Poliovirus modulates Bcl-xl expression in the human U937 promonocytic cell line

Poliovirus induces bcl-2-independent apoptosis in the human U937 promonocytic cell line [28]. Here we describe that this cell death, induced after viral infection, correlates with the modulation of the protooncoprotein Bcl-xl. Furthermore, poliovirus infection decreases the detected Bcl-xl in a U937 clone that overexpresses this protein (U937bcl-xl). Although in U937bcl-xl cells, Bcl-xl was not as highly regulated as in parental U937 cells, correlation between Bcl-xl modulation and the cleavage of poly(ADP-ribose)polymerase could be observed. Nevertheless, the induction of shutoff after infection of transfected control U937neo or U937bcl-xl clones was not significantly altered. Finally, production of new viral particles was slightly restricted in Bcl-xl-overexpressing U937 cells. Taken together, these results suggest that Bcl-xl is modulated during the induction of apoptosis in the promonocytic cell line U937 after poliovirus infection, although modulation of this protooncogene was not sufficient to modify the course of infection.     

Biochemical and functional alterations induced by CD23 ligation in the human promonocytic cell line U937.

The early events triggered in interleukin-4 (IL-4)-stimulated U937 cells by ligation of CD23/Fc epsilon RII with specific monoclonal antibodies (mAb) were analysed, as a model of the action of this molecule on the differentiation of promonocytic cells. As well as IL-4-activated human monocytes, addition of anti-CD23 mAb to IL-4-treated U937 cells triggered cAMP accumulation but did not evoke significant polyphosphoinositide hydrolysis. However, by a microspectrofluorometric technique allowing single cell analysis, anti-CD23 mAb was found to elicit calcium mobilization in these cells. In addition, the treatment induced phenotypic alterations in these cells, as evidenced by the acquisition of the monocyte marker CD14 and the increase of the alpha-chain (CD11a) and of the common beta-chain (CD18) of the leucocyte function-associated antigen 1 (LFA-1) family antigens. Although weaker than in monocytes, CD23 ligation evoked a small secretion of the pro-inflammatory mediators IL-6 and thromboxane B2. These data suggest that a significant maturation of promonocytic cells towards a more mature monocytic phenotype can be achieved through successive exposure to IL-4 and CD23 ligation.     

甘露聚糖结合凝集素诱导人白血病细胞系U937细胞凋亡

目的研究甘露聚糖结合凝集素（MBL）对白血病细胞系U937凋亡的影响。方法不同浓度MBL处理U937细胞后,应用CCK-8法分析细胞增殖情况,Hoechst33258染色观察细胞核及染色质,AnnexinV/PI双染流式细胞术分析细胞凋亡率,real-timePCR和免疫印迹分析凋亡相关基因及蛋白表达。结果 30～50μg/mlMBL培养72h,U937细胞增殖明显受抑,出现不同程度核固缩、核碎裂;随MBL浓度升高或作用时间延长,凋亡细胞数逐渐增多;Fas、Caspase-3mRNA、Fas和FasL蛋白表达量升高,Caspase-3和多腺苷二磷酸多聚酶（PARP）蛋白被剪切激活或失活。结论 MBL可诱导白血病细胞U937细胞凋亡,其机制与上调Fas表达、剪切Caspase-3和PAPR有关。     

Poliovirus infection interferes with the phorbol ester-induced differentiation of the monocytic U937 cell line

Monocytic U937 cells can differentiate in vitro into macrophage-like cells by treatment with phorbol esters such as phorbol 12-myristate 13-acetate (PMA). We have analyzed the effect of poliovirus infection in this pathway of differentiation. Poliovirus RNA replication took place in both untreated and PMA-treated U937 cells infected before or after PMA addition, although a slight reduction in poliovirus RNA levels was observed in PMA-treated cells at late times postinfection. Total protein synthesis remained unchanged during the first 5 h of infection both in normal and PMA-treated cells. However, an inhibition on total RNA synthesis was observed early in infection. PMA-induced c-myc mRNA expression was abolished when infection took place 1 h before PMA addition but was just partially inhibited when poliovirus was added 1 h after PMA stimulation. Fluorescence flow cytometry analysis revealed that poliovirus infection induced an increase in the number of 4F2 molecules per cell in normal U937 cells and a slight decrease in the number of positive cells for the antigens CD14, CD4 and CD11c in both untreated or PMA-treated U937 cells. These findings suggest that poliovirus infection of U937 cells interferes at various levels with monocyte maturation yielding cells which are unable to undergo the complete pathway of differentiation to macrophages.     

Karyotypic analysis of the human monoblastic cell line U937

Karyotypes of three sublines of the human cell line U937 showed considerable variation, but all contained four consistent marker chromosomes (i.e., 3q−, 11q−, 16p+, and 17p− chromosomes). The 11q− chromosome appeared to be derived from either an interstitial deletion in bands 11q21–23 or from a translocation with an unidentified chromosome. The presence of this chromosome was of particular interest because rearrangements of chromosome #11 at band 11q23 are often associated with malignancies of the monocytic lineage. The possible significance of these findings is discussed.     

Hydrogen sulfide induces the synthesis of proinflammatory cytokines in human monocyte cell line U937 via the ERK-NF-kappaB pathway

Hydrogen sulfide (H2S) is now considered an endogenous, gaseous mediator, which has been demonstrated to be involved in many inflammatory states. However, the mechanism of its proinflammatory function remains unknown. In the present study, we used IFN-gamma-primed human monocytic cell line U937 to investigate the effects of H2S in vitro on monocytes. We found that treatment with the H2S donor, sodium hydrosulfide, led to significant increases in the mRNA expression and protein production of TNF-alpha, IL-1beta, and IL-6 in U937 cells. H2S-triggered monocyte activation was confirmed further by the up-regulation of CD11b expression on the cell surface. We also observed that H2S could induce a rapid degradation of IkappaBalpha and subsequent activation of NF-kappaB p65, and this effect was attenuated by Bay 11-7082, a specific inhibitor of NF-kappaB. Furthermore, pretreatment of cells with Bay 11-7082 substantially inhibited the secretion of TNF-alpha, IL-1beta, and IL-6 induced by H2S. We also found that H2S stimulated the phosphorylation and activation of ERK1/2, but not of p38 MAPK and JNK, and pretreatment with PD98059, a selective MEK1 antagonist, could inhibit H2S-induced NF-kappaB activation markedly. Together, our findings suggest for the first time that H2S stimulates the activation of human monocytes with the generation of proinflammatory cytokines, and this response is, at least partially, through the ERK-NF-kappaB signaling pathway.     

替米沙坦抑制U937细胞增殖及诱导凋亡

目的:探讨替米沙坦对U937细胞株的生长抑制及凋亡诱导作用。方法:分别以不同浓度的替米沙坦处理人类急性髓系白血病细胞U937;以CCK-8法检测不同浓度替米沙坦对U937细胞的生长抑制作用;以集落形成实验观察不同浓度替米沙坦对U937细胞集落形成能力的影响;以Annexin V-PI双染法及Hoechst 33342染色法检测不同浓度替米沙坦作用前后U937细胞凋亡程度的变化;以流式细胞术检测U937细胞表面抗原CD11b的阳性表达率,瑞氏染色后倒置显微镜进行细胞形态学观察,了解U937细胞的分化情况;以Western blot法检测不同浓度替米沙坦作用U937细胞后凋亡相关蛋白表达量的改变。结果:CCK-8实验结果证实替米沙坦呈时间和剂量依赖性抑制U937细胞的生长;集落形成实验显示低剂量替米沙坦可以完全抑制U937细胞的集落形成能力;Annexin V-PI双染法及Hoechst 33342法结果证实替米沙坦可以诱导U937细胞凋亡;细胞表面抗原流式检测术及瑞氏染色结果证实替米沙坦可以促进部分U937细胞分化;Western blot实验结果证实替米沙坦作用于U937细胞72 h后,促凋亡相关蛋白cleaved PARP及cleaved caspase-3蛋白的水平明显增高。结论:替米沙坦可以抑制细胞增殖以及诱导U937细胞部分分化,并通过caspase依赖的凋亡途径触发U937细胞凋亡。     

Prostaglandin biosynthesis by a human macrophage-like cell line, U937

The human macrophage-like cell line, U937, produced significant amounts of prostaglandin (PG) E2 when incubated with exogenous arachidonic acid (AA). The synthesis of PGE2 was completely inhibited by pretreatment with indomethacin (20 micrograms/ml). Another major metabolite, unidentified, which was released during incubation with AA, was not inhibited by indomethacin, but was decreased by nordihydroguaiaretic acid (NDGA) (10(-5)M) or BW755C (10(-4)M). These results confirm the presence of cyclooxygenase and perhaps lipoxygenase activities in this macrophage-like cell line. Challenge of U937 cells with zymosan, opsonized zymosan, phorbolmyristate acetate (PMA), heat-aggregated human IgG (AHG), or calcium ionophore A23187 failed to stimulate synthesis and release of either PGE2 or the above mentioned metabolite. The inability of U937 cells to release endogenous AA from cell lipid for PG synthesis constitutes an important functional difference between these cells and normal macrophages.     

Caffeine induces apoptosis in human neuroblastoma cell line SK-N-MC

Caffeine is one of the most widely consumed neuroactive drugs, coming mostly from everyday beverages such as coffee and tea. To investigate whether caffeine induces apoptosis in the central nervous system, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, flow cytometric analysis, DNA fragmentation assay, and caspase-3 enzyme assay were performed on SK-N-MC human neuroblastoma cells. Cells treated with caffeine at concentrations as high as 10 mM exhibited several characteristics of apoptosis. In addition, caffeine was shown to increase the caspase-3 activity. These results suggest that high-dose of caffeine induces apoptosis in human neuroblastoma cells, probably by increasing the caspase-3 enzyme activity.     

Replication of human cytomegalovirus in the cells of the U937 monocytoid cell line

Human cytomegalovirus (HCMV) infection in immunocopromized hosts sometimes occurs as a result of reactivation. Cells of the monocytemacrophage linkage are suggested to be a site of latency and persistence for HCMV. The human monocytic cell line U937 was infected with the AD169 strain and a clinical isolate of HCMV. The expression of surface antigens on the cells was assessed by flow cytometry. The polymerase chain reaction (PCR) was used to detect viral DNA from infected cells. CMV immediate early antigen, early antigen, and late antigen (LA) were detected from both clinical isolate- and AD169-inoculated U937 cells by flow cytometry. CMV DNA which code major immediate early gene (US3) and LA gene (US14) were detected from the clinical isolateinoculated U937 over a period of 31 days as tested by PCR. These U937 cells proliferated as well as uninfected U937 cell, but only a small number of AD169-inoculated U937 cells survived after 14 days of inoculation. Interleukin-2 activities were detected in the media on days 24–40 after inoculation with AD169. This chronic CMV infection model of U937 might be utilized to study the mechanisms of persistence and reactivation.     

Growth of measles virus in a human macrophagelike cell line: U937

Measles virus infection was established in U937, a continuous human macrophagelike cell line. Unlike cultured human peripheral macrophages, infection resulted in prominent giant cell formation, indicating that these cells are susceptible to viral-induced fusion. Although a high proportion of cells in culture contained measles viral antigen by immunofluorescent assay a relatively small amount of infectious virus was produced. In contrast to continuously cultured human lymphoblastoid cell lines, infection of U937 was lytic, and persistent infection could not be established. The U937 cell line may be useful for further studies of viral interaction with macrophages, including those related to the induction of cell fusion by measles or other syncytium-forming viruses.     

Bioactivity-guided identification and cell signaling technology to delineate the immunomodulatory effects of Panax ginseng on human promonocytic U937 cells

Background  

Ginseng is believed to have beneficial effects against human diseases, and its active components, ginsenosides, may play critical roles in its diverse physiological actions. However, the mechanisms underlying ginseng's effects remain to be investigated. We hypothesize some biological effects of ginseng are due to its anti-inflammatory effects.     

Glucocorticoids inhibit IgE receptor expression on the human monocyte cell line U937.

Cells of a human monocyte-like cell line (U937) were analysed for IgE Fc receptors (Fc epsilon R) before and after glucocorticoid treatment. Specific binding of human myeloma IgE (Sha) was measured by 125I-labelled IgE, and by fluorescein-labelled IgE monitored by flow cytometry. Treatment of cells with dexamethasone or other steroids with glucocorticoid activity caused a significant decrease in Fc epsilon R expression. The inhibition was dose dependent, with a half-maximal effect at 20 nM dexamethasone, a concentration which is near to the dissociation constant of glucocorticoid receptors for dexamethasone. Inhibition of Fc epsilon R was significant beginning 8 h following glucocorticoid treatment and reached a plateau at 24 hr. The Ka for IgE binding was similar for control and dexamethasone-treated cells, while the number of IgE binding sites was decreased by 50-60%. Culture supernatants from dexamethasone-treated U937 cells which were concentrated 10-fold and depleted of free steroid did not affect Fc epsilon R expression. These results demonstrate that glucocorticoids can directly decrease the number of Fc epsilon R. This effect could participate in the glucocorticoid-induced suppression of IgE-mediated allergic reactions.     

Growth of Legionella pneumophila in a human macrophage-like (U937) cell line

We established a model of the bacteria-macrophage interaction to study the cellular basis of Legionella pneumophila pathogenesis and to characterize avirulent L. pneumophila. We found that U937 cells, which are derived from a human histiocytic lymphoma cell line, support intracellular growth of L. pneumophila with a doubling time of 6 h, and that sustained intracellular growth is associated with a cytopathic effect (CPE) that can be detected by microscopic examination and quantified with the vital stain 3-(4,5-dimethyl thiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (MTT). An L. pneumophila isolate obtained directly from infected guinea-pig spleens can grow and produce CPE in these cells, destroying most of the cell layer after 72 h of growth. Only 10(6) organisms of this strain are required to kill 50% of guinea-pigs inoculated by the intraperitoneal route. In contrast, an avirulent isolate derived by 203 successive plate passages of the same strain can neither kill guinea-pigs at an intraperitoneal inoculum of 10(7) nor grow or produce CPE in U937 cells. Since the cells were able to differentiate between a virulent and an avirulent strain of L. pneumophila, we conclude that U937 cells are an appropriate model system for study of the bacteria-macrophage interaction.     

姜黄素诱导永生化人HaCaT细胞凋亡

目的 探讨姜黄素对人永生化上皮细胞凋亡的诱导作用.方法 应用细胞计数、流式细胞术、琼脂糖凝胶电泳、Hoechst33258染色、苏木素-伊红(HE)染色和透射电镜检测经姜黄素诱导处理后人永生化上皮HaCaT细胞的凋亡.结果经7.5mg/L姜黄素诱导处理后,人永生化上皮HaCaT细胞的增殖活动受到明显的抑制,细胞生长抑制率达83.03%;细胞周期检测出现亚二倍体(亚G1期)细胞峰值,细胞凋亡率达13.1%,并发生G2/M期阻滞;琼脂糖凝胶电泳出现细胞凋亡典型的DNA"梯状"条带;细胞核经Hoechst33258染色出现浓染致密的固缩形态和颗粒状荧光;光镜和电镜观察结果显示,姜黄素处理组细胞体积缩小,细胞核固缩,染色质凝聚,线粒体肿胀,有凋亡小体形成等显著的凋亡特征.结论姜黄素对人永生化上皮HaCaT细胞凋亡有显著的诱导作用,从而为进一步研究表皮细胞衰老、凋亡机理提供了重要基础和研究依据.     

Expression of complement factor H on the cell surface of the human monocytic cell line U937

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.     

稳定表达CD200的U937细胞株的建立

目的：构建稳定表达CD200蛋白的人单核细胞系U937细胞株,为研究该基因的作用机制奠定基础。方法：用电转染基因法将含有人CD200 cDNA的真核表达载体pcDNA3-CD200质粒和pcDNA3质粒分别转入人单核细胞系U937细胞中,经G418筛选,转染阳性细胞连续传代培养,并应用RT-PCR及流式细胞术检测CD200 mRNA和蛋白表达情况。结果：经G418筛选,U937细胞全部死亡,转染pcDNA3质粒和pcDNA3-CD200重组质粒的U937细胞存活,并可连续传代培养30代;转染pcDNA3-CD200重组质粒的U937细胞RT-PCR检测有一特异性扩增带;流式细胞术检测CD200表达结果显示：转染pcD-NA3-CD200重组质粒的U937细胞CD200表达阳性细胞率（77.20%）显著高于转染pcDNA3质粒（3.20%）和U937细胞组（2.10%）（F=133996.40,P〈0.01）;各代间差异无显著性（F=1.03,P〉0.05）。结论：用基因转染法成功地建立了稳定表达CD200蛋白的人单核细胞株U937-pcDNA3-CD200。     

Binding of multivalent CD147 phage induces apoptosis of U937 cells

CD147 is a broadly expressed cell-surface molecule and serves as a signaling receptor for extracellular cyclophilins. CD147 also appears to interact with immune cells, but its counter-receptor on these cells has not been clearly described. In the present report, we displayed multiple copies of the CD147 extracellular domain (CD147Ex) on VCSM13 phage to study the interaction of CD147 with its ligand. Recognition of phage containing fusion protein of CD147Ex and gpVIII (CD147Ex phage) by four different anti-CD147 mAbs indicated that at least parts of the CD147 are properly folded. Specific binding of CD147Ex phage to various cell types was demonstrated by flow cytometry. Morphological changes, however, were observed only in U937, a monocytic cell line, after 24 h incubation with multivalent CD147Ex phage. After 48 h, U937 cell propagation ceased. Staining with annexin V and the presence of cleaved caspase-3 indicated that many of the CD147Ex phage-treated cells had lost viability through apoptotic cell death. The above results suggest that CD147 induces apoptosis in U973 cells and that at least a portion of this cell death program involves a caspase-dependent pathway.     

Interactions of promonocytic U937 cells with proteins of the extracellular matrix.

Monocyte interaction with proteins of the extracellular matrix (ECM) is regulated by expression of specific cell-surface receptors. 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been shown to induce the promonocytic cell line U937 to a more differentiated monocyte-like state. In this study we have analysed the attachment of U937 cells to ECM proteins and the effects of treatment with TPA on this process. Non-induced U937 cells attach to fibronectin- and Matrigel-coated surfaces without TPA stimulation, but TPA further increases adherence to these substrates as measured by an enhanced binding and by the lower concentration of proteins needed in the substrate to achieve 50% of maximal cell adhesion. Attachment to type I collagen was seen only with activated U937 cells, whereas no measurable attachment to bovine serum albumin, vitronectin, and type IV collagen was detected. TPA-activated U937 cells showed a two-fold increase in the expression of the RGD-dependent integrin receptors alpha 3 and alpha 5, and a reduction in the expression of alpha 4, another fibronectin-specific receptor, whereas the common beta 1 chain was unchanged. Attachment of U937 cells to fibronectin was primarily mediated by the alpha 3 and alpha 5 integrins, as revealed by the ability of GRGDS peptides to inhibit attachment, whereas the CS-1 peptide, containing the alpha 4 binding site, was largely ineffective in blocking attachment.