DNA repair in human promyelocytic cell line, HL-60.

The human promyelocytic cell line, HL-60, shows large changes in endogenous poly(ADP-ribose) and in nuclear ADP-ribosyl transferase activity (ADPRT) during its induced myelocytic differentiation. DNA strand-breaks are an essential activator for this enzyme; and transient DNA strand breaks occur during the myelocytic differentiation of HL-60 cells. We have tested the hypothesis that these post-mitotic, terminally differentiating cells are less efficient in DNA repair, and specifically in DNA strand rejoining, than their proliferating precursor cells. We have found that this hypothesis is not tenable. We observe that there is no detectable reduction in the efficiency of DNA excision repair after exposure to either dimethyl sulphate or gamma-irradiation in HL-60 cells induced to differentiate by dimethyl sulphoxide. Moreover, the efficient excision repair of either dimethyl sulphate or gamma-irradiation induced lesions, both in the differentiated and undifferentiated HL-60 cells, is blocked by the inhibition of ADPRT activity.     

The retinoic acid receptors alpha and beta are expressed in the human promyelocytic leukemia cell line HL-60

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into granulocytes upon exposure to retinoids. Previously we have shown that extracts of undifferentiated HL-60 cells possess a specific retinoid-binding activity (RSBP-1) corresponding to an approximate 95 kilodalton (kDa) protein as determined by size-exclusion chromatography. We now extend these observations to reveal a second approximate 95 kDa retinoic acid-binding component (RSBP-2), which is separable from RSBP-1 using anion exchange chromatography. We further show that the chromatographic properties of RSBP-1 and RSBP-2 are identical to those found for the retinoid-binding activities present in extracts of HeLa cells transfected with the human retinoic acid receptor (RAR) expression vectors RAR-beta phi and RAR-alpha phi, respectively. Moreover, an antiserum preparation directed against RAR-beta selectively immunoprecipitated both the retinoid-binding activity in extracts of HeLa cells transfected with RAR-beta phi and that corresponding to RSBP-1 in HL-60 cell extracts. Similarly, an antiserum preparation directed against RAR-alpha immunoprecipitated the retinoid-binding activity in extracts from RAR-alpha phi transfected HeLa cell as well as that corresponding to RSBP-2 in HL-60 cell extracts. Using these antisera, Western blot analyses of extracts from HL-60 cells, and from HeLa cells transfected with either RAR-alpha phi or RAR-beta phi, confirmed that RSBP-2 and RSBP-1 are identical to RAR-alpha and RAR-beta, respectively. However, RAR-alpha, RAR-beta, RSBP-1, and RSBP-2 appeared as an approximate 51 kDa species in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in contrast with an apparent approximate 95 k mol wt as estimated from size-exclusion chromatography in the presence of 0.6 M KCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of HL-60 cells with dimethyl sulphoxide inhibits the formation of beta-N-acetylhexosaminidase S.

beta-N-Acetylhexosaminidase of HL-60 cells was separated into two main forms, A and S, by chromatography on DEAE-cellulose. Analysis of developmental changes in the isoenzyme pattern was complicated by the fact that the specific activity of beta-N-acetylhexosaminidase underwent a 6-fold change during the normal growth cycle. Two other lysosomal enzymes, beta-galactosidase and alpha-mannosidase, behaved similarly. Induction of differentiation of HL-60 cells with dimethyl sulphoxide at a low cell density (3 x 10(5) cells/ml) had a greater effect on the abundance of alpha-subunits of beta-N-acetylhexosaminidase, measured with 4-methylumbelliferyl-beta-N-acetylglucosaminide 6-sulphate, than of beta-subunits, measured with 4-methylumbelliferyl-beta-N-acetylglucosamine, and resulted in an isoenzyme profile in which A and B were the major forms, with the levels of form S greatly decreased.     

Expression of immunoglobulin lambda light chain by the promyelocytic cell line HL-60

The HL-60 cell line, established from a patient with acute promyelocytic leukemia, can be induced to undergo differentiation along the granulocyte or monocyte/macrophage line, depending on the particular inducer that is used. In this communication we provide evidence that HL-60 cells also have B lymphoid characteristics because by flow cytometry and clonal excess calculations, these cells are found to express immunoglobulin (Ig) lambda light chains on their surface. Furthermore, HL-60 cells contain poly(A)+ RNA that hybridizes with a DNA fragment encoding the constant region of Ig lambda chains and comigrates with lambda mRNA on RNA blots. Treatment of HL-60 cells with a phorbol ester that induces monocyte/macrophage differentiation resulted in the loss of surface Ig lambda chains and lambda RNA.     

Evidence of multifactorial mechanisms in an adriamycin-resistant HL-60 promyelocytic leukemia cell line

HL-60/AR leukemia cells, which were 60-fold resistant to the growth inhibitory activity of adriamycin, remained sensitive to the antiproliferative and differentiation-inducing activities of aclacinomycin A. The replication of HL-60/AR and of adriamycin sensitive parental HL-60 cells was inhibited by greater than 80% by 30 nM aclacinomycin A and the majority of cells (about 60 to 70%) of each line underwent granulocytic differentiation when treated with this agent, as assessed by the reduction of nitroblue tetrazolium. Measurement of the initial rates of uptake of daunorubicin and steady-state levels of adriamycin in sensitive and resistant lines indicated that transport differences do not fully account for the insensitivity of HL-60/AR cells to these anthracyclines. Furthermore, 30-fold greater levels of cell-associated adriamycin were required in HL-60/AR cells for toxic effects equivalent to those occurring in parental HL-60 cells. Analysis of DNA histograms of adriamycin treated HL-60 cells indicated that cell-cycle progression was blocked in G2-M, while this antibiotic blocked progression of resistant HL-60/AR cells in the S phase. These results suggest that, in addition to alterations in membrane permeability, differential sensitivity of multiple biochemical targets may be important in the toxicity and the development of resistance to anthracyclines. Furthermore, the finding that HL-60/AR cells do not exhibit cross-resistance to aclacinomycin A indicates that this oligosaccharide-containing anthracycline may have utility in the treatment of adriamycin resistant neoplasms.     

Induction of differentiation of the human promyelocytic cell line HL-60 by activin/EDF.

A human promyelocytic cell line, HL-60, treated with activin/EDF was found to differentiate into monocyte/macrophage-like cells. This was shown not only by morphology but by the loss of myeloperoxidase granules and the appearance of nonspecific esterase. Dose-dependent inhibition of the differentiation by follistatin, an activin-binding protein, confirmed that it was indeed caused by activin. Thus, activin/EDF exerts its effect on hematopoietic cells not only on erythroid differentiation but also on at least a part of myeloid cell differentiation.     

Susceptibility of the human promyelocytic cell line HL-60 to an endogenous antileukaemic factor in suspension cultures

HL-60 human leukaemic cell line a suitable homogeneous target population for the selective endogenous inhibitor of myelopoiesis, isolated in our laboratory, was submitted to multiparameter analysis of cell proliferation in suspension cultures. As detected by 3H-TdR incorporation, a single dose of the regulator elicited a 6 to 8 hours arrest of DNA synthesis. The inhibition could be prolonged by repeated applications. As affected by the factor, alteration of population kinetics is characterized, revealed by flow cytofluorometric analysis, in G1 arrest of a fraction of cells, and diminishing those in hyperdiploid and tetraploid stage. 51Cr-release detection of vitality proved, that the endogenous factor, chemically determined as nucleopeptide, affected non-toxically and reversibly HL-60 cell proliferation.     

Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.     

Correlation between plasma membrane potential and second messenger generation in the promyelocytic cell line HL-60.

The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.     

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.     

Stimulation of sialidase activity during cell differentiation of human promyelocytic leukemia cell line HL-60

Sialidase activity of human promyelocytic leukemia cell line HL-60 was assayed by a modification of the fluorometric method using 4MU-NANA as a substrate. The pH optimum was 4.1 and the apparent Km value was 0.10 mM. When the cells were induced to differentiate into granulocytes by either retinoic acid or DMSO, sialidase activity increased markedly. After incubation of HL-60 cells with 1 μM retinoic acid for 6 days and with 1.3% DMSO for 8 days, 91% and 75% of total cells, respectively, differentiated into morphologically mature myeloid cells and the sialidase activity increased to 2.5–2.7 times as much as that of the corresponding controls. In other human myeloid leukemia cell lines, K562 and KG-1, the sialidase activity was found to be 1.5- and 3.8-fold that of HL-60, respectively.     

Development of the superoxide-generating system during differentiation of the HL-60 human promyelocytic leukemia cell line

Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.     

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.     

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     

Effects of dimaprit on growth and differentiation of human promyelocytic cell line, HL-60

The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyryl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE2, and cholera toxin). HL-60 is also known to be rich in H2 -histamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H2-stimulation on growth and differentiation of HL-60 using the potent H2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30-60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10(-7)M to 10(-4)M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxide (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10(-7)M to 10(-4)M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyelocyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, ii) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H2 antagonist. Dimaprit-induced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation.     

Differentiation-linked expression of the plasminogen activator inhibitor type-2 gene in the human HL-60 promyelocytic cell line

HL-60 is a human promyelocytic cell line which was found to be capable of differentiating toward a macrophage-like or granulocyte-like phenotype. Histochemical analysis demonstrated that incubation of cells in the presence of phorbol myristate acetate (PMA) or 1,25-dihydroxyvitamin D3 induced varying degrees of monocytic differentiation, while incubation in the presence of retinoic acid (RA) or dimethyl sulfoxide (DMSO) induced granulocytic differentiation. The differentiation induced by PMA, RA, and to a lesser extent DMSO, was accompanied by the induction of plasminogen activator inhibitor expression. mRNA analysis of control and PMA-induced cultures revealed the induction of a 2-kb message in treated cells which hybridized with a PAI-2-specific oligonucleotide probe. This is consistent with the literature concerning the expression of PAI by macrophages and granulocytes. No hybridization was detected with a PAI-1 specific probe. Expression of PAI by cells of hematopoietic origin appears to be associated with differentiation or stimulation of committed cells. Furthermore, PAI-2 expression by HL-60 cells is not restricted to one specific hematopoietic lineage. Since other cells of hematopoietic origin such as platelets express PAI-1, future studies using pluripotential cell lines could provide information on the initial events of lineage commitment and gene expression.     

Expression of CD38 in human promyelocytic leukemia HL-60 cell line during differentiation by niacin-related compounds

It was found that three niacin-related compounds, isonicotinic acid, nicotinamide, and nicotinamide N-oxide, induced granulocytic differentiation in HL-60 cells. We investigated the expression of CD38, which catalyzes the synthesis of cyclic ADP-ribose, a Ca2+ mobilizer, during differentiation by niacin-related compounds. It was found that CD38 was induced by isonicotinic acid, whereas nicotinamide and nicotinamide N-oxide containing an amino group did not induce it. The difference in expression of CD38 may provide some useful information for the elucidation of the mechanisms of cell differentiation.     

NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.     

Biosynthesis, transport and processing of myeloperoxidase in the human leukaemic promyelocytic cell line HL-60 and normal marrow cells.

The processing and intracellular transport of myeloperoxidase were studied in the human promyelocytic leukaemia cell line HL-60 and in normal marrow cells labelled with [35S]methionine or [14C]leucine. Myeloperoxidase was precipitated with antimyeloperoxidase serum; the immunoprecipitates were subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and radiolabelled myeloperoxidase visualized by fluorography. During a 1 h pulse, myeloperoxidase was labelled in a chain of apparent Mr 90 000. With a subsequent chase, the Mr 90 000 polypeptide disappeared and was replaced by chains of Mr 62 000 and 12 400 corresponding roughly to the size of neutrophil myeloperoxidase subunits. The identification of the radioactive polypeptides as different forms of myeloperoxidase was established also by the similarity in patterns generated by partial proteolysis with V8 proteinase from Staphylococcus aureus. Processing of myeloperoxidase in HL-60 was slow; mature polypeptides were significantly increased only after 6 h. Another myeloperoxidase chain of apparent Mr 82 000 was an intermediate precursor or degradation form. Pulse-chase experiments in combination with sucrose-density-gradient separations of homogenates showed that the Mr 90 000 precursor was located in light density organelles only and not in granule fractions, whereas the Mr 82 000 precursor was located only in intermediate density organelles, suggesting that the latter is a product of the former. Processed mature myeloperoxidase was concentrated in the granule fraction, but some occurred in lower density organelles, which may indicate processing during intracellular transport. Only the Mr 90 000 polypeptide was secreted into the culture medium; this was also the only form found in the cytosol fraction.