Chemistry of a human monocyte-derived cell line (U937): identification of the angiotensin I-converting activity as leukocyte cathepsin G

Angiotensin-converting enzyme, a dipeptidyl carboxypeptidase, catalyzes the conversion of angiotensin I to the vasoactive peptide angiotensin II. The finding of angiotensin-converting enzyme in dexamethasone- stimulated cultured monocytes and alveolar macrophages prompted the examination of a human monocyte-like cell line (U937) for angiotensin I- converting activity. Conversion of angiotensin I (5 X 10(-5) mol/L) to angiotensin II by U937 cell extracts (10(4) - 4 X 10(6) cells) was detected, and the pH optimum for the reaction was 7.0 to 8.0. The U937 cell angiotensin I-converting activity was purified to homogeneity by carboxymethylcellulose chromatography and trasylol affinity chromatography. The purified protein performed similarly to purified human neutrophil cathepsin G on sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis (SDS-gradient PAGE), elicited a reaction of complete identity with neutrophil cathepsin G when diffused against anti-cathepsin G antiserum, and had quantitatively similar angiotensin I-converting activity as neutrophil cathepsin G. Neutrophils and U937 cells had 143 and 52 times greater angiotensin I- converting capability than cultured monocytes or peripheral blood mononuclear cells, suggesting the relative importance of mobile cells containing cathepsin G in the local generation of angiotensin II. These data identify the angiotensin I-converting activity of the U937 cell as leukocyte cathepsin G and provide evidence that the U937 cell has neutrophil-like as well as monocyte-like characteristics.     

Existence of tartrate-resistant acid phosphatase activity in differentiated lymphoid leukemic cells

Acid phosphatase (AcP) in neoplastic cells from various lymphoid leukemias was examined. In the cytochemical studies, tartrate-resistant AcP (T-rAcP) activity was observed in the neoplastic cells from well-differentiated lymphoid leukemias such as adult T-cell leukemia (ATL), B-cell chronic lymphocytic leukemia (B-CLL), T-cell chronic lymphocytic leukemia (T-CLL), and hairy-cell leukemia (HCL). T-rAcP activity was also detected in a small number of leukemic cells obtained from T-cell acute lymphoblastic leukemia (T-ALL), while it was not detected in the neoplastic cells from null-ALL, macroglobulinemia, and multiple myeloma (MM). In the electrophoretical studies, fraction 1 (F-1), F-3, F-3b, and F-4 were completely tartrate-sensitive, while F-2 was partially resistant and F-5 was completely resistant. T-rAcP activity (F-5) was observed in ATL cells, B-CLL cells, and HCL cells, while it was not detected in ALL cells, macroglobulinemia cells, and MM cells. The present study indicates that T-rAcP activity is observed not only in HCL cells but also in the well-differentiated lymphoid cells such as ATL cells, B-CLL and T-CLL cells except the most highly differentiated forms of B-cells of MM and macroglobulinemia.     

Tumor necrosis factor induces tissue factor-like activity in human leukemia cell line U937 and peripheral blood monocytes

The induction of procoagulant activity (PCA) by human recombinant tumor necrosis factor (rTNF) was studied in human monoblastic leukemia cell line U937 and human peripheral blood monocytes. Using a one-step recalcificating clotting assay, PCA in cell lysates or whole cell preparations was measured by comparison to a rabbit brain thromboplastin standard. There was a dose- and time-dependent increase in PCA when U937 cells were cultured with rTNF. The effect of rTNF was not enhanced by recombinant human interferon-gamma (rIFN gamma). Cycloheximide inhibited the expression of PCA by U937 cells, showing that protein synthesis was necessary to mediate the effects of rTNF. Whole cell preparations demonstrated that greater than 80% of the PCA was expressed on the surface of the cells. The PCA functioned as a tissue factor-like substance, since it required coagulation factor VII and factor X. rTNF also increased PCA in human monocytes in a dose- and time-dependent manner. This effect was abrogated by boiling the rTNF for ten minutes, and was not inhibited by adding polymyxin-B to the cultures, making it unlikely that endotoxin accounted for the observed effects. These results suggest that TNF-induced expression of tissue factor by mononuclear phagocytes may modulate immunologic, inflammatory, and hemostatic processes.     

Melatonin prevents apoptosis induced by UV-B treatment in U937 cell line

Melatonin influences circadian rhythms and acts as antioxidant and free radical scavenger. UV irradiation triggers multiple cellular events which lead to cell death, in particular to apoptosis; this process involves reactive oxygen species. Apoptotic machinery involves several pathways, in which mitochondria play crucial roles. In this work we have evaluated by means of cytometric, biochemical and ultrastructural approaches, if incubation of U937 promonocytic leukemia cells with melatonin may affect apoptotic behavior induced by UV-B. The cell line was treated with 1 mm melatonin before and after UV-B exposure. Melatonin pretreatment significantly reduced the number of apoptotic cells, as revealed by FITC Annexin-V and propidium iodide assays (P < 0.005), as well as attenuated mitochondria alterations, as shown by ultrastructural morphology, Mito Tracker and JC-1 staining, and cytochrome c (cyt c) release (P < 0.005). On the contrary, incubation with melatonin after UV-B exposure significantly protect U937 cells from UV-B induced alterations, showing a possible delay of the apoptotic machinery (as revealed by the presence of earlier stages of apoptosis and significant cyt c release). Our results suggest that, in our experimental model, melatonin may play a role as noncytotoxic anti-apoptotic compound and, at least in part, may protect U937 cells from UV-B induced mitochondria dysfunction/damage.     

Effect of cisplatin treatment of human monocyte cell line U937 on the induction of tumoricidal activity.

U937, a human monocyte-like, cell line was checked for cytotoxic activity against tumor target cells. Untreated U937 cells showed little cytotoxicity against tumor cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) and LPS significantly activated the U937 cells to tumoricidal state. Treatment of U937 cells with cisplatin did not enhance the tumoricidal activity. Similarly, interferon gamma (IFN-Y) and macrophage colony stimulating factor (M-CSF) could also not activate either the tumoricidal activity of U937 cells. Pretreatment of U937 cells with GM-CSF for 24 h and then the treatment with cisplatin significantly augmented the tumoricidal activity as compared to that of GM-CSF alone.     

Complement-mediated enhancement of HIV-1 infection of the monoblastoid cell line U937

To assess the role of complement and complement receptors in HIV-1 infection of monocytes and macrophages, we studied the infectivity of HIV-1, isolated from the peripheral blood of a patient with subacute AIDS-related encephalopathy, on the human monoblastoid cell line U937. HIV-1 and HIV-1-infected cells were capable of activating the complement system via the classical and the alternative pathways, respectively. Low concentrations of HIV-1 were able to infect U937 cells more easily in the presence than in the absence of complement. At higher virus concentrations, infectivity was no longer facilitated by the presence of complement. Infection of U937 cells was reduced in the presence of any of the monoclonal antibodies (MAbs), OKT4a (anti-CD4), OKM1 (anti-CR3), or M522 (anti-CR3). A combination of all three of these MAbs reduced the infection by an even greater amount. These data indicate that complement receptors may be a port of entry for complement-coated HIV-1.     

Chemotaxis of the monocyte cell line U937: Dependence on cholesterol and early mevalonate pathway products

In the present study we investigated the influence of cholesterol depletion and hydroxymethylglutaryl-coenzyme A reductase (HMG-CoA reductase) inhibition on chemotaxis of the human monocytic cell line U937. Chemotaxis was nearly completely depressed after incubation for 24 h in the absence of lipoproteins. This was accompanied by a significant decrease in cellular cholesterol. Addition of 10 μg/ml low density lipoprotein (LDL) for 2 h to the cholesterol-depleted cells restored chemotaxis. Free cholesterol had no effect under these conditions. Inhibition of HMG-CoA reductase by pravastatin (0.01–1.0 mM) for 20 or 72 h also reduced chemotaxis. However, this effect was not accompanied by a decrease in cellular cholesterol when cells were grown in the presence of lipoproteins. The effect of pravastatin could be reversed by the addition of mevalonate. Addition of LDL did not change the response to pravastatin. We propose that the availability of cholesterol plays an important role in cellular chemotaxis. Furthermore, it can be suggested that other products of the mevalonate pathway apart from cholesterol may contribute to the regulation of chemotaxis.     

Study of the effects and mechanism of alltrans retinoic acid on leukemic cell line U937 cells with NPM1 mutation

正Objective To investigate the effect and mechanism of all-trans retinoic acid(ATRA)on leukemic cell line U937 cells with NPM1 mutation.Methods Human acute myeloid leukemia cell line U937 was explored,NPM1mutated(A type)plasmids were transfected into U937 to form stable clones A1 and A2,which were identified by Western blot and Co-immunoprecipitation.The cell proliferation was measured by methylthiazolyl tetrazolium     

Human mononuclear phagocyte differentiation: a study of the U-937 cell line by ultrastructural cytochemistry and surface antigen analysis

U-937 represents a well-established permanent human haematopoietic cell line, which exhibits characteristics of the monocyte/macrophage series. U-937 cells were investigated by peroxidase ultrastructural cytochemistry in order to determine the normal developmental stage to which they correspond. This study was performed in non- and TPA-stimulated cells, in conjunction with surface analysis by monoclonal antibodies. It is concluded: (1) peroxidase-positive U-937 cells are monoblasts and promonocytes involved in myeloperoxidase synthesis; (2) TPA-stimulation caricatures transformation of these cells into monocytes but not into resident macrophages, as far as peroxidase cytochemistry is concerned; (3) the reactivity of myeloperoxidase present in the endoplasmic reticulum of synthesizing cells is inhibited by glutaraldehyde fixation.     

Rheumatoid arthritis synovial fibroblast and U937 macrophage/monocyte cell line interaction in cartilage degradation

Objective. To examine the interaction between synovial fibroblasts and macrophages in the context of cartilage degradation. Methods. An in vitro model of human cartilage degradation was used, in which purified populations of fibroblasts and macrophages were added to a radio-labeled cartilage disc. Cartilage destruction was measured by the percentage of radiolabel release. Results. Fibroblasts, obtained from either rheumatoid arthritis (RA) or osteoarthritis synovial tissue, could mediate cartilage degradation if cocultured with the U937 macrophage cell line. Skin and RA bone marrow fibroblasts had no degradative effect on cartilage. Fibroblast—macrophage contact was not required for cartilage degradation. Cartilage degradation by synovial fibroblasts was inhibited by antibodies to tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-6. Cartilage degradation was almost completely abrogated by a combination of antibodies to TNFα and IL-1β. Contact between fibroblasts and cartilage was shown to be essential. Antibodies to CD44, but not to intercellular adhesion molecule 1, markedly inhibited cartilage degradation. Conclusion. TNFα, IL-1β, and IL-6 were involved in the activation of synovial fibroblasts to cause cartilage degradation. Cartilage degradation occurred only when fibroblasts were in contact with cartilage. CD44 was demonstrated to be involved in the fibroblast—cartilage interaction.     

桂皮醛对U937细胞增殖和凋亡的影响及其机制探讨

目的:探讨桂皮醛对急性髓系白血病细胞株U937的增殖和凋亡的影响及其相关机制.方法:以U937细胞为研究对象,CCK-8法测定细胞增殖活性;流式细胞术检测细胞周期、凋亡率、线粒体膜电位水平;ELISA法检测细胞上清液中VEGF浓度.结果:桂皮醛呈时间和剂量依赖性影响U937细胞生长.桂皮醛处理后U937细胞阻滞于G2/...     

Growth of Leishmania donovani amastigotes in the continuous human macrophage cell line U937: studies of drug efficacy and metabolism

We have developed a simple and reproducible system for infecting a human macrophage cell line (U937) with stationary-phase Leishmania donovani promastigotes. Four days after infection, greater than 90% of the promastigotes had transformed to amastigotes. The antileishmanial agents allopurinol riboside, formycin B, 9-deazainosine, and sodium stibogluconate effectively inhibited the growth of L. donovani amastigotes in this cell line. To study the capability of amastigotes in the U937 cell line to carry out biochemical reactions that could be monitored experimentally, we incubated the cells with radiolabeled 9-deazainosine. This purine analogue underwent metabolism in the amastigote phase similar to that occurring in the promastigote phase. This cell line should be useful for studies of parasite maturation and differentiation, parasite-human interactions, and antiparasitic drugs.     

抗酒石酸酸性磷酸酶测定及在骨质疏松症诊断中的应用

目的：建立抗酒石酸酸性磷酸酶测定方法及在原发性骨质疏松症诊断中的临床应用。方法：以1－萘酚磷酸为基质,测定肝素抗凝血抗酒石酸,氟化物抑制酸性磷酸酶,并以双能X线骨密度仪测定戎腰椎2－4的骨密度,结果：抗酒石酸酸性磷酸酶测定方法在本地区老年妇女的参考值为2．9－5．3U／L,该方法的平均批内和批间变异分别为2．7％和5．0％,骨质疏松症该酶活性明显高于健康对照组（P＜0．001）,并与骨密度测定呈显著的相关性（r=0.82),结论：抗酒石酸性磷酸酶测定方法简便,快速,是早期原发性骨质疏松症的筛选,诊断及观察疗效的敏感指标。     

人胆固醇酯水解酶真核表达载体的构建及U937细胞系的转染

目的构建人胆固醇酯水解酶（hCEH）绿色荧光蛋白真核表达载体pEGFP-N1-hCEH,并观察其转染单核巨噬细胞株U937后,细胞中hCEH的表达情况。方法以人肝细胞L-O2总RNA为模板,采用RT-PCR技术扩增hCEH编码区序列,将扩增片段插入到pMD19-T载体中,回收、纯化目的片段后亚克隆到pEGFP-N1真核表达载体上,经双酶切、测序鉴定后,转染U937,观察U937中绿色荧光蛋白的表达情况。结果 RT-PCR扩增hCEH基因的编码区序列获得1条约1.7 kb的片段,与预期片段大小相符;以pEGFP-N1为载体,成功构建重组表达质粒pEGFP-N1-hCEH,该质粒可以在U937中表达。结论成功构建pEGFP-N1-hCEH,用其转染U937后,细胞中pEGFP-N1-hCEH大量表达。