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.     

Identification of the differentiation-associated p93 tyrosine protein kinase of HL-60 leukemia cells as the product of the human c-fes locus and its expression in myelomonocytic cells

A differentiation-associated 93-kDa tyrosine kinase (p93) was purified previously from the human promyelocytic leukemia cell line HL-60. The present study conclusively identifies p93 as the c-fes proto-oncogene product and shows that expression of p93c-fes and its associated tyrosine kinase activity are marked in mature granulocytes, monocytes, and human myeloid leukemia cell lines. Antisera to peptides obtained by expression of c-fes cDNA fragments in Escherichia coli reacted strongly with p93 purified from HL-60 cells. Western blots using one of these antisera demonstrated high levels of p93c-fes protein in normal human granulocytes and monocytes, as well as the cell lines KG-1, THP-1, HEL, and U-937, all of which can be induced to differentiate along the myelomonocytic pathway. Conversely, in cell lines resistant to myeloid differentiation, p93c-fes expression was either very low or absent. Expression of immunoreactive p93c-fes in these cell lines showed a strong positive correlation with p93c-fes tyrosine kinase activity, which was measured in cell extracts using a nondenaturing gel assay. Finally, the expression of p93c-fes, its tyrosine kinase activity, and the binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) were all coordinately increased in HL-60 cells treated with the granulocytic differentiation inducer dimethyl sulfoxide, while all three parameters were low in untreated or differentiation-resistant HL-60 cells. These results suggest that expression of p93c-fes tyrosine kinase activity may be an essential component of myeloid differentiation and responsiveness to granulocyte-macrophage colony-stimulating factor.     

Induction of leukemia cell differentiation and apoptosis by recombinant P48, a modulin derived from Mycoplasma fermentans

P48 is a 48-kDa monocytic differentiation/activation factor which was originally identified in the conditioned medium of the Reh and other leukemia cell lines and has recently been shown to be a Mycoplasma fermentans gene product. Previously, conditioned medium P48 has been shown to induce differentiation of HL-60 (human promyelocytic leukemia) cells. Recently our laboratory isolated cDNA clones for P48 from Reh cells and genomic clones from Mycoplasma fermentans and expressed the recombinant protein as a maltose binding protein (MBP) fusion protein in E. coli. In this report we present the initial characterization of this recombinant P48 fusion protein (rP48-MBP). We show that rP48-MBP induces differentiation of HL-60, U937 (human histiocytic lymphoma), and M1 (mouse myeloid leukemia) cell lines. Interestingly, rP48-MBP also induces apoptosis of U937 and HL-60 cells as assessed by terminal transferase (TUNEL) assays. This is the first report of induction of apoptosis by a Mycoplasma gene product. P48 is a Mycoplasma-derived immunomodulatory molecule which has differentiation and apoptosis-inducing activities and may be important in the pathophysiology of Mycoplasma infections. The recombinant protein may be useful in studying the mechanisms of differentiation, cytokine production, and apoptosis in malignant and nonmalignant hematopoietic cells.     

Regulation of myeloperoxidase gene expression during differentiation of human myeloid leukemia HL-60 cells

When human myeloid leukemia HL-60 cells were induced to differentiate into mature cells by dimethyl sulfoxide or retinoic acid, the amount of myeloperoxidase activity per cell decreased to 20 to 30% of that of uninduced cells, and the rate of myeloperoxidase biosynthesis decreased to an undetectable level in 19 h after induction of differentiation. After 19-h exposure to an inducer, the cells could not resume myeloperoxidase synthesis on further incubation in inducer-free medium. When polysomes and mRNAs prepared from untreated and treated cells were translated in rabbit reticulocyte lysates, the former showed myeloperoxidase polypeptide synthesis, and the latter did not. These results indicate that the inability of induced cells to synthesize myeloperoxidase is due to the absence of myeloperoxidase mRNA.     

The role of interferon-gamma in induction of differentiation of human myeloid leukemia cell lines, ML-1 and HL-60

Highly purified natural interferon-gamma (IFN-gamma) induced differentiation having characteristics that are associated with the human promyelocytic leukemia cell line, HL-60. Monoclonal antibody to INF-gamma neutralized its activity. However, the natural IFN-gamma had almost no inducing activity in ML-1, a human myeloblastic leukemia cell line. Similar results were obtained using recombinant IFN-gamma. Mitogen stimulated human leukocyte conditioned medium (LCM) induced differentiation of both ML-1 and HL-60 cells. After treatment of LCM with monoclonal antibody to IFN-gamma, LCM activity was reduced more than 50% in ML-1 cells, and 80% in HL-60 cells. Even if IFN-gamma was eliminated from LCM by affinity chromatography, the LCM induced differentiation of ML-1 and HL-60 cells, but IFN-gamma markedly enhanced the ML-1 cell differentiation induced by IFN-gamma free LCM. The results suggest that leukocytes produce differentiation inducing factor(s) other than IFN-gamma, and that IFN-gamma is both an inducer and an enhancer of induction of human myelogenous leukemia cells.     

Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells

We have previously reported that Brazilian propolis extracts inhibited growth of HL-60 human myeloid leukemia cells, which is partly attributed to the induction of apoptosis associated with granulocytic differentiation. In this study, we isolated three compounds which induce granulocytic differentiation evaluated by nitroblue tetrazolium (NBT)-reducing assays from the water extract of propolis and identified as 4,5-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 3,4-di-O-caffeoylquinic acids by NMR analysis. Cell growth inhibitory activity of these caffeoylquinic acids was found in HL-60 cell, which was mainly attributed to the induction of apoptosis. Furthermore, the potency of caffeoylquinic acid derivatives to induce granulocytic differentiation was examined in HL-60 cells. Caffeic, quinic, and chlorogenic acids had no effects on the NBT-reducing activity, while 3,4,5-tri-O-caffeoylquinic acid induced more than 30% of NBT-positive cells. These results suggest that the number of the caffeoyl groups bound to quinic acid plays an important role in the potency of the caffeoylquinic acid derivatives to induce granulocytic differentiation. This is the first report demonstrating that the caffeoylquinic acid derivatives induce granulocytic differentiation of HL-60 cells.     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

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.     

Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL-60, U937, and THP-1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL-60, U937, and THP-1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL-60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL-60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.     

Membrane origin for a signal eliciting a program of cell differentiation

Evidence is presented to indicate that the retiNOic acid (RA)-induced program of myeloid differentiation and growth arrest by HL-60 human promyelocytic leukemia cells was initiated by a signal originating at the cell membrane. Free RA and RA covalently immobilized on a solid substrate elicited similar kinetics of differentiation and G1/0-specific growth arrest. No evidence of cell-induced RA detachment from the solid substrate was found. The data explain why HL-60 cells which are deficient in cellular RA-binding protein (CRABP) nevertheless respond to RA.     

Biosynthesis of glycosphingolipids by human myeloid leukemia cells

We have performed comparative studies of the neutral glycosphingolipids synthesized by three human myeloid leukemia cell lines, K562, KG1, and HL-60, which were metabolically labeled with [14C]galactose, to evaluate changes in neutral glycosphingolipid synthesis with myeloid cell differentiation. Individual neutral glycosphingolipids containing one to four sugars were purified by a combination of the following methods: diethylaminoethyl-Sephadex column chromatography, acetylation-Florisil column chromatography, and high-performance liquid chromatography using an Iatrobead column. Compounds with one sugar were analyzed by thin-layer chromatography on borate plates. This analysis showed that HL-60 cells synthesize only glucosylceramide, whereas K562 and KG1 cells synthesize predominately glucosylceramide, but also a small amount of galactosylceramide. Compounds with two to four sugars were characterized by treatment with exo- and endoglycosidases. The results showed that K562 and KG1 cells are similar to cells from patients with acute leukemia in expressing two series (globo and neolacto) of natural glycosphingolipids, whereas the HL-60 cells are similar to mature human myeloid cells in expressing only one series (neolacto). Therefore, human myeloid leukemia cells blocked at different stages of differentiation vary in their ability to synthesize neutral glycosphingolipids.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin

Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells.     

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.     

Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation

Sphingolipid metabolism was examined in human promyelocytic leukemia HL-60 cells. Differentiation of HL-60 cells with 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3; 100 nM) was accompanied by sphingomyelin turnover. Maximum turnover of [3H]choline-labeled sphingomyelin occurred 2 h following vitamin D3 treatment, with sphingomyelin levels decreasing to 77 +/- 6% of control and returning to base-line levels by 4 h. Ceramide and phosphorylcholine were concomitantly generated. Ceramide mass levels increased by 55% at 2 h following vitamin D3 treatment and returned to base-line levels by 4 h. The amount of phosphorylcholine produced equaled the amount of sphingomyelin hydrolyzed, suggesting the involvement of a sphingomyelinase. Vitamin D3 treatment resulted in a 90% increase in the activity of a neutral sphingomyelinase from HL-60 cells. The inferred role of sphingomyelin hydrolysis in the induction of cell differentiation was investigated using an exogenous sphingomyelinase. When a bacterial sphingomyelinase was added at concentrations that caused a similar degree of sphingomyelin hydrolysis as 100 nM vitamin D3, it enhanced the ability of subthreshold levels of vitamin D3 to induce HL-60 cell differentiation. This study demonstrates the existence of a "sphingomyelin cycle" in human cells. Such sphingolipid cycles (Hannun, Y., and Bell, R. (1989) Science 243, 500-507) may function in a signal transduction pathway and in cellular differentiation.     

A new endogenous differentiating factor (myelopeptide-4) for myeloid cells

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.     

Tumor necrosis factor alpha up-regulates in an autocrine manner the synthesis of plasminogen activator inhibitor type-1 during induction of monocytic differentiation of human HL-60 leukemia cells

Tumor necrosis factor-alpha (TNFalpha) critically regulates several cellular functions during monocyte/macrophage differentiation. We therefore investigated during the phorbol ester (phorbol 12-myristate 13-acetate (PMA))-induced monocyte/macrophage differentiation of the human HL-60 leukemia cells, if TNFalpha contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis that is initiated by a protein kinase Cbeta-extracellular signal-regulated kinase 2-dependent pathway (Lopez, S., Peiretti, F., Morange, P., Laouar, A., Fossat, C., Bonardo, B., Huberman, E., Juhan-Vague, I., and Nalbone, G. (1999) Thromb. Haemostasis 81, 415-422). Following PMA treatment, the level of TNFalpha mRNA strongly increased and appeared earlier than PAI-1 mRNA. An anti-TNFalpha antibody significantly inhibited the PMA-induced PAI-1 mRNA and protein levels. The recombinant human TNFalpha, which is inactive on native HL-60 cells in terms of PAI-1 synthesis, optimally potentiates it once HL-60 cells are committed into the differentiation process. The use of 1) the HL-525 cell line, a clone issued from HL-60 cells rendered resistant to PMA-induced differentiation, and 2) the transforming growth factorbeta-1/vitamin D3 differentiative mixture confirmed the relationships between the induction of differentiation and the potency of TNFalpha to up-regulate PAI-1 synthesis. In conclusion, we showed that during the induction of monocyte/macrophage differentiation, TNFalpha and PAI-1 gene expressions are activated and that synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, the synthesis of PAI-1.     

Lipids of human promyelocytic leukemia cell HL-60: increasing levels of ether-linked phospholipids during retinoic acid-induced differentiation

Cells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulocytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70% of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkenyl-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the untreated cells, while 1,2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1,2-diacyl GPE decreased to 63%, while those of 1-alkenyl-2-acyl GPE, choline-containing glycerophospholipids, and phosphatidylinositol increased to 169, 154, and 153%, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.     

Effects of protein kinase A and calcium/phospholipid-dependent kinase modulators in the process of HL-60 cell differentiation: their opposite effects between HL-60 cell and K-562 cell differentiation.

We have previously shown that HL-60 cells treated with 1 alpha, 25-(OH)2D3 in magnesium-deficient medium are committed to differentiate but do not express differentiation-related phenotypes. In the present study, we demonstrated that Mg2+ deprivation blocked the process of differentiation before the induction of lysozyme mRNA and that the process of HL-60 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step. We studied the effects of protein kinase A (PKA) and calcium/phospholipid-dependent protein kinase (PKC) modulators at each step. The results indicated that agonists of PKA enhanced both steps but that N-(2-[methylamino]ethyl-5-isoquinolinesulfonamide inhibited them. On the other hand, 1-oleyl-2-acetylglycerol and 12-O-tetradecanoylphorbol-13-acetate enhanced the commitment step but inhibited that of phenotypic expression. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine inhibited the commitment step and enhanced that of phenotypic expression. These results indicate that PKA acts as a positive regulatory signal and that PKC has a dual role in the process of HL-60 cell differentiation, i.e., as a positive regulatory signal in the commitment step and as a negative one in the phenotypic expression step. Recently, we have also shown that in K-562 cell differentiation into erythroid lineage, PKA may serve as a negative regulatory signal in both steps; however, PKC may act dually, namely as a negative regulatory signal in the commitment step and as a positive one in the phenotypic expression step.(ABSTRACT TRUNCATED AT 250 WORDS)