Induction of the differentiation of HL-60 promyelocytic leukemia cells by L-ascorbic acid

The present study was undertaken to examine the effect of L-ascorbic acid (LAA) on the growth of HL-60 promyelocytic leukemia cells, besides induction of apoptosis. LAA (> or = 10(-4) M) was found to markedly inhibit the proliferation of HL-60 in liquid culture and clonogenicity in semisolid culture. Moreover, LAA-treated HL-60 showed activity to produce chemiluminescence and expressed CD 66b cell surface antigens, indicating that LAA induces the differentiation of HL-60 mainly into granulocytes. The results are supported by morphological changes of LAA-treated HL-60 into segmented neutrophils. Therefore, the inhibitory effect of LAA on the growth of HL-60 cells seems to arise from the induction of differentiation. To assess the potential role of LAA, cells were exposed to oxygen radical scavengers in the absence or presence of LAA. Catalase abolished and superoxide dismutase promoted LAA-induced differentiation of HL-60. Thus, H2O2 produced as a result of LAA treatment seems to play a major role in induction of HL-60 differentiation.     

Induction of differentiation in human promyelocytic leukemia HL-60 cell line by niacin-related compounds

Water-soluble vitamin, niacin, and its related compounds were examined for their differentiation-inducing activity in human promyelocytic leukemia cells (HL-60). Among the compounds, which inhibited cell proliferation measured by MTT assay, isonicotinic acid, nicotinamide N-oxide, and nicotinamide induced NBT reducing activity. HL-60 cells were differentiated into granulocyte-like cells by these compounds, judging from morphological changes and loss of nonspecific esterase activity. The differentiation-inducing activity of water-soluble vitamin and its related compounds suggest that these compounds may be applicable for medical use.     

Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin

Tiazofurin is an effective inducer of the maturation of HL-60 promyelocytic leukemia cells, as monitored by increased phagocytic ability and the capacity to reduce nitroblue tetrazolium (NBT). The antimetabolite acts as a potent inhibitor of IMP dehydrogenase, which results in a profound depression in the cellular levels of guanine nucleotides. Flow cytometric analysis of DNA histograms indicated that the commitment of HL-60 cells to differentiate when exposed to tiazofurin was preceded by a transient delay in the G1 phase of the cell cycle. HL-60 leukemia cells enriched in the various phases of the cell cycle by centrifugal elutriation were utilized to further characterize the relationship between the phase of the cell cycle and the commitment to enter a pathway of differentiation. Fractions composed mainly of G1 cells demonstrated an increased capacity to mature when exposed to tiazofurin, whereas fractions containing cells from the S and G2 + M phases of the cell cycle had a lower ability to enter a differentiation pathway. The findings suggest that the commitment of HL-60 cells to mature when exposed to tiazofurin is mediated during the G1 phase of the cell cycle.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work     

Development of capping ability during differentiation of HL-60 human promyelocytic leukemia cells

Developmental changes in cell surface and cytoskeletal elements have been studied in human promyelocytic leukemia cells (line HL-60) which differentiate into functionally mature myeloid cells when grown in dimethyl sulfoxide (DMSO)-supplemented medium. Both differentiated and undifferentiated HL-60 cells bind fluorescent concanavalin A (F-Con A) in a diffuse pattern over the entire cell surface. As with normal neutrophils, pretreatment of the differentiated HL-60 cells with colchicine before incubation with Con A causes the formation of large cytoplasmic protrusions over which the lectin associates into a cap. On the other hand, similarly treated undifferentiated HL-60 cells do not form the cytoplasmic protuberances and are unable to cap the Con A. Transmission electron microscopy reveals that the number and distribution of microtubules and microfilaments change during differentiation. Thus, developing myeloid cells undergo important alterations in the structure and function of the cytoskeleton as they differentiate into mature phagocytes.     

Expression of leukocyte adherence-related glycoproteins during differentiation of HL-60 promyelocytic leukemia cells

We used the HL-60 human promyelocytic leukemia cell line to analyze the surface expression of a family of adherence-related leukocyte surface antigens during myeloid differentiation. These antigens are composed of discrete alpha subunits, designated alpha L, alpha M, and alpha X, that are each noncovalently associated with a common beta subunit. Monoclonal antibodies directed against the individual subunits served as markers in both indirect immunofluorescence studies and immunoprecipitations from HL-60 cells differentiated preferentially towards mature granulocytes (DMSO, retinoic acid) or monocyte/macrophages (PMA, vitamin D3). In undifferentiated HL-60 cells, the alpha L and alpha X subunits were constitutively expressed, whereas the alpha M subunit was not. Differentiation of HL-60 cells along the granulocytic pathway with DMSO resulted in a marked increase in alpha M and minimal increases in alpha L and alpha X. The phenotypic expression of these antigens on DMSO-treated HL-60 cells closely resembled that on normal circulating PMN. Differentiation along the monocyte/macrophage pathway when using PMA or vitamin D3 resulted in major increases in alpha L and alpha X expression, as well as alpha M. These changes resulted in a surface phenotype characteristic of that present on human monocyte-derived macrophages. Triggering of undifferentiated HL-60 cells with PMA caused no increase in subunit expression, whereas stimulation of DMSO-differentiated HL-60 cells with PMA produced more than a 1.5-fold enhancement of both the alpha M and alpha X subunits, and stimulation of human PMN with PMA increased the surface expression of alpha M more than fourfold and alpha X subunit twofold. Stimulation with PMA produced no change in expression of the alpha L subunit in any of the three cell populations. These results indicate that the alpha subunits of this glycoprotein family can be selectively regulated during in vitro differentiation of a human promyelocytic leukemia cell line. Second, DMSO-differentiated HL-60 cells and human PMN possessed an intracellular pool of alpha M and alpha X, but not alpha L, that could be translocated to the surface. Thus, despite structural and functional relationships among the alpha subunits in this glycoprotein family, they undergo disparate surface expression and intracellular regulation during differentiation.     

Loss of transferrin receptors following induced differentiation of HL-60 promyelocytic leukemia cells

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.     

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

Fatty acid changes during the induction of differentiation of human promyelocytic leukemia (HL-60) cells by phorbolmyristate acetate.

We studied fatty acid changes that are likely to occur during phorbolmyristate acetate (PMA)-induced differentiation of HL-60 cells. It was observed that PMA-induced differentiation is associated with increased uptake, but not synthesis, of fatty acids. Fatty acid analysis revealed that arachidonic acid (AA), 20:5 n-3 and 22:6 n-3 levels are reduced with a concomitant increase in 22:5 n-6 in the phospholipid fraction. In the FFA fraction there are increases in free AA, free 20:5 n-3, 22:5 n-3 and 22:6 n-3, and a fall in free 22:5 n-6 in PMA-treated cells. PMA-induced differentiation and nitroblue tetrazolium reduction by PMA-treated cells was only partially inhibited (about 20-30%) by indomethacin and nordihydroguiaretic acid (cyclooxygenase and lipoxygenase inhibitors respectively), but not by superoxide dismutase, catalase or mannitol. These results indicate that PMA-induced differentiation of HL-60 cells is accompanied by specific changes in the fatty acid composition of the cells.     

Induction of cyclo-oxygenase synthesis in human promyelocytic leukaemia (HL-60) cells during monocytic or granulocytic differentiation.

Cyclo-oxygenase (COX) production in human promyelocytic leukaemia (HL-60) cells was studied during monocytic differentiation induced by 1 alpha, 25-dihydroxyvitamin D3 (24 nM; 3 days) or phorbol 12-myristate 13-acetate (100 nM; 1 day), or during granulocytic differentiation induced by retinoic acid (1 microns; 4 days). Undifferentiated or differentiated HL-60 cells were labelled with [35S]methionine, and membrane-bound COX was solubilized and quantified by SDS/PAGE. Immunoprecipitated 35S-labelled COX from cells induced to differentiate into monocytic or granulocytic lineage were clearly detected on the autoradiograms as a protein of approx. 70 kDa molecular size, whereas only a very faint COX band was detected in untreated HL-60 cells. During both monocytic and granulocytic differentiation, COX activity (measured by the conversion of exogenous arachidonic acid into prostaglandin E2) was dramatically increased. In addition, thromboxane synthesis was preferentially enhanced during monocytic differentiation. HL-60 cells, induced to differentiate into the monocytic or granulocytic lineage, provide a useful tool to investigate the cellular mechanisms involved in regulation of the synthesis of individual prostanoid-metabolizing enzymes.     

Induction of differentiation in HL-60 promyelocytic cells: a comparative study in two sublines

Two variants of HL-60 promyelocytic leukemia cells (HSC, OCI) that were indistinguishable by morphology, cell surface markers, DNA histograms, and by their inability to reduce nitroblue tetrazolium, were induced to differentiate by retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and by phytohemagglutinin-leucocyte conditioned medium (PHA-LCM). Only OCI cells were induced to differentiate to mature granulocytes by TPA. Both cell lines expressed, however, the monocytic associated cell surface antigen detected by MO1 monoclonal antibody in response to TPA. MO1 expression was detected as early as 32 hours after initiation of differentiation by TPA, whereas partial morphologic changes were apparent only after 72 hours. Induction of differentiation by retinoic acid led to a significant inhibition of colony formation in HSC variant (from 1522 +/- 60 to 523 +/- 20/10(4) cells plated) and in the OCI variant (from 628 +/- 20 to 185 +/- 33 colonies/10(4) cells plated). The addition of PHA-LCM further inhibited colony growth of both RA-induced cell lines (155 +/- 7/10(4) cells plated in HSC, and 59 +/- 4 in OCI). PHA-LCM by itself reduced HL-60 colony numbers in a dose-related manner, and also increased the expression of MO1 on noninduced HSC and OCI cells. These observations suggest that differentiation of HL-60 cells is not necessarily accompanied by concomitant change in morphology, cell surface characteristics, and proliferation potentials, and may be dependent on different degrees of cellular commitment. They also suggest a role for growth factors in the induction to maturation of leukemic cells.     

Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells

Triphenyltin (TPT) is an environmental endocrine disruptor and toxic substance, but little information is available on its immunological effects. To assess the effect of TPT on leukocyte differentiation, we investigated its effect on the neutrophilic differentiation of HL-60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At a low concentration, 10(-7)M, TPT increased superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 45% and increased expression of CD18, a component of the OZ-receptor, by about 90%. Real-time PCR analysis revealed that TPT augmented the expression not only of CD18 but also of components of superoxide-generating NADPH-oxidase, p47phox, 2.7-fold, and p67phox, 2.0-fold, and of granulocyte colony-stimulating factor receptor (G-CSFR), 3.0-fold, whereas various other endocrine disruptors, including parathion, vinclozolin, and bisphenol A, had no such enhancing effects. The results of a DNA macroarray analysis showed that TPT enhanced the expression of G-CSFR and certain other neutrophil functional proteins, including CD14 and myeloid leukemia cell differentiation protein (MCL-1), and that TPT induced a decrease in expression of LC-PTP, leukocyte protein-tyrosine phosphatase, to about half the control level. The TPT-dependent suppression of LC-PTP was confirmed by real-time PCR analysis, and the results of immunoblotting indicated that TPT enhances the expression of myeloid specific tyrosine kinase hck by about 30% at the protein level, and this together with the reduction of LC-PTP may enhance tyrosine phosphorylation, in turn resulting in enhancement of superoxide production. These findings suggest that TPT may have an enhancing effect on the neutrophilic maturation of leukocytes.     

Detection of a novel cyclooxygenase metabolite produced by human promyelocytic leukemia (HL-60) cells

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.     

Characterization of human alpha-dystrobrevin isoforms in HL-60 human promyelocytic leukemia cells undergoing granulocytic differentiation

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.     

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.     

Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.     

Cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) in human promyelocytic leukemia cells HL-60

12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumor promoter and is known to induce terminal differentiation of human promyelocytic leukemia cells HL-60 to mature monocytes. To investigate the molecular mechanism of TPA actions, TPA-specific binding proteins in HL-60 were analyzed. Anion exchange high-performance liquid chromatography revealed that HL-60 cells possess TPA-specific binding proteins other than protein kinase C (PKC). One of these TPA-specific binding proteins exists in the cytosolic fraction of HL-60 cells, but translocates into the nuclear fraction of HL-60 cells after the treatment of the cells with TPA. The results suggest that HL-60 cells take up TPA into the nuclei via the TPA-specific binding protein. The TPA-specific binding protein binds TPA, phorbol 12,13-di-butylate, teleocidin B-2, teleocidin B-3, and debromoaplysiatoxin in a mutually competitive manner. However, the protein does not bind to okadaic acid, olivoretin C, retinoic acid, or dioxin. This cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) might play an essential role in the action of tumor promoters.     

Sialic acid lyase in human promyelocytic leukemic cells (HL-60) during phorbol-ester-induced differentiation

There is a marked increase in the activity of sialic acid lyase (N-acetylneuraminate lyase; EC 4.1.3.3; also known as sialic acid aldolase) in HL-60 cells induced to differentiate into macrophages by the phorbol ester, tetradecanoylphorbol 12-myristate 13 acetate (TPA). Exposure of HL-60 cells to retinoic acid, butyric acid or dimethyl sulfoxide has little or no effect. The level of the enzyme remains unaltered in HL-60 cells grown in the presence of an inactive analog of TPA, nor does it change in variants of HL-60 cells resistant to TPA.     

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.