Biological characteristics of interferon-r-induced resistant histiocytic lymphoma cell line U937

A brief exposure (for 6 h) of U937 cells to interferon (IFN)-gamma (500 U/ml) followed by a long term incubation of cells in normal medium for 8 or more weeks resulted in the induction of cells that were refractory to the anticellular and differentiating effects of not only IFN-gamma but also IFN-alpha and IFN-beta at concentrations up to 10(4) U/ml. In addition, the cells became insensitive to the potent differentiating effect of the phorbol ester--tumor promoting agent (TPA). However, the resistant cells retained their sensitivity to the antiviral effect of different IFNs and were fullu responsive to the induction of endonuclease 2',5'-oligoadenylate (2-5A) synthetase by IFN. Furthermore, the resistant cell population appeared to be homogeneous because clones derived from single cells from this population all exhibited the same resistant phenotype to IFN and TPA. These results suggest that induction of resistant U937 cells may involve a dedifferentiation process which results in the formation of an immature cell population that do not respond to the differentiating and/or anticellular effects of various types of IFNs.     

Role of putrescine in interleukin 1 beta production in human histiocytic lymphoma cell line U937

Treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and incubation with lipopolysaccharide (LPS) induces interleukin 1 beta (IL-1 beta) production in the histiocytic lymphoma cell line U937. Here we investigated the effect of treatment with both TPA and 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) on LPS-induced IL-1 beta production in U937 cells. To clarify the mechanism of IL-1 beta production, the possible role of polyamines in this process was examined. Combined treatment with TPA and 1,25(OH)2D3 for 72 h followed by incubation with LPS for 24 h caused synergistic induction of both IL-1 beta release and mRNA expression. On the other hand, TPA increased the numbers of vitamin D3 receptors, which may be one mechanism of this synergistic induction. Ornithine decarboxylase (ODC), a rate-limiting enzyme for polyamine biosynthesis, was also induced by these compounds biphasically: the first peak of ODC activity was observed at 4 h of the incubation with the two compounds and the second peak was at 4 h after the addition of LPS. To find whether these peaks were related to IL-1 beta production, DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, was added together with TPA and 1,25(OH)2D3. DFMO decreased the cellular levels of putrescine and spermidine and suppressed IL-1 beta release and IL-1 beta mRNA expression by 65%. Exogenous putrescine, but not spermidine, abrogated these kinds of inhibition. Similar results were obtained with DFMO and the polyamines during the differentiation of the cells up to the monocyte or macrophage stage. These results thus suggest that changes in either of these intracellular polyamines, especially putrescine, help to regulate the differentiation of U937 cells, resulting in partial control of the regulation of IL-1 beta production.     

Induction of differentiation of the human histiocytic lymphoma cell line U937 in the absence of vimentin expression

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.     

Monokine-producing activity of human monocyte-like cell line U937 induced by Yersinia pestis EV antigens

The data on a study of the monokine-producing ability of human monocyte-like cell line U937 are presented. Antigens of Yersinia pestis EV (lipopolysaccharide and fraction 1A) induce monokine production by cell line U937. The obtained monokines essentially enhance neutrophil killer and chemotactic activities, stimulate FcR expression, increase the number of lysosomes, and the lability of lysosomal membranes in neutrophils. F1A significantly suppresses LPS in respect to the ability to induce monokine production, which stimulate neutrophil functional activity.     

Activation of human monocyte cell line U937 via cell surface calreticulin

U937 cells were found to be activated by an antibacterial peptide, KLKLLLLLKLK-NH2 (L5), to generate superoxide anion (O2-)-like peripheral neutrophils. However, the state of cell surface calreticulin, a possible receptor for L5, was suggested to differ between neutrophils and U937 cells. Unlike the former, the latter ones were activated by anti-C-domain peptide antibody of calreticulin even in the absence of L5 and generated O2- in a GTP-binding protein (G-protein)-dependent manner.     

Patterns of glycosidases in the histiocytic cell line U-937. Effects of agents inducing cell differentiation

1. The cell bound glycosidases in sublines and clones of the histiocytic cell line U-937 have previously been shown to display characteristic patterns. 2. In this paper the effects of differentiation inducing agents upon glycosidase patterns of one subline, U-937 GTB, are presented. 3. Teleocidin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), dimethyl-sulfoxide (DMSO), dihydroxyvitamin D3 and supernatants from mixed lymphocyte culture (MCL) all induce cellular differentiation of U-937 GTB. 4. Significant changes of the levels of cell bound glycosidases were seen after addition of inducing agents. 5. Alterations have been monitored as relative effects upon the absolute glycosidase activities and as effects upon selected ratios of different glycosidases. 6. The separate inducing agents show distinct enzyme patterns.     

Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937

We report the production, purification, characterization, and partial amino acid sequence of a plasminogen inhibitor (PA-I). The starting material is culture fluid from phorbol myristate 13-acetate-treated U-937 cells and the isolation steps consist of preparative isoelectric focusing followed by affinity chromatography on Cibacron Blue-Sepharose. PA-I migrates as a closely spaced doublet of 47-kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forms covalent complexes with urokinase and two-chain tissue-type plasminogen activator, displaying second order rate constants of 0.9 X 10(6) M-1 s-1 and 0.2 X 10(6) M-1 s-1, respectively. Upon treatment with 1 M NH4OH, the covalent complexes were hydrolyzed, yielding a 35-kDa inhibitor fragment. A partial amino acid sequence of PA-I showed that it belongs to the antithrombin III family of inhibitors. PA-I is immunologically related to a PA-inhibitor from human placenta. mRNA from phorbol myristate 13-acetate-treated U-937 cells directed, in a rabbit reticulocyte derived cell-free system, the biosynthesis of only one 47-kDa protein that could be immunoprecipitated with anti-PA-I IgG, indicating that the two molecular forms of PA-I are the products of post-translational processing.     

DNA induces apoptosis in electroporated human promonocytic cell line U937

Experimental gene transfer has permitted a wide variety of studies on gene regulation and function. However, possible effects of the introduced DNA on cellular metabolism are not well understood. Here we demonstrated that introduction of DNA into a promonocytic cell line, U937, by electroporation caused extensive cell death. The toxicity of DNA was concentration-dependent. Various DNAs including plasmid and genomic DNAs all caused cell death, indicating that the toxicity is nucleotide sequence-independent. DNA-induced cell death was associated with internucleosomal DNA fragmentation, a decrease in cell size, and a considerable proportion of cells outside cell cycle. From these results, we concluded that cells died by apoptosis. Our findings have experimental implication for an important issue concerning the interpretation of experiments using gene transfer. In addition, we propose that our observed phenomenon may be relevant to an important immune defense mechanism in monocytes/macrophages that facilitates a response to certain viral infections.     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.     

The appearance of phospholipase activity in the human macrophage-like cell line U937 during dimethyl sulfoxide induced differentiation

The human histiocyte cell line, U937, with monocyte characteristics, can be induced to differentiate into macrophage-like cells when exposed to growth medium containing 1.5% DMSO. Following three days of exposure, DMSO-treated but not control U937 cells can be stimulated to release endogenous arachidonic acid from their phospholipids. Maximum release of the unsaturated fatty acid occurs with 10 microM calcium ionophore in the presence but not in the absence of exogenously added calcium ion. In addition, DMSO-treated but not control U937 cells exhibit phospholipase activity when exposed to human IgG and then anti-human immunoglobulin. These data suggest that with respect to arachidonic acid metabolism U937 cells differentiate into functional macrophage-like cells when exposed to DMSO.     

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937

Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation.     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

Porphyromonas gingivalis fimbriae induce adhesion of monocytic cell line U937 to endothelial cells

This study used the human monocytic cell line U937 to examine whether or not Porphyromonas gingivalis fimbriae could induce the adhesion of monocytes to endothelial cells. An in vitro adhesion assay was used to investigate the effects of the fimbriae on U937 cell adhesion to human umbilical vein endothelial cells (HUVEC). The fimbriae enhanced U937 cell adhesion to HUVEC in a dose-dependent manner. U937 cells adhered better to HUVEC pretreated with the fimbriae for a minimum of 2 hr than to untreated HUVEC. The enhanced adhesion was inhibited by a monoclonal antibody against P. gingivalis 381 fimbriae. Pretreatment of U937 cells with the fimbriae for 24 hr enhanced U937 cell adhesion to HUVEC approximately 4-fold. This phenomenon was inhibited by an anti-CD11b antibody, suggesting the involvement of CD11b. These results indicate that P. gingivalis fimbriae can induce monocyte adhesion to the endothelial cell surface. They also suggest that the fimbriae may be involved in the initial event for infiltration of monocytes into the periodontal tissues of individuals with adult periodontitis.     

Differentiation and dedifferentiation of the human monocytic leukemia cell line, U937.

U937 cells were differentiated into macrophages after being treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) for the first two days and dedifferentiated with daily medium renewal for 10 days. Cell proliferation slowed down and the number of cells reached the maximum level on day 2. By day 4, all of the cells had spread and attached firmly to the culture dish, and more than 90% of the cells expressed the Fc-receptor and produced superoxide anion. From there on, the number of adherent, living cells decreased gradually to about half the initial count. Most of the cells eliminated from the culture by cell death were in the S phase at the time of TPA treatment. After day 8, the number of cells expressing macrophage-specific phenotypes gradually decreased, cell adhesion was weakened, and at the same time, DNA synthesis was initiated anew. The cells became round and began to proliferate as floating cells on days 9 to 10, and thereafter they became sensitive to the second round of TPA treatment. On the basis of all the results taken together, it is suggested that fully differentiated U937 cells were dedifferentiated after being cultured with frequent medium renewal.     

Altered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937

The human cell line U937 was used as a model for differentiation along the mononuclear phagocyte lineage. Following treatment with the phorbol ester TPA, PGE2 and TxB2 secretion was induced 50-100-fold, and both PGF2 alpha and PGI2 levels became detectable in the supernatant of TPA-differentiated U937 cells. The content of the prostaglandin precursor, arachidonic acid, remained unchanged in the cellular phospholipids of undifferentiated and TPA-differentiated U937 cells. Of the enzymes involved in the availability and metabolism of arachidonic acid, phospholipase A2 activity was increased 2-fold in the membranes of TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase activity remained unaltered. Cyclooxygenase activity, however, was enhanced 5-10-fold, which was due to enhanced expression of the enzyme as demonstrated by dot-blot analysis. The data suggest that the capacity to secrete prostaglandins is acquired during differentiation with TPA and results mainly from an increased cyclooxygenase activity. Despite the capacity of TPA-differentiated U937 cells to synthesize prostaglandins, none of the known monocytic stimuli further stimulated prostaglandin secretion in TPA-differentiated U937 cells. Generation of leukotrienes appears to represent a later state in the differentiation along the monocyte-macrophage lineage, since neither LTB4 nor cysteinyl-leukotrienes were detectable in the supernatants of either undifferentiated or TPA-differentiated U937 cells.     

Anti-oxidant activity and effect of Pinus morrisonicola Hay. on the survival of leukemia cell line U937

The free radical scavenging and anti-cancer activites of Pinus morrisonicola Hay. were studied using different parts of the pine, namely, needle, bark and cone. Results showed that pine needle water extract has the highest scavenging superoxide anion activity and the lowest IC50 value in inhibiting superoxide anion formation; however, the bark water extract showed the best anti-lipid peroxidation activity. Additionally, needle water extract displayed the highest inhibition of leukemia cell line U937 growth. The results indicated that P. morrisonicola Hay. possesses potential chemopreventative and therapeutic properties.     

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.