变化,试图为临床阿糖胞苷的用药和评判化疗药效提供一些理论上的依据和指导。方法采用不同浓度Ara-C作用相同时间、同一浓度Ara-C作用不同时间来处理HL-60细胞,通过流式细胞仪观察其凋亡和坏死细胞比例,RT-PCR方法检测端粒酶各组分基因hTERT、hTR和hTP1的mRNA水平变化。结果①体外小剂量0~0.2μg/ml的Ara-C诱导HL-60细胞12h,端粒酶各组分基因在mRNA水平上无变化;②2μg/ml和10μg/mlAra-C组,端粒酶hTERTmRNA水平由0.80±0.07分别下调至0.50±0.04和0.39±0.03而hTR、hTP1mRNA无变化;③10μg/ml浓度的Ara-C作用HL-60细胞不同时间,随时间推移,细胞凋亡比例增加,12h处达到最大值(18.16±4.25)%,随后降低;而细胞坏死比率一直随时间的延长而增加,48h处的坏死比例在本实验的时间段处最大,为(57.94±12.03)%,同时hTERT基因mRNA水平也随着时间的延长而逐渐降低,48h处达最低值0.18±0.03,而hTR、hTP1mRNA水平不随时间的延长而改变。结论①阿糖胞苷下调hTERT基因的mRNA水平,且有浓度和时间依赖性,而对hTR基因、hTP1基因的mRNA水平无影响;②阿糖胞苷下调hTERT基因mRNA水平可能与阿糖胞苷诱导细胞坏死有关,而与诱导细胞凋亡无关。     

雷公藤多甙诱导HL-60细胞凋亡

目的：探讨雷公藤多甙对ＨＬ－６０细胞凋亡的作用。方法：应用细胞形态学检查,ＤＮＡ凝胶电泳及流式细胞仪分析检测。结果：ＨＬ－６０细胞加上雷公藤多甙１０ｍｇ／Ｌ孵育７２ｈ,流式细胞仪检测细胞凋亡率为２９．４％（与空白对照组比较,Ｐ〈０．０１）。电检查和光镜检查均可见细胞核固缩、碎裂等。ＤＮＡ电泳显示明显的梯状条带。结论：雷公藤多甙能诱导ＨＬ－６０细胞凋亡,提示雷公藤多甙可能具有抗白血病作用。     

P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL-60 cells

Purpose One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3.Materials and methods Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 M or with UVC to induce apoptosis. Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting.Results Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation. No active caspase-8 was detected. Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells.Conclusions Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells. However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.