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。     

曲古抑菌素A对HL-60细胞分化的影响

目的 探讨曲古抑菌素A(TSA)在体外诱导急性早幼粒白血病细胞HL-60分化的作用.方法 采用溴化四甲基偶氮唑蓝法检测TSA对HL-60细胞增殖的影响,流式细胞术检测细胞周期;通过检测细胞分化特异性抗原CD11b的表达研究细胞分化作用;逆转录-聚合酶链反应(RT-PCR)方法检测c-myc基因mRNA的表达.结果 经TSA作用后,细胞增殖受抑制,细胞周期阻滞在G0和G1期,P<0.01;TSA作用48 h后,细胞分化率达15.24%;c-myc表达随TSA作用时间延长而降低.结论 TSA对HL-60细胞具有抑制增殖和诱导分化作用.     

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.     

视黄酸依赖反义cyclin D1对HL-60细胞细胞周期素依赖性激酶的影响

目的探讨视黄酸(retinoic acid,RA)及其诱导的反义(anti-sense,AS)cyclin D1对HL-60细胞的细胞周期素激酶CDK4的影响.方法通过构建含有视黄酸反应元件(retinoic acid response element, RARE)的反义cyclin D1 RNA表达载体pCI-neo/RARE3-TK/Ascyclin D1,使反义cyclin D1的表达可受视黄酸诱导调控.以脂质体转染HL-60白血病细胞后,运用MTT比色法检测细胞增殖功能、NBT还原实验观测细胞分化状况,RT-PCR以及western-blot等方法观测视黄酸处理后CDK4的mRNA和蛋白表达水平的改变.结果 RA处理后,细胞生长显著减慢,分化能力明显增强,CDK4 mRNA和蛋白表达水平均有所降低,其中,以反义cyclin D1转染细胞经RA处理后变化更为显著.结论 RA及其诱导的反义cyclin D1对HL-60细胞的抑制增殖与诱导分化效应可能与CDK4表达下调有关.     

抗CD44单抗A3D8对HL-60细胞Cyclin D1 CDK4 p21 cip1表达的影响*

目的:探讨CD44单克隆抗体A3D8对人急性髓系白血病细胞株HL-60细胞增殖分化影响的分子作用机制.方法:采用FCM、RT-PCR及Western blot方法检测A3D8作用前后HL-60细胞Cyclin D1、CDK4、p21cip1表达的变化.结果:A3D8作用使HL-60细胞发生G0/G1期阻滞.A3D8下调HL-60细胞Cyclin D1及CDK4的表达,上调HL-60细胞p21cip1 mRNA及P21蛋白表达.结论:A3D8抑制HL-60细胞增殖、诱导其分化的分子机制可能与p21cipl表达上调及Cyclin D1、CDK4表达下调有关.     

视黄酸依赖反义cyclin D1对HL-60细胞增殖和分化的影响及其机制

目的：探讨视黄酸(retinoic acid.RA)及其诱导的反义(anti-sense,AS)cyclin D1对肿瘤细胞的协同抑制增殖与诱导分化效应及其机制。方法：通过构建的含有视黄酸反应元件(retinoic acid response element．RARE)的反义cyclin D1 RNA表达载体pCI-nco/RARE3-TK/Ascyclin D1,使反义cyclin D1的表达可受RA诱导调控。以脂质体转染HL-60白血病细胞后．运用生长曲线、MTT比色法和流式细胞仪分析检测细胞增殖功能，NBT还原实验和透射电镜观测细胞分化状况，western-blot检测p16基因蛋白表达水平。结果：反义cyclin D1转染和未转染的HL-60细胞经RA处理后．生长减慢．细胞被阻滞在G0/G1期．分化能力明显增强，p16基因蛋白表达增加．其中,以反义cyclin D1转染细胞经RA处理后变化更为明显。结论：RA及其诱导的反义cvclin D1可协同抑制HL-60细胞增殖和诱导细胞分化成熟．上调p16基因表达可能是其分子机制之一。     

HL-60细胞淫羊藿甙诱导后信号传导因子表达变化与意义

目的检测急性早幼粒白血病细胞HL-60经过淫羊藿甙(ICA)诱导后信号传导与转录激活因子(STAT1、STAT3)和有丝分裂原激活蛋白激酶(p38MAPK、p42MAPK)表达变化在分化中的作用。方法建立ICA诱导分化模型,用瑞氏染色观察细胞形态,MTT实验测定细胞增殖的变化,NBT还原实验测定细胞分化状态,RT-PCR方法检测STAT1、STAT3、p38MAPK、p42MAPK 的mRNA的表达。结果 HL-60细胞经ICA作用24 h后,随着细胞增殖降低和分化的发生, p42MAPK的mRNA表达增加,STAT3的mRNA表达降低,STAT1和p38MAPK的mRNA未见明显的表达。结论 p42MAPK和STAT3与ICA诱导HL-60细胞分化有关,而p38MAPK和STAT1则与ICA诱导HL-60细胞分化无关。     

HL-60细胞淫羊藿甙诱导后信号传导因子表达变化与意义

目的检测急性早幼粒白血病细胞HL-60经过淫羊藿甙（ICA）诱导后信号传导与转录激活因子（STAT1、STAT3）和有丝分裂原激活蛋白激酶（p38MAPK、p42MAPK）表达变化在分化中的作用。方法建立ICA诱导分化模型，用瑞氏染色观察细胞形态，MTF实验测定细胞增殖的变化，NBT还原实验测定细胞分化状态，RT-PCR方法检测STATI、STAT3、p38MAPK、p42MAPK的mRNA的表达。结果HL-60细胞经ICA作用24h后，随着细胞增殖降低和分化的发生，p42MAPK的mRNA表达增加，STAT3的mRNA表达降低，STAT1和p38MAPK的mRNA未见明显的表达。结论p42MAPK和STAT3与ICA诱导HL-60细胞分化有关，而p38MAPK和STAT1则与ICA诱导HL-60细胞分化无关。     

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。     

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。     

Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte

We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC50, approximately 30 microM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G1 phase in HL-60 human promyelocytic leukemia cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21(CIP1/WAF1) and p27(KIP1), were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21(CIP1/WAF1) and p27(KIP1) to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G1 arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21(CIP1/WAF1) and p27(KIP1) levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.     

Gallic acid induces G₀/G₁ phase arrest and apoptosis in human leukemia HL-60 cells through inhibiting cyclin D and E, and activating mitochondria-dependent pathway

Gallic acid (GA) induces apoptosis in different types of cancer cell lines. In this study, we investigate the apoptotic effects induced by GA in human promyelocytic leukemia HL-60 cells, and clarify the underlying mechanism. Our results showed that GA reduced the viability of HL-60 cells in a dose- and time-dependent manner. GA led to G(0)/G(1) phase arrest in HL-60 cells through promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. GA caused DNA damage and fragmentation in HL-60 cells as assayed using DAPI staining and Comet assay. Flow cytometric analysis revealed that GA increased Ca(2+) levels and reduced the mitochondrial membrane potential (ΔΨ(m)) in HL-60 cells. Apoptotic protein expressions were determined by Western blotting. The results indicated that GA-mediated apoptosis of HL-60 cells mainly depended on mitochondrial pathway, by promoting the release of cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) and by up-regulating the protein expression of Bcl-2-associated X protein (BAX), caspase-4, caspase-9 and caspase-3. In addition, GA also activated the death receptor-dependent pathway by enhancing the protein expressions of fatty acid synthase (FAS), FAS ligand (FASL), caspase-8 and BCL-2 interacting domain (BID). We determined the mRNA expression of the gene levels of these proteins by real-time PCR. The results showed that GA-mediated apoptosis of HL-60 cells mainly depended on up-regulation of the mRNA of caspase-8, caspase-9, caspase-3, AIF and Endo G. In conclusion, GA-induced apoptosis occurs through the death receptor and mitochondria-mediated pathways. The evaluation of GA as a potential therapeutic agent for treatment of leukemia seems warranted.     

HL-60细胞高三尖杉酯碱诱导后CD44表达变化及机制的研究

目的:研究高三尖杉酯碱(homohar-ringtonine,HHT)诱导HL-60细胞分化后CD44表达变化及其与细胞周期调控的关系,探讨HHT的作用机制。方法:建立HHT诱导分化模型,瑞氏染色观察细胞形态,MTT法检测细胞增殖变化,NBT还原实验测定细胞分化状态,流式细胞术检测细胞表面CD11b表达及细胞周期变化,RT-PCR检测CD44、p27、p21和Cyclin E基因在mRNA水平的表达变化。结果:MTT实验显示,HHT对HL-60细胞产生增殖抑制作用,HL-60细胞G0/G1期细胞百分比明显上升,S期细胞百分比明显下降,P<0.05。半定量分析PCR扩增产物光密度相对表达量显示,HHT诱导后HL-60细胞中CD44基因表达逐渐减弱,p27和p21基因逐渐增强,Cyclin E基因逐渐减弱。CD44与p27表达呈明显负相关,r=-0.686,P<0.05。结论:HHT可能通过下调CD44基因,进而提高p27和p21表达,抑制Cyclin E活性而对HL60细胞产生诱导分化作用。     

Effect of a novel vitamin D3 analog, EB1089, on G1 cell cycle regulatory proteins in HL-60 cells

Progression of cell cycle in eukaryotes is regulated by a series of the cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs). It has been shown that 1,25(OH)2D3 is able to arrest cell cycle at G1 phase in malignant cells including HL-60 cells. EB1089 is a novel 1,25(OH)2D3 analog that has more potent antileukemic properties with reduced hypercalcemic effect in vitro and in vivo than 1,25(OH)2D3. In the present study, we examined the effect of EB1089 on HL-60 cells at the protein levels of several G1 regulatory proteins. Exposure of HL-60 cells to EB1089 (1x10-8 M) for 3 days showed the G1 block by FACS analysis. The level of p21 was markedly induced in HL-60 cells treated with EB1089 at 24 h, and p27 were progressively increased in a time-dependent manner. The expressions of CDK2 and CDK6 were down-regulated during G1 block of HL-60 cells, and CDK4 is progressively elevated. In addition, level of cyclin D1 was increased in a time-dependent manner, however, no change of cyclin E was noted through the G1 to S traverse. Immunoprecipitation study demonstrated that p27 did not bind to CDK2, CDK4 and CDK6 in EB1089-treated HL-60 cell extracts. In contrast, complexes immunoprecipitated from EB1089-treated HL-60 cells with antibodies CDK2 and CDK6 contained higher amounts of immunodetectable p21 protein compared to untreated HL-60 cells, whereas no detectable change was noted with anti-CDK4 antibody. Furthermore, the kinase activities of CDK2 and CDK6 were decreased while little change was observed in CDK4 activity. These data indicated that p21 protein is a strong candidate for the control of G1 progression in EB1089-treated HL-60 cells, and its major target molecules are CDK2 and CDK6.     

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.