DADS对DJ-1核定位高表达人白血病HL-60细胞的影响（英文）

最近研究表明,DJ-1在许多肿瘤中过表达,而且DJ-1的核表达与肿瘤的生物学行为有关.本文主要研究二烯丙基二硫(DADS)对DJ-1核定位高表达人白血病HL-60细胞生物行为学的影响,阐明DJ-1在细胞核内的功能,为临床诊断及治疗过程提供一个潜在的治疗靶点.通过基因转染技术建立核内高表达HL-60细胞株(DJ-1/HL-60),利用软琼脂集落形成实验、 MTT法、间接免疫荧光细胞化学实验、硝基蓝四氮唑(NBT)还原比色实验评估HL-60细胞的增殖与分化,DJ-1核定位过表达促进HL-60细胞的增殖并抑制其分化. Transwell迁移侵袭小室实验表明DJ-1核定位过表达可以促进HL-60的迁移和侵袭能力. Western blot结果表明DADS具有抑制HL-60细胞中核内DJ-1蛋白表达的能力.说明DJ-1核定位高表达具有促进HL-60细胞增殖和迁移侵袭及抑制HL-60细胞分化的作用,DADS可以诱导DJ-1核定位高表达HL-60细胞分化及抑制迁移侵袭, DJ-1核定位高表达可减弱DADS抑制HL-60细胞增殖的作用.     

DADS对DJ-1核定位高表达人白血病HL-60细胞的影响

最近研究表明,DJ-1在许多肿瘤中过表达,而且DJ-1的核表达与肿瘤的生物学行为有关. 本文主要研究二烯丙基二硫（DADS）对DJ-1核定位高表达人白血病HL-60细胞生物行为学的影响,阐明DJ-1在细胞核内的功能,为临床诊断及治疗过程提供一个潜在的治疗靶点. 通过基因转染技术建立核内高表达HL-60细胞株（DJ-1/HL-60）,利用软琼脂集落形成实验、 MTT法、间接免疫荧光细胞化学实验、硝基蓝四氮唑( NBT)还原比色实验评估HL-60细胞的增殖与分化,DJ-1核定位过表达促进HL-60细胞的增殖并抑制其分化. Transwell迁移侵袭小室实验表明DJ-1核定位过表达可以促进HL-60的迁移和侵袭能力. Western blot结果表明DADS具有抑制HL-60细胞中核内DJ-1蛋白表达的能力. 说明DJ-1核定位高表达具有促进HL-60细胞增殖和迁移侵袭及抑制HL-60细胞分化的作用,DADS可以诱导DJ-1核定位高表达HL-60细胞分化及抑制迁移侵袭, DJ-1核定位高表达可减弱DADS抑制HL-60细胞增殖的作用.     

CD44v6对急性白血病细胞HL-60和THP-1增殖和迁移的影响

目的:探讨CD44变异亚型对急性白血病细胞增殖和迁移的影响。方法:选择对数生长期的急性白血病细胞株HL-60、THP-1和慢性白血病细胞株K562,采用荧光定量PCR法检测CD44v6mRNA的表达。通过电转的方法转染CD44v6siRNA到HL-60和THP-1细胞抑制细胞的CD44v6表达,通过western方法检测CD44v6蛋白的抑制情况。将实验分成HL-60+N-siRNA、HL-60+CD44V6-siRNA、THP-1+N-si RNA、THP-1+CD44V6-siRNA共4组,培养24、48、72 h后分别取细胞悬液用台盼蓝染色后计数活细胞数检测细胞的增殖情况;使用Transwell小室培养法观察HL-60和THP-1细胞的迁移率。结果:通过荧光定量PCR方法检测THP-1和HL-60细胞均高表达CD44v6(分别为0.0037±0.0007和0.00292±0.0002),明显高于K562的表达(P0.01);转染后的HL-60和THP-1细胞株中CD44v6蛋白表达水平明显下调,细胞计数结果显示转染CD44v6-siRNA的HL-60和THP-1细胞在24、48和72 h增殖均明显下降。迁移实验结果显示THP-1+N-si RNA和HL-60+N-si RNA细胞的迁移率为17%和23%,与相应对照组相比THP-1+CD44v6-siRNA和HL60+CD44v6-siRNA组细胞24 h迁移率明显下降(分别降至11%和14%)。结论:CD44v6可以通过干预白血病细胞的增殖和迁移能力,参与调解白血病细胞的增殖和髓外进展。     

维生素A酸诱导HL-60细胞分化后银染核仁形成区的研究

用Ag-NOR、Brachet反应和作者改进的悬浮培养细胞Ag-aNOR的TEM技术等,综合地对RA诱导HL-60细胞分化后银染核仁形成区的变化进行了研究。实验结果表明,银染NOR的均值在给药组与对照组细胞中差异不显著。对间期细胞进行Ag-aNOR反应,结果??表明其数量在给药组比对照组明显减少,同时发现Brachet反应显示富含rRNA的核仁数与Ag-aNOR数改变极为一致,提示虽然活性的rRNA基因的数量在RA诱导分化的HL-60细胞中不变,但其转录明显受抑,使rRNA合成减少,进而在细胞水平出现了恶性表达的核仁变得少而小、甚至消失的分化表型。HL-60细胞的Ag-aNOR电镜观察更进一步证明了这一结论。     

GPI-PLD调节CEA的释放对白血病HL-60细胞的增殖和凋亡的影响

糖基化磷脂酰肌醇特异性磷脂酶D(glycosyl phosphatidyl inositol specific phospholipase D,GPI-PLD)是人体内唯一可水解细胞膜表面GPI结构、调节GPI锚定蛋白释放的酶.将GPI-PLD转染入急性粒细胞白血病(AGL)的HL-60细胞株,采用实时荧光定量PCR法和Western blot法确定转染后HL-60细胞内GPI-PLD的表达水平;并检测GPI-PLD活性;噻唑蓝(MTT)检测HL-60细胞的增殖;流式细胞仪检测HL-60细胞的凋亡.ELISA检测GPI锚定癌胚抗原(CEA)的表达和释放情况.转染GPI-PLD后,HL-60细胞株中GPI-PLD表达量与活性增加;MTT检测显示,GPI-PLD过表达后HL-60细胞株增殖生长受到抑制;流式检测证实HL-60细胞凋亡增加;且GPI锚定的蛋白质CEA释放增加.该结果提示GPI-PLD基因有抗肿瘤的作用,过表达GPI-PLD后能抑制HL-60细胞增殖且促进其凋亡,所涉机制可能与GPI-PLD释放GPI锚定蛋白,增强白血病细胞对补体杀伤的敏感性有关.     

TGFβ1参与视黄酸诱导的HL-60细胞分化

为研究内源性TGF-β１对全反式视黄酸（ATRA）作用HL60细胞的影响,应用定量RT-PCR和ELISA方法,研究ATRA诱导HL-60细胞分化过程中TGF-β１ 表达的变化.并构建TGF-β１ RNA干扰表达质粒,抑制HL-60细胞内源性TGF-β１表达,进而研究ATRA诱导内源性TGF-β１表达下降的HL-60细胞分化的情况．结果发现,ATRA诱导HL-60细胞分化过程中,TGF-β１ 表达明显增高．得到4个针对不同靶位点的RNA干扰表达质粒.其中,针对起始编码区的质粒转染48 h后对HL-60细胞的TGF-β１蛋白抑制率为73~2％．内源性TGF-β１表达下降后,ATRA作用的HL-60细胞NBT还原试验的光密度值降低,CD33抗原阳性的细胞比例较对照组升高,CD11b抗原阳性的细胞比例较对照组降低.表明内源性TGF-β１表达下降后,ATRA诱导HL-60细胞分化的作用有所减弱,提示内源性TGF-β１在ATRA诱导HL-60细胞分化中起一定的作用．     

PHA—T细胞条件培养液对白血病细胞的作用

本文研究了培养18h收获的PHA-T细胞条件培养液(PHA-TCM)对人白血病细胞系HL-60和U937的作用。结果表明,PHA-TCM能够显著地抑制U937细胞的增殖,并能降低其~3H-TdR摄入能力和自发集落形成能力。而且,经PHA-TCM作用一定时间后,部分U937细胞获得了NBT还原能力和乳胶颗粒吞噬能力。与此相反,PHA-TCM对HL-60细胞无此作用,提示PHA-TCM对白血病细胞增殖的抑制具有某种选择性。     

NADPH氧化酶源性活性氧介导epinodosin诱导HL-60细胞分化及细胞骨架重组

该文分析研究了对映-贝壳杉烷二萜epinodosin通过调节NADPH氧化酶源活性氧诱导急性早幼粒细胞白血病细胞HL-60向成熟粒细胞分化的特征。结果显示,epinodosin在4.0~8.0μmol/L浓度时抑制HL-60细胞生长,导致其S期阻滞,诱导HL-60细胞形成肾形核和多叶核细胞,使细胞吞噬能力和对硝基四氮唑蓝(nitrotetrazolium blue chloride,NBT)的还原力显著增强、细胞表面抗原CD11b表达上调,表明epinodosin可诱导HL-60细胞向成熟粒细胞分化;epinodosin还诱导HL-60细胞骨架重组,显著促进微管的成束组装以及提高波形纤维含量,上述分化特征与临床诱导分化治疗剂全反式维甲酸(all-trans retinoic acid,ATRA)对HL-60细胞的分化诱导特性极为相似。进一步检测显示,抗氧化剂NAC(N-acetyl-L-cysteine)以及NADPH氧化酶抑制剂夹竹桃麻素(apocynin,APO)和联苯基三价碘(diphenyleneiodonium,DPI)均可抑制epinodosin对HL-60细胞的分化诱导作用,表明epinodosin导致HL-60细胞活性氧浓度的升高与NADPH氧化酶活性关联,提示epinodosin通过调节NADPH氧化酶源性活性氧诱导HL-60向粒细胞分化。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

脂氧素对K562和HL-60白血病细胞增殖和凋亡的影响(英文)

炎症在肿瘤的发生发展过程中扮演重要角色,脂氧素是一类重要的内源性抗炎介质。但是迄今为止,脂氧素对肿瘤的影响报道极少。为此,本文研究了脂氧素对HL-60和K562白血病细胞增殖和凋亡的影响。体外培养白血病细胞株HL-60和K562,Western印迹和实时荧光定量PCR检测脂氧素受体的表达情况;CCK-8法(cell counting kit-8 assay)检测HL-60和K562的增殖能力;PI染色后利用流式细胞仪进行细胞周期分析;膜联蛋白V试剂盒检测脂氧素对细胞凋亡的影响。实验结果表明脂氧素抑制HL-60和K562白血病细胞增殖(P0.05);脂氧素处理组S期细胞比例明显减少而G_0/G_1期细胞比例增加;脂氧素还可以诱导HL-60和K562白血病细胞凋亡。由此可见,脂氧素抑制HL-60和K562白血病细胞增殖,其机制可能与诱导白血病细胞G_0/G_1期阻滞和细胞凋亡有关。     

Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

Recombinant human leukocyte interferon (IFN-alpha A) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-alpha A is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-alpha A and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represents a novel means of reducing resistant leukemic cell populations.     

Effect of retinoic acid on the phosphorylation of endogenous proteins in HL-60 cells

HL-60 cells were incubated with [32P]-Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two-dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic-acid-differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL-60 cells with [35S]-methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL-60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO-arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two-dimensional gels revealed that the phosphorylation of the 53-kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic-acid-induced differentiation process.     

Magnesium deprivation inhibits the expression of differentiation-related phenotypes in human promyelocytic leukemia HL-60 cells

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.     

Control of HL-60 myeloid differentiation. Evidence of uncoupled growth and differentiation control, S-phase specificity, and two-step regulation

Myeloid differentiation of HL-60 human promyelocytic leukemia cells was studied during DMSO-induced differentiation. G 1/0-specific growth arrest could occur without the usual associated subsequent phenotypic differentiation into mature myeloid cells, suggesting that growth arrest and phenotypic differentiation are separately regulated. In the course of differentiating, the cells achieved a semi-stable intermediate state where they had a labile, pre-commitment memory of exposure to inducer, but were not yet committed to differentiation. This state was associated with a nuclear structural change previously found to be associated with the precommitment memory state. The process of differentiation could thus be resolved into two steps, early events up through development of pre-commitment memory and late events subsequents to pre-commitment memory. The kinetics of terminal cell differentiation indicated that the cellular regulatory event initiating a program of differentiation in response to inducer was S phase-specific. A comparison of the present results for DSMO to previous results for retinoic acid (RA)-induced HL-60 myeloid differentiation showed that the two inducers effect different cellular pathways for differentiation of HL-60 cells to mature myeloid cells, but with certain common features including the above S-phase specificity and pre-commitment memory.     

维生素A类化合物和二甲基亚砜诱导人早幼粒白血病细胞(HL-60)分化的显微与亚显微结构研究

用光镜和电镜技术研究了HL-60细胞在诱导分化过程中的显微与亚显微结构变化,10~(-6)M的维A酸处理6天,细胞按粒系途径定向分化,其核质比例降低,核浓缩、分叶,核仁减少或消失。经RA处理的细胞在电镜下出现下列明显的变化:细胞核浓缩和分叶,异染色质区域增加,约46%细胞显示出类似成熟粒细胞核的亚显微形态特征,胞质中嗜天青颗粒减少,特异颗粒显著增加,两种颗粒的比率发生明显变化;细胞质中微管、微丝的量增加;多聚和单个分散的游离核糖体减少,有些??细胞胞质空泡化;出现主要以微丝为筑架的大型钝形伪足和不规则的表面突起。上述这些变化似可作为HL-60细胞形态分化的标志。维A酸诱导HL-60细胞形态分化具有明显的时间效应关系。1.4%DMSO对HL-60细胞分化的诱导作用类似于10~(-6)MRA,而等剂量的(10~(-6)M)R Ⅰ、RⅡ其作用弱于RA。     

Membrane origin for a signal eliciting a program of cell differentiation

Evidence is presented to indicate that the retiNOic acid (RA)-induced program of myeloid differentiation and growth arrest by HL-60 human promyelocytic leukemia cells was initiated by a signal originating at the cell membrane. Free RA and RA covalently immobilized on a solid substrate elicited similar kinetics of differentiation and G1/0-specific growth arrest. No evidence of cell-induced RA detachment from the solid substrate was found. The data explain why HL-60 cells which are deficient in cellular RA-binding protein (CRABP) nevertheless respond to RA.     

Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin

Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells.     

Nuclear CD38 in retinoic acid-induced HL-60 cells

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD(+) and hydrolysis of either NAD(+) or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD(+) glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a approximately 43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the approximately 43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.     

Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60)

More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for self-renewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator-the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonaI homogeneity of the population with respect to differentiation capacity. The loss of self-renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self-renewal potential without being able to complete their program of differentiation.