Effect of sera from acute lymphoblastic leukemia children on proliferation and differentiation of HL-60 promyelocytic leukemia cell line

Influence of sera of children with ALL on character (dispersed or compact) and composition (granulocyte or macrophage) of colonies, formed from the human promyelocytic leukemia cell line HL-60, was estimated. An increased activities stimulating formation of dispersed G and M colonies was found in the sera of patients before therapy. Dynamics of colonies formation and their composition in the course of treatment point to some changes in the serum the levels of these activities with a tendency to full normalization in remission.     

Studies of leukotoxin from Actinobacillus actinomycetemcomitans using the promyelocytic HL-60 cell line

The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease.     

兔中性粒细胞防御素对HL—60细胞毒性及对病毒的直接灭活作用

本研究从新西兰家兔腹腔渗出中性粒细胞溶酶体内提取酸溶性组分，经制备性ＡＵ－ＰＡＧＥ获得纯化的防御素（ＮＰ１和ＮＰ２），ＳＤＳ－ＰＡＧＥ测定分子量为３ｋｄ左右。以ＨＬ－６０细胞作靶细胞，当ＮＰ１或ＮＰ２在１００μｇ／ｍｌ浓度时，可杀死６０％以上的ＨＬ－６０细胞。ＮＰ１（２６０μｇ／ｍｌ）还可直接灭活单纯疱疹病毒Ｉ型和乙型流感病毒，分别使病毒感染滴度降度９８．２％和９０％。本研究结果提示，中性粒细胞可     

The loss of IAP expression during HL-60 cell differentiation is caspase-independent

Human promyelocytic leukaemia cells (HL-60) differentiate into neutrophil-like cells that die spontaneously by apoptosis when treated with retinoic acid (RA). Inhibitors of apoptosis proteins (IAP) bind to and inhibit caspases 3, 7, and 9 activity and the induction of apoptosis. In this study, we demonstrate that undifferentiated HL-60 cells express IAP. During their differentiation, IAP expression is decreased at the mRNA and protein levels. In addition, we show that there is a corresponding increase in the expression and functional activity of active caspases 3 and 9. This activity was associated with the cleavage of XIAP, NAIP, and cIAP-2. Most importantly, we demonstrate that blocking caspase activity does not alter the decrease in IAP protein expression during differentiation but prevents caspase activation, IAP cleavage, and the induction of apoptosis. This result shows that the loss of IAP expression is independent of the induction of apoptosis and is solely related to the differentiation process. However, IAP cleavage is caspase-dependent. Terminal differentiation results in an altered apoptotic phenotype that is associated with the induction of HL-60 cell apoptosis.     

一氧化氮诱导HL-60细胞凋亡及其机制探讨

目的：观察一氧化氮对人类白血病细胞是否具有致凋亡作用，并研究Bcl-2基因和P53基因在此过程中的作用。方法：将不同浓度的外源性一氧化氮供体亚硝基铁氰化钠与HL-60细胞作用，观察其作用的时间效应和剂量效应；用MTT法观察NO对细胞的抑制作用，用透射电镜观察细胞结构改变，用DNA凝胶电泳、细胞DNA含量、DNA末端标记、Annexin-V/PI法等分析细胞凋亡，并用流式细胞法进一步观察在NO作用过程中凋亡调控因子Bcl-2和P53蛋白及线粒体膜蛋白表达变化。结果：NO能抑制HL-60细胞生长，并在一定的剂量范围内显示作用时间和剂量的量效关系；典型的细胞形态改变、DNA片段化、亚G1峰检出、DNA末端标记、Annexin-V/PT^-表达增加等证实。NO能诱导白血病细胞凋亡；在此过程中，P53蛋白表达上调，而Bcl-2蛋白表达下调，线粒体膜蛋白表达增加。结论：NO对HL-60细胞有强的致凋亡作用，Bcl-2和P53参与NO诱导HL-60细胞凋亡的调控。     

Experimental model of hemorrhagic stroke: Rabbit immunization with HL-60 promyelocytic cell differentiation factor

Rabbits immunized with synthetic HLDF differentiation factor developed hemorrhagic stroke with thrombosis of small cerebral vessels and destruction of vascular endotheliocytes. The severity of stroke correlated with serum level of antibodies to differentiation factor. The role of different sites of HLDF molecule in the induction of clinical signs of hemorrhagic stroke was studied. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 2, pp. 237–240, February, 2006     

茶多酚对HL-60细胞株体外增殖及细胞周期的影响

目的 探讨茶多酚对人急性早幼粒白血病细胞株HL-60细胞增殖和细胞周期的影响.方法 采用四甲基偶氮唑盐微量酶反应比色法(MTT比色法)观察茶多酚对体外培养的HL-60细胞增殖活性的影响.采用HE染色、荧光染色观察用茶多酚后细胞形态变化.用流式细胞仪(FACS)检测细胞凋亡率及细胞周期.结果 (1)MTT比色法检测显示茶多酚能抑制HL-60细胞增殖,在一定范围内呈剂量和时间依赖性(P＜0.05).当茶多酚浓度达到400和800 mg/L时,48 h抑制率分别为(58.90±1.19)%和(72.57±0.70)%.(2)流式细胞仪分析,茶多酚处理组出现一特征性的亚二倍体凋亡峰.其凋亡率呈时间、剂量依赖性.(3)流式细胞术发现茶多酚可使HL-60细胞阻滞于S期,其阻滞细胞的数量与药物浓度呈正相剂量关系,以作用24 h对细胞周期的阻滞作用最强.结论 茶多酚能有效抑制HL-60细胞增殖,在一定范围内具有时间和剂量依赖性.茶多酚可体外诱导HL-60细胞凋亡,并使细胞周期阻滞于S期.     

SU11248对白血病细胞HL-60增殖及凋亡作用的实验研究

目的:探讨新型抗肿瘤药物苹果酸舒尼替尼(SU11248)对白血病细胞HL-60的生物学效应的影响及其作用机制。方法:应用MTT法检测SU11248对HL-60细胞增殖能力的影响;用AnnexinV/PI双染流式细胞术检测细胞凋亡;用流式细胞技术分析细胞的DNA倍体及细胞周期变化;用凝胶电泳分析DNA片段化;以Western blot法检测2.0μg/ml SU11248作用HL-60后bcl-2、bax、caspase-3蛋白水平的变化。结果:SU11248可明显抑制HL-60细胞增殖(P<0.05),呈剂量和时间依赖性,半数抑制浓度(IC50)约为2.00μg/ml。SU11248可促进细胞凋亡,并呈剂量依赖性;能将HL-60细胞阻滞于G0/G1期,并呈时间依赖性;诱导HL-60细胞呈典型的DNA梯带;SU11248作用后bcl-2蛋白表达随时间依赖性降低,caspase-3蛋白表达升高,bax蛋白表达无明显变化。结论:SU11248能抑制HL-60细胞增殖,诱导其凋亡,调节bcl-2家族蛋白的表达,并裂解caspase-3是其可能作用机制之一。     

阿糖胞苷联合光动力疗法对人白血病HL-60细胞作用的研究

目的 探讨阿糖胞苷（Ara-c）在癌光啉（PSD-007）介导的光动力疗法（PDT）杀伤人早幼粒白血病HL-60细胞中的联合作用.方法 实验分为空白对照组、PDT单独作用组（PDT l～4组,为光敏剂剂量（5、7.5μg/ml）和激光能量密度（1.2、2.4 J/cm^2）的两两组合）、Ara-c单独作用组（Ara-c A组和Ara-c B组中Ara-c质量浓度分别为0.3 μg/ml和1.2μg/ml）和联合作用组即上述PDT单独作用组和Ara-c单独作用组的两两组合.所有分组分别按3种时序即P24A时序（PDT作用24h后再加入Ara-c共同作用24h）、A24P时序（Ara-c作用24h后进行PDT再共同作用24h）和PA24时序（PDT作用的同时加入Ara-c再共同作用24h）进行处理.采用CCK-8法检测各组细胞活性,用金氏公式分析联合效应,并用流式细胞术检测细胞周期变化.结果 小剂量PSD-007介导的PDT和Ara-c联合时,3种时序的联合作用均表现为协同效应;而大剂量PSD-007介导的PDT和Ara-c联合时,P24A和A24P 2种时序的联合作用效应为协同或相加,PA24时序则主要为相加或拮抗.流式细胞仪检测细胞周期变化结果显示,Ara-c和PSD介导的PDT均能引起细胞周期G0/G1期阻滞.结论 Ara-c与PSD-007介导的PDT联合作用对HL-60细胞的杀伤有协同作用,其协同作用与剂量和作用时序相关,小剂量比大剂量协同效果明显,且Ara-c和PDT间隔24h作用比2者同时作用于该细胞的协同效果明显.     

Factors influencing basophilic differentiation of HL-60 cells

Lineage-specific hematopoietins apparently act in concert with multipotent factors in an orderly sequence of growth and differentiation. We have used the human acute promyelocytic leukemia cell line HL-60 to examine basophilic differentiation, using radioenzymatic assay of histamine content as an end point. Recombinant human interleukin 1 (rhIL-1), rhIL-2, rhIL-4, and recombinant human alpha and gamma interferons did not stimulate basophilic differentiation either in the presence or absence of sodium butyrate, an important cofactor for induction of differentiation. In contrast, rhG-CSF (granulocyte colony-stimulating factor), rhGM (granulocyte-macrophage) CSF, rhIL-3, rhIL-5, nerve growth factor, conditioned medium (CM) from the hairy T cell leukemic line Mo, and nasal polyp epithelial CM stimulated significant increases in histamine content in HL-60 cells at day 5 in vitro. GM-CSF did not account for all of the basophilic differentiating activity in Mo-CM. The data suggest that a unique, lineage-specific, basophilic cell differentiation factor is produced by T cells and point to the possible diagnostic and therapeutic relevance of in situ hematopoietic mechanisms in human respiratory disease.     

Histamine-induced differentiation of eosinophilic subclone of HL-60 cells

Inflammation Research -     

Induction of HLA-DQ antigen expression on a human promyelocytic leukemia cell line HL-60

A variant strain of HL-60, which is positive for HLA-DR antigen, was induced to express HLA-DQ antigen following treatment with phorbol esther. It was preceded by cell cycle arrest in the G1 phase and was accompanied by augmented phagocytosis. This differential expression of HLA class II antigens on this subline may contribute to understanding the functional role of HLA class II antigens in the hematopoietic differentiation of macrophage cells.     

Histamine-induced bi-directional differentiation of HL-60 cells towards neutrophils and eosinophils

HL-60 cells, treated under alkaline conditions (pH 7.6) or acidic conditions (pH 7.2) for 2 months, were stimulated with histamine for 7 days. From the morphological examination and cytochemical characterization, it became clear that one of the clones treated in acidic pH differentiated to neutrophils and the other clone treated in alkaline medium differentiated to eosinophils after histamine-stimulation. The growth curve reached a maximum 4 days after stimulation. By means ofin situ hybridization, it has been shown that the mRNA of major basic protein increased after histamine treatment only in the eosinophilic subclone, starting 4 days after stimulation. From the present study, it is suggested that when HL-60 cells were cultured under different pH conditions, commitment of lineages to the direction of either eosinophils or neutrophils takes place. Histamine may potently stimulate the further differentiation of both eosinophilic and neutrophilic clones.

     

In vitro detection of apoptotic stimuli by use of the HL-60 myeloid leukemic cell line.

The human histiocytic lymphoma line HL-60 has served as a model of myeloid cell differentiation and can be induced to differentiate along the neutrophil or monocytic lineage, depending on the external stimulus. The nondifferentiated cell line retains a premyeloid leukemic phenotype and is capable of anchorage-independent growth and proliferation. The role of apoptosis in the regulation of immunologic and inflammatory events associated with homeostasis and disease has been most intensively studied in lymphocytes. In the present study, nondifferentiated HL-60 has served as a model for studying myeloid cell apoptosis by investigating apoptotic changes induced by camptothecin, a DNA topoisomerase inhibitor, as well as physiologic stimuli, including ceramide analogs and a monoclonal antibody against the Fas antigen. Multiparameter flow cytometry was used to evaluate apoptosis by measuring changes in both side scatter and propidium iodide staining. The appearance of apoptotic cells was confirmed biochemically by measuring DNA endonuclease activity by both enzyme-linked immunosorbent assay quantitation and DNA ladder formation on agarose gels and morphologically with the detection of micronuclei by confocal laser microscopy. These studies demonstrate that HL-60 can serve as an in vitro model for the detection of physiologic and pharmacologic apoptotic stimuli and for understanding the early and late cellular changes associated with induction of the apoptotic program.     

Replication of human cytomegalovirus in the cells of the U937 monocytoid cell line

Human cytomegalovirus (HCMV) infection in immunocopromized hosts sometimes occurs as a result of reactivation. Cells of the monocytemacrophage linkage are suggested to be a site of latency and persistence for HCMV. The human monocytic cell line U937 was infected with the AD169 strain and a clinical isolate of HCMV. The expression of surface antigens on the cells was assessed by flow cytometry. The polymerase chain reaction (PCR) was used to detect viral DNA from infected cells. CMV immediate early antigen, early antigen, and late antigen (LA) were detected from both clinical isolate- and AD169-inoculated U937 cells by flow cytometry. CMV DNA which code major immediate early gene (US3) and LA gene (US14) were detected from the clinical isolateinoculated U937 over a period of 31 days as tested by PCR. These U937 cells proliferated as well as uninfected U937 cell, but only a small number of AD169-inoculated U937 cells survived after 14 days of inoculation. Interleukin-2 activities were detected in the media on days 24–40 after inoculation with AD169. This chronic CMV infection model of U937 might be utilized to study the mechanisms of persistence and reactivation.     

三七皂甙R1诱导HL—60细胞系分化的形态和功能变化

用三七皂甙R_1(80μg/ml)处理HL-60细胞,发现68%的HL-60细胞向中性粒细胞分化,其中晚幼粒细胞32%,杆状核粒细胞30%,分叶核粒细胞6%;进一步实验显示分化后细胞硝基蓝四氮唑(NBT)还原能力、吞噬功能、细胞膜补体受体以及酸性磷酸酶(ACP)和β葡萄糖醛酸酶(β-Gr)活性都明显增高。结果表明,三七皂甙R_1能诱导HL-60细胞向中性粒细胞分化。