Myeloid Cell Lines U937 Research Articles

Effects of exposure to alternating low-intensity, intermediate-frequency electric fields on the differentiation of human leukemic cell line U937.

Studying the bioeffects of electric fields have been the subject of ongoing research which led to promising therapeutic effect, particularly in cancer treatment. Here, we investigated the impact of low-intensity, intermediate-frequency alternating electric fields on the differentiation of human myeloid leukemia cell line U937. The results showed a near twofold increase in differentiation of U937 cells treated for 24 h by alternating 600 kHz, 150 V/m electric fields, in comparison to their control groups. This measure was evaluated by latex bead phagocytosis assay, nitro blue tetrazolium test, and cell cycle analysis which revealed a significant shift in the number of cells from G2 +M to G0 +G1 phases. The simulation result for the intracellular field intensity showed around 50% attenuation with respect to the applied external field for our setup which ruled out masking of the applied field by the internal electric noise of the cell. Based on previous studies we postulate a possible calcium-related effect for the observed differentiation, yet the exact underlying mechanism requires further investigation. Finally, our results may offer a potential therapeutic method for leukemia in the future.

A new antibody for the study of human CD157 expression and function

Abstract CD157 is an ectoenzyme that has both cyclic ADP-ribose hydrolase and ADP-ribosyl cyclase activities. In the human hematopoietic system, CD157 is prevalently expressed by cells of the myelomonocytic lineage, and interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signaling. CD157 is implicated in the pathophysiology of several neurological disorders. Although it lacks a cytoplasmatic domain, CD157 is able to transduce intracellular signals by establishing functional and structural crosstalk with β1 (CD29) and β2 (CD18) integrins. CD157 ligation causes integrin– dependent cytoskeletal remodeling and increase in F-actin content. Activation of downstream signaling pathways leads to cell polarization, with CD157 migrating prevalently to the rear of the cells whereas F-actin is localized at the opposite pole. However, the molecular and cellular functions of CD157 still remain poorly understood. Here, we describe the generation of a monoclonal antibody specific for human CD157 (clone W21007F) that can be used in flow cytometry, immunofluorescence and blocking of CD157 mediated adhesion of monocytes to fibronectin. Treatment of the pro-monocytic, myeloid leukemia cell line U937 with clone W21007F induces cell polarization and a marked increase in filamentous actin, with CD157 clustering at the uropod. The majority of F-Actin fibers are located at the opposite end of CD157, and CD157 co-localizes with integrin beta-1 in distinct membrane domains. The reference clone SY/11B5 and isotype control antibody do not show this effect. In conclusion, anti-human CD157 (clone W21007F) is a valuable tool for the study of CD157 function and expression in human cells.

Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 μM) close to that of Nelarabine (IC50 = 3 μM) and was evaluated as active.

Comparative Analysis of In Vitro Models to Study Antibody-Dependent Enhancement of Zika Virus Infection.

During the 2015-2016 outbreak of Zika virus (ZIKV) in the Americas, a previously unknown severe complication of ZIKV infection during pregnancy resulting in birth defects was reported. Since the ZIKV outbreak occurred in regions that were highly endemic for the related dengue virus (DENV), it was speculated that antibody-dependent enhancement (ADE) of a ZIKV infection, caused by the presence of cross-reactive DENV antibodies, could contribute to ZIKV disease severity. Emerging evidence indicates that, while in vitro models can show ADE of ZIKV infection, ADE does not seem to contribute to congenital ZIKV disease severity in humans. However, the role of ADE of ZIKV infection during pregnancy and in vertical ZIKV transmission is not well studied. In this study, we hypothesized that pregnancy may affect the ability of myeloid cells to become infected with ZIKV, potentially through ADE. We first systematically assessed which cell lines and primary cells can be used to study ZIKV ADE in vitro, and we compared the difference in outcomes of (ADE) infection experiments between these cells. Subsequently, we tested the hypothesis that pregnancy may affect the ability of myeloid cells to become infected through ADE, by performing ZIKV ADE assays with primary cells isolated from blood of pregnant women from different trimesters and from age-matched non-pregnant women. We found that ADE of ZIKV infection can be induced in myeloid cell lines U937, THP-1, and K562 as well as in monocyte-derived macrophages from healthy donors. There was no difference in permissiveness for ZIKV infection or ADE potential of ZIKV infection in primary cells of pregnant women compared to non-pregnant women. In conclusion, no increased permissiveness for ZIKV infection and ADE of ZIKV infection was found using in vitro models of primary myeloid cells from pregnant women compared to age-matched non-pregnant women.

Pancreatic Ductal Adenocarcinoma (PDAC) circulating tumor cells influence myeloid cell differentiation to support their survival and immunoresistance in portal vein circulation.

The portal venous circulation provides a conduit for pancreatic ductal adenocarcinoma (PDAC) tumor cells to the liver parenchyma sinusoids, a frequent site of metastasis. Turbulent flow in the portal circulation promotes retention of PDAC shed circulating tumor cells (CTC) and myeloid-derived immunosuppressor cells (MDSC). Excessive colony stimulating factor-1 receptor (CSF1R) signaling can induce myeloid differentiation to MDSC and transformation of MDSC to myeloid-derived fibroblasts (M-FB). Interactions between PDAC CTC and M-FB in the portal blood promotes the formation of immunoresistant clusters that enhance CTC proliferation, migration, and survival. Analysis of portal and peripheral blood samples collected intraoperatively from 30 PDAC patients undergoing pancreatico-duodenectomy showed that PDAC patient plasma contained high levels of macrophage colony stimulating factor (M-CSF/CSF1), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interleukin-8 (IL-8), and interleukin-34 (IL-34) compared to healthy control levels. Moreover, the level of M-CSF in portal blood was significantly higher than that detected in the peripheral blood of PDAC patients. PDAC CTC aseptically isolated by fluorescence activated cell sorting (FACS) out of freshly collected patient portal blood mononuclear cells (PortalBMC) had elevated RNA expression of IL34 (IL-34 gene) and CSF1 (M-CSF/CSF1 gene) which both signal through CSF1R. PDAC CTC also had high levels of RNA expression for CXCL8, the gene encoding chemokine interleukin-8 (IL-8) which can attract myeloid cells through their CXCR2 receptors. FACS-isolated portal PDAC CTC and M-FB co-cultured ex vivo had increased CTC proliferation, motility, and cluster formation compared to CTC cultured alone. CSF1R and CXCR2 cell surface expression were found on PDAC portal blood CTC and M-FB, suggesting that both cell types may respond to M-CSF, IL-34, and IL-8-mediated signaling. Portal PDAC CTC displayed enhanced RNA expression of CSF1 and IL34, while CTC+M-FB+ clusters formed in vivo had increased RNA expression of CSF2 and IL34. Portal M-FB were found to have high CSF1R RNA expression. CTC isolated from ex vivo 7-day cultures of PDAC patient portal blood mononuclear cells (PortalBMC) expressed elevated CSF1, IL34, and IL8 RNA, and CSF1 expression was elevated in M-FB. Treatment with rabbit anti-CSF1R antibodies decreased CTC proliferation. Treatment of PortalBMC cultures with humanized anti-CSF1R, humanized anti-IL-8, or anti-IL-34 antibodies disrupted CTC cluster formation and increased CTC apoptosis. U937 myeloid precursor cell line cultures treated with conditioned media from PortalBMC ex vivo cultures without treatment or treated with anti-IL-8 and/or anti-CSF1R did not prevent myeloid differentiation in the myeloid precursor cell line U937 to macrophage, dendritic cell, MDSC, and M-FB phenotypes; whereas, U937 cultures treated with conditioned media from PortalBMC ex vivo cultures exposed to anti-IL-34 were significantly inhibited in their myeloid differentiation to all but the M-FB phenotype. PDAC patient T cells that were found phenotypically anergic (CD3+CD25+CTLA4+PD1L1+) in PortalBMC could be re-activated (CD3+CD25+CTLA4-PD1L1-), and displayed increased interferon gamma (IFNγ) production when PortalBMC ex vivo cultures were treated with anti-CSF1R, anti-IL-8, and anti-IL-34 antibodies alone or in combination. These findings suggest that PDAC CTC have the potential to influence myeloid differentiation and/or antigen presenting cell activation in the PDAC portal blood microenvironment, and that disruption of CTC/M-FB interactions may be potential targets for reversing the immunosuppression supporting CTC survival in the portal blood.

Effects of SIDT2 on the miR-25/NOX4/HuR axis and SIRT3 mRNA stability lead to ROS-mediated TNF-α expression in hydroquinone-treated leukemia cells.

Our previous studies indicated that the benzene metabolite hydroquinone (HQ) evokes the ROS/p38 MAPK/protein phosphatase 2A/tristetraprolin axis, leading to increased TNF-α expression in human acute myeloid leukemia cell lines U937 and HL-60. In this study, we aimed to identify the upstream pathway involved in ROS-mediated TNF-α expression. HQ treatment increased SIDT2 expression, which subsequently decreased miR-25 and SIRT3 expression in U937 cells. Notably, miR-25 downregulation promoted SIDT2 expression in HQ-treated U937 cells. SIDT2 induced lysosomal degradation of SIRT3 mRNA, but inhibited miR-25 expression through a lysosome-independent pathway. MiR-25 inhibition reduced NOX4 mRNA turnover, resulting in increased NOX4 protein levels. NOX4 induces mitochondrial ROS production and HuR downregulation. Restoration of HuR expression increased SIRT3 expression, suggesting that NOX4-mediated HuR downregulation promotes SIDT2-mediated degradation of SIRT3 mRNA. Inhibition of NOX4 or SIRT3 overexpression abolished HQ-induced ROS production, thereby abolishing TNF-α upregulation. Overall, these results indicate that SIDT2 regulates the miR-25/NOX4/HuR axis and SIRT3 mRNA destabilization, leading to ROS-mediated TNF-α upregulation in HQ-treated U937 cells. HQ-induced increase in TNF-α expression in HL-60 cells was also mediated through a similar pathway.

Daemonorops draco Blume Induces Apoptosis Against Acute Myeloid Leukemia Cells via Regulation of the miR-216b/c-Jun.

Daemonorops draco Blume (DD), also called dragon’s blood, has been used as a traditional Korean medicine, especially for relieving pain caused by wound infection. Recently, it has been described that DD has antibacterial and analgesic effects. In this study, the underlying anticancer effect of DD associated with apoptosis was investigated in acute myeloid leukemia cell lines U937 and THP-1. DD exhibited cytotoxic effects and induced apoptosis in U937 and THP-1 cells. Moreover, DD treatment significantly reduced mitochondrial membrane potential (ΔΨ). The protein expression of cleaved poly(ADP-ribose) polymerase, cleaved caspase-3, p-H2A.X, CCAAT/enhancer-binding protein (CHOP), and activating transcription factor 4 was upregulated by DD treatment. Consistently, DD-treated cells had increased reactive oxygen species (ROS) level in a concentration-dependent manner via miR-216b activation in association with c-Jun inhibition. N-acetyl-L-cysteine pretreatment reversed the cytotoxic effect of DD treatment as well as prevented ROS accumulation. Collectively, the results of this study suggest that the anticancer effect of DD in AML was mediated by CHOP-dependent apoptosis along with ROS accumulation and included upregulation of miR-216b followed by a decrease in c-Jun.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

BackgroundThe U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements.ResultsSNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances. Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified. There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss.ConclusionsCentromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of cell types.

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.

Cytidine deaminase enables Toll-like receptor 8 activation by cytidine or its analogs.

Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.

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.

Inhibitory effect of JQ1 on proliferation of acute myeloid leukemia U937 cell line and its effect on the signal transduction pathway of STAT-5

Objective To study the suppressive mechanism of the bromo structural domain inhibitor JQ1 on the acute myeloid leukemia cell line U937 and its regulation of the STAT-5 signaling pathway. Methods U937 cells in the logarithmic growth phase were tested. Each experimental group was fed with 0.2, 1.0 and 4.0 μmol/L of JQ1, and a blank control group without JQ1 was set up. U937 cells were cultured for 48 h, and the cell proliferation inhibitory rate was measured by MTT assay. Flow cytometry (FCM) was used to detect the apoptotic rate. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of focal adhesion kinase (FAK) and P21 activated kinase (PAK1). The expression of STAT-5 protein was analyzed by Western blot. Results Different concentration of JQ1 could inhibit the proliferation of U937 cells in a time and dose-dependent manner, and different concentration of JQ1 could induce the apoptosis of U937 cells in a dose-dependent manner. Treatment of U937 cells with different concentrations of JQ1 (0.2, 1.0 and 4.0 μmol/L) reduced the expression of FKA mRNA and PAK1 mRNA after 48 h, the expression of FAK mRNA was 0.417±0.066, 0.140±0.026, and 0.027±0.006 (F = 454.651, P = 0.000), and the expression of PAK1 mRNA was 0.533±0.045, 0.080±0.010, and 0.010±0.001 (F = 2 434.610, P = 0.000), and STAT-5 protein expression was also significantly inhibited. The expression of STAT-5 protein in U937 cells after treated with different concentrations of JQ1 (0.2, 1.0 and 4.0 μmol/L) for 48 h were 0.71±0.19, 0.62±0.16, 0.53±0.14, and 1.00±0.21 in the control group (F = 263.135, P = 0.000). Conclusion JQ1 can effectively inhibit the proliferation of the acute myeloid leukemia cell line U937 cells and induce their apoptosis, and the possible mechanism of action is regulated via STAT-5 signaling pathway. Key words: Leukemia, myeloid, acute; Cell line, U937; JQ1; STAT-5

High expression of lnc-CRNDE presents as a biomarker for acute myeloid leukemia and promotes the malignant progression in acute myeloid leukemia cell line U937.

To detect the expression of long non-coding RNA-CRNDE in patients with acute myeloid leukemia and its effect on proliferation and apoptosis in acute myeloid leukemia cell line U937. 81 cases of newly diagnosed acute myeloid leukemia (AML) were enrolled, and 35 non-malignant hematological patients were selected as controls. Quantitative RT-PCR (qRT-PCR) was performed to detect the expression of lncRNA-CRNDE in the bone marrow specimens of the subjects, and the difference between the two groups was also compared. The correlation between the expression of lncRNA-CRNDE and the sex, age, classification and total survival of clinical patients was analyzed according to the clinical data. U937 cells and monocytes isolated from normal people were cultured, and the expression of lncRNA-CRNDE in acute myeloid leukemia cell line U937 and normal monocytes was compared. SiRNA-CRNDE and pcDNA-CRNDE were transfected into U937 cells, and cell counting kit-8 (CCK-8) assay was performed to detect proliferation of U937 cells, Annexin V/PI flow cytometry was carried out to detect cell apoptosis. Cell cycle was measured by flow cytometry. The expression of lncRNA-CRNDE in patients with AML and U937 cells was significantly higher than that in non-malignant hematological controls. Results of clinical data showed that the expression of lncRNA-CRNDE was associated with the classification and total survival of myeloid leukemia in clinical patients. After transfection of siRNA-CRNDE, the proliferation and cloning ability of U937 cells decreased, while the apoptosis increased (p < 0.01) and cells were arrested in G0-G1 phase. Meanwhile, after transfection of pcDNA-CRNDE, the proliferation ability of U937 cells increased significantly, which indicated that the expression of lncRNA-CRNDE might play an essential role in promoting the proliferation of U937 cells. LncRNA-CRNDE is highly expressed in the bone marrow tissues of AML patients, and the expression level is negatively correlated with the total survival of those clinical patients. Meanwhile, the expression is higher in FAB type M4 and M5 than that in M1, M2 and M3. LncRNA-CRNDE promotes the proliferation and cell cycle of U937 cells, and inhibits cell apoptosis, which is expected to become a molecular marker for predicting and treating AML.

Adhesion of human monocytes to oxygen- and nitrogen- containing plasma polymers: Effect of surface chemistry and protein adsorption

The interactions between monocytes and biomaterials can potentially be modulated by controlling the chemical and structural surface properties of biomaterials. The objective of this study was to determine the effect of plasma-deposited functional organic coatings on monocyte adhesion and differentiation into macrophages. Organic coatings with varying oxygen and nitrogen concentration were prepared by low-pressure plasma co-polymerization of binary gas mixtures combining a hydrocarbon (butadiene/ethylene) and a heteroatom-containing gas (carbon dioxide/ammonia) to deposit either oxygen or nitrogen-containing coatings. The deposition parameters controlled the composition of the coatings and, consequently, the surface charge (between 26 mV and −28 mV) and wettability. The adhesion of myeloid leukemia cell lines U937 and NB4 as well as human monocytes to plasma polymerized coatings, was tested using cell culture medium with and without fetal bovine serum. The results showed that the concentration of [-NH2] and [-COOH] on the surface of the plasma polymers, controls the adhesion of U937 and NB4 cell lines to the coatings. Thus, above a certain composition threshold, i.e. [-NH2]=2.6–3.0% and [-COOH]=1.2–1.57 nmol/cm2, the surface facilitates adhesion of both cell lines, irrespective of the culture medium used. Based on qualitative observations the number of monocytes adhering to the coatings was proportional to the concentration of functional groups at the surface of the coatings. The surface plasmon resonance results, in line with cell culture experiments, indicated that the presence of albumin on the surfaces with [-NH2] and [-COOH] above the determined critical concentration may be an indicator of monocyte adhesion to these plasma polymers.

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.

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

目的探讨全反式维甲酸（ATRA）对NPM1突变的白血病细胞系U937细胞的作用及其机制。方法转染NPM1突变型（A型）质粒至U937细胞系构建稳定克隆A1和A2，利用Western blot法和荧光共聚焦技术鉴定细胞。MTT法检测细胞增殖活性，流式细胞术检测细胞周期和细胞凋亡，显微镜下计数检测集落形成能力，Western blot法检测细胞增殖相关信号通路蛋白表达。结果①NPM1突变型U937细胞A1和A2经ATRA处理后，细胞增殖活性分别为对照组（未经ATRA处理组）水平的52.6%和35.8%，差异均有统计学意义（t=3.649，P=0.010；t=4.906，P=0.002）。②突变型细胞A1和A2的G0/G1期比例经ATRA处理后较对照组分别增加20.1%和35.8%，差异均有统计学意义（t=4.544，P=0.010；t=15.850，P<0.001）。③U937细胞经ATRA处理后集落生长受抑制，其中空载体组集落数目下降了32.7%，野生型组下降了57.9%，突变型组（A1）下降了90.9%，差异均有统计学意义（t=8.507，P=0.010；t=22.090，P<0.001；t=90.200，P<0.001）。④突变型U937细胞经ATRA处理后p-ERK水平明显降低。⑤突变型U937细胞接受化疗药物联合ATRA处理后细胞凋亡率明显高于化疗药物单药处理组，在化疗药物处理后添加ATRA能导致更多的细胞凋亡。结论ATRA能够抑制NPM1突变的U937细胞增殖和克隆形成，将细胞阻滞于G0/G1期，显著降低其ERK磷酸化水平。在NPM1突变的U937细胞中，当ATRA在化疗药物处理后添加能够与化疗药物发挥更强的协同杀伤效果。

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.

Identification of the Oncogene SKI As a New Target Gene of the Myeloid Transcription Factor c-MYB in AML

Background:Acute myeloid leukemia (AML) accounts for 80% of acute leukemias and arises through clonal expansion and arrest of differentiation of hematopoietic progenitor cells in the bone marrow. Depending on the AML subtype and age of the patient, AML patients have a 5-year survival rate of about 25%. AML is a genetically heterogenous disease, where chromosomal aberrations, point mutations in critical oncogenes as well as aberrant expression of key regulatory factors of hematopoiesis collude during transformation. c-MYB is an important hematopoietic transcription factor involved in proliferation and differentiation of progenitor cells of the myeloid and lymphoid lineages. It was first described as a viral oncogene of avian leukemia viruses and is upregulated or mutated in many leukemic subtypes as well as solid tumors. c-MYB interacts with other transcription factors or co-factors, which are essential for its transcriptional activity. In this regard, c-MYB transactivation ability is inhibited by a histone deacetylases recruiting corepressor complex containing SKI, TIF1BETA, NCOR and mSIN3A. c-SKI is a proto-oncogene and an inhibitor of TGFβ signalling. However, it acts not only as a transcriptional co-repressor but also as a transcriptional co-activator. Like c-MYB, c-SKI is upregulated in different solid tumors and leukemias. Though SKI activity is well described, transcriptional regulation of the SKI gene itself still remains unknown. Here, we deliver insight into the transcriptional regulation of the human SKI gene via the transcription factor c-MYB.Methods:In silico analyses were performed to identify potential MYB binding sites in the SKI regulatory region. Chromatin immunoprecipitation (ChIP) assays were used to validate the interaction between MYB and the potential SKI regulatory regions. Reporter gene assays were engaged to analyze MYB regulatory potential regarding SKI expression. RNAi experiments were performed to further examine transcriptional regulation of SKI by MYB. Since MYB is known to be downregulated by the histone deacetylase inhibitor (HDACi) valproic acid (VPA), MYB and SKI protein levels were analyzed in AML cell lines treated with VPA via Western Blot. Correlated protein levels of MYB and SKI were examined in the myeloid leukemia cell lines HL60, U937, THP1, NB4 and K562 as well as in primary cells of AML patients (n=27). Correlation of MYB and SKI transcript levels were performed in three different data sets of primary AML samples. The first cohort (n=7) was analyzed via RT-qPCR. Cohort 2 consists of cDNA microarray data of AML patients (n=17). Furthermore, principal component analysis (PCA) of the gene expression profile of MYB and SKI of AML patients (n=542) were performed with the leukemia gene atlas (LGA).Results:In silico analyses revealed four putative MYB binding sites (MBS1-4) in the SKI regulatory region. Direct binding of MYB to the regulatory sites MBS2-4 of the SKI gene could be confirmed via ChIP experiments. Dual luciferase reporter gene assays comprising c-MYB binding sites present in the SKI gene locus further show c-MYB-dependent transcriptional activation of the reporter. RNAi experiments depleting c-MYB in leukemic cell lines resulted in the decrease of SKI protein levels and thereby reveal that c-MYB is essential for the induction of SKI gene expression. Accordingly, treatment of the AML cell lines with the HDACi VPA led to a decrease of MYB and consequently SKI protein levels. Consistently, we observed a positive correlation of MYB and SKI protein expression in leukemic cell lines and in samples of AML patients. Moreover, a highly positive correlation of MYB and SKI transcript levels could be observed in three different cohorts of AML patients, further confirming regulation of SKI expression by the transcription factor MYB.Conclusion:Our findings provide new insights in the transcriptional regulation of the proto-oncogene c-SKI by the oncogenic transcription factor c-MYB during leukemogenesis. c-MYB and c-SKI expression and functions are highly positively correlated in human AML suggesting that c-SKI is a mediator of c-MYB oncogenic potential. So far, various therapeutic approaches targeting MYB failed to be transferred to patients. In this regard, c-MYB and c-SKI represent promising marker and target proteins for novel HDACi-based therapeutic approaches in AML. DisclosuresNo relevant conflicts of interest to declare.

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.

A Tissue-Specific Transcriptional Element for C/ebpa Is Required for HSC Maintenance and Neutrophil Development

Background: C/EBPa is a lineage determining transcription factor, critical for terminal cell differentiation in different tissues including the bone marrow (BM), lung, liver, and adipocytes. Adequate CEBPA levels are needed to maintain the haematopoietic stem cell (HSC) pool and to promote neutrophilic differentiation. This knowledge points towards the importance of CEBPA dosage at different stages of differentiation.Aim: To investigate how CEBPA dosage is regulated at the transcriptional level by specific enhancer(s) and to study their role in haematopoiesis.Results: Chromatin immunoprecipitation for the active mark H3K27ac followed by deep sequencing (ChIP-seq), revealed eight putative regulatory elements within the CEBPA locus. One enhancer showed marked H3K27ac enrichment at +42Kb downstream of CEBPA particularly in CD34+ haematopoietic stem cells (HSCs), implying a role for CEBPA regulation at earlier stages of haematopoiesis. ChIP-Seq experiments revealed binding of RUNX1, ERG, PU.1, FLI1 and GATA2 to this +42Kb enhancer in CD34+ BM cells. Moreover, myeloid cell lines MOLM1 and U937 also showed H3K27ac enrichment at this enhancer, indicating that its activity is maintained upon myeloid differentiation. In contrast, active histone marks were completely devoid at this element in CEBPA expressing lung (A549) and liver (HepG2) cell lines, indicating hematopoietic specificity of this enhancer. Chromatin looping between the +42Kb enhancer and the CEBPA promoter was demonstrated by 4C-Seq, highlighting the specificity of this enhancer in CEBPA regulation. Furthermore, using CRISPR/Cas9 technology we deleted the +42Kb enhancer in the myeloid cell line THP-1 and showed 70% reduction of CEBPA mRNA levels.The +42Kb enhancer is conserved and located at +37Kb near Cebpa in mice. Using CRISPR/Cas9 system, we deleted the +37Kb enhancer in one-cell stage zygotes. Heterozygous mice were inter-crossed and F1 generation mice were born at normal mendelian ratios. Morphological and flow-cytometric analysis of peripheral blood and BM at 8-10 weeks of age, showed 10-20 fold decrease in (MAC1+/GR1+) neutrophil counts in homozygous +37Kb-/- mice, compared to +37Kbwt/wt and +37Kb-/wt controls. In line with the block of neutrophil development, flow-cytometric analysis revealed an increase (2 fold) in CD34+CD16/32low common myeloid progenitors and a decrease (2 fold) in the CD34+CD16/32high granulocyte/ monocyte progenitorsof +37Kb-/- mice. From these findings we hypothesized that the absence of the +37Kb enhancer disturbs the myeloid differentiation program via reduced Cebpa levels. In fact, Cebpa expression levels were reduced by 60-80% in bone marrow of +37Kb-/- mice, but unchanged in other Cebpa -expressing tissues such as lung and liver, indicating tissue specificity of this enhancer. Diminished Cebpa expression levels were accompanied by decreasedexpression of Cebpa target genes, including Csf3r. In line with this, bone marrow progenitor cells from +37Kb-/- mice were completely unresponsive to Csf3 in a colony forming assay.Given the importance of Cebpa in HSCs maintenance, we investigated the HSC population and found that long-term HSCs (CD48- CD150+) and short-term HSCs (CD48- CD150-) were depleted in the bone marrow of the +37Kb-/- mice. HSC depletion was accompanied by an increase in the CD48+/CD150- multipotent progenitors (MPPs). The +37Kb-/- MPPs, unlike controls, were able to serially replate in vitro under IL-3, GM-CSF, IL-6, SCF growth factor conditions with minimal evidence of differentiation, suggesting a leukemogenic potential. Reintroduction of Cebpa cDNA into +37Kb-/- MPPs fully recovered neutrophil development.Conclusion: We conclude that the +37Kb enhancer is tissue-specific and plays a central role in haematopoiesis regulating Cebpa dosage. Our study reveals that the bone marrow maintains its integrity through the activity of the +37Kb enhancer, which (1) prevents HSC exhaustion and (2) preserves neutrophilic development. The in vitro replating capacity of MPPs isolated from +37Kb-/- animals suggests that aberrant control of this enhancer may be a primary leukaemogenic event. In line with this, it is important to note that the conserved enhancer in humans (+42 KB) is a frequent target for oncogenic transcription factors such as AML1-ETO or EVI1, two oncogenes which are found in two distinct subtypes of AMLs with very low C/EBPa expression. DisclosuresNo relevant conflicts of interest to declare.