CCAAT增强子结合蛋白α在甾醇类新药NSC67657诱导HL60细胞向单核系分化中的作用

目的 探讨CCAAT增强子结合蛋白α(C/EBPα)在甾醇类新药NSC67657诱导HL60细胞向单核系分化中的作用.方法 采用NSC67657诱导HL60细胞分化,流式细胞仪检测细胞表面分化抗原CD14的表达.应用逆转录聚合酶链反应(RT-PCR)和Western blot方法连续检测C/EBPα基因和蛋白在细胞分化前后的表达情况.构建pDsRed-C/EBPα真核表达载体,基因测序后采用电转染技术,将真核表达载体转入HL60细胞中,并进行表达验证.采用四甲基偶氮唑蓝(MTT)法、瑞氏染色和流式细胞技术,观察转染细胞在NSC67657作用前后的增殖和分化情况.结果 10 μmol/L NSC67657可以诱导HL60细胞向单核系分化,连续诱导5 d后,有93.9%的细胞有CD14阳性表达.分化后的HL60细胞中,C/EBPα基因和蛋白表达均先升高后下降,分别在第2天和第3天达到峰值.真核表达载体构建成功,通过电转染和G418筛选,可获得90%以上阳性克隆.C/EBPα高表达可使HL60细胞增殖明显减缓,表面抗原CD11b的表达可达到(50.44±4.92)%.在NSC67657处理的重组质粒转染HL60细胞中,细胞单核系分化受到抑制,保留部分粒系分化.结论 NSC67657诱导HL60细胞向单核系分化不一定通过C/EBPα蛋白的协调作用,但C/EBPα蛋白高表达可以阻止NSC67657诱导HL60细胞向单核系分化的进程.     

小剂量As_2O_3体外诱导HL-60细胞发生非终末分化分子机制的研究

目的探讨增强子结合蛋白C／EBPs在小剂量三氧化二砷(As_2O_3)诱导HL-60细胞发生非终末分化中的作用。方法采用瑞式染色观察细胞形态,WST1实验测定细胞增殖变化,测定和分析细胞周期,NBT还原实验测定细胞分化状态,RT-PCR方法检测C／EBPα和C／EBPε的 mRNA表达。结果HL-60细胞经全反式维甲酸(ATRA)作用24 h后,随着细胞分化的发生, C／EBPε mRNA的表达明显增加,C／EBPα mRNA的表达降低。HL-60细胞经As_2O_3作用24 h后, C／EBPα mRNA的表达降低,C／EBPε mRNA的表达轻微增加。结论经As_2O_2和ATRA诱导后不同分化状态HL-60细胞,C／EBPα mRNA的表达降低,二者差异无显著意义;C／EBPε的表达差异较大(P<0．05),二者差异有显著意义。小剂量As_2O_3诱导HL-60细胞发生非终末分化可能是由于C／EBPε不能持续表达升高引起的。     

小剂量As2O3体外诱导HL-60细胞发生非终末分化分子机制的研究

目的探讨增强子结合蛋白C／EBPs在小剂量三氧化二砷（As2O3）诱导HL-60细胞发生非终末分化中的作用。方法采用瑞式染色观察细胞形态，WSTl实验测定细胞增殖变化，测定和分析细胞周期，NBT还原实验测定细胞分化状态，RT-PCR方法检测C／EBPct和C／EBPε的mRNA表达。结果HL-60细胞经全反式维甲酸（ATRA）作用24h后，随着细胞分化的发生，C／EBPε mRNA的表达明显增加，C／EBPα mRNA的表达降低。HL-60细胞经As2O3作用24h后，C／EBPα mRNA的表达降低，C／EBPε mRNA的表达轻微增加。结论经As2O3和ATRA诱导后不同分化状态HL-60细胞，C／EBPα mRNA的表达降低，二者差异无显著意义；C／EBPε的表达差异较大（P〈0．05），二者差异有显著意义。小剂量As2O3诱导HL-60细胞发生非终末分化可能是由于C／EBPε不能持续表达升高引起的。     

全反式维甲酸对HL-60细胞VEGF表达与分泌影响的分子机制研究

目的 探讨人髓系白血病细胞株HL-60在诱导分化过程中血管内皮生长因子(VEGF)表达及分泌水平变化的分子机制.方法 四甲基偶氮唑蓝(MTT)法检测HL-60细胞被全反式维甲酸(ATRA)诱导分化后的增殖水平变化,流式细胞术测定HL-60细胞CD11b表达和细胞周期的改变,半定量逆转录聚合酶链式反应(RT-PCR)检测HL-60细胞VEGF、c-myc、缺氧诱导因子(HIF)-1α、基质金属蛋白酶(MMP)-9和MMP-2 mRNA表达水平,应用酶联免疫吸附试验(ELISA)法检测药物作用前后HL-60细胞培养上清液中VEGF蛋白的含量.结果 经ATRA诱导分化后,HL-60细胞增殖水平明显降低,CD11b表达水平明显升高,出现粒系定向分化趋势,且分化程度明显升高(P<0.05),c-myc与VEGFmRNA表达水平均明显下调(P<0.05),HIF-1α mRNA表达水平则明显升高(P<0.05).用MMP-2及MMP-9抑制因子Ⅰ阻断HL-60细胞MMP-9和MMP-2表达后,细胞培养上清中VEGF蛋白分泌量明显降低(P<0.05).结论 HL-60细胞被诱导分化过程中VEGF的表达与c-myc表达正相关,与HIF-1α表达负相关.MMP-9、MMP-2可能是调控HL-60细胞分泌VEGF的主要原因之一.     

蟾蜍灵联合ATRA诱导HL-60细胞分化并上调Syk表达

目的：观察低剂量蟾蜍灵、全反式维甲酸（All—trans retinoic acid,ATRA）单药或联合应用对HL-60细胞增殖及分化的影响,同时检测上游通路Syk表达的变化。方法：流式细胞仪技术检测CD11b表达;Western Blot检测Syk和Tubulin表达。结果：与对照组相比,低剂量蟾蜍灵、ATRA或联合用药不同程度地抑制HL-60细胞增殖,同时蟾蜍灵联合ATRA以时间依赖性的方式诱导HL-60细胞分化,并伴有Syk表达上调,但是Syk的酪氨酸磷酸化并未受影响。结论：Syk表达上调可能参与调节低剂量蟾蜍灵联合ATRA对HL-60细胞的分化诱导作用。     

肿瘤坏死因子α增强全反式维甲酸诱导早幼粒白血病细胞凋亡

目的:分析肿瘤坏死因子α增强全反式维甲酸诱导早幼粒白血病细胞凋亡的作用。方法:选择NB4人急性早幼粒白血病细胞,利用全反式维甲酸单独作用,并选择肿瘤坏死因子α联合全反式维甲酸作用,测定细胞增殖以及细胞凋亡情况。结果:随着早幼粒白血病细胞分化的逐渐进行,单一组、联合组的细胞增殖速度均慢慢下降;随着早幼粒白血病细胞分化的逐渐加深,单一组、联合组细胞凋亡率慢慢升高;分化2天,联合组细胞CD11b阳性率明显高于单一组,P<0.05。结论:全反式维甲酸具有诱导早幼粒白血病细胞分化、凋亡的作用,而肿瘤坏死因子α会增强全反式维甲酸的作用效果。     

蟾蜍灵联合ATRA诱导HL-60细胞分化并上调Syk表达

目的:观察低剂量蟾蜍灵、全反式维甲酸(All-trans retinoic acid,ATRA)单药或联合应用对HL-60细胞增殖及分化的影响,同时检测上游通路Syk表达的变化.方法:流式细胞仪技术检测CD11b表达;Western Blot检测Syk和Tubulin表达.结果:与对照组相比,低剂量蟾蜍灵、ATRA或联合用药不同程度地抑制HL-60细胞增殖,同时蟾蜍灵联合ATRA以时间依赖性的方式诱导HL-60细胞分化,并伴有Syk表达上调,但是Syk的酪氨酸磷酸化并未受影响.结论:Syk表达上调可能参与调节低剂量蟾蜍灵联合ATRA对HL-60细胞的分化诱导作用.     

全反式维甲酸对HL-60细胞分化和凋亡的影响

背景与目的：用全反式维甲酸(all trans retinoid acid,ATRA)治疗早幼粒细胞性白血病(premyeloid leukemia,M3)在肿瘤治疗史上是一个重要里程碑。已有研究表明,ATRA治疗白血病的机理是诱导白血病细胞向成熟细胞方向分化,然而人们对分化的肿瘤细胞最终去向还不完全清楚。本实验进一步探讨ATRA诱导HL-60细胞分化和凋亡的关系。方法：以分化诱导剂ATRA(终浓度为10μmol／L)作用不同时间的HL-60细胞为检测对象,应用流式细胞仪分析细胞表面的分化标志物及细胞周期;经碘化丙啶(propidium iodide,PI)染色后,激光共聚焦显微镜下对已分化细胞进行形态学确认;应用流式细胞仪检测药物诱导后不同时间点的细胞凋亡。结果：(1)随着药物诱导时间的延长,被诱导的HL-60细胞体积逐渐增大,在72h后,被诱导细胞开始表达分化标志物CD11b并出现细胞核型的变化;(2)在药物诱导96h后,细胞开始出现了凋亡峰,而在药物诱导72h后脱药培养8h,则可以出现高于96h的凋亡峰。结论：ATRA并不能诱导HL-60细胞完成终端分化,但是该药可以诱导白血病细胞向成熟方向分化,而且已分化的肿瘤细胞易于发生凋亡。     

应用mRNA差异显示方法克隆维甲酸诱导的人肺腺癌GLC-82细胞分化相关基因

目的克隆维甲酸诱导的人肺腺癌ＧＬＣ８２细胞分化相关基因。方法在全反式维甲酸诱导人肺腺癌ＧＬＣ８２细胞系分化的基础上,采用ｍＲＮＡ差异显示技术,对这个细胞系以全反式维甲酸诱导前及诱导后８小时、２４小时和４天的细胞基因表达差异情况进行分析。结果在全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异。有些基因经维甲酸（ＲＡ）诱导后持续表达或瞬时表达,而有些基因经ＲＡ作用后表达水平降低或完全被抑制。经克隆筛选,获得了一些经全反式维甲酸诱导激活或抑制的差异表达基因片段,对其中的３个片段进行了序列分析和同源性比较。结论全反式维甲酸具有调控分化相关基因表达与否的重要作用,由ＲＡ诱导的肿瘤细胞再分化是一个多基因参与的过程     

全反式维甲酸诱导HL60细胞分化分子机制探讨

目的 探讨全反式维甲酸(alltrans retinoic acid,ATRA)诱导HL-60细胞分化过程中PADI4的变化与作用机制.方法 ATRA诱导HL-60细胞24、48、72和96 h后,采用Wright-Gimesa染色观察细胞形态学变化;采用流式细胞术检测细胞表面分化抗原CD11b的表达变化;采用RT-PCR分析PADI4在基因水平的表达趋势,蛋白质印迹法检测PADI4在蛋白水平的表达变化;RNAi技术干扰PADI4后流式细胞仪检测CD11b变化,蛋白质印迹法检测PADI4、p42/44、p65、p105和p55等蛋白水平变化.结果 HL-60细胞经ATRA诱导后与对照组相比,Wright-Gimesa染色显示核质比明显减小,核有凹陷及分叶,杆状核、分叶核现象明显增多.流式细胞术检测结果显示,细胞表面分子CD11b表达由2.1％升高到48.9％,升高了346.8％,t=411.51,P＜0.01;RT-PCR结果显示,PADI4在mRNA水平表达随时间延长逐渐升高,F＝ 318.046,P＜0.001,r=0.595;PADI4与内参基因GAPDH灰度值的比值从0.122±0.033升高到0.464±0.077,差异有统计学意义,F＝16.002,P＜0.001.蛋白质印迹法检测结果显示,PADI4在蛋白水平表达随时间延长逐渐升高,F=16.002,P＜0.001,r=0.873;PADI4与内参GAPDH灰度值的比值分析从0.077±0.045升高到0.263±0.095,差异有统计学意义,F＝221.8,P＜0.000 1.蛋白质印迹法检测结果显示,P-p44/42与内参GAPDH灰度值的比值分析从0.470±0.023下降到0.320±0.012,差异有统计学意义,F=92.942,P＜0.001.结论 ATRA诱导HL-60细胞定向粒系分化的过程中,PADI4促进HL-60细胞向粒系分化,而且PADI4可能是通过促进MAPK信号通路中的p42/44磷酸化而发挥作用.     

抑制c-myc基因表达对HL-60细胞表面分化抗原CD13、CD33的影响

目的：利用重组反义c-myc腺病毒载体（Ad-AS-c-myc）抑制HL-60细胞c-myc基因表达,检测细胞表面分化抗原CD13、CD33变化,了解抑制HL-60细胞c-myc表达,对细胞分化及分化抗原的影响。方法：重组Ad-AS-c-myc病毒按感染强度（MOI）为100的转染剂量,转染HL-60细胞。RT-PCR检测c-myc表达。流式细胞仪检测CD13、CD33阳性细胞率。同时对转染细胞行Wright＇s染色,观察细胞形态变化。结果：Ad-AS-c-myc转染HL-60细胞后,c-myc基因表达降低,CD13阳性细胞率升高,CD33阳性细胞率降低。结论：抑制c-myc基因表达,使HL-60细胞CD13表达增强,CD33表达降低。细胞形态向成熟粒细胞方向分化。     

miR-146a对氢醌抑制HL-60细胞分化的调控作用

目的： 观察氢醌对HL-60细胞向单核细胞分化的影响,并初步探讨miR-146a的调控作用。方法：设miR-146a-5p 抑制剂和阴性对照处理的HL-60细胞,分别以不同浓度(0、0.5、1.0、2.5和5.0 μmol/L)的氢醌处理3 h后,接种于含豆蔻酰佛波醇(PMA)的培养基中,培养72 h诱导其分化。采用实时荧光定量PCR检测细胞内miR-146a及其靶基因TRAF6的表达量,流式细胞术检测CD11b和CD14的表达,Western blot检测TRAF6蛋白及其下游调控因子IκBα蛋白的表达水平。结果：与对照组相比(0 μmol/L), 氢醌可抑制PMA诱导的HL-60细胞CD11b和CD14的表达,5.0 μmol/L的氢醌使其表达量分别减少44%和38% (P均<0.01);同时miR-146a的表达比对照组升高近2倍(P<0.01);TRAF6 mRNA的表达下降约40%,其蛋白表达下降74%(P均<0.01);磷酸化IκBα蛋白的表达减少约45%,IκBα总蛋白表达升高约73%(P均<0.01)。当抑制miR-146a的表达后,氢醌对TRAF6、IκBα和磷酸化IκBα蛋白表达的影响不明显。结论：miRNA-146a是氢醌影响HL-60细胞分化的调控因子之一。     

Identification of a S100 calcium-binding protein expressed in HL-60 cells treated with all-trans retinoic acid by two-dimensional electrophoresis and mass spectrometry

To characterize the alteration of protein expression during tumor cell differentiation induced by all-trans retinoic acid (ATRA) and to understand downstream signaling and molecular mechanism of ATRA action, we compared the protein expression profiles in HL-60 cells with ATRA treatment using two-dimensional electrophoresis (2-DE). Although many changes in protein expression were found in 2-DE maps, here we identified two protein spots remarkably expressed in the differentiated cells by nanoelectrospray ionization mass spectrometry and database searching. These two protein spots were found to be the same protein, namely S100 calcium-binding protein A9 (S100A9). Further study will be done to ascertain whether S100A9 plays a role in the regulation of differentiation or just a consequence of differentiation.     

Cell cycle-independent down-regulation of BCL-2 protein expression in differentiating HL-60 cells

To understand the relationship between bcl-2 protein expression and the cell cycle during the processes of differentiation, we examined bcl-2 protein levels expressed during cell cycle phases in differentiating HL-60 human leukemia cells by using a two-color flow cytometric method. In exponentially proliferating HL-60 cells bcl-2 protein was constitutively expressed throughout the cell cycle phases, but a small population of G0/G1 cells expressed decreased levels of protein as compared with other cell cycle phases. HL-60 cells can be induced to differentiate to granulocytic pathway by retinoic acid or dimethylsulfoxide, and to monocytic/macrophagic pathway by 1, 25 dihydroxyvitamin D3 or 12-O-tetradecanoylphorbol -13-acetate. During treatment with any of these inducing agents, bcl-2 protein expression was time-dependently down-regulated after 24 h. A two-color flow cytometric analysis revealed that this down-regulation occurred throughout cell cycle phases, indicating that bcl-2 protein expression was down-regulated in cell cycle-independent manner during induction of differentiation in HL-60 cells. To our knowledge, this is the first demonstration suggesting that the regulation of bcl-2 protein expression is not related to the cell cycle during induction of differentiation in HL-60 cells.     

Cell Cycle-Independent Down-Regulation of BCL-2 Protein Expression in Differentiating HL-60 Cells

To understand the relationship between bcl-2 protein expression and the cell cycle during the processes of differentiation, we examined bcl-2 protein levels expressed during cell cycle phases in differentiating HL-60 human leukemia cells by using a two-color flow cytometric method. In exponentially proliferating HL-60 cells bcl-2 protein was constitutively expressed throughout the cell cycle phases, but a small population of G0/G1 cells expressed decreased levels of protein as compared with other cell cycle phases. HL-60 cells can be induced to differentiate to granulocytic pathway by retinoic acid or dimethylsulfoxide, and to monocytic/macrophagic pathway by 1, 25 dihydroxyvitamin D3 or 12-O-tetradecanoylphorbol-13-acetate. During treatment with any of these inducing agents, bcl-2 protein expression was time-dependently down-regulated after 24h. A two-color flow cytometric analysis revealed that this down-regulation occurred throughout cell cycle phases, indicating that bcl-2 protein expression was down-regulated in cell cycle-independent manner during induction of differentiation in HL-60 cells. To our knowledge, this is the first demonstration suggesting that the regulation of bcl-2 protein expression is not related to the cell cycle during induction of differentiation in HL-60 cells.     

Modifications in phospholipase D activity and isoform expression occur upon maturation and differentiation in vivo and in vitro in human myeloid cells.

Activation of phospholipase D (PLD) occurs in response to various stimuli and results from the activity of two isozymes, hPLD1 and hPLD2. PLD activity appears to be involved in several myeloid cell processes during their development and activation, including proliferation of myeloblasts in the bone marrow and secretion, phagocytosis and NADPH oxidase activation, essential functions of differentiated neutrophils. The present work studies PLD characteristics, activity and both isozyme expression during maturation and differentiation of myeloid cells by using three different systems: leukemic myeloblasts at different stages of maturation, terminally differentiated neutrophils ex vivo and four human myeloid cell lines, NB4, HL-60, PLB 985 and U937, induced to differentiate with alltrans retinoic acid (ATRA), a cyclic adenosine monophosphate (cAMP) analogue or both agents together. HL-60, a bipotential cell line has also been differentiated along the granulocytic pathway with DMSO and the monocytic pathway with 1,25-dihydroxy vitamin D3. In all these systems, PLD activity increases with maturation and differentiation whatever the inducer used and the granulocytic or monocytic pathways. Increase in basal activity which reflects the expression during development of both hPLD1 and hPLD2 appears to be mainly related to the former isozyme expression. Association of PLD characteristic changes with maturation and differentiation was also confirmed using two NB4 clones resistant to these processes. Comparison between PLD characteristics in myeloblasts during maturation and differentiation ex vivo and in vitro in the different cell lines demonstrated that NB4 induced to differentiate with ATRA represents the best model for further studies on the specific roles of each PLD isoform in various functions of differentiated myeloid cells.     

Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines.

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 microM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.     

HL－60细胞诱导分化后血管内皮生长因子表达变化分子机制的探讨

目的探讨全反式维甲酸（ATRA）诱导HL-60细胞分化模型中血管内皮生长因子（VEGF）表达调控的分子机制,为白血病的抗血管新生治疗提供新的靶点。方法Wright—Gimesa染色观察细胞形态,硝基四唑氮蓝（NBT）还原实验检测HL-60细胞分化,逆转录－聚合酶链反应（RT—PCR）、蛋白质印记（Westernblot）分别从mRNA和蛋白水平检测细胞VEGF、信号转导和转录激活因子-3（STA33）、c-myc的表达变化。结果1μmolATRA作用于HL-60细胞96h后可明显抑制细胞增殖,并出现明显的分化现象,倒置显微镜下可观察到细胞向成熟粒细胞方向分化;NBT阳性率为82．59％（t=－24．157,P〈0．01）;VEGFmRNA表达水平下降（t=7．339,P〈0．05）、STAT3mRNA表达水平下降（t=3．667,P〈0．05）、c—mycmRNA表达水平下降（t=6．858,P〈0．05）。VEGF蛋白表达水平下降（t=3．386,P〈0．05）、STA33蛋白表达水平下降（t=4．074,P〈0．05）、c-myc蛋白表达水平下降（t=3．333,P〈0．05）。结论在ATRA诱导HL-60细胞分化的模型中VEGF的表达水平随诱导分化的进程而下降,其下调机制可能与STAT3、c—myc具有相关性。     

PBK/TOPK expression during TPA-induced HL-60 leukemic cell differentiation

Objective: This study concerns expression of PBK/TOPK during differentiation of HL-60 leukemic cellsinduced by tetradecanoyl phorbol acetate (TPA). Methods: Wright-Giemsa staining was performed to observemorphological changes in the HL-60 cells, and flow cytometry was used to assess the cell cycle and CD11b,CD14, CD13, and CD33 expression. PBK/TOPK levels were determined by Western blot analysis. Results: Aftertreating HL60 cells with 5.1 × 10-9 mmol/L of TPA for three days, the number of nitroblue-tetrazolium-positivecells and CD11b, CD13, and CD14 expression increased, whereas the PBK/TOPK levels decreased. Conclusions:TPA can inhibit proliferation and induce differentiation of HL60 cells of the granulocytic or monocytic lineage.PBK/TOPK expression was downregulated during this process, whereas the Pho-PBK/TOPK expression wasincreased.