EphA4 is required for cell adhesion and rhombomere-boundary formation in the zebrafish

The formation of boundaries between or within tissues is a fundamental aspect of animal development. In the developing vertebrate hindbrain, boundaries separate molecularly and neuroanatomically distinct segments called rhombomeres. Transplantation studies have suggested that rhombomere boundaries form by the local sorting out of cells with different segmental identities. This sorting-out process has been shown to involve repulsive interactions between cells expressing an Eph receptor tyrosine kinase, EphA4, and cells expressing its ephrinB ligands. Although a model for rhombomere-boundary formation based on repulsive Eph-ephrin signaling is well established in the literature, the predictions of this model have not been tested in loss-of-function experiments. Here, we eliminate EphA4 and ephrinB2a proteins in zebrafish with antisense morpholinos (MO) and find that rhombomere boundaries are disrupted in EphA4MO embryos, consistent with a requirement for Eph-ephrin signaling in boundary formation. However, in mosaic embryos, we observe that EphA4MO cells and EphA4-expressing cells sort from one another, an observation that is not predicted by the Eph-ephrin repulsion model but instead suggests that EphA4 promotes cell adhesion within the rhombomeres in which it is expressed. Differential cell adhesion is known to be an effective mechanism for cell sorting. We therefore propose that the well-known EphA4-dependent repulsion between rhombomeres operates in parallel with the EphA4-dependent adhesion within rhombomeres described here to drive the cell sorting that underlies rhombomere-boundary formation.     

Leukemic cell maturation: phenotypic variability and oncogene expression in HL60 cells: a review

Leukemic cells in vitro and in vivo demonstrate an unstable phenotype. We have observed in HL60 cells maintained in culture over long periods of time such phenomena as emergence of drug resistance, oncogene amplification, loss of granulocyte and monocyte lineage markers, and alteration in cell growth parameters. As summarized in this report, a comprehensive review of the literature on HL60 cells shows a wide diversity of phenotype for these cells. We have developed and acquired from other laboratories a series of HL60 sublines with varying phenotypic characteristics. The time in continuous log phase culture for HL60 cells (passage 13 ATCC, Rockville MD) to undergo phenotypic drift from a heavily granulated promyelocytic cell to a more undifferentiated agranular blast form on four occasions varied from 3 to 18 months. The actual loss of promyelocytic phenotype occurred rapidly (within less than 1 month) following a variable period of apparent stable phenotype, The change in morphology was invariably accompanied by decreased sensitivity to ARA-c (5- to 20-fold increase in LD50 and dose necessary to induce NSE positive cells). The c-myc gene is variably amplified in sublines of HL60 cells. The expression of c-myc primarily reflects alterations in cell cycle kinetics and was not found to be correlated with a switch between proliferation and maturation. These results suggest that phenotypic drift may be due to loss of response to regulatory signals that affect the expression of a number of cellular genes.     

Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells

Cell death was induced by electroporation in HL60 cells, a human promyeloid leukaemia strain, in order to determine by both morphological and biochemical criteria whether necrotic or apoptotic processes occurred. Cells sampled at several times after electroporation were analyzed for the assessment of the following end-points: (i) chromosomal DNA fragmentation; (ii) cell viability; (iii) mono- and oligonucleosomes in the cytoplasmic fraction; (iv) apoptotic index; and (v) morphology of treated cells. The results indicate that about 50% of the cells killed by electroporation die through necrosis, while the remaining 50% of the cells undergo apoptosis. Chromosome damage was also studied by cytogenetic analysis at metaphase. The possibility of killing tumour cells by electroporation, as a variant of electrotherapy, constitutes, in our opinion, a promising procedure in cancer therapy, avoiding the undesirable side effects normally derived from treatment with cytotoxic drugs.     

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.     

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.     

Antileukemic agent alkyllysophospholipid regulates phosphorylation of distinct proteins in HL60 and K562 cells and differentiation of HL60 cells promoted by phorbol ester

The tumor-promoting 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulated phosphorylation of several proteins in block I (including protein Ia) and protein 3 in HL60 cells. The antileukemic agent alkyllysophospholipid (ALP) inhibited the TPA-stimulated phosphorylation of these proteins and the TPA-induced differentiation of the cells. In comparison, TPA only stimulated phosphorylation of protein 3 in K562 cells which, in contrast, were not induced to differentiate by TPA and lacked protein Ia and had a very high basal phosphorylation of protein B. ALP inhibited phosphorylation of protein 3 as well as protein B in K562 cells. The data suggest that the presence of distinct phosphoproteins and regulation of their phosphorylation may be related to the selective susceptibility of the two leukemia cell lines to the maturating effect of TPA and cytotoxicity of ALP.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).     

Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes.

HL60 cells were adapted to grow in a serum-free medium containing 1 mg l-1 inositol, in which they differentiated normally towards neutrophils (in 0.9% by volume dimethylsulphoxide) and towards monocytes (in 10 nM phorbol myristate acetate). Cells that had been equilibrium-labelled with [2-3H]myo-inositol contained a complex pattern of inositol metabolites, several of which were at relatively high concentrations. These included InsP5 and InsP6, which were present at concentrations of about 25 microM and 60 microM, respectively. Striking and different changes occurred in the levels of some of the inositol polyphosphates as the cells differentiated towards either neutrophils or monocytes. Most notable were a large but gradual accumulation of Ins(1,3,4,5,6)P5 as HL60 cells decreased in size and acquired neutrophil characteristics, and much more rapid and sequential declines in InsP4, InsP5 and InsP6 as the cells started to take on monocyte character. There was a marked accumulation of free inositol and of phosphatidylinositol in the cells during neutrophil differentiation, probably caused at least in part by an increased rate of inositol uptake providing an increased intracellular inositol supply. The same accumulation of Ins(1,3,4,5,6)P5 occurred during neutrophil differentiation, whether it was induced by dimethylsulphoxide or by a combination of retinoic acid and a T-lymphocyte cell line-derived differentiation factor. Ins(1,4,5)P3, a physiological intracellular mediator of Ca2+ release from membrane stores, did not change in concentration during these differentiation processes. These observations suggest that some of the more abundant cellular inositol polyphosphates play some important, but not yet understood, role either in the processes of haemopoietic differentiation or in the expression of differentiated cell character in myeloid cells.     

Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway and AP-1 activity in monocytic but not in macrophage or granulocytic differentiation of HL60 cells

Monocytic differentiation of HL60 cells induced by 1,25-dihydroxyvitamin D(3) (1,25 D(3)) has been recently shown (Exp Cell Res 258, 425, 2000) to be enhanced by an exposure to SB203580 or to SB202190, specific inhibitors of p38MAP kinase, with concomitant up-regulation of the c-jun N terminal kinase (JNK) pathway. In the present study we inquired if this enhancement and the JNK up-regulation are limited to 1,25 D(3)-induced differentiation, or if they occur more generally in HL60 cell differentiation. We found that dimethylsulfoxide (DMSO)-induced differentiation, and to a lesser extent tetradecanoylphorbol acetate (TPA)-induced macrophage differentiation were also potentiated by the p38MAPK inhibitors, but that granulocytic differentiation in response to all-trans retinoic acid (RA) was not. The enhancement of differentiation by p38MAPK inhibitors was accompanied by an activation of the JNK MAPK pathway, as shown by the phosphorylation levels of these kinases and by AP-1 binding, but only in 1,25 D(3)-treated cells. This shows that an up-regulation of the JNK stress pathway during 1,25 D(3)-induced monocytic differentiation occurs selectively in this lineage of differentiation and is not necessary for the expression of the differentiated phenotype.     

ICAT as a potential enhancer of monocytic differentiation: implications from the comparative proteome analysis of the HL60 cell line stimulated by all‐trans retinoic acid and NSC67657

A novel sterol mesylate compound (NSC67657) was recently identified and reported by National Cancer Institute that could efficiently induce the differentiation of HL60 cells into monocytes in vitro and in vivo. The expression of many proteins would have been changed during the differentiation process, and some proteins may have played key roles in the differentiation of HL60 cell line induced by this drug. Therefore, we treated HL60 cells with NSC67657 and all‐trans retinoic acid (ATRA) to identify the differentially expressed proteins and determine their functions in cellular differentiation. Of the 45 differentially expressed protein spots investigated, 24 were either elevated or decreased in both the monocytic and granulocytic differentiating HL60 cells, 8 showed significant changes only when induced by NSC67657, and 13 showed significant changes only when induced by ATRA. After verification by RT‐PCR, Western blotting, and immunocytochemistry, only the protein ICAT was found to be elevated by NSC67657 treatment alone. Although the over‐expression of ICAT is not sufficient to induce the differentiation of HL60 cells into monocytes, it did increase the proportion of CD14+ cells in cells pretreated with NSC67657. Successful application of multiple techniques including two‐dimensional gel electrophoresis, matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry, Western blotting, and eukaryotic electroporation revealed that proteomic and molecular biological analyses provide valuable tools in drug development research. Copyright © 2009 John Wiley & Sons, Ltd.     

Glucocorticoid receptors and corticosensitivity in the human monocytic cell line CM-S: changes during phorbol ester-induced differentiation

CM-S is a line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Untreated CM-S cells expressed a single class of high-affinity (KD = 4.5 +/- 2.3 nM) glucocorticoid receptor sites (GCr) (27,530 +/- 3752 sites/cell) as measured by a whole-cell assay at 37 degrees C using [3H]triamcinolone acetonide as tracer, while CM-S cells induced to macrophage differentiation by 10(-7) M TPA showed reduced levels of GCr (10,729 +/- 2135 sites/cell). Kinetic studies indicated that this reduction was progressive, reaching about 34% of the original value 96 hr after TPA addition. The GCr in untreated and TPA-induced cells were similar in their specificity for corticosteroids. In the precursor cell population dexamethasone enhanced both the rate of protein synthesis and the production of autostimulatory growth factor(s), while in TPA-induced cells it inhibited the rate of protein synthesis in a dose-dependent manner. Our data with the CM-S cell line thus suggest that in the monocytic cell line lineage both the number of GCr and the cell response to glucocorticoids depend on the degree of cell maturation.     

Apolipoprotein A-I induces IL-10 and PGE2 production in human monocytes and inhibits dendritic cell differentiation and maturation

Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoprotein, exhibits anti-inflammatory activity in atherosclerosis. In this study, we demonstrate that apoA-I inhibits DC differentiation and maturation. DC differentiated from monocytes in the presence of apoA-I showed a decreased expression of surface molecules such as CD1a, CD80, CD86, and HLA-DR. In addition, these DC exhibited decreased endocytic activity and weakened allogeneic T-cell activation. During DC differentiation in the presence of apoA-I, PGE(2) and IL-10, which are known to be DC differentiation inhibitors and/or modulators of DC function, were produced at remarkable rates, whereas IL-12 production in the cells after stimulation with CD40 mAb and IFN-gamma was significantly decreased in comparison with the control DC. T cells stimulated by apoA-I-pretreated DC produced significantly low levels of IFN-gamma, and apoA-I inhibited cross-talk between DC and NK cells, in terms of IL-12 and IFN-gamma production. Therefore, apoA-I appears to play an important role in modulating both innate immune response and inflammatory response. The novel inhibitory function of apoA-I on DC differentiation and function may facilitate the development of new therapeutic interventions in inflammatory diseases.     

Class A scavenger receptors mediate cell adhesion via activation of G(i/o) and formation of focal adhesion complexes

Class A macrophage scavenger receptors (SR-A) are multifunctional receptors with roles in modified lipoprotein uptake, innate immunity, and macrophage adhesion. Our previous studies conducted in mouse peritoneal macrophages demonstrated that pertussis toxin (PTX) mediated inhibition of G(i/o) attenuated SR-A-dependent uptake of modified lipoprotein. The finding that SR-A-mediated lipoprotein internalization was PTX-sensitive led us to hypothesize that SR-A-mediated cell adhesion might be similarly regulated by G(i/o)-dependent signaling pathways. To test this hypothesis, SR-A was expressed in HEK cells under inducible control. Relative to HEK cells that lack SR-A, SR-A expressing cells displayed enhanced adhesion to tissue culture dishes. SR-A-mediated adhesion was significantly reduced following PTX treatment and was insensitive to chelating divalent cations with EDTA. SR-A-expressing cells exhibited a distinct cell morphology characterized by fine filopodia-like projections. Both polymerized actin and vinculin were codistributed with SR-A in the filopodia-like projections indicating the formation of focal adhesion complexes. Overall, our results indicate that the ability of SR-A to enhance cell adhesion involves G(i/o) activation and formation of focal adhesion complexes.     

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.