To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.
Gene Library ; Humans ; RNA, Messenger ; analysis ; U937 Cells ; metabolism

Bcl-2 protects tumor cells from the apoptotic effects of various anti-neoplastic agents. Increased expression of Bcl-2 has been associated with a poor response to chemotherapy in various malignancies, including leukemia. Hence, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. This study was undertaken to examine whether the anticancer drug, cisplatin and the synthetic chenodeoxycholic acid (CDCA) derivative, HS-1200 show anti-tumor activity in U937 and U937/Bcl-2 cells. Viability assays revealed that HS-1200 overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells. Various apoptosis assessment assays further demonstrated that HS-1200 overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells by inducing apoptosis. In addition HS-1200, but not cisplatin, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2 over-expressing human leukemic cells (U937/Bcl-2 cells). Notably, we observed that the HS-1200-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with a suppression of the anti-apoptotic effects of Bcl-2 in human leukemic cells over-expressing this protein (U937/Bcl-2 cells). Furthermore, HS-1200 was found to induce the association between PML and SUMO-1, Daxx, Sp100, p53 or CBP in the aggregated PML-NBs of U937/Bcl-2 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that may help to bypass the resistance to apoptosis conferred by Bcl-2. Elucidating the exact mechanism by which PML regulates Bcl-2 will require further work.
Apoptosis ; Chenodeoxycholic Acid ; Cisplatin ; Humans ; Leukemia ; U937 Cells

BACKGROUND: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 cells. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. RESULTS: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. CONCLUSION: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.
Blood Cells ; Dendritic Cells ; Erythrocytes ; Hematopoiesis ; Humans ; Megakaryocytes ; Transcription Factors ; U937 Cells

Apoptosis ; drug effects ; Asparaginase ; pharmacology ; HL-60 Cells ; Humans ; K562 Cells ; U937 Cells

OBJECTIVE: To investigate the effects of high dose vitamin C on proliferation and apoptosis of acute myeloid leukemia (AML) cell lines including HL-60, U937 and primary CD34 leukemia cells in AML. METHODS: CD34 cells were sorted by using immunomagnetic cell sorting system, then the primary CD34 leukemia cells, including HL-60 and U937 cell lines were cultured in vitro. Cells in each group were treated with different concentrations of vitamin C, the survival rate of cells was determined by MTT assay, the apoptosis rate of cells was evaluated by Annexin V/PI double staining, the expression of apoptotic proteins-including cleaved caspase 3, cleaved caspase-9 and cleaved PARP were detected by Western blot. RESULTS: The proliferation of HL-60 and U937 cells could be inhibited by high dose vitamin C, which showed a concentration-dependent manner (r=-0.9664; r=-0.9796). HL-60 and U937 cells were treated with different concentrations of vitamin C (8 and 20 mmol/L) for 24 hours, respectively, it was found that with the increasing of vitamin C concentration, cell apoptosis rate was significantly increased (r=0.9905; r=0.9971), and the expression of apoptosis related proteins including cleaved caspase 3, cleaved caspase-9 and cleaved PARP was aslo significantly increased with the increasing of concentration. In addition, it was found that with or without the mutation of TET2, high dose vitamin C could inhibit the proliferation (r=-0.9719; r=-0.9699) and promote the apoptosis (r=0.9998; r=0.9901) of primary CD34 leukemia cells in AML, which showed a dose-dependent manner, but it showed no effect on the proliferation (r=-0.2032) and apoptosis (r=0.1912) of normal CD34 cells. CONCLUSION High dose vitamin C can inhibit the proliferation and promote the apoptosis of acute myeloid leukemia cells, and selectively kill primary CD34 leukemia cells in AML.
Apoptosis ; Ascorbic Acid ; Cell Proliferation ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; U937 Cells

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CDl4 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with 10 microM, 50 microM or 100 microM of TPA. The cell morphology change was observed and the expression of the CD11b and CDl4 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 h and 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.
Binding Sites ; Cytomegalovirus* ; Fireflies ; Humans* ; Luciferases ; Macrophages ; Permissiveness ; Transfection ; U937 Cells*

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CDl4 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with 10 microM, 50 microM or 100 microM of TPA. The cell morphology change was observed and the expression of the CD11b and CDl4 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 h and 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.
Binding Sites ; Cytomegalovirus* ; Fireflies ; Humans* ; Luciferases ; Macrophages ; Permissiveness ; Transfection ; U937 Cells*

OBJECTIVETo explore the expression of B7 co-stimulator in human malignant hematological cell lines and its significance.

METHODSAmplified and purified plasmid DNA was transfected by liposome and detection of B7 gene expression by flow cytometry and RT-PCR.

RESULTEight human malignant hematological cell lines U937, K562, CEM, Daudi, GM, PEER, Jurkat, Raji express high level of HLA molecules, low or undetectable level of B7-1 gene, and high level of B7-2 gene. Expression level of B7-1 gene was enhanced after B7-1 transfection. T cell expressed high level of IL-2 mRNA after B7-1 transfection.

CONCLUSIONMultiple human malignant hematological cell lines express low or undetectable level of B7-1 gene, suggesting that B7-1 plays a critical role in tumor immunity.

B7-1 Antigen ; Cell Line ; Flow Cytometry ; Humans ; Interleukin-2 ; T-Lymphocytes ; immunology ; Transfection ; U937 Cells

OBJECTIVETo investigate the cytotoxicity and its mechanism of ZnO nanoparticles on human leukemic monocyte lymphoma cell line U937.

METHODSFour different size ZnO (10, 30, 60, 500 nm) were carefully characterized. The survival rate and viability were measured by trypan blue assay and MTT assay for each size ZnO particles at different concentrations (12, 120, 240, 600, 1200 µmol/L). The zinc probe, Fluozin-3, was used to detect the intracellular free zinc. Transmission electron microscopy was adopted to observe the cellular ultrastructure and the uptake of ZnO.

RESULTSAll four kinds of ZnO were rod shape, with a purity of > 99.9 wt%, and they were classified as zincite phase crystal and their surface areas were in accordance with the sizes. The viability (ZnO-n10: (97 ± 19)%, (91 ± 4)%, (24 ± 4)%, (15 ± 2)%; ZnO-n30: (111 ± 4)%, (81 ± 3)%, (24 ± 2)%, (27 ± 8)%; ZnO-n60: (105 ± 11)%, (73 ± 20)%, (43 ± 11)%, (28 ± 14)%; ZnO-µm: (88 ± 16)%, (62 ± 7)%, (22 ± 4)%, (13 ± 5)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r values were 0.965, 0.979, 0.998, 0.992, and the t values were 19.8, 25.3, 76.3, 40.9, respectively, P < 0.05). The liability (ZnO-n10: (98 ± 1)%, (67 ± 2)%, (59 ± 7)%, (13 ± 13)%, (5 ± 4)%; ZnO-n30: (98 ± 1)%, (97 ± 2)%, (50 ± 3)%, (20 ± 14)%, (7 ± 2)%; ZnO-n60: (97 ± 2)%, (88 ± 5)%, (48 ± 10)%, (12 ± 5)%, (4 ± 1)%; ZnO-µm: (96 ± 1)%, (76 ± 3)%, (58 ± 3)%, (19 ± 5)%, (20 ± 10)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r valued at 0.982, 0.956, 0.972, 0.980, and the t valued at 19.3, 12.1, 15.6, 18.5, respectively, P < 0.05). The increase of the zinc concentration showed by the zinc fluorescence probe was 121 ± 11, which was similar to the fluorescence of cells treated with ZnAc(2) (132 ± 14, F = 0.6, P > 0.05) at the Zn-equivalent concentration. There was no statistic difference for the percents of high zinc content cells in total cells exposed to ZnO-n30 (87.6 ± 2.6)% and these exposed to ZnAc(2) (86.9 ± 3.2)% (F = 1.5, P > 0.05).

CONCLUSIONZnO nanoparticles are highly cytotoxic to U937 cells and the solubilization of ZnO is the main toxicological mechanism.

Cell Survival ; Humans ; Monocytes ; drug effects ; ultrastructure ; Nanoparticles ; toxicity ; U937 Cells ; Zinc Oxide ; toxicity

This study was aimed to explore the potential therapy of Gambogic acid (GA) combined with magnetic nanoparticle of Fe3O4 (Fe3O4-MNP) on leukemia. The proliferation of U937 cells and the cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscopy and flow cytometry respectively. The expressions of gene and protein were detected by quantitative real-time polymerase chain reaction and Western blot respectively. The results showed that GA enhanced the cytotoxicity for U937 cells in dose- and time-dependent manners. The Fe3O4-MNP itself had not cytotoxicity, but could enhance the inhibitory effect of GA on proliferation of U937 cells. The apoptotic rate of U937 cells induced by combination of GA with Fe3O4-MNP was higher than that by GA alone. The typical apoptotic features of cells treated with GA and Fe3O4-MNP were observed. The expression levels of caspase-3 and bax after co-treatment of GA and Fe3O4-MNP were higher than that exposed to GA or Fe3O4-MNP alone, but the expressions of bcl-2, NF-kappaB and survivin were down-regulated. It is concluded that Fe3O4-MNP can promote GA-induced apoptosis in U937 cells, and the combination of GA with Fe3O4-MNP may be a safer and less toxic new therapy for leukemia.
Apoptosis ; drug effects ; Humans ; Iron Compounds ; administration & dosage ; pharmacology ; Magnetics ; Nanoparticles ; U937 Cells ; Xanthones ; pharmacology