Synchronization of the human promyelocytic cell line HL 60 by thymidine

Cultures of the promyelocytic cell line HL 60 were synchronized with thymidine. A concentration of 0.05 mM thymidine and an exposure time of 24 hr was found optimal for blocking about 90% of the cells in S phase. Following release from the thymidine block the cell cultures were followed intermittently over 40 hr for fluctuation in cell numbers, labelling with radioactive thymidine and nuclear DNA distributions. Mathematical evaluation of the results revealed a cycling time of 18.6 hr and a duration of specific cell phases of 8.6 hr, 7.1 hr and 2.9 hr for G1, S and G2 + M, respectively. The doubling time was 26 hr and the growth fraction was estimated as 1.     

Changes in cell kinetics associated with differentiation of a human promyelocytic cell line (HL60)

Abstract. Terminal cell differentiation results in an irreversible arrest in the G₁ phase of the cell cycle and loss of the capacity for cell renewal. In the murine erythroleukaemia cell line (MELC), commitment to erythroid differentiation was found also to be preceded by an early, transient, phase of inhibition of growth due to prolongation of the G₁ phase. We determined the effect of differentiation-inducing agents on the growth kinetics of a human promyelocytic cell line (HL60) which undergoes differentiation into mature granulocyte. At concentrations of inducers optimal for cell differentiation, an early, transient stimulation of cell multiplication was found. DNA synthesis was enhanced in HL60 cells as early as 5 hr after exposure to inducer. Nevertheless, HL60 cell maturation eventually also resulted in a loss of the multiplication ability. The duration of exposure to inducer required for irreversible loss of the potential for self-renewal was determined by the fall in the cloning efficiency of induced cells; the results indicate that it preceded the switch-off of the replication mechanism; the majority of the cells lost their ability to form large colonies at the time of peak DNA synthesis and were able to complete an additional two to three cell cycles at a rate similar to uninduced cells. These changes occurred before HL60 cells became committed and might play a pivotal role in the process of cell differentiation.     

Characterization of insulin receptors in human promyelocytic leukemia cell HL60

Highly specific insulin receptors have been identified on human promyelocytic leukemia cells HL60. Insulin binding increased progressively with time to reach a maximum at 2 h at 22° and was proportional to the number of cells in the incubation mixture. Insulin degradation as assessed by TCA precipitation and reincubation studies was negligible. Scatchard analysis of the binding data was curvilinear and the total number of insulin receptor sites per cell was around 45,000. The average affinity profile gave an “unoccupied site” affinity constant of 3.5 × 10⁸ M^?1. The promyelocytic cells HL60, thus, have specific binding sites and binding characteristics similar to more mature human myeloid cells.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectin-like molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Synthesis of cathepsin B by cells derived from the HL60 promyelocytic leukaemia cell line

Cells of the human promyelocytic HL60 line were induced to differentiate into granulocyte-like cells with dimethylsulphoxide (DMSO) or macrophage-like cells with 12-0-tetradecanoylphorbol-13-acetate (TPA). The synthesis of Cathepsin B by these cells was studied by immunoperoxidase staining and assay of cell lysates using the fluorimetric substrate benzoyloxycarbonyl-phenylanalyl-arginine-4-methyl-7-coumarylamide. Only 2–5% of the uninduced HL60 cells and DMSO-induced cells were immunohistochemically positive for Cathepsin B, compared with over 80% of the TPA-induced cells. Cathepsin B activity was lowest in the lysates of uninduced HL60s. DMSO-induced cells contained 1.5–2-fold the enzyme activity of HL60s and TPA-induced cell lysates demonstrated 5–14-fold the activity of uninduced HL60s. Induction of Cathepsin B synthesis was therefore associated with differentiation of the promyelocytes into cells of the monocyte/macrophage type, but not granulocyte-like cells. Cathepsin B was located immunohistochemically in human palatine tonsils. The enzyme was only demonstrated within macrophages in these tissues. Cathepsin B may therefore be a useful immunohistochemical marker for malignant and nonmalignant cells of the monocyte/macrophage lineage.     

Acute regulation of glucose transport in a human megakaryocytic cell line: difference between growth factors and H(2)O(2)

The present study was undertaken to: (i) compare the effect of some hematopoietic growth factors, like interleukine-3, thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, stem cell factor, and reactive oxygen species such as H(2)O(2) on glucose uptake in a human leukemic megakaryocytic cell line, M07; (ii) investigate the changes in kinetic parameters of the transport activity induced by these stimuli; and (iii) evaluate the effect of genistein, a tyrosine kinase inhibitor, on the glucose uptake activation by the cited agents. The results are as follows: (i) exposure of M07 cells to thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, and stem cell factor resulted in a rapid stimulation of glucose transport; interleukine-3-treated cells exhibited no increase in the rate of glucose uptake, although M07 proliferation is interleukine-3 dependent; a rapid glucose transport enhancement was also observed when M07 cells were exposed to low doses of H(2)O(2); (ii) the transport kinetic parameters point out that an important difference exists between the effect of cytokines and that of H(2)O(2): cytokines increased predominantly the affinity for glucose, while H(2)O(2) raised both the V(max) and K(m) values; (iii) the isoflavone genistein, at a very low concentration, inhibited the stem cell factor- or H(2)O(2)-induced stimulation of hexose transport, reversing the variations of K(m) and V(max), but it did not affect the transport activity of granulocyte-megakaryocyte colony-stimulating factor-treated cells; and (iv) catalase completely abolished the stimulatory action of H(2)O(2) on glucose transport and slightly prevented the effect of stem cell factor, while caffeic acid phenethyl ester was only able to affect the activation due to stem cell factor.     

Commitment to differentiation of human promyelocytic leukemia cells (HL60): an all-or-none event preceded by reversible losses of self-renewal potential

A method for clonal analysis has been developed which allows the characterization of the number and type of progeny cells produced by each single cell arising during clonal evolution. The method is based on a symmetry of self-renewal exhibited by sister cells of the human promyelocytic leukemia cell line -HL60-. This permits the use of one of the sister cells to measure the potential for self renewal of the other. Using a system of sequential daughter cell transfers in semisolid medium, we have analysed self-renewal and differentiation in individual clones exposed to all-trans retinoic acid or dimethylsulfoxide (DMSO). We find that in clones exposed to chemical inducers of differentiation commitment occurs as an all-or-none event which is preceded by coordinated but reversible losses of self-renewal potential. It is concluded that the differentiation pathway of HL60 cells has two distinct portions. These are, first, a predeterministic portion, reflected by coordinated but reversible losses of self-renewal potential, and second, a deterministic portion, reflected by irreversible phenotypic differentiation.     

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.     

Development of calcium and secretory responses in the human promyelocytic leukemia cell line HL60

We have begun to characterize the development of the excitation-response coupling sequence in the human promyelocytic leukemia cell line HL60. Using the recently developed fluorescent calcium probe quin-2, it was found that DMSO induced myeloid differentiation of the HL60 cells is accompanied by the development of a calcium response to the addition of the chemotactic factors fMet-Leu-Phe and leukotriene B4. The characteristics (time course, concentration dependence, stereospecificity, and metabolic dependence) of the calcium response are extremely similar to those previously described in human neutrophils. These results imply that functional receptors for leukotriene B4 appear in HL60 cells upon the induction of differentiation and also lend strong support to the use of these HL60 cells as a model of human myeloid differentiation. We have also characterized the emergence of a secretory response to fMet-Leu-Phe and leukotriene B4 in cytochalasin B treated HL60 cells. In addition, it is found that differentiation was required for the calcium ionophore A23187 to express its secretory activity toward the HL60 cells. This last set of results implies that differentiation is accompanied by the coordinated appearance of surface receptors and cytoplasmic factors required for the expression of cellular responsiveness.     

Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells.

The promyelocytic leukemia (HL60) cell line differentiates into monocyte-like cells after treatment with phorbol dibutyrate (PBt2). In contrast, bryostatin 1 (bryo), a structurally distinct protein kinase C (PKC) activator, does not induce differentiation and blocks the cytostatic effect of PBt2. The divergent responses to these agents correlate with activation of a PKC-like activity at the nucleus in response to bryo but not PBt2 (Fields, A. P., Pettit, G. R., and May, W.S. (1988) J. Biol. Chem. 263, 8253-8260). In the present study, this nuclear PKC-like activity (termed PKCn) was isolated from HL60 cells and shown to phosphorylate its known nuclear substrate, lamin B. PKCn-mediated phosphorylation of nuclear envelope-associated lamin B in vitro is calcium-dependent and is stimulated by bryo and 1,2-dioctanoylglycerol (DiC8), but not PBt2. In contrast, PKCn-mediated phosphorylation of histone IIIS is stimulated equally by all three activators. PKCn mediates calcium- and phosphatidylserine-dependent phosphorylation of both histone IIIS and partially purified lamin B. PKCn activity can be inhibited by an anti-PKC monoclonal antibody which specifically inhibits PKC. Isotype-specific PKC antibodies identify PKCn as beta II-PKC. Immunoblot analysis indicates that HL60 cells express both alpha- and beta II-PKC but no beta I- or gamma-PKC. Treatment of intact cells with bryo for 30 min leads to complete translocation of both alpha- and beta II-PKC from the cytosol to the membrane fractions. Approximately 8-10% of the total beta II-PKC (and less than 0.3% of the alpha-PKC) is found associated with the nuclear membrane of bryo-treated cells. In contrast, PBt2 treatment leads to complete translocation of alpha-PKC, but only partial translocation of beta II-PKC to the plasma membrane fraction. Neither PKC isotype is found associated with the nuclear membrane of PBt2-treated cells. These data demonstrate that alpha- and beta II-PKC differ with respect to activator responsiveness, intracellular distribution, and substrate specificity and indicate that their selective activation at distinct intracellular sites, including the nucleus, can have a dramatic effect on resulting cellular responses.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type I cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type I cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.     

Large-scale purification of myeloperoxidase from HL60 promyelocytic cells: characterization and comparison to human neutrophil myeloperoxidase

A large-scale purification procedure was developed for the isolation of myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils.     

Oxidative stress enhances granulocytic differentiation in HL 60 cells,an acute promyelocytic leukemia cell line

Abstract

This paper studied the effects of physiologically available oxidants on HL 60 differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Hydrogen peroxide (15 μM) and taurine chloramine (200 μM) induced HL 60 differentiation, which was detected by CD11b expression and superoxide production. Cd11b and p67^phox mRNA expression was also augmented by these oxidants. In contrast, reducing chemicals, such as dithiothreitol, 2,3-dimercapto-1-propanol and N-acetylcysteine inhibited CD11b expression. Notably, DMSO inhibited methionine sulfoxide reductase activity, induced heme oxygenase-1 (ho-1) mRNA and enhanced oxidant-induced cell death, which indicated that DMSO intensified oxidative stress. After the addition of oxidants, ho-1 expression preceded the cd11b expression. Vicinal dithiol-reactive phenylarsine oxide (50 nM) also increased CD11b expression induced by DMSO or ATRA. These observations suggested that oxidative stress enhanced granulocytic differentiation of HL 60 cells and that leukaemic cell differentiation was affected by cellular redox status.