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.     

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.     

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.     

Germ cell differentiation and synaptonemal complex formation are disrupted in CPEB knockout mice.

CPEB is a sequence-specific RNA binding protein that regulates translation during vertebrate oocyte maturation. Adult female CPEB knockout mice contained vestigial ovaries that were devoid of oocytes; ovaries from mid-gestation embryos contained oocytes that were arrested at the pachytene stage. Male CPEB null mice also contained germ cells arrested at pachytene. The germ cells from the knockout mice harbored fragmented chromatin, suggesting a possible defect in homologous chromosome adhesion or synapsis. Two CPE-containing synaptonemal complex protein mRNAs, which interact with CPEB in vitro and in vivo, contained shortened poly(A) tails and mostly failed to sediment with polysomes in the null mice. Synaptonemal complexes were not detected in these animals. CPEB therefore controls germ cell differentiation by regulating the formation of the synaptonemal complex.     

Suppression of cytokine production and cell adhesion molecule expression in human monocytic cell line THP-1 by Tripterygium wilfordii polysaccharide moiety

Extracts of the vine-like plant Tripterygium wilfordii (TW) have been widely used in China as an immunosuppressant and anti-inflammatory drug for the treatments of rheumatoid arthritis, lupus erythematosus and other inflammatory disorders. In this study the molecular mechanisms of action of three TW extracts (ethanol, aqueous, polysaccharide) on the expression of inflammatory cytokines and adhesion molecules were investigated by RT-PCR and immunofluorescence binding techniques. The lipopolysaccharide (LPS)-mediated stimulatory effects of tumor necrosis factor-alpha (TNF-alpha) cytokine production and cell adhesion molecule (CD11c, CD18, CD14, CD54) expression in human monocytic THP-1 cells were modulated by treatments of the TW extracts or tacrolimus (FK506). The TW polysaccharide moiety exhibited more profound immunosuppressive properties than the aqueous and ethanol extracts. Biochemical characterization of the polysaccharide moiety revealed a major molecular weight of 22 kDa (viz. PSP22). The PSP22 was found to be a potential immunosuppressant that manifests the necessary immunomodulating properties.     

Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.     

Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines

Expression of receptors for IL 2 was believed initially to be restricted to T cells after their activation by IL 1 and antigen. However, recently IL 2 receptors (IL 2R) were demonstrated on activated B cells by using an anti-IL 2R monoclonal antibody (anti-Tac). In this study, we examined the capacity of cultured human alveolar macrophages, blood monocytes, and myelomonocytic (HL-60) or monoblast (U937) cell lines to bind three different anti-IL 2R monoclonal antibodies before or after stimulation with the monocyte-activating agents IFN-gamma, LPS, phorbol ester, or lymphokine-containing conditioned medium. For each of the four cell populations examined, resting unstimulated cells bound little or no anti-IL 2R antibody, as shown independently by quantitative cell binding assay and by immunoperoxidase labeling. By contrast, incubation with recombinant IFN-gamma, conditioned medium, or to a lesser extent, native or recombinant IL 2 itself, resulted in a significant enhancement of anti-IL 2 receptor monoclonal antibody binding by all four populations, whereas LPS, PMA, or IL 1 had no effect. In addition, membrane binding of anti-Tac antibody, similar to that seen after stimulation of normal lung macrophages with IFN-gamma, was detected by using macrophages obtained by bronchoalveolar lavage of five patients with active pulmonary sarcoidosis. These findings are consistent with the expression of a functional IL 2R on activated cells of the monocyte lineage, since anti-Tac binding to IFN-gamma-treated HL-60 cells was inhibited by addition of excess IL-2; specific binding of anti-IL 2 monoclonal antibodies was detected in the presence of exogenous IL 2; and a 50 to 55 kD molecule was immunoprecipitated from both activated lung macrophages and T lymphoblasts by using anti-Tac antibody. We conclude that human mononuclear phagocytes can be induced by lymphokines to express IL 2R, and that such IL 2R+ macrophages can be detected in vivo during inflammation.     

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)