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.     

Failure of F-Met-Leu-Phe to induce chemotaxis in differentiated promyelocytic (HL-60) leukemia cells

Treatment of HL-60 promyelocytic leukemia cells with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) induced these cells to differentiate toward mature neutrophilic granulocytes (PMN). DbcAMP was found to produce a rapid time- and dose-dependent inhibition of HL-60 cell proliferation, reaching a maximum after 48 hr treatment of the cells with 250-500 microM. This was associated with morphologic alterations consistent with maturing PMN. Using immunofluorescence and flow cytometry, we found that dbcAMP-treated HL-60 cells expressed some, but not all, surface markers characteristic of mature phagocytes. Thus receptors for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP); the monocyte/granulocyte markers My-8, Mo-1, and Leu-15; as well as the monocyte markers Mo-2 and My-4 were identified on the cells. In contrast, dbcAMP-treated cells did not express the PMN-specific antigen DL1.2. We also found that dbcAMP-treated HL-60 cells produced hydrogen peroxide in response to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, the magnitude of this response was significantly less than that observed in mature PMN. In contrast, fMLP-stimulated levels of hydrogen peroxide production were comparable in both cell types. In addition, despite the fact that dbcAMP-treated cells expressed fMLP receptors, they did not exhibit directed migration toward this chemotactic peptide. Taken together, these data suggest that dbcAMP-induced differentiation of HL-60 cells is incomplete.     

Relationship between3H-histamine uptake and H2-receptors in the human promyelocytic leukemia cell line HL-60

Association of³H-histamine (³H-H) with HL-60 cells was a time- and temperature-dependent process. At 37°C, the association of³H-H (50 fmole/10⁶ cells) was maximal and constant 60 min after the addition of HL-60 cells, while there is no significant radioactivity associated with cells incubated at 4°C during 120 min incubation. Under these conditions, the cell-associated radioactivity remains unchanged, even after a washout period and addition of large excess (10^–3 M) of histamine, indicating an irreversible interaction between histamine and HL-60 cells. The interaction of³H-H with HL-60 cells depends on cell viability, cell concentration and was reduced by various metabolic inhibitors such as iodacetamide, dinitrophenol, sodium azide and ouabain. Histamine uptake by HL-60 cells was inhibited by a series of H₁, H₂ histamine agonists (impromidine, histamine, 4-MH, AET, PEA) and antagonists (cimetidine, oxmetidine, ranitidine, diphenhydramine), according to the following order of potencies: 1>H, 4-MH>AET>PEA and H, C>DPH. Among the histamine analogs tested (acidN-acetyl histamine, imidazole, acid imidazole acetic and histidine), only theN-acetyl histamine acid was able to inhibit³H-H uptake by HL-60 cells. Three antidepressant drugs (amitriptyline, imipramine, clomipramine) also antagonize³H-H uptake by HL-60 cells, but this effect was only observed at toxic concentrations (10^–5 M and above) and was related to a loss of the cell viability. It was concluded that the promyelocytic leukemia cells HL-60 take up histamine by a specific energy-dependent mechanism, preferentially inhibitable by histamine H₂-receptor agonists and antagonists (H₂>H₁). Therefore, the uptake of³H-H by HL-60 cells accounts for an H₂-receptor-mediated process, consistent with the pharmacological specificity of the histamine H₂-receptor linked to the cAMP generating system in this cell line.     

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.     

Biosynthesis of proteochondroitin sulfate by HL-60 human promyelocytic cells

Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/10(5) viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCl gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCl gradient (rho greater than 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/10(5) cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.     

The bone marrow peptide (myelopeptide-2) abolishes induced by human leukemia HL-60 cell suppression of T lymphocytes

Myelopeptide-2 (MP-2) Leu-Val-Val-Tyr-Pro-Trp originally isolated from the supernatant of porcine bone marrow cell culture was examined for its capacity to restore the mitogen responsiveness of human T lymphocytes inhibited by conditioned media from HL-60 leukemia cells (HL-60 CM). MP-2 added to phytohemagglutinin (PHA)-stimulated T lymphocytes together with HL-60 CM abolished the suppression of T-lymphocyte proliferative response in a dose-dependent manner. Another bone marrow hexapeptide Phe-Leu-Gly-Phe-Pro-Thr, MP-1, did not display this action in that experimental system. MP-2 was also effective being added after T-lymphocyte exposure to HL-60 CM which suggests its recovery but not protective effect on T-lymphocytes treated with tumor cell products. Flow cytometry analysis revealed HL-60 CM influence on the expression of CD3 and CD4 T-cell surface antigens. It decreased the content of CD3- and CD4-positive cells and induced the appearance of T lymphocytes with reduced density of CD3 and CD4 antigens. MP-2 was able to restore the T-cell phenotype altered by HL-60 CM. MP-2 seems to be promising in anti-tumor therapy.     

Dexamethasone affects cytokine-mediated adhesion of HL-60 human promyelocytic leukemia cells to cultured dermal microvascular endothelial cells

Leukocyte endothelial adhesion (LEA) is the prelude to a complex cascade of reactions following an immunological challenge. Recently, LEA has been implicated in the molecular basis of several dermatological disorders. While the role of proinflammatory cytokines, such as interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), in LEA has been investigated using nondermal models, limited data exist regarding their effects on LEA in dermal models. This study shows that cotreatment of cultured human dermal endothelial cells (CADMEC) with IL-1beta and TNF-alpha resulted in a marked increase in the adherence of human promyelocytic leukemia (HL-60) cells to CADMEC and an increase in expression of intercellular adhesion molecule-1 and E-selectin. Pretreatment of CADMEC with dexamethasone, a long-lasting glucocorticoid, resulted in a decrease in both HL-60 cell adhesion to CADMEC and adhesion molecule expression. Taken together, these data demonstrate that LEA may play a role in inflammatory skin conditions and in the mechanisms underlying the potential use of glucocorticoids as a treatment option.     

Characterization of the defect in a variant of HL-60 promyelocytic leukemia cells with reduced transferrin receptor expression

The mechanism by which a clone of HL-60 human promyelocytic leukemia cells designated Tf-Gel-1 expresses reduced levels of the transferrin receptor (TfR) was investigated. Tf-Gel-1 was developed by continuous exposure of HL-60 cells to human iron-saturated transferrin covalently linked to the plant toxin gelonin (Tf-Gel); this variant was five- to sixfold more resistant to Tf-Gel than parental HL-60 cells. The amount of cell surface, as well as of solubilized, TfR and the cycling pools of TfR in Tf-Gel-1 cells, as measured by the binding of [¹²⁵I]Tf, were all decreased to 20–30% of the levels present in parental cells. The growth of Tf-Gel-1 cells was independent of exogenous Fe³⁺ and was comparable to that of parental HL-60 cells. Despite the lower levels of TfRs, the Tf-Gel-1 clone retained the capacity to alter receptor expression, depending upon the phase of growth and the intracellular iron concentration, and to down-regulate TfRs in response to inducers of differentiation. Southern hybridization of cellular DNA with TfR cDNA did not reveal differences between parental and Tf-Gel-1 cells in the level and arrangement of the TfR gene. Basal and inducible (repressible) levels of TfR mRNA from Tf-Gel-1 cells, as measured by northern hybridization of cellular RNA with TfR cDNA, were comparable to those of parental cells. Metabolic labeling of cells with [³⁵S]methionine, followed by immunoprecipitation of TfRs, demonstrated that the amount of radioactivity incorporated into TfRs in Tf-Gel-1 cells was reduced to a degree that approximated the decrease in [¹²⁵I]Tf binding. Cell surface TfRs prepared from exponentially growing parental cells labeled with¹²⁵I by the solid-phase lactoperoxidase-glucose oxidase method existed as a doublet, with one form being phosphorylated and the other not phosphorylated. In contrast, Tf-Gel-1 cells not only contained diminished amounts of TfRs but also contained only the phosphorylated form of TfRs in the surface membrane. The decrease in the surface membrane concentration of the TfR in Tf-Gel-1 cells was specific for this glycoprotein, since the levels of other cell surface antigens, such as CD13, CD15 and CD45, were normal in Tf-Gel-1 cells. A reduction in the incorporation of [³H]mannose into the acid-insoluble fraction of cells and an increase in sensitivity to ricin suggested that Tf-Gel-1 cells possessed an aberration in carbohydrate metabolism.     

Autoinduction of differentiation in human myelocytic leukemia cells (HL-60-Y3)

We conducted a study on autoinduction of differentiation in human myelocytic leukemia cells (HL-60-Y3) in which the effects of serum cytodifferentiation were excluded by the use of a serum-free semisolid culture. In the culture dish the HL-60-Y3 colony count per dish was kept at 100 or below, and only the formation of clumping-type colonies, which consisted of blastoid cells, was observed. The formation of spreading-type colonies increased with the colony count and when the colony count reached 500 per dish, more than 90% of the colonies formed were spreading-type colonies. The main component cells of the spreading-type colonies were mature monocytoid cells, which were positive for alpha-naphthyl butyrate esterase. Moreover, a marked reduction in the recloning ability was observed in differentiated colonies compared to undifferentiated colonies. These results indicate the autoinduction of differentiation in human myelocytic leukemia cells. Furthermore, a single cell study that excluded the effect of colony to colony interactions suggested the presence of a differentiation autoinducing factor in the medium.     

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.     

Oncogene mobility in a human leukemia line HL-60

HL-60, a cell line derived from a human promyelocytic leukemia, shows amplification of the oncogene c-myc. Chromosome aberrations reported in HL-60 include double minutes (DMs) and an abnormally banded region (ABR) on chromosome #8. A relationship between these chromosomal aberrations and amplification of c-myc DNA has been suggested. We report the localization by cytologic hybridization of amplified c-myc DNA to a marker chromosome, M3q+, in an early passage of HL-60. The localization of c-myc to an ABR on an 8q+ chromosome was confirmed in later passage clones. The most probable derivation of the M3q+ chromosome is t(5p;17q) with additional material associated with c-myc amplification inserted into 17q. This localization is of interest in light of the association between t(15:17) and promyelocytic leukemia. The results indicate that amplification and chromosome integration can occur at a site other than the native gene locus and at different integration sites in different lineages of the same tumor.     

Activation of human neutrophils by the air pollutant sodium sulfite (Na(2)SO(3)): comparison with immature promyelocytic HL-60 and DMSO-differentiated HL-60 cells reveals that Na(2)SO(3) is a neutrophil but not a HL-60 cell agonist

Sulfite exposure can induce inflammatory responses characterized by an influx of neutrophils into the airways leading to lung malfunctions. Studies focusing on sodium sulfite (Na(2)SO(3))/neutrophil interactions have shown that this chemical possesses proinflammatory properties based on its ability to induce a respiratory burst. Information regarding how this chemical could alter other neutrophil responses/functions as well as its role on immature promyelocytic cells is currently lacking in the literature. In this study, we report that Na(2)SO(3) can induce tyrosine phosphorylation events in human neutrophils but not in both HL-60 and HL-60 + DMSO. As a positive control, GM-CSF was found to induce tyrosine phosphorylation of a particular protein of 120-130 kDa in both HL-60 and HL-60 + DMSO cells testifying that these cells were responsive. In addition, we report that Na(2)SO(3) does not alter neutrophil phagocytosis and that this chemical increases the release of the proinflammatory cytokine IL-8 but not TNF-alpha. Paradoxically, we found that Na(2)SO(3) acts as a potent inhibitor of de novo neutrophil protein synthesis in a concentration-dependent fashion (0.1, 1, or 10 mM) as assessed by SDS-PAGE from metabolically [(35)S]-labeled cells. In contrast to mature neutrophils, we found that Na(2)SO(3) does not modulate de novo protein synthesis in HL-60 cells treated with low concentrations (0. 1 or 1 mM) and that this pollutant was toxic at 10 mM as judged by a drastic decrease of total protein content stained with Coomassie blue. We conclude that Na(2)SO(3) can activate human neutrophils and that its proinflammatory potential is further supported by its ability to increase IL-8 production. In addition, our results clearly indicate that HL-60 and HL-60 + DMSO respond differently than mature human neutrophils to the inflammatory pollutant Na(2)SO(3). Extrapolation of data obtained with HL-60 (and/or HL-60 + DMSO) to neutrophils should be taken with caution. Our data obtained with Na(2)SO(3) are an example.     

Infection of undifferentiated and differentiated promyelocytic cells (HL-60) with varicella-zoster virus

HL-60 cells can be induced to differentiate into macrophage-like cells by treatment with 12-O-tetra-decanoylphorbol-13-acetate (TPA). The relationship between virus replication and cell differentiation was investigated using HL-60 cells that had been induced to differentiate by TPA (dHL-60 cells) and undifferentiated cells (udHL-60 cells). On infection of these cells with cell-free varicella-zoster virus (VZV), virus antigens were detected in dHL-60 cells but not in udHL-60 cells, and the percentage of antigen-positive dHL-60 cells increased during incubation. Similar results were obtained by infectious center assay, and the percentage of antigen-positive cells correlated with the stage of cell differentiation. No significant difference was found in the binding of VZV to dHL-60 cells and udHL-60 cells. Furthermore, trypsin treatment after adsorption suggested that VZV penetrated into udHL-60 cells. These findings indicate that VZV may be able to replicate in mature monocytes but may be harbored in immature monocytes in vivo.     

1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.     

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.     

Acrylamide-induced molecular mutation spectra at HPRT locus in human promyelocytic leukaemia HL-60 and NB4 cell lines

Acrylamide (AA) is a compound widely used in many industries around the world. The recent finding that it is formed naturally in foods by heating raises human health concerns. AA is a proven carcinogen in animals and a probable carcinogen in humans, while its mutagenicity detected using in vitro mammalian gene mutation assays is still inconsistent in different cell systems. In the present study, we investigated the mutagenicity of AA in human promyelocytic leukaemia cells, HL-60 and NB4 cells, by examining the mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus. In a 6-h treatment without the exogenous activation, AA exerted a weak mutagenic effect at the highest concentration used in the study (700 mg/l) in HL-60 cells (P < 0.01) as well as in NB4 cells (P < 0.05). Molecular analysis of AA-induced mutants revealed a different mutation spectrum, when compared to that of spontaneous mutants. The most frequent spontaneous mutations were point mutations, whereas AA-induced mutations were mainly single exon deletions besides point mutations, and an increase in the proportion of partial deletion was associated with the increase of AA treatment. There was no obvious difference in the mutation spectra observed between the HL-60 and NB4 cell lines. These results showed that AA has a weak mutagenic effect at HPRT gene locus in human promyelocytic leukaemia HL-60 and NB4 cell lines and those molecular mutation spectra (single exon deletions and point mutations) may be related to some specific and precise mechanism.     

人参皂甙单体Rb1对人急性早幼粒白血病细胞系增殖及糖皮质激素受体α表达的影响

目的:观察人参皂甙(GS)Rb1对人急性早幼粒白血病细胞(HL60)糖皮质激素受体α(GRα)mRNA及受体蛋白表达的调节作用和对细胞生长的影响。方法:以四唑盐比色法(MTT法)观察细胞生长,用蛋白印迹法(Western blot)和反转录-聚合酶链反应(RT-PCR)分析G-Rb1对细胞的GR蛋白和GRα mRNA的改变。结果:G-Rb1能够抑制HL60细胞的生长,呈时间和剂量依赖性;经G-Rb1作用后RT-PCR和Western blot分析,细胞内GRα mRNA以及GR蛋白表达增加。结论:G-Rb1可上调HL60细胞GRα mRNA及GR蛋白的表达并抑制细胞生长。