Differential cell fates induced by all-trans retinoic acid-treated HL-60 human leukemia cells

HL-60 human leukemia cells, differentiated into a neutrophil lineage by all-trans retinoic acid (ATRA) treatment, express three members of the carcinoembryonic antigen (CEA) gene family, CEA-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM3 (CD66d), and CEACAM6 (CD66c). CD66d is a neutrophil lineage-specific marker, and CD66a and CD66c are found on epithelial and other cells. HL-60 cells continuously treated with ATRA underwent apoptosis, and cells transiently treated for 1 day underwent cell-cycle arrest, entered into senescence, and exhibited reduced apoptosis with CD66-positive cells accounting for the majority of live cells. CD66 antigens were also induced in NB4 leukemic cells upon continuous treatment with ATRA. NB4 cells underwent apoptosis with a higher frequency in transient versus continuous-treated cells (38% vs. 19% at Day 5), in contrast to HL-60 cells that underwent cell-cycle arrest and senescence when transiently treated with ATRA. CD66 antigens were not induced in transient, ATRA-treated NB4 cells compared with HL-60 cells. Cell-cycle arrest in HL-60 cells involved reduction in expression levels of p21, cyclins D and E, while Rb1 exhibited reduction in protein levels without changes in mRNA levels over the time course of ATRA treatment. Analysis of several proapoptotic proteins implicated the activation of calpain and cleavage of Bax in the intrinsic apoptotic pathway, similar to published studies about the apoptosis of neutrophils. CD1d expression was also induced by ATRA in HL-60 cells and ligation with anti-CD1d antibody-induced apoptosis. In contrast, CD1d-positive primary monocytes were protected from spontaneous apoptosis by CD1d ligation. These studies demonstrate distinct cell fates for ATRA-treated HL-60 cells that provide new insights into ATRA-induced cell differentiation.     

抑制mel18表达增强桂皮醛诱导的HL60细胞分化

目的:研究下调mel18基因表达对桂皮醛(CA)诱导HL60细胞分化的影响。方法:用低浓度CA和反式维甲酸(ATRA)处理HL60细胞。用shRNAmel18质粒及对照质粒shRNALuc包装慢病毒,用病毒感染HL60细胞。低浓度CA和ATRA作用于病毒感染的HL60细胞,流式细胞术检测细胞周期和细胞表面分化抗原表达,Western blot法检测MEL18蛋白、cyclin D1、p27的表达和Akt的磷酸化水平。结果:低浓度CA和ATRA增加HL60细胞的粒细胞分化抗原CD11b表达,MEL18蛋白表达下降。shmel18病毒感染的HL60细胞(shmel18-HL60细胞)MEL18蛋白表达下降,而对照病毒感染的HL60细胞(sh Luc-HL60细胞)MEL18蛋白表达无明显变化。shmel18-HL60细胞的CD11b表达率明显增高,细胞阻滞于G1期;经低浓度CA处理后,shmel18-HL60细胞的CD11b表达率进一步增高。shmel18-HL60细胞Akt的磷酸化水平及cyclin D1表达明显下降,而p27表达则显著升高。结论:抑制mel18基因表达导致HL60细胞向成熟粒细胞方向分化,mel18基因可能通过PI3K/Akt信号途径调控cyclin D1和p27的表达,影响HL60细胞分化。而CA可能通过抑制mel18基因表达从而通过PI3K/Akt途径促进HL60细胞分化。     

Anti-leukemic effect of a synthetic compound, (±) trans-dihydronarciclasine (HYU-01) via cell-cycle arrest and apoptosis in acute myeloid leukemia

(±) trans-Dihydronarciclasine, isolated from Chinese medicinal plant Zephyranthes candida, has been shown to possess quite potent anti-tumoral effect against selected human cancer cell lines. However, little is known about the anti-tumoral effect of (±) trans-dihydronarciclasine in acute myeloid leukemia (AML). This study was performed to investigate the effect of a novel synthetic (±) trans-dihydronarciclasine (code name; HYU-01) in AML. The HYU-01 inhibited the proliferation of various AML cell lines including HL-60 as well as primary leukemic blasts in a dose-dependent manner. To investigate the mechanism of the anti-proliferative effect of HYU-01, cell-cycle analysis was attempted in HL-60 cells, resulting in G1 arrest. The expression levels of CDK2, CDK4, CDK6, cyclin E, and cyclin A were decreased in a time-dependent manner. In addition, HYU-01 up-regulated the expression of the p27, and markedly enhanced the binding of p27 with CDK2, 4, and 6, ultimately resulting in the decrease of their kinase activities. Furthermore, HYU-01 induced the apoptosis through the induction of proapoptotic molecules and reduction of antiapoptotic molecules in association with the activation of caspase-3, -8, and -9. These results suggest that HYU-01 may inhibit the proliferation of HL-60 cells, via apoptosis, as well as G1 block in association with the induction of p27.     

Study on the synergic effect of all-trans retinoic acid combined with IFN-alpha on the proliferation and differentiation of HL-60 cells]

AIM: To investigate the synergic effect of all-trans retinoic acid (ATRA) combined with IFN-alpha on the proliferation and differentiation of HL-60 cells. METHODS: HL-60 cell's differentiation and apoptosis were assessed by NBT reduction and TUNEL in situ apoptosis assay kit. CYP26 mRNA expression was detected by in situ hybridization assay kit. Metabolism of ATRA was measured by high performance liquid chromatography. RESULTS: Either IFN-alpha or ATRA induced HL-60 cell differentiation and apoptosis, which was enhanced when combining ATRA with IFN-alpha. The level of ATRA and the expression of CYP26 were higher in HL-60 cells treated with both ATRA and IFN-alpha than in the cells treated with ATRA alone. CONCLUSION: ATRA has remarkable synergic effect with IFN-alpha on HL-60 cells, probably because IFN-alpha inhibits CYP26 mRNA expression and thus reduces the metabolism of ATRA.     

HL-60细胞ATRA诱导后MMP-9/TIMP-1表达的变化

为了检测HL-60细胞经过全反式维甲酸(ATRA)诱导后基质金属蛋白酶(MMP-9、MMP-2)及金属蛋白酶组织抑制剂1(TIMP-1)表达变化,并探讨其变化的意义。采用瑞氏染色观察细胞形态,WST1实验测定细胞增殖变化,NBT还原实验测定细胞分化状态,RT-PCR方法检测MMP-9、MMP-2、TIMP-1、VEGF的mRNA的表达。结果显示,HL-60细胞经ATRA作用24h后,随着细胞增殖降低与分化的发生,MMP-9mRNA表达增加,TIMP-1mRNA和VEGF表达降低,MMP-2mRNA未见明显表达。ATRA诱导HL-60细胞分化后MMP-9表达增加,而TIMP-1表达降低,MMP-9不促进HL-60细胞表达VEGF。     

视黄酸依赖反义cyclin D1对HL-60细胞分化效应研究

目的 检测视黄酸(Retinoic acid，RA)依赖反义cyclin D1表达载体转染HL-60细胞后启动的诱导分化效应，并探讨其分子机制。方法 成功构建视黄酸依赖反义cyclin D1表达载体，以脂质体转染HL-60白血病细胞，经NBT还原实验、免疫荧光检测CD14表面抗原表达、RT-PCR以及Western blot等方法观测视黄酸处理后细胞分化效应的发生以及Rb基因的mRNA和蛋白表达水平的改变。结果 反义eyelin D1转染和未转染的HL-60细胞经RA处理后，NBT还原能力明显增强、CD14表面抗原表达显著增加，Rb基因的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处理后变化更为明显。结论 RA及其诱导的反义cyclin D1可协同诱导HL-60细胞分化成熟，下调Rb基因表达可能是其分子机制之一。     

乌苯美司下调c-myc表达与其增强全反式维甲酸诱导NB4细胞

目的： 了解氨肽酶抑制剂乌苯美司（bestatin）能否增强全反式维甲酸（ATRA）对NB4细胞的诱导分化作用，及此过程中NB4细胞c-myc mRNA表达水平的改变。 方法： MTT法检测药物抑制细胞生长的作用。流式细胞仪测细胞表面分化抗原CD11b及四氮唑蓝（NBT）还原实验检测NB4细胞的分化。RT-PCR检测细胞c-myc mRNA表达水平。 结果： 50 mg/L、75 mg/L、100 mg/L乌苯美司与10 nmoL/L ATRA联合处理72 h，均能明显增强NB4细胞的NBT还原能力，与10 nmoL/L ATRA组差异显著（P<0.05，P<0.01）。从48 h到96 h，100 mg/L乌苯美司时间依赖性地增强10 nmoL/L ATRA诱导NB4细胞的NBT还原能力，与相应时点10 nmoL/L ATRA组差异明显（P<0.01）。100 mg/L乌苯美司与10 nmoL/L ATRA联合应用72 h，NB4细胞CD11b表达率明显高于10 nmoL/L ATRA组（P<0.01）。50 mg/L、75 mg/L、100 mg/L乌苯美司与10 nmoL/L ATRA联合处理4 h，NB4细胞c-myc mRNA表达水平明显低于单用ATRA组（P<0.05，P<0.01）；药物联合应用各组NB4细胞的c-myc mRNA表达水平与NBT还原能力之间呈负相关（r=-0.917,P<0.05）。 结论： 乌苯美司可能通过下调NB4细胞c-myc mRNA的表达，从而增强ATRA诱导NB4细胞分化的作用。     

黄芩苷对HL-60白血病细胞的诱导分化作用

目的： 观察中药单体黄芩苷(baicalin) 对人髓系白血病（AML）细胞株HL-60细胞的诱导分化作用。方法: 应用细胞形态学方法、细胞克隆形成试验、流式细胞术分析和NBT还原实验检测黄芩苷诱导HL-60细胞分化的能力。结果: 黄芩苷可诱导HL-60细胞向成熟阶段分化，低浓度黄芩苷可显著抑制HL-60细胞克隆的形成；HL-60细胞经黄芩苷处理后CD11b表达显著增高，CD33表达显著降低；NBT还原实验示黄芩苷处理组的分化成熟细胞阳性率明显高于未加药组。结论: 黄芩苷具有诱导HL-60白血病细胞向成熟粒细胞分化的作用。     

5-aza-2dC诱导白血病细胞分化及对Annexin A1/A2表达和甲基化状态的影响

目的：观察甲基化转移酶抑制剂5-杂氮-2’-脱氧胞苷(5-aza-2’-deoxycitydine, 5-aza-2dC)对人急性髓系白血病细胞系HL-60细胞分化及对膜联蛋白A1/A2(Annexin A1/A2)表达和甲基化状态的影响。 方法：瑞氏染色和流式细胞术检测5-aza-2dC对HL-60细胞分化的影响；RT-PCR法检测药物处理HL-60细胞前后Annexin A1和A2基因mRNA的表达水平；甲基化特异性PCR(methylation-specific PCR，MSP)检测药物处理HL-60细胞前后Annexin A1和A2基因启动子区域CpG岛的甲基化水平。 结果：5-aza-2dC处理后HL-60细胞的髓系分化抗原CD11b的表达增强，细胞向成熟分化，且在0.5 μmol/L时其促分化作用最明显；Annexin A1和A2基因在HL-60细胞中低表达，0.5 μmol/L 5-aza-2dC处理HL-60细胞72 h后，Annexin A1和A2基因mRNA表达水平明显上调，而其启动子区域CpG岛甲基化水平明显降低。结论：5-aza-2dC具有促进白血病细胞分化的作用，Annexin A1和A2基因启动子去甲基化可能与5-aza-2dC诱导白血病细胞分化有关。     

初发急性髓性白血病患者外周血中高表达TAL1基因及其意义

目的:分析调控造血分化的重要转录因子TAL1在急性髓性白血病(AML)细胞株及原代AML细胞中的表达特点。方法:利用real-time PCR法分别检测47例初发急性髓性白血病患者外周血单个核细胞以及急性白血病细胞株(Jurkat、CCRF-CEM、HL-60和NB4细胞株)中TAL1 mRNA的水平,并以12例健康志愿者外周血样本作为对照组。结果:TAL1 mRNA在AML细胞株(HL-60和NB4)、T细胞急性淋巴细胞白血病(T-ALL)细胞株(Jurkat和CCRF-CEM)和原代AML细胞中的水平均显著高于健康对照组(P0.05)。各AML亚型中TAL1的mRNA表达水平均较对照组显著升高,并以AML-M1和AML-M5 2个亚型升高最为明显(P0.05)。结论:AML细胞中TAL1异常高表达可能与其影响粒系的分化发育有关,其能否作为AML发病相关分子标志物有待进一步研究。     

视黄酸对CDK2组成性表达HL—60细胞增殖的影响

探讨细胞周期素依赖性激酶在视黄酸对急性早幼粒细胞白血病ＨＬ－６０细胞的增殖抑制和分化调控过程中的作用。方法应用真核细胞基因转染技术将外源ＣＤＫ２基因转入人ＨＬ－６０早幼粒细胞白血病细胞中,进而以视黄酸处理１－４ｄ。结论ＣＤＫ２在视黄酸诱导的增殖抑制和分化过程中起重要作用,由于ＣＤＫ２的组成性超过表达,导致视黄酸对ＨＬ－６０细胞的增殖抑制和诱导分化作用下降。     

Modulation of death receptor-mediated apoptosis in differentiating human myeloid leukemia HL-60 cells

Differentiating myeloid cells may become resistant to various apoptotic stimuli. In the present study, dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA) were found to modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis in a time-dependent manner. During the early stages of differentiation, DMSO treatment increased the response of HL-60 cells to tumor necrosis factor alpha; (TNF-alpha), but enhanced responsiveness was lost during later differentiation stages. In contrast, ATRA treatment induced resistance to TNF-alpha-induced apoptosis. HL-60 cells were resistant to Fas-mediated apoptosis but were sensitized by culturing in serum-free conditions. Similar to its effect on TNF-alpha sensitivity, DMSO pretreatment augmented the response to Fas-mediated signaling, which coincided with increased expression of Fas on DMSO-pretreated cells. However, during the later stages of DMSO-induced differentiation, sensitivity to anti-Fas antibody-induced apoptosis declined significantly, although Fas expression was still elevated. The reduced sensitivity to anti-Fas treatment partially correlated with increased Fas-associated phosphatase-1 mRNA expression. Thus, regardless of either Fas up-regulation or potentiation of TNF-alpha-mediated apoptosis during early DMSO-induced differentiation, a slow increase in resistance to apoptosis mediated through these death receptors occurs during DMSO-induced differentiation, which contrasts with the rapid induction of resistance following treatment with ATRA.     

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D₃ (VitD₃) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD₃. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD₃ increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD₃-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

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维甲酸诱导HL-60细胞分化过程中WT1基因表达及其异构体比例变化的研究

目的 探讨全反式维甲酸(ATRA)诱导HL-60细胞分化过程中WT1基因及其异构体表达水平及比例变化.方法 采用ATRA诱导HL-60细胞分化,以硝基四氮唑蓝(NBT)还原实验及细胞免疫标记CD11b检测判断细胞分化程度;即时荧光定量RT-PCR方法检测HL-60细胞在诱导分化过程中总WT1、WT1(17AA+)及WT1(KTS+)的表达,并计算出WT1(+/+)、WT1(+/-)、WT1(-/+)、WT1(-/-)四种异构体的比例.结果 ATRA诱导HL-60细胞分化过程中NBT阳性率及CD11b表达阳性率与诱导分化前(0 h)相比均显著增加(P<0.05,P<0.001).随着细胞分化,WT1表达水平由0 h的(4.17±2.21)× 10~(-3)降低至96 h的(7.53±2.30)×10~(-4);17AA+和KTS+两类异构体的比例也逐渐下降,17AA+异构体比例由0 h的0.60±0.05降到96 h的0.42±0.08(P<0.05).KTS+异构体比例由0.53±0.08降至96 h的0.41±0.04(P<0.05);四种异构体的比例变化表现不一致,WT1(+/+)比例由0 h的0.32±0.06降至96 h的0.17±0.03,而WT1(-/-)比例由0 h的0.19±0.04升至96 h的0.34±0.05,另外两类异构体比例变化差异无统计学意义.结论 ATRA诱导HL-60细胞分化过程中总WT1表达水平逐渐降低,分化前异构体以WT1(+/+)为主,而分化后以WT1(-/-)为主,提示WT1基因可能通过调节四种异构体比例而发挥抑制或促进分化的作用.     

Induction of differentiation of the human myeloid cell line, ML3, by tumour necrosis factor and interferon-gamma is accompanied by enhanced expression of the CD4 protein and messenger RNA.

Tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma) induce differentiation of human myeloid cell lines along the monocytic lineage. In this study we investigated the effects of TNF and IFN-gamma on the expression of the CD4 protein and messenger RNA (mRNA) in the two myeloid cell lines, ML3 and HL-60. We observed that CD4 antigen expression on ML3 cells is almost undetectable and that TNF and IFN-gamma induced CD4 antigen expression on these cells. HL-60 cells express surface CD4 antigen at high density and treatment with TNF and IFN-gamma caused a decrease of CD4 expression. We also investigated the expression of CD4 mRNA in ML3 and HL-60 cells. ML3 constitutively express, albeit at low levels, CD4 mRNA. TNF induced CD4 mRNA in ML3 cells and IFN-gamma synergistically potentiated the effect of TNF, thus indicating that the enhanced expression of the CD4 protein on ML3 cells is due, at least in part, to an enhanced accumulation of the CD4 mRNA. CD4 mRNA is constitutively expressed in HL-60 cells at high levels. TNF and IFN-gamma, alone or in combination, did not cause any significant change of CD4 mRNA expression in HL-60 cells, thus indicating that decrease of surface CD4, which accompanies differentiation with these cytokines, is likely due to alterations of the CD4 protein synthesis and/or transport to the plasma membrane. These results provide evidence that myeloid cell lines are heterogeneous in expression of CD4, and that in ML3 cells, which constitutively express low levels of CD4 mRNA and undetectable amounts of surface CD4, the predominant effect of the two cytokines is to induce both CD4 mRNA and protein.     

Differentiation of HL-60 myeloid leukemia cells induced by all-trans retinoic acid is enhanced in combination with caffeic acid

We investigated a possible enhancement of all-trans retinoic acid (ATRA)-induced differentiation of HL-60 human myeloid leukemia cells by caffeic acid (CA), a widely distributed plant phenolic compound. Our results showed that CA, in the concentration of 13 or 52 micro M, had no or minimal influence on cell differentiation, whereas the differentiating activity of ATRA was potentiated by CA treatment. We proved, using flow cytometric detection of the CD66b surface molecule, a synergistic effect of CA: at day 10, 18.3% of CD66b-positive cells were detected after treatment with ATRA only, and 33% when CA and ATRA were combined together. NBT-assay confirmed that this additive effect of CA on ATRA-induced differentiation. Proliferating activity as assessed by MTT-assay was generally not affected by CA at given concentrations. However, cell proliferation was significantly reduced by 52 micro M CA at 96-h intervals. This effect was markedly enhanced when CA, at both concentrations, and ATRA were combined. The possibility to enhance the differentiation potential of ATRA by CA may improve outcomes in the therapy of acute promyelocytic leukemia.     

Trdmt1 3'-untranslated region functions as a competing endogenous RNA in leukemia HL-60 cell differentiation

Severe blockage in myeloid differentiation is the hallmark of acute myeloid leukemia (AML). Trdmt1 plays an important role in hematopoiesis. However, little is known about the function of Trdmt1 in AML cell differentiation. In the present study, Trdmt1 was up-regulated and miR-181a was down-regulated significantly during human leukemia HL-60 cell differentiation after TAT-CT3 fusion protein treatment. Accordingly, miR-181a overexpression in HL-60 cells inhibited granulocytic maturation. In addition, our “rescue” assay demonstrated that Trdmt1 3′-untranslated region promoted myeloid differentiation of HL-60 cells by sequestering miR-181a and up-regulating C/EBPα (a critical factor for normal myelopoiesis) via its competing endogenous RNA (ceRNA) activity on miR-181a. These findings revealed an unrecognized role of Trdmt1 as a potential ceRNA for therapeutic targets in AML.