Human Myeloid Cell Line U937 Research Articles

An immune cell spray (ICS) formulation allows for the delivery of functional monocyte/macrophages

Macrophages are key cells of the innate immune system and act as tissue resident macrophages (TRMs) in the homeostasis of various tissues. Given their unique functions and therapeutic use as well as the feasibility to derive macrophages in vitro from hematopoietic stem cell (HSC) sources, we propose an “easy-to-use” immune cell spray (ICS) formulation to effectively deliver HSC-derived macrophages. To achieve this aim, we used classical pump spray devices to spray either the human myeloid cell line U937 or primary murine HSC-derived macrophages. For both cell types used, one puff could deliver cells with maintained morphology and functionality. Of note, cells tolerated the spraying process very well with a recovery of more than 90%. In addition, we used osmotic preconditioning to reduce the overall cell size of macrophages. While a 800 mosm hyperosmolar sucrose solution was able to reduce the cell size by 27%, we identified 600 mosm to be effective to reduce the cell size by 15% while maintaining macrophage morphology and functionality. Using an isolated perfused rat lung preparation, the combinatorial use of the ICS with preconditioned and genetically labeled U937 cells allowed the intra-pulmonary delivery of cells, thus paving the way for a new cell delivery platform.

Effect of Native and Modified Apolipoprotein A-I on DNA Synthesis in Cultures of Different Cells.

Culturing of bone marrow cells in serum-free RPMI-1640 medium for 24 h was accompanied by a decrease in the rate of [3H]-thymidine incorporation into DNA. Addition of native apolipoprotein A-I (apoA-I) or plasma LDL and HDL to the culture medium increased this parameter. In contrast to native apoA-I, its modified form decelerated DNA synthesis in bone marrow cells. A similar inhibitory effect of modified protein was observed in cultures of human embryonic kidney cells (HEK293) and in rapidly proliferating mouse macrophage cell line ANA-1. The only exclusion was human myeloid cell line U937: neither native nor modified apoA-I affected DNA synthesis in these cells. Thus, the regulatory effects of apoA-I are tissue-specific; this protein can produce either stimulatory or inhibitory effect on DNA biosynthesis in cells depending on its conformation.

Affinity Binding of EMR2 Expressing Cells by Surface-Grafted Chondroitin Sulfate B.

The propensity of glycosaminoglycans to mediate cell-cell and cell-matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS)-B is of particular interest, since it interacts with the receptor (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells preincubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types.

Combination of Homoharringtonine with Arsenic Trioxide Induces Apoptosis of Human Acute Myeloid Leukemia Cell Line U937

To investigate the effects and mechanisms of the combination of homoharringtonine (HHT) with arsenic trioxide(As2O3) on human myeloid cell line U937 in vitro. MTT method was used to determine the antiproliferating effect of different concentrations of HHT, As2O3 and their combination on U937 cells; the flow cytometry with Annexin-V-FITC/PI double staining was used to determine the apoptosis-induced effect of HHT and As2O3 alone or their combination; Western blot method was used to detect the protein expression of P-AktSer473,P-AktThr308,BCL-XL, BID,MCL-1,P-MCL-1 and so on. HHT and As2O3 could significantly inhibit proliferation of U937 cells and induce their apoptosis. The combination of these 2 drugs could significantly enhance the early apoptosis of U937 cells. After combination of these 2 drugs was used, the protein expressions of P-AktSer473,P-AktThr308,MCL-1,P-MCL-1 and BCL-XL were obviously down-regulated, but the expression of BID protein did not change. The combination treatment of HHT and As2O3 can synergistically inhibit the growth of U937 cells through inhibition of PI3K/Akt signal way and MCL-1 protein.

Identification of two distinct cell binding sequences in the vitamin D binding protein

The vitamin D binding protein (DBP) is a multifunctional, albumin-like plasma protein that often requires cell surface binding to mediate some of its diverse functions. DBP binds to several different molecules on the external face of the plasma membrane indicating that it may possess distinct cell binding sequences. In this report, surface plasmon resonance was utilized to evaluate the relative binding of the human myeloid cell line U937 to immobilized recombinant expressed DBP in order to identify cell localization sequences. U937 cells showed robust binding to immobilized native DBP, but essentially no interaction when sensor chips were coated with β 2-microglobulin or BSA. The cell–DBP interaction was completely eliminated if cells were pretreated with soluble DBP. Recombinant DBP domains and truncated domains were next evaluated to determine the location of cell binding regions. Domains I (amino acids 1–191) and III (379–458), but not domain II (192–378), could support cell binding. Further evaluation of domain I, using truncated proteins and overlapping peptides, demonstrated that a single amino acid sequence, residues 150–172 (NYGQAPLSLLVSYTKSYLSMVGS), mediated cell binding. The domain III cell binding region was investigated using truncated versions of domain III fused to full-length domain II that served as a scaffold. These experiments indicated that the cell binding sequence is located in the first portion of that domain (379–402: ELSSFIDKGQELCADYSENTFTEY). Overlapping peptides spanning this sequence could partially block cell binding only when used in combination. We conclude that DBP contains two cell localization sequences that may be required for some of the multiple functions of this protein.

Inducible expression of EVI1 in human myeloid cells causes phenotypes consistent with its role in myelodysplastic syndromes

The oncogene EVI1 has been implicated in the etiology of AML and MDS. Although AML cells are characterized by accelerated proliferation and differentiation arrest, MDS cells hyperproliferate when immature but fail to differentiate later and die instead. In agreement with its roles in AML and in immature MDS cells, EVI1 was found to stimulate cell proliferation and inhibit differentiation in several experimental systems. In contrast, the variant protein MDS1/EVI1 caused the opposite effect in some of these assays. In the present study, we expressed EVI1 and MDS1/EVI1 in a tetracycline-regulable manner in the human myeloid cell line U937. Induction of either of these proteins caused cells to accumulate in the G0/G1-phase of the cell cycle and moderately increased the rate of spontaneous apoptosis. However, when EVI1- or MDS1/EVI1-expressing cells were induced to differentiate, they massively succumbed to apoptosis, as reflected by the accumulation of phosphatidylserine in the outer leaflet of the plasma membrane and increased rates of DNA fragmentation. In summary, these data show that inducible expression of EVI1 in U937 cells causes phenotypes that may be relevant for its role in MDS and provides a basis for further investigation of its contribution to this fatal disease.

Assessment of the U937 cell line for the detection of contact allergens

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1beta and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times. Test item-specific modulations of the chosen activation markers (CD86, IL-1beta and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.

A CD33‐specific single‐chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells

A novel single-chain immunotoxin was constructed by combining a CD33-specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C-terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL-60 and THP-1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen-specific and occurred by apoptosis. A control protein, consisting of a CD19-specific scFv antibody fragment fused to the ETA-KDEL toxin, failed to induce death of the CD19-negative cell lines U937, HL-60 and THP-1. The CD33-ETA toxin also mediated apoptosis of fresh patient-derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen-restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.

Changes in Sensitivity of a Human Myeloid Cell Line (U937) to Metal Toxicity after Glutathione Depletion

Glutathione (GSH) concentrations have been determined in the human myeloid cell line U937. The effects of modulating GSH concentration on sensitivity to metal toxicity has also been examined. Intracellular concentrations of GSH increased as the cells entered into the cell cycle, reaching a maximum level after 24 hours of cell culture, after which levels declined. Cell concentration was also observed to influence intracellular GSH concentrations. A reciprocal relationship was observed with higher maximum intracellular GSH concentrations being measured in cultures initiated with smaller cell number. The relative toxicity's determined for five metal chlorides were mercury < cadmium < cobalt < zinc < gold. Treatment of cells with N-acetylcysteine (NAC) increased intracellular GSH but had little effect on the absolute or relative toxicity's of the metals. Treatment of cells with L-buthionine-(S-R)-sulfoximine (BSO) depleted intracellular GSH and resulted in increased sensitivity of the cells to gold, 40 fold, cadmium 8 fold and mercury 3 fold.

Degradation of hammerhead ribozymes by human ribonucleases.

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

Human BST-1 Expressed on Myeloid Cells Functions as a Receptor Molecule

We have previously identified and cloned BST-1 as a molecule which is overexpressed on the bone marrow stromal cell lines derived from patients with rheumatoid arthritis and which has the ability to support the pre-B cell growth. BST-1 is a glycosylphosphatidylinositol-anchored ectoenzyme having ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities. In this report, we demonstrate that human BST-1 was expressed on monocytes, granulocytes in the peripheral blood of healthy donors, and macrophages maturedin vitro.Cross-linking of BST-1 with a polyclonal anti-BST-1 antibody induced tyrosine phosphorylation of a 130-kDa protein (p130) in the human myeloid cell lines U937 and THP-1. Cross-linking of BST-1 overexpressed on a transfectant induced tyrosine phosphorylation of p130, dephosphorylation of the 100-kDa protein, and growth inhibition. These results suggest that BST-1 can deliver signals into cells and function as a receptor.

The human lysozyme gene

We have isolated two overlapping recombinant lambda-phage clones from a genomic lambda-EMBL3 library containing 25 kb of the human lysozyme gene region. Furthermore a full-lenght human lysozyme cDNA clone of 1.5 kb was isolated from a human placenta cDNA library. Nucleotide sequences of the entire structural gene and the cDNA clone were determined. The human lysozyme gene spans 5856 bp and its sequence organization with four exons and three introns is homologous to the chicken lysozyme gene and the human alpha-lactalbumin gene. Human and chicken lysozyme genes differ mainly in the size of their introns and 3' non-coding region. Four Alu repetitive elements were found in the human lysozyme gene, one in each intron and one on the fourth exon. Lysozyme transcripts of 1.6 kb and 0.6 kb in size were detected in human myeloid cell lines U-937, HL-60 and THP-1 and surprisingly in human hepatoma cell lines HepG2 and Hep3B. The lysozyme gene locus was assigned to human chromosome 12 by hybridization to a panel of DNAs from human-rodent somatic cell hybrids.