Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.     

Changes in the kinetics of inositol transport during TPA-induced differentiation of HL60 cells towards monocytes.

When exposed to the phorbol ester TPA, HL60 cells undergo growth arrest and differentiate towards monocytes. During TPA-induced differentiation there was a 2.6-fold increase in the rate of inositol transport (Vmax), a 2.1-fold increase in intracellular inositol and a 1.5-fold increase in inositol lipid. An increase in the Vmax of inositol transport did not occur when the variant cell line HL60Ast3 was exposed to TPA, which has been shown in this cell line to induce growth arrest but not differentiation. This observation suggests that the change in inositol transport during HL60 monocyte differentiation is specifically associated with the process of cell differentiation as opposed to growth arrest.     

Modification of the glyoxalase system in human HL60 promyelocytic leukaemia cells during differentiation to neutrophils in vitro

The glyoxalase system of human promyelocytic leukaemia HL60 cells was substantially modified during differentiation to neutrophils. The activity of glyoxalase I was decreased and the activity of glyoxalase II was markedly increased relative to the level in control HL60 promyelocytes. There was a decrease in the apparent maximum velocity, Vmax, of glyoxalase I, and an increase in the Vmax of glyoxalase II. The apparent Michaelis constants for both enzymes remained unchanged. The flux of intermediates metabolised via the glyoxalase system increased during differentiation, as judged by the formation of D-lactic acid, whereas the percentage of glucotriose metabolised via the glyoxalase system remained unchanged. The cellular concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, were markedly decreased during differentiation. The maturation of HL60 promyelocytes is associated with an increased ability to metabolise S-D-lactoylglutathione by glyoxalase II and a concomitant decrease in the mean intracellular concentrations of S-D-lactoylglutathione and methylglyoxal. The maintenance of a high concentration of S-D-lactoylglutathione in HL60 promyelocytes may be related to the status of the microtubular cytoskeleton, since S-D-lactoylglutathione potentiates the GTP-promoted assembly of microtubules.     

Metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells

The metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells was studied. LiCl, BeCl2 and NaF inhibited the hydrolysis of both monophosphates with half maximal inhibition occurring at 1.2 mM, 0.3 microM, 0.25 mM (Ins 1P) and 0.14 mM, 0.56 microM, 0.28 mM (Ins 4P) respectively. Lithium was an uncompetitive inhibitor with respect to both substrates. Ins 4P inhibited the hydrolysis of Ins 1P in a concentration dependent manner, suggesting that it acts as a competing substrate for the same enzyme. Half maximal inhibition occurred at 120 microM Ins 4P. The lithium sensitive activity responsible for the metabolism of both monophosphates was present in a soluble fraction made from the cells. Taken together these data suggest that Ins 1P and Ins 4P are hydrolysed by a single soluble enzyme activity which is sensitive to inhibition by lithium, beryllium and fluoride.     

Levels of inositol metabolites within normal myeloid blast cells and changes during their differentiation towards monocytes.

A homogeneous population of undifferentiated myeloid blast cells was purified from human fetal liver by rosette sedimentation of erythroblasts and macrophages, after coating these cells with monoclonal antibodies, followed by a cell elutriation step. The undifferentiated blast cells were maintained in culture, in a serum-free medium containing 1 mg l-1 inositol, by the presence of a high concentration of interleukin-3 (100 U ml-1). This allowed equilibrium labelling of cells with [2-3H]myo-inositol and analysis of the concentrations of inositol metabolites. The myeloid blast cells contained high concentrations of an unidentified inositol metabolite, possibly sn-glycero-3-phospho-1-inositol (GroPIns, 22 microM), inositol monophosphate (InsP, 16 microM), an unidentified inositol bisphosphate (InsP2, 9.4 microM), inositol pentakisphosphate (InsP5, 37 microM) and inositol hexakisphosphate (InsP6, 31 microM). These high concentrations are similar to those reported in the promyeloid cell line, HL60. Treatment of the blast cells with 10 nM phorbol myristate acetate (PMA) resulted in rapid differentiation of 48% of the cells towards monocytes. Notable changes in the levels of inositol metabolites included an increase in the putative GroPIns peak (to 73 microM) and decreases in the concentrations of InsP4 (from 4 microM to 1 microM) and InsP5 (to 21 microM). These changes in response to PMA, with the exception of the rise in the putative GroPIns, are similar to those reported in HL60 cells undergoing monocyte differentiation. These observations suggest that the abundant inositol polyphosphates may have an as yet unknown role in myeloid differentiation.     

Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells.

Immunoprecipitated retinoblastoma protein from HL60 cells migrated as a series of bands during electrophoresis. The heterogeneity appeared to be generated by phosphorylation of the retinoblastoma protein. Treatment of the cells with the phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in both a loss of the heterogeneity of the pRB species and a significant decrease in the level of pRB phosphorylation. These changes accompanied differentiation of the HL60 cells into macrophages. Treatment of the cells with dibutyryl cAMP also resulted in dephosphorylation of pRB as well as cell cycle arrest, although no recognizable differentiation occurred. These results are consistent with a model in which TPA and dibutyryl cAMP dependent pathways can activate pRB by altering its phosphorylation.     

Phorbol esters inhibit inositol phosphate and diacylglycerol formation in proliferating HL60 cells. Relationship to differentiation

Phorbol 12-myristate 13-acetate (PMA) induces the differentiation of the human promyelocytic cell line, HL60, towards adherent macrophage-like cells within 2 days. We have examined the early effects of PMA on inositol phosphates and on diacylglycerol production, two second messengers derived from inositol lipids. In proliferating HL60 cells, PMA induced a time- and concentration-dependent decrease in inositol phosphate levels. Maximal effects were seen after 1 h at 10 nM PMA. PMA also induced the translocation of protein kinase C from the cytosol to the membrane. Comparison between the differentiating effects of several phorbol esters and of 1-oleoyl-2-acetylglycerol with their ability to inhibit inositol phosphate formation suggests that the two effects are correlated.     

Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

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.     

Development of distinct clonal patterns of carbohydrate-binding activity in human promyelocytic HL 60 cells and histiocytic U 937 cells during DMSO-or PMA-induced differentiation

Increased ability to recognize carbohydrate structures on particles was observed in promyelocytic HL 60 cells and histiocytic U 937 cells during differentiation inducedin vitro with dimethylsulfoxide (DMSO) or phorbol myristate acetate (PMA). The size of the cells and increased capacity to bind and ingest IgG-or complement-coated yeast particles were used as indicators of phagocytic maturation. Carbohydrate affinities were assessed by the binding of glycolipid-containing liposomes displaying mannose, galactose, lactose,N-acetylgalactosamine, fucose, inositol, or ganglioside residues. With DMSO, HL 60 cells showed greater affinity for mannose and ganglioside residues, and with PMA also for fucosyl ligands. U 937 cells displayed a slightly different pattern; mannose binding was present before induction and by DMSO affinity was clearly augmented for galactose, fucose, ganglioside and inositol residues. With PMA these effects were smaller except for increased binding of lactosyl liposomes.Subclones of cells derived from U 937 (Cl 1, Cl 2 and Cl 3) appeared more mature already in the absence of inducing agent, and the lectin activity was barely affected by DMSO or PMA. Incidentally, Cl 1 lacked mannose affinity, which was fully expressed in Cl 2. With respect to inositol and ganglioside residues the reverse pattern was observed.In conclusion, DMSO- or PMA-mediated maturation in HL 60 and U 937 cells is accompanied by increased carbohydrate binding similar to what has been found in mature macrophages and granulocytes, indicating that these cellular systems can be used for further assessment of the molecular origin of lectin-like membrane components in phagocytic cells.     

Nuclear inositol lipids in Friend erythroleukemia cells. Changes related to differentiation induced by hexamethylenebisacetamide

Subcellular distribution of inositol lipids has been studied in Friend Erythroleukemia Cells following induction to erythroid differentiation with hexamethylenebisacetamide, after labelling with [3H]myo-inositol. In situ autoradiography indicated that inositol-derived molecules were present also in the nuclear compartment of uninduced and induced cells. Fractionation studies showed that the nuclear polyphosphoinositides were deeply changed after short induction times, while the whole cell inositol lipids resulted only slightly modified by the inducer. The nuclear recovery of phosphatidylinositol 4,5-bisphosphate was largely increased after 2 hrs of induction, suggesting that inositol lipid metabolism is involved in the early differentiation events occurring at the nuclear level.     

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.     

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.     

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.     

The appearance of phospholipase and cyclo-oxygenase activities in the human promyelocytic leukemia cell line HL60 during dimethyl sulfoxide-induced differentiation

The human promyelocytic leukemia cell line HL60 can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. During differentiation a phospholipase activity, which releases arachidonic acid from membrane phospholipids, is expressed. Similarly, fatty acid cyclo-oxygenase activity increases 10-fold. In addition, there is a 40-fold increase in chemotactic formyl peptide receptor binding and a dramatic increase in glucose oxidation via the hexosemonophosphate shunt. The addition of indomethacin, a potent cyclo-oxygenase inhibitor, to the culture medium reduced the cyclo-oxygenase activity of HL60 cells exposed to dimethyl sulfoxide by 97%. However, the presence of indomethacin did not block the dimethyl sulfoxide induced increases in chemotactic formyl peptide receptor binding and hexosemonophosphate shunt activity.