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.     

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.     

Phosphorylation and dephosphorylation of human promyelocytic leukemia cell (HL-60) proteins by tumor promoter

Human promyelocytic leukemia cell line (HL-60) has been shown to be induced to the terminal differentiation into macrophage-like cells by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The present studies describe the effects of TPA on the phosphorylation of HL-60 cell proteins. A rapid decrease in the phosphorylation of a 75 kD protein was observed within a few minutes after treatment with TPA. On the other hand, TPA treatment of HL-60 cells caused rapid increase in the phosphorylation of a 67 kD protein and other minor proteins. Phorbol and 4α-phorbol-12,13-dodecanoate, both of which are biologically inactive derivatives of TPA, failed to cause any changes in protein phosphorylation in HL-60 cells. These results suggest that changes in protein phosphorylation are involved in mechanisms of the differentiation in HL-60 cells induced by TPA. Cell fractionation experiments revealed that 67K protein was located in cytosol. Though 75K protein also seemed to be located in cytosol, the phosphate moiety of 75K protein was almost lost during cell fractionation, suggesting that the phosphorylation of 75K protein was specifically regulated in HL-60 cells. Dimethyl sulfoxide (DMSO), retinoic acid (RA) and 1,25-dihydroxy-vitamin D₃, all of which induce the differentiation in HL-60 cells, did not cause any changes in protein phosphorylation. These results suggest that the changes in protein phosphorylation are specific for TPA. The possible mechanisms of changes in protein phosphorylation by TPA were discussed.     

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.     

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.     

Pharmacological characterization of the histamine uptake system in HL-60 human promyelocytic leukemia cells

Serotonin and histamine H₁, H₂ receptor agonists or antagonists inhibited [³H]histamine uptake by HL-60 cells, according to the following order of potency: impromidine >4-MH>histamine>AET>PEA and: cimetidine, histamine>diphenhydramine, serotonin. It is concluded that histamine uptake by HL-60 cells was specifically controlled by the H₂ type histamine receptor and that this active process might be involved in pathophysiological regulations in leukemic and normal granulocytic precursors and in the control of histamine levels in peripheral blood and tissues in man.     

Matrix metalloproteinases expression in HL-60 promyelocytic leukemia cells during apoptosis

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrix-metalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.     

Characterization of the binding of S-adenosyl-L-methionine to plasma membranes of HL-60 promyelocytic leukemia cells

S-Adenosyl-L-methionine (AdoMet) has been found to bind specifically to the plasma membrane of promyelocytic leukemia cells, HL-60. The Kd for AdoMet is 4.2.10(-6) M and the Bmax is 4.0.10(-12) mol/10(7) HL-60 cells. The binding is not related to the adenosine receptor since neither adenosine, ADP, nor ATP affect the ligand-receptor reaction. When HL-60 cells were incubated with physiological concentrations of [methyl-3H]AdoMet (20 microM) at 36 degrees C, AdoMet did not equilibrate with the intracellular pool, nor were any [3H]methyl groups incorporated into nucleic acids or proteins. In contrast, significant amounts of [3H]methyl groups were incorporated into membrane phospholipids. When cells were incubated with 20 microM [methyl-3H]AdoMet, [3H]methyl groups were transferred to phosphatidylethanolamine, -monomethylethanolamine, and -dimethylethanolamine yielding phosphatidylcholine. However, the rate of methyl transfer with AdoMet was only 22% of that observed when cells were incubated with a comparable amount of [methyl-3H]methionine. Both the binding of AdoMet and the methylation of phospholipids were inhibited by exogenous S-adenosyl-L-homocysteine. Therefore, the binding may be linked to a phospholipid methyltransferase.     

Asymmetric cell division in human leukemic promyelocytic cells (HL-60).

Small cells accounted for 8-9% of the human leukemic promyelocytic cells (HL-60). The diameter of the small cells was 8.44 μm, whereas that of the large cells, which were heterogeneous in cell size, was 11.0 μm. The small cells were produced from the large cells through asymmetric cell division, which was demonstrated by cloning experiments and by microscopy.     

ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.     

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.     

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.     

Studies on changes in nucleolar organizer region of human promyelocytic leukemia cells (HL-60) treated with retinoic acid

Changes of nucleolar organizer region in HL-60 cells after treated with retinoic acid (RA) were studied with techniques of silver-staining nucleolar organizer region (Ag-NOR) in metaphase karyotypes, Brachet's reaction and with our improved TEM techniques for studying silver-stained active nucleolar organizer region (Ag-aNOR) in interphase nucleoli. Number of Ag-NOR in HL-60 cells is 4.5/cell on average. The Ag-NOR number of cells treated with RA showed no remarkable difference from that of control group. Ag-aNOR number treated with RA was reduced obviously as compared with that of control group. Meanwhile, the changes of nucleolus number showed by Brachet's reaction were in accordance with those of Ag-aNOR. Therefore, it may be concluded: (1). Though the number of active rRNA genes did not changed after the differentiation of HL-60 cells induced by RA, their expression was clearly inhibited: (2). The relationship between the changes of Brachet-No and Ag-aNOR is in positive correlation (r = 0.98, p less than 0.01). EM examination of Ag-aNOR of HL-60 cells reveals that Ag-protein (RNA polymerase I) only presented in fibrillar centers (FC) and the dense fibrillar components (DFC) of nucleolus. In addition, in control group, large amount of Ag-protein, FC, DFC and granular components (GC) were observed, and there were many large nucleoli in a nucleus, meanwhile, the cells of the treated group tended to be mature, with a decrease in the amount of Ag-protein, FC, DFC and GC accordingly, and the nucleoli reduced both in size and number significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells

Curcumin, a well-known dietary pigment derived from the food flavoring turmeric (Curcuma longa) exhibits anti-proliferative, anti-inflammatory, and anti-oxidative activities. Recently, studies have shown that a chemopreventive effect of curcumin could be due to the hyperproduction of reactive oxygen species (ROS) inducing apoptosis in tumor cells. In our previous studies, ornithine decarboxylase (ODC) overexpression prevented tumor necrosis factor alpha (TNF-alpha)- and methotrexate-induced apoptosis via reduction of ROS. Furthermore, ODC is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. In this study, we found that enzyme activity and protein expression of ODC were reduced during curcumin treatment. Overexpression of ODC in human promyelocytic leukemia HL-60 parental cells could reduce curcumin-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsi(m)), through reducing intracellular ROS. Moreover, ODC overexpression prevented cytochrome c release and the activation of caspase-9 and caspase-3 following curcumin treatment. These results demonstrate that curcumin-induced apoptosis occurs through a mechanism of down-regulating ODC and along a ROS-dependent mitochondria-mediated pathway.     

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.     

An artificial protein,possessing biological activity of HL-60 human promyelocytic leukemia differentiation factor

The ABB-df artificial protein was prepared by inserting the TGENHR biologically active peptide corresponding to the 41-46 sequence of the differentiation factor for the HL-60 cell line of the human promyelocyte leukemia into the N-terminus of the polypeptide chain of albebetin, an artificial protein with the preset structure. The ABB-df protein was found to induce the differentiation of HL-60 cells and to inhibit their proliferation; its efficiency was almost the same as that of the starting peptide. According to CD spectroscopy, the inclusion of the peptide fragment into albebetin exerts virtually no effect on the regular secondary structure of albebetin.     

Effect of phorbolmyristate acetate on fatty acid uptake and distribution in human promyelocytic leukemia (HL-60) cells in vitro

Human promyelocytic leukemia (HL-60) cells can be induced to differentiate to macrophages in vitro by phorbolmyristate acetate (PMA). HL-60 cells, unlike normal cells incorporated a major portion of linoleic acid (LA) and arachidonic acid (AA) in the ether lipid fraction. On exposure to PMA, similar to the normal cells tested, the fatty acids were incorporated mainly in the phospholipid fraction. Since, ether lipid pool is metabolically inert and considered as a storage pool where as the phospholipid fraction is a metabolically active pool this may explain, at least in part, the low metabolic rate of AA and the low phospholipase A2 activity in HL-60 cells.