The U937 cell culture is a pro-monocytic, human histiocytic lymphoma cell line. These monocytes can differentiate into either macrophages or dendritic cells (antigen-presenting cells) depending on the initiators. The U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) change their morphology into macrophage-like cells creating pseudopodia and adhering generously. Macrophages are known to produce reactive oxygen species (ROS) mostly during phagocytosis of foreign particles, an important non-specific immune response. Recently, we have focused on the role of hydroxyl radical (HO∙) and provide evidence on its importance for differentiation in U937 cells. Based on electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM), formation of HO∙ was confirmed within the cells undergoing differentiation and/or apoptosis during the PMA treatment. This study aims to increase our knowledge of ROS metabolism in model cell lines used in human research.

The objective of this study was to investigate the use of chloroquine (CLQ) as an antiviral agent against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of U937 cells infected with dengue virus type 2 (DENV-2). Viral replication was assessed by quantification of virus produced through detection of copy numbers of DENV-2 RNA, plaque assay and indirect immunofluorescence. qRT-PCR and plaque assays were used to quantify the DENV-2 load in infected U937 cells after CLQ treatment. It was found that a dose of 50 μg/mL of CLQ was not toxic to the cells and resulted in significantly less virus production in infected U937 cells than occurred in untreated cells. In the present work, CLQ was effective against DENV-2 replication in U937 cells, and also caused a statistically significant reduction in expression of proinflammatory cytokines. The present study indicates that CLQ may be used to reduce viral yield in U937 cells.

Nigella sativa is from botanical Ranunculaceae family and commonly known as black seed. Apoptotic effect of N. sativa and its apoptotic signaling pathways on U937 lymphoma cells are unknown.

Farnesyltransferase inhibitor tipifarnib (R115777) has been used for treatment of hematological malignancies; however, its observed anticancer effect was limited. This prompted us to search for inhibitors that would show synergic, proapoptotic effect when combined with R115777. We decided to study LY294002, which inhibits PI-3 kinase, and tanespimycin (17AAG), which inhibits Hsp90--a chaperone for a number of proteins, including Akt kinase.

Acute myeloblastic leukemia (AML) is the most common type of acute leukemia in adults. Despite numerous treatment strategies including chemotherapy and radiotherapy, a large number of patients do not respond to treatment and experience relapse. The main problem of these patients is the development of resistance to anti-cancer drugs. Therefore, any endeavor to reduce drug resistance in these patients is of high priority. In general, several mechanisms such as changes in drug metabolic pathways, drug inactivation, drug target alterations and reduced drug accumulation in the cells contribute to drug resistance of cancer cells. In this context, evidence suggests that exosomes could reduce drug resistance by removing drugs from their parent cells. In the present study, we aimed to investigate the effects of exosome release inhibition on the resistance of U937 cells to PEGylated liposomal doxorubicin (PLD).

Phorbol ester (TPA) treatment of human U937 myeloid leukemia cells is associated with increasing adherence and monocyte-like maturation whereby the role of β2 integrin-mediated attachment for subsequent growth properties and the differentiation program remains unclear. Here, stably-transfected U937 cells with a pMTH1 vector containing the β2 integrin gene of CD11b in antisense orientation (asCD11b-U937) demonstrated a significantly reduced proliferative capacity in contrast to control vector transfectants (pMTH1-U937) or wild-type U937 cells. Phorbol ester exposure induced adherence and growth arrest in more than 90% of pMTH1-U937 and wild-type U937 cells after 72 h. In contrast, TPA-treated asCD11b-U937 failed to attach and the proliferation continued in more than 30% of the cells. Moreover, increased apoptosis appeared in asCD11b-U937 after TPA induction in contrast to pMTH1-U937 cells. In addition, non-specific inhibition of adherence on an agarose surface demonstrated internucleosomal DNA fragmentation in both, pMTH1-U937 and asCD11b-U937 after TPA treatment indicating a functional relationship between abolished adherence, regulation of proliferation and induction of apoptosis. Western blot analysis revealed differences in the expression levels and altered phosphorylation patterns of Pyk-2, pp60src and p42/p44 MAP kinases between pMTH1-U937 and asCD11b-U937 following TPA exposure which was also substantiated by Pyk-2 immunoprecipitation. These findings suggested that induced adherence predominantly mediated by a functional CD11b/CD18 integrin in U937 cells is involved in the activation of downstream signaling kinases and contributes to cell cycle regulation and apoptosis during monocytic maturation.

Additional sex combs-like 1 (ASXL1) is a well‑known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis.

Annexin 1 (An 1), a phospholipid and calcium binding protein, is strongly expressed in differentiated U 937 cells. In attempting to correlate the expression of An 1 with phospholipase A2 (PLA2) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA2 activity was measured by Flow cytometry analysis utilizing the bis-Bodipy-C11-PC fluorescent probe. U 937 cells stably transfected with the sense or antisense vectors were differentiated for 24 h with phorbol 12-myristate 13-acetate (PMA, 6 ng ml(-1)). Both in undifferentiated and differentiated cells, the Antisense clone (36.4 AS) showed consistently higher PLA2 activity than the control Sense clone (15 S). Since the fluorescent probe measures the total PLA2 activity, we used two different stimuli, PMA: (100 ng ml(-1)) or lipopolysaccharide (LPS, 10 ng ml(-1)), and two different inhibitors, to discriminate the PLA2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA2, and SB 203347 specific for the secretory PLA2). In the Antisense clone the inhibitory effect of AACOCF was stronger [68%, P<0.025] than in the Sense, which may reflect the lower endogenous level of An 1 present in the cells. On the contrary, the inhibitory effect of SB 203347 [60% of inhibition] was identical in both clones. Since cPLA2 activity is correlated with its phosphorylation, Western and shift blot analysis were performed. They did not show any significative difference between the phosphorylated and non phosphorylated form of the enzyme in both the differentiated or not, Sense and Antisense clones. Furthermore the tyrosine phosphorylation analysis of An 1 showed that less than 10% of An 1 was phosphorylated irrespective of PMA presence or absence. From the pattern of inhibition observed, we propose that the endogenous unphosphorylated form of An 1 may act intracellularly to block the activity of a cytosolic PLA2.

To investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response.

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.

Deficiency in vitamin A has been associated with adverse clinical outcomes in drug users with HIV-1 infection. Retinoids have been demonstrated to suppress proinflammatory cytokine production by immune cells in vitro. These effects are induced by ligand-mediated activation of the retinoid receptors--retinoic acid receptor (RAR) and retinoid X receptor (RXR). In these studies, the effects of all-trans-retinoid acid (ATRA, a RAR agonist), 9-cis-retinoic acid (9cis RA; RAR and RXR agonist), LG101305 (RXR agonist), LG100815 (RAR antagonist) and LG101208 (RXR antagonist) on TNF-alpha production by phytohemagglutanin-activated U937 cells and the modulation of these effects by morphine were examined. TNF-alpha production was suppressed in all cultures exposed to retinoid agonist and antagonist agents. For cells exposed to RXR agonists or RAR antagonist, incubation with morphine resulted in the reversal of TNF-alpha suppression and this effect was inhibited by naloxone. These data suggest that interactions between RXR and morphine are involved in the immune effects of retinoids on TNF-alpha production by activated U937 cells. Such information may be important for understanding interactions between drugs of abuse and immune function in individuals with chronic proinflammatory states such as HIV-1 infection.

Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer properties of these drugs. However, cardiac glycosides have also been shown to stimulate cell growth in several cell types. In the present investigation we analyzed the pro-death and pro-survival properties of ouabain in the human lymphoma derived cell line U937.

Mesenchymal stem cells (MSCs) are key components of the hematopoietic stem cells (HSCs) niche. They control the process of hematopoiesis by secreting regulatory cytokines, growth factors and expression of important cell adhesion molecules for cell-tocell interactions. In this research, we have investigated the effect of bone marrow derived MSCs on monocytic differentiation of U937 cells line.

Plasma therapy offers an exciting and novel way of cancer treatment. Specifically, it is shown that Jurkat death rates are closely governed by the plasma treatment time. However, apart from time, alterations to different parameters of treatment process may yield better results. Here, Dielectric barrier discharge (DBD) reactors excited by a nanosecond-pulse energy source are used to investigate cell viability for longer exposure times as well as the effects of polarity of reactor on treatment. Plasma discharge regimes are discussed and assessed using imaging and thermal imaging methods. We found that by changing the polarity of reactor i.e. changing the direction of plasma discharge, the plasma discharge regime changes influencing directly the effectiveness of treatment. Our results showed that ns-DBD- reactor could induce both apoptosis and necrosis of human Jurkat and U937 cells, and this cytotoxic effect of plasma was not completely antagonized by N-acetyl cysteine. It indicates that plasma could induce ROS-independent cell death. Gene expression analyses revealed that p53, BAD, BID and caspase 9 may play vital roles in plasma caused cell death. In addition, our findings demonstrate how different parameters can influence the effectiveness of our reactors. Our assay reveals the custom ability nature of plasma reactors for hematologic cancer therapy and our findings can be used for further development of such reactors using multi-objective optimisation techniques.

The gravitational force has been constant throughout Earth's evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth's gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.

Monocytes and macrophages play essential role in innate immunity. Understanding the underlying mechanism of macrophage differentiation and the identification of regulatory mechanisms will help to find new strategies to prevent their harmful effects in chronic inflammatory diseases and sepsis.

α-Mangostin (α-MG) is a main constituent of the fruit hull of the mangosteen. Previous studies have shown that α-MG has pharmacological activities such as antioxidant, antitumor, anti-inflammatory, antiallergic, antibacterial, antifungal and antiviral effects. This study aims to investigate the anti-inflammatory molecular action of α-MG on gene expression profiles.

The role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated.

Quercetin, a natural flavonoid, inhibits the growth of leukemia cells and induces apoptosis. Heat shock protein 27 (HSP27) has been reported to promote the development of leukemia by protecting tumor cells from apoptosis through various mechanisms. The present study investigated the effects of small hairpin (sh)RNA-mediated HSP27 knockdown on the anti‑cancer effects of quercetin in U937 human leukemia cells. Cells were transfected with recombinant lentiviral vector pCMV‑G‑NR‑U6‑shHSP27 (shHSP27), which expressed shRNA specifically targeting the HSP27 gene, alone or in combination with quercetin. The results showed that shHSP27 and quercetin synergistically inhibited U937 cell proliferation and induced apoptosis by decreasing the Bcl2-to-Bax ratio. Furthermore, this combined treatment significantly suppressed the infiltration of tumor cells and the expression of angiogenesis‑associated proteins HIF1α and VEGF. Compared with shHSP27 or quercetin alone, shHSP27 plus quercetin markedly decreased the protein expression of cyclinD1 and thus blocked the cell cycle at G1 phase. The Notch/AKT/mTOR signaling pathway is important in tumor aggressiveness; quercetin plus shHSP27 significantly decreased Notch 1 expression and the phosphorylation levels of the downstream signaling proteins AKT and mTOR. The inhibitory effects of quercetin plus shHSP27 on this pathway may thus have been responsible for the cell cycle arrest, inhibition of proliferations and infiltration as well as enhancement of apoptosis. Therefore, these findings collectively suggested that suppression of HSP27 expression amplified the anti‑cancer effects of quercetin in U937 human leukemia cells, and that quercetin in combination with shHSP27 represents a promising therapeutic strategy for human leukemia.

In mammals, methylation of DNA within regulatory sites and histone deacetylase recruitment in transcriptional repressing domains are involved in the loss of the expression of retroviral DNA or repeat arrays transferred in cells for therapeutic purposes. Various investigation results suggest that methylation/deacetylation events are modulated by extracellular and cytoplasmic signal transduction pathways closely involved in regulating cell differentiation. To analyse gene silencing mechanisms and assess if potential pharmacological treatment affects gene silencing kinetics we transduced U937 myelomonocytic cells with a bicistronic retroviral construct carrying the herpes simplex virus thymidine kinase (HSV-TK) and beta-galactosidase (Lac-Z) genes. This vector can be employed in vivo and in vitro to render transduced cell populations susceptible to ganciclovir (GCV). We verified the effect of the histone deacetylase inhibitor Trichostatin A (TSA) alone or combined with 5'-azacytidine (5'aza-C) on transcription downmodulation. Our results indicate that in our in vitro model TSA is able to reactivate transgene expression, more efficiently and with quicker kinetics (12-24h) than 5'aza-C (36-48 h). The effect is dose dependent (between 1 and 50 nM), with no relevant toxicity. Treatment with both drugs is synergistic in gene reactivation in terms of extension and persistence, with low toxicity and no relevant differentiating effects. The cells in which transgene expression has been reactivated undergo progressive silencing, but once weekly drug treatment can maintain high transgene expression levels for more than 90 days with no evidence of selection. The results obtained by treating U937 transduced clones with TSA and/or 5'aza-C together with IL-3, G-CSF or GM-CSF cytokines suggest that transduced U937 differentiation levels do not affect basal expression, but render these cells more responsive to reactivation by TSA or TSA plus 5'aza-C, but not to 5'aza-C alone. In conclusion, the results suggest that in vitro inhibition of histone deacetylase by TSA can interfere with gene silencing mechanisms affecting 5' Moloney murine leukaemia virus long terminal repeat (MoMuLV-LTR) driven transgene expression thus providing the rationale for TSA and/or 5'aza-C administration in animal models for the translation on gene therapy applications.