Myeloid Leukemia 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.

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.

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.

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。

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.

Discovery and preliminary evaluation of 2-aminobenzamide and hydroxamate derivatives containing 1,2,4-oxadiazole moiety as potent histone deacetylase inhibitors

Using Entinostat as a lead compound, 2-aminobenzamide and hydroxamate derivatives have been designed and synthesized. The entire target compounds were investigated for their in vitro antiproliferative activities using the MTT-based assay against five human cancer cell lines including U937, A549, NCI-H661, MDA-MB-231 and HCT116. 2-Aminobenzamide series of compounds (10a–10j) demonstrated the most significant inhibition against human acute monocytic myeloid leukemia cell line U937, but no or poor activities against two human lung cancer cell lines. Furthermore, the target compounds were screened for their inhibitory activities against HDAC 1, 2, and 8. 2-Aminobenzamide derivatives (10) manifested a higher selectivity for HDAC 1 over HDAC 2, but were not active against HDAC 8. In contrast, most hydroxamate derivatives (11) inhibit HDAC 8 with lower IC50 values than SAHA and Entinostat. Docking study with selected compounds 10f and 11a revealed that the compounds might bind tightly to the binding pockets in HDAC 2 and HDAC 8, respectively. The results suggest that they may be promising lead compounds for the development of novel anti-tumor drug potentially via inhibiting HDACs.

Effects of emodin on apoptosis and cell cycle related genes in U937 cells

This study was aimed to observe the effects of emodin on apoptosis and cell cycle related genes in human myeloid leukemia cell line U937 cells. U937 cells were exposed to 60 µmol/L emodin for 24, 48, 72 h. The expressions of C-MYC, h-TERT, PIM-2, Survivin, wild type P53, P21, TGF β-1 and MCL-1 genes before and after treatment with emodin were determined and quantitated by using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the expressions of C-MYC, h-TERT, PIM-2, Survivin in treated U937 cells decreased, but the expressions of WTp53, P21 and TGFβ1 increased, while the expression of MCL-1 gene had no obvious change. It is concluded that multiple pathways may be involved in the processes of emodin-induced U937 cell apoptosis.

Design, synthesis, and in vitro antiproliferative activity of novel Dasatinib derivatives

Two series of novel Dasatinib derivatives have been designed and synthesized, with their in vitro cytostatic effect screened on human chronic myeloid leukemia cell line K562 and human myeloid leukemia cell line U937. Some target compounds demonstrated significant inhibitory activities against both cell lines. Compared to the contrast drug Dasatinib, 1b, 1c, 1d, 1e and 1f were found to demonstrate more potent antitumor activities. The structures of all the newly synthesized compounds were determined by (1)H NMR and (13)C NMR.

Effects of human bone marrow stromal cell line (HFCL) on the proliferation, differentiation and apoptosis of acute myeloid leukemia cell lines U937, HL-60 and HL-60/VCR

This study investigated the effects of human bone marrow fibroblastoid stromal cell line (HFCL) on the proliferation, differentiation and chemosensitivity of acute myeloid leukemia cells (AML) in vitro coculture. By setting up coculture system of sensitive U937, HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cells in direct contact with human bone marrow fibroblastoid stromal cell line HFCL, or separated by transwell, the proliferation of AML cells cocultured with HFCL cells was inhibited, compared with AML cells alone. And NBT positive cells increased slightly. The percentage of G1 phase cells of AML cells cocultured with HFCL cells was higher than that without HFCL cells, and that of S phase cells was lower. The expression of CD11b and CD14 increased. Meanwhile HL-60 and HL-60/VCR cells treated by TPT were observed to have apoptosis characteristic morphological changes. The proportion of G0/G1 HL-60 and HL-60/VCR cells treated with TPT increased and the sub-G1 increased. The percentage of Annexin V-positive cells and apoptotic cells increased with expression of activated Caspase-3 and the reduced expression of Bcl-2. But when they were cocultured with HFCL cells, the percentage of Annexin V-positive cells and apoptotic cells decreased and sub-G1 reduced. After indirect contact with HFCL cells the expression of activated Caspase-3 decreased and the expression of Bcl-2 increased. After direct contact with HFCL cells for 96 h, the expression levels of 582 genes in HL-60 cells were up-regulated, and 1,323 genes were down-regulated at least twofold by Affymetrix GeneChip Human Genome U133 set A. The expression change in some genes, such as HL14, was confirmed by RT-PCR and northern blot. In a word, HFCL cells could inhibit the proliferation, induce the monocytic differentiation of U937, HL-60 and HL-60/VCR cells, and prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via modulation of Bcl-2 and active Caspase-3. Many genes might take part in the influence of HFCL cells on AML cells, which may give important insights into the interaction of bone marrow stromal cells and leukemic cells.

Antitumor effect of interferon-on U937 human acute leukemia cells in vitro and its molecular mechanism

In order to investigate the antitumor effect and molecular mechanism of interferon-alpha (IFN-alpha) on human acute myeloid leukemia cell line U937 cells in vitro, the proliferation of U937 cells was determined by MTT assay, the apoptosis rate was analyzed by flow cytometry (FCM), and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR. The results showed that IFN-alpha could inhibit the proliferation of U937 cells significantly in a dose-and time-dependent way (P<0.01), and induce the apoptosis of U937 cells also in a dose-and time-dependent manner at the concentration of 1000-4000 U/L (P<0.01). The apoptosis rate of U937 cells was even over 50% when cultured with IFN-alpha for 36-48 h at the concentration of 2000-4000 U/L. Moreover, the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-alpha, and the inhibition was time-dependent (P<0.01). It was concluded that the anti-leukemia mechanism of IFN-alpha might be correlated with its antiproliferative and apoptotic inducing effects, and the down-regulation of the cyclin E expression might be one of its molecular mechanisms.

Hypericin‐induced Photocytotoxicity is Connected with G2/M Arrest in HT‐29 and S‐Phase Arrest in U937 Cells

Susceptibility of the HT-29 human colon adenocarcinoma cell line and human myeloid leukemia cell line U937 to hypericin-mediated photocytotoxicity was investigated and compared in this study. Cellular parameters as viability, cell number, metabolic activity and total protein amount were monitored in screening experiments with subsequent cell-cycle analysis and apoptosis detection to determine the cellular response of the different tumor types to various concentrations of photoactivated hypericin. The results show concentration dependence of the photosensitizer's cytotoxicity on the studied cell lines, with higher sensitivity of U937 cells. Whereas the two extreme hypericin concentrations (1 x 10(-9) M and 1 x 10(-6) M) resulted in similar changes in all tested cellular parameters on the two studied cell lines, 1 x 10(-8) M and 1 x 10(-7) M hypericin treatment resulted in different responses of the cell lines in all monitored parameters except for viability. Although leukemic cells proved sensitive to both 1 x 10(-8) M and 1 x 10(-7) M hypericin, significant changes on HT-29 cells were detected only after the 1 x 10(-7) M hypericin concentration. Cell-cycle arrest was related to simultaneously occurring apoptosis in colon cancer. Remarkable is the difference in cell-cycle profile where G2/M arrest in colon cancer cells versus accumulation of leukemic cells in the S phase appears. This suggests that hypericin treatment affecting the cell-cycle machinery of different cancer cells is not universal in effect.

Effect of novel modulators of protein kinase C activity upon chemotherapy-induced differentiation and apoptosis in myeloid leukemic cells.

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol. U937 cells displayed a slight reduction in the number of cells in G(2)/M cell cycle phase after exposure to FTT, SC-10, R59022 and R59949, respectively. In contrast, HL-60 cells demonstrated a largely unaltered cell cycle distribution. Whereas TPA treatment resulted in a strong induction of p21(WAF/CIP1), c-Fos and c-Jun levels, neither one of the novel PKC activators altered expression of these proteins. Consequently, we tested the ability of the activators to cause membrane translocation of PKC. While TPA treatment resulted in translocation of the PKC isoforms alpha, delta and epsilon, SC-10 and FTT failed to induce alterations in the PKC content of the membrane and cytosolic fractions, respectively. Expression of the beta(2)-integrin CD11c that is induced during TPA-mediated differentiation remained unaltered after exposure to SC-10 and was partly reduced after treatment with FTT. To further investigate the effect of these activators upon apoptosis in leukemic cells, HL-60 and U937 cells were treated with 1-beta-D-arabinofuranosylcytosine (Ara-C) or etoposide (VP-16). Whereas TPA strongly reduced apoptosis in Ara-C- or VP-16-treated U937 cells, little if any reduction was observed after pretreatment with either FTT, SC-10, R59022 or R59949, respectively, in these cells. In contrast, TPA enhanced apoptosis in Ara-C- or VP-16-treated HL-60 cells. Interestingly, FTT and SC-10 demonstrated a protective effect in Ara-C-treated HL-60 cells. Taken together, these data suggest that the novel PKC activators FTT, SC-10, R59022 and R59949 exhibit modest biological effects upon leukemic blast cells, and are not capable of enhancing the apoptotic response of these cells to cytotoxic drugs.

Differential Effects of Histone Deacetylase Inhibitors on Interleukin-18 Gene Expression in Myeloid Cells

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.

The combination of apoptotic U937 cells and lupus IgG is a potent IFN-alpha inducer.

Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-alpha production, that may be of pathogenic significance in the disease. We previously showed that SLE patients have an IFN-alpha-inducing factor in blood, probably consisting of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells, because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-alpha inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell lines (MonoMac6, H9, Jurkat, U266) could induce IFN-alpha production in PBMC when combined with IgG from SLE patients. The IFN-alpha production by PBMC was much enhanced when PBMC were costimulated by IFN-alpha2b. The ability of IgG from different SLE patients to promote IFN-alpha induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs, but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can cause production of a clearly immunostimulatory cytokine, which is IFN-alpha. This mechanism for induction of IFN-alpha production could well be operative also in vivo, explain the IFN-alpha production seen in SLE patients, and be important in the pathogenesis of SLE.