Monoblastic Leukemia Cell Line U937 Research Articles

PH-Sensitive Nanomicelles for High-Efficiency siRNA Delivery in Vitro and in Vivo: An Insight into the Design of Polycations with Robust Cytosolic Release.

The extremely low efficient cytosolic release of the internalized siRNA has emerged recently as a central issue for siRNA delivery, while there is a lack of guidelines to facilitate the cytosolic release of internalized siRNA. To address these concerns, we studied the contribution of the pH-sensitive inner core on handling the cytosolic release of siRNA delivered by a series of PG-P(DPAx-co-DMAEMAy)-PCB amphiphilic polycation nanomicelles (GDDC-Ms) with extremely low internalization (<1/4 of lipofactamine 2000 (Lipo2000)). Significantly, just by varying the mole ratio of DPA and DMAEMA to adjust the initial disassembly pH (pHdis) of the core near to 6.8, GDDC4-Ms/siRNA could get nearly 98.8% silencing efficiency at w/w = 12 with 50 nM siRNA and ∼78% silencing efficiency at w/w = 30 with a very low dose of 5 nM siRNA in HepG-2 cell lines, while Lipo2000 only got 65.7% with 50 nM siRNA. Furthermore, ∼98.4% silencing efficiency was also realized in the hard-to-transfect human acute monoblastic leukemia cell line U937 by GDDC4-Ms/siRNA (at w/w = 15, 50 nM siRNA), in the inefficient case for Lipo2000. Additionally, the high silencing efficiency (∼80%) in skin tissue in vivo was discovered. Undoubtedly, the robust potential of GDDC4-Ms in handling the cytosolic release paves a simple but efficient new way for the design of the nonviral siRNA vector.

Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48h and 72h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC50 values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC50 values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt.In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates.

Characterization of TNF-induced caspase-independent necroptosis

Caspase-independent programmed necrotic cell death (necroptosis) has recently been described. Previously described models of necroptosis required 16h or more of induction, which made the interpretation of findings somewhat difficult. In human monocytic leukemia cell line U937 necroptosis could be induced within 6h by combination of TNF and Z-VAD-fmk. Here we show that the reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that necroptosis is associated with the loss of mitochondrial membrane potential, but not the activation of Bak/Bax or calcineurin.

Design, Synthesis, and In vitro Antitumor Evaluation of Novel Phenylaminopyrimidine Derivatives

Two series of novel phenylaminopyrimidine derivatives were designed and synthesized. All target compounds were determined against the human acute monocytic leukemia cell line U937 and the human chronic myeloid leukemia cell line K562 in vitro. Some of the target compounds demonstrated significant inhibitory activity against both cell lines. Compared with the control drug VX-680, 8a, 8e, 8g, 8h, 8j, and 8k demonstrated more potent antitumor activity. The structures of all target compounds were identified by 1H-NMR, 13C-NMR, IR, MS, and EA.

Identification of aldo-keto reductases as NRF2-target marker genes in human cells

Transcription factor NF-E2-related factor 2 (NRF2) plays a crucial role in the cellular defense against oxidative/electrophilic stress by up-regulating multiple antioxidant genes. Numerous studies with genetically modified animals have demonstrated that Nrf2 is a sensitivity determining factor upon the exposure to environmental chemicals including carcinogens. Moreover, recent studies have demonstrated that polymorphism in the human NRF2 promoter is associated with higher risks for developing acute lung injury, gastric mucosal inflammation, and nephritis. Therefore, the identification of reliable and effective human target genes of NRF2 may allow the monitoring of NRF2 activity and to predict individual sensitivity to environmental stress-induced damage. For this purpose, we investigated genes that are tightly controlled by NRF2 to establish markers for NRF2 activity in human cells. Firstly, in the normal human renal epithelial HK-2 cells, the measurement of the expression of 30 previously reported NRF2 target genes in response to NRF2 inducers (sulforaphane, tert-butylhydroquinone, cinnamic aldehyde, and hydrogen peroxide) showed that the aldo-keto reductase (AKR) 1C1 is highly inducible by all treatments. Accordantly, the basal and inducible expressions of AKRs were significantly attenuated in NRF2-silenced HK-2 cells. Whereas, cells with stable KEAP1 knockdown, which causes a modest NRF2 activation, demonstrated substantially increased levels of AKR1A1, 1B1, 1B10, 1C1, 1C2, and 1C3. Secondly, the linkage between NRF2 and the AKRs was confirmed in human monocytic leukemia cell line U937, which can be a model of peripherally available blood cells. The treatment of U937 cells with NRF2 inducers including sulforaphane effectively elevated the expression of AKR1B1, 1B10, 1C1, 1C2, and 1C3. Whereas, the levels of both the basal and sulforaphane-inducible expression of AKR1C1 were significantly reduced in NRF2-silenced stable U937 cells compared to the control cells. Similarly, the inducible expression of AKR1C1 was observed in another human monocytic leukemia cell line THP-1 as well as in human primary blood CD14+ monocytes. In conclusion, together with the high inducibility and NRF2 dependency shown in renal epithelial cells as well as in peripherally available blood cells, current findings suggest that AKRs can be utilized as a marker of NRF2 activity in human cells.

Molecular Mechanisms of Pro-Survival and Differentiating Function of Bone Marrow-Derived Adipocytes On Acute Monoblastic Leukemia Cells.

AbstractAbstract 2582Survival of adult patients with acute monocytic leukemia remains dismal. We proposed the hypothesis that bone marrow (BM) adipocytes abundant in aging marrow microenvironment play a role in chemoresistance of AML cells. We have reported that BM adipocytes promote monocytic differentiation, activation, and survival of monoblastic leukemia cells (ASH, 2012).In this study, we examined the molecular mechanism of BM adipocytes causing survival and differentiation of monoblastic leukemia cells. Monoblastic leukemia cell line U937 and primary samples from acute monoblastic leukemia patients (n=6) were co-cultured with BM-derived marrow stromal cells (MSC) and MSC-differentiated adipocytes. Under limiting serum conditions, MSCs and adipocytes inhibited spontaneous cell death of U937 cells with decrease in subG1 fractions, more prominently in co-cultures with adipocytes than with MSCs (% subG1 fraction; cultured alone 23.9+9.0, co-cultured with MSC 15.7+1.9%, co-cultured with adipocytes 11.9+1.2%, p=0.04). Adipocytes but not MSC moderately reduced spontaneous apoptosis in primary leukemic blasts (n=6, p=0.02).We next focused on adipocytokines, lepin and plasminogen activator inhibitor 1(PAI-1), both implicated in cancer progression, as potential mediators of the protective effects by adipocytes. mRNA levels of leptin, leptin receptor and PAI-1 were up-regulated in MSC-derived adipocytes during adipocytic differentiation (relative expression to GAPDH, leptin: MSC 0, adipocyte 123.3+35.0; leptin receptor: MSC 0.16+0.05, adipocyte 4.47+0.37; PAI-1: MSC 314.9+46.5, adipocyte 3766.1+656.2). High level of secreted leptin was confirmed by ELISA in adipocyte conditioning medium (MSC 23.1+2.9, adipocyte 110.0+4.6 pg/mL). In serum-starved conditions, human recombinant leptin (5ng/mL) stimulated cell growth of monoblastic U937 cells known to express leptin receptor (35.8+7.9% increase of viable cell number at 48 hours, p=0.02) and inhibited apoptosis (%subG1 fraction; control 26.2+3.9, leptin16.4+4.1%, p=0.04).Human recombinant PAI-1 inhibited spontaneous and ara-C-induced apoptosis in U937 cells (11.2+1.1% and 15.1+1.7% decrease of Annexin V posivitity p=0.01). These results suggest that BM adipocytes produced leptin and PAI-1contribute to support myeloid leukemia cells survival within BM microenvironment.We next focused on molecular changes in U937 cells co-cultured with adipocytes. We found up-regulation of monocyte/macrophage differentiation gene PPARg and of PPARg target genes, CD36 and FABP4, by quantitative RT-PCR (relative expression to GAPDH, control vs co-culture with adipocytes: PPARg: 8.8+0.9 v.s.18.8+2.6, CD36; 105.0+11.8v.s.425.7+35.6; FABP4: 0.27+0.03v.s.153.7+12.8). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. PPARγ is known to promote monocytic differentiation and uptake of oxidized cholesterol through the induction of scavenger receptor CD36, a protein marker of mature monocytes/macrophages. The up-regulation of cell surface CD36 expression by co-culture with adipocytes was indeed found in U937 cells and in 4 of the 6 primary AML samples by flow cytometory (increase in CD36 MFI: U937; 230.28, Pt samples; 46.4+20.2). These data suggest that BM adipocytes promote monocytic differentiation of monoblastic leukemia cells.In summary, our findings indicate that secretion of leptin and PAI-1 by BM adipocytes supports leukemic cell survival, while PAI-1 primarily and directly inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of monocytic leukemia cells to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

A Plant Triterpenoid Avicin D Stimulates Adipocytic Differentiation of Bone Marrow Stromal Cells and Promotes Their Pro-Survival Effects On Acute Monoblastic Leukemia Cells

Abstract 4315A natural triterpene Avicin D induces apoptosis in various tumor cells and regulates cellular metabolism via Glucocorticoid receptor (GR) signalling, known to be involved in adipocyte differentiation (Tang, Annu Rev Biochem. 2012;81:715). Since adipocytes represent an essential component of the aging bone marrow (BM) microenvironment (Tabe, Blood 2004;103:1815), and promote monocytic differentiation and survival of monoblastic leukemia cells (Tabe, ASH, 2011), we examined the potential of Avicin D to modulate BM adipocytes and its effects on the survival of the leukemic cells.In mature adipocytes derived from BM mesenchymal stem cells (MSCs), Avicin D (1ƒE′M, 48 hrs) significantly increased the number of lipid vesicles without apoptosis induction or significant cell cycle arrest. This was associated with upregulation of adipose-specific genes, PPARG and fatty acid binding protein 4 (FABP4), and inflammation-related haptoglobin (HP) gene (p<0.005, Figure 1). Avicin D further enhanced leptin release from adipocytes and inflammatory cytokines production, IL-6 and IL-8, from both MSCs and adipocytes (Figure 2). Of note, Avicin D increased pro-survival Bcl-2 mRNA expression in adipocytes (fold increase compared to MSCs: MSCs+Avicin D 1.0±0, adipocytes 1.8±0.1, adipocytes+Avicin D 4.4±0.6, p=0.02).We next investigated the effects of MSCs and adipocytes pre-treated with 1mM Avicn D for 24 hours on co-cultured monoblastic leukemia cell line U937. Avicin D (1ƒE′M) moderately (26±5 %) inhibited cell proliferation of U937 cells cultured alone as detected by MTT assay. Serum-starvation induced cell death in U937 cells was inhibited by MCS or adipocyte co-culture, and further blocked by Avicin D-pretreated adipocytes, which was accompanied with cell cycle arrest (sub-G1 fraction; control U937 36.1±7.1%, co-culture with MSC 17.9±4.1%, MSC+Avicin D 20.9±3.9%, adipocyte 16.9±2.9, adipocyte+Avicin D 11.5±0.8%, p=0.03, G0/G1 phase; control 39.1±5.9%, with MSC 44.3±1.0%, MSC+Avicin D 40.3±1.3%, adipocyte 40.6±1.3, adipocyte+Avicin D 50.4±2.3%, p=0.03). Co-culture of U937 cells with Avicin D-pretreated adipocytes induced upregulation of PPARG and CD36, markers of monocytes/macrophages maturation in U937 cells (fold increase compared to control U937: PPARG: co-culture with adipocytes 2.1±0.4, adipocytes+Avicin D 13.8±6.2; CD36: adipocytes 2.2±0.4, adipocytes+Avicin D 4.7±2.9). Co-culture with Avicin D-pretreated adipocytes also caused 3.1 ±0.4 fold increase of BCL2 mRNA in U937 cells compared to the one of co-cultured with control adipocytes (p=0.02). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. Utilization of the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry allowed the identification of 1,634 proteins. We found changes in 14 proteins in U937 cells co-cultured with Avicin D-pretreated adipocytes compared to untreated adipocytes. Among 13 down-regulated proteins, three are involved in pyruvate metabolism (Glyoxalase I; p=0.01, Lactate dehydrogenase B; p=0.02, Pyruvate kinase; p=0.03), and four proteins participate in cell cycle progression (DNA replication licensing factor MCM4, MCM5; p=0.02, p=0.03, DNA-dependent protein kinase; p=0.02, Interleukin enhancer-binding factor 3; p=0.005). Voltage-dependent anion-selective channel protein 2, which is involved in the mitochondrial apoptotic pathway via regulation of Bcl-2, and an enzymatic antioxidant Superoxide dismutase were up-regulated (p=0.04, p=0.03). These data indicate that U937 cells co-cultured with Avicin D pretreated adipocytes were induced to undergo cell cycle arrest, escape apoptosis with down-regulation of glucose metabolism.Altogether, these results suggest that Avicin D induces adipocytic differentiation of BM-derived stromal cells, promotes production of leptin and inflammatory cytokines, which in turn supports survival of monocytic leukemia cells. These findings suggest a contributory role of the aging “pro-inflammatory” bone marrow microenvironment in the reduced efficacy of cytotoxic chemotherapy which is prevalent in elderly AML patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bone Marrow-Derived Adipocytes Promote Differentiation, Proliferation and Survival of Acute Monoblastic Leukemia Cells

Abstract 1501Acute monoblastic leukemia occurs most commonly in young individuals, whereas acute monocytic leukemia is common in adults (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008). Adipocytes are the prevalent stromal cell type in adult bone marrow (BM) and play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103: 1815–22). BM stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the BM space with increasing age. In this study, we examined whether BM-derived adipocytes participate in the molecular events associated with proliferation, differentiation and apoptosis of monoblastic leukemia cells. To this end, we performed gene expression microarray analysis of 339 genes associated with lipid metabolism (GeneSQUARE, Kurabo, Japan) using RNA from monoblastic leukemia cell line U937 co-cultured with MSC or MSC-derived adipocytes. Five genes were upregulated (>2.0 fold) and 2 genes down-regulated in U937 cells co-cultured with adipocytes compared to U937 cultured alone. The up-regulated genes included monocyte/macrophage differentiation specific genes, such as scavenger receptor CD36, adipsin or fatty acid binding protein 4 (FABP4). CD36 and FABP4 are classical target genes of PPARγ which was also upregulated in U937 co-cultured with adipocytes. Upregulation of the inflammation-related haptoglobin gene was observed in U937 cells co-cultured with either adipocytes or MSC. These findings were confirmed by quantitative RT-PCR assay with concordant results. Down-regulated genes included monocyte chemoattractant protein-1 (MCP-1) (0.1 fold) and glycolytic enzyme hexokinase 2 (0.4 fold). In turn, U937 cells co-cultured with premature or mature adipocytes accumulated in G2 cell cycle phase compared to U937 cultured alone or co-cultured with MSC (% G2/M-phase fraction; U937 cultured alone, 14.6, co-culture with MSC 25.1, co-culture with preadipocyte 32.0, with mature adipocyte 31.9) and were protected from spontaneous cell death under serum-starved conditions (% subG1 fraction: U937 cultured alone 23.9%, co-cultured with MSC 15.7%, with preadipocytes 3.9% and with mature adipocytes 11.6%). In summary, results suggest that BM adipocytes, abundant in adult BM space, promote monocytic differentiation, proliferation and survival of monoblastic leukemia cells. We propose that these cells represent an essential component of the BM microenvironment that contributes to malignant phenotypes of adults with monocytic leukemia. Disclosures:No relevant conflicts of interest to declare.

Protective effects of fucoidan against γ-radiation-induced damage of blood cells

Fucoidan, a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and Laminaria japonica, has a variety of biological activities, including antioxidant and antitumor activities. Here, we investigated the radioprotective effects of fucoidan on human monoblastic leukemia cell line U937. Further, animal tests were carried out using Balb/c mice in order to determine the radiation-induced changes in the counts of blood cells, including thrombocytes, erythrocytes, leukocytes and hematocrit. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wherein fucoidan (1, 10, and 100 μg/mL) was observed to improve recovery from damage caused by 8-Gy radiation in a dose dependent manner. The viability of U937 cells pre-treated with fucoidan also increased in a dose dependent manner. Furthermore, fucoidan at 100 mg/kg was found to protect against changes in the counts of blood cells as follows: on day 28 after irradiation, the thrombocyte count in the irradiated controls decreased to 45% compared with the non-irradiated controls, while that in the fucoidan-treated group was 60%. The hematocrit in the fucoidan-treated group recovered to 75% on day 28, while that in the irradiated control was 68%. The erythrocyte count in the irradiated controls consistently ranged from 64% to 67% throughout the experiment, but that in the fucoidan-treated group increased gradually, ranging from 75% to 80%. The mean number of survival days and 50-day actuarial survival rate increased dose dependently in the fucoidan-treated group. The mean number of survival days and the 50-day actuarial survival rate in this group was 16, 21, and 29 days and 12%, 20%, and 30% at fucoidan doses of 1, 10, and 100 mg/kg. The values of these parameters in the control group were 9 days and 0%, although the difference between the test and control groups was not statistically significant. Our results may prove valuable in the field of radioprotection.

Agreement among experts in ratings of emotion in dogs

Caspase-independent programmed necrotic cell death (necroptosis) has recently been described. Previously described models of necroptosis required 16 h or more of induction, which made the interpretation of findings somewhat difficult. In human monocytic leukemia cell line U937 necroptosis could be induced within 6 h by combination of TNF and Z-VAD-fmk. Here we show that the reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that necroptosis is associated with the loss of mitochondrial membrane potential, but not the activation of Bak/Bax or calcineurin.

HSP90 inhibitor, celastrol, arrests human monocytic leukemia cell U937 at G0/G1 in thiol-containing agents reversible way

BackgroundBecause some of heat shock protein 90's (HSP90) clients are key cell cycle regulators, HSP90 inhibition can affect the cell cycle. Recently, celastrol is identified both as a novel inhibitor of HSP90 and as a potential anti-tumor agent. However, this agent's effects on the cell cycle are rarely investigated. In this study, we observed the effects of celastrol on the human monocytic leukemia cell line U937 cell cycle.ResultsCelastrol affected the proliferation of U937 in a dose-dependent way, arresting the cell cycle at G0/G1 with 400 nM doses and triggering cell death with doses above 1000 nM. Cell cycle arrest was accompanied by inhibition of HSP90 ATPase activity and elevation in HSP70 levels (a biochemical hallmark of HSP90 inhibition), a reduction in Cyclin D1, Cdk4 and Cdk6 levels, and a disruption of the HSP90/Cdc37/Cdk4 complex. The observed effects of celastrol on the U937 cell cycle were thiol-related, firstly because the effects could be countered by pre-loading thiol-containing agents and secondly because celastrol and thiol-containing agents could react with each other to form new compounds.ConclusionsOur results disclose a novel action of celastrol-- causing cell cycle arrest at G0/G1 phase based upon thiol-related HSP90 inhibition. Our work suggests celastrol's potential in tumor and monocyte-related disease management.

A Peptide Against the N-Terminus of Myristoylated Alanine-Rich C Kinase Substrate Inhibits Degranulation of Human LeukocytesIn Vitro

Leukocytes synthesize a variety of inflammatory mediators that are packaged and stored in the cytoplasm within membrane-bound granules. Upon stimulation, the cells secrete the granule contents via an exocytotic process whereby the granules translocate to the cell periphery, the granule membranes fuse with the plasma membrane, and the granule contents are released extracellularly. We have reported previously that another exocytotic process, release of mucin by secretory cells of the airway epithelium, is regulated by the myristoylated alanine-rich C kinase substrate (MARCKS) (Li Y, Martin LD, Spizz G, Adler KB. MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro. J Biol Chem 2001;276:40982-40990; Singer M, Martin LD, Vargaftig BB, Park J, Gruber AD, Li Y, Adler KB. A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med 2004;10:193-196). In those studies, mucin secretion in vitro and in vivo was attenuated by a synthetic peptide identical to the N-terminus of MARCKS, named the MANS peptide (Li and colleagues, 2001). In this study, we used the MANS peptide to investigate possible involvement of MARCKS in secretion of leukocyte granule proteins. In neutrophils isolated from human blood, phorbol 12-myristate 13-acetate-induced myeloperoxidase release was attenuated in a concentration-dependent manner by MANS but not by equal concentrations of a missense control peptide. In additional studies using human leukocyte cell lines, secretion of eosinophil peroxidase from the eosinophil-like cell line HL-60 clone 15, lysozyme from the monocytic leukemia cell line U937, and granzyme from the lymphocyte natural killer cell line NK-92 were attenuated by preincubation of the cells with MANS but not with the missense control peptide. The results indicate that MARCKS protein may play an important role in the secretion of membrane-bound granules from different leukocytes. MARCKS may be an important component of secretory pathways associated with release of granules by different cell types.

Down-regulation of 7SL RNA Expression and Impairment of Vesicular Protein Transport Pathways by Leishmania Infection of Macrophages

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

Presence of a peptide component of thymosin fraction-5 manifesting discrete cytostatic properties in HL-60 human promyelocytic leukemia cells

Thymosin fraction-5 (TF5), an array of small molecular weight peptides present in crude extracts of the adult bovine thymus, contains numerous constituents with demonstrable biological activity. Because TF5 generally enhances immune reactivity in a variety of settings, and additionally restricts proliferation of certain neoplasms, we examined the effects of TF5 on proliferative capacity in the human promyelocytic leukemia cell line HL-60. Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were monitored to assess rates of cellular proliferation; our results demonstrate that TF5 restricted HL-60 cell growth, an influence that exhibited comparable potency and efficacy among all three indices. This antiproliferative activity was labile, insofar as medium conditioned in HL-60 cells for 24 h became devoid of the initial growth-suppressive activity after 24-h culture when subsequently administered to naïve cultures. Review of cytoarchitectural traits, chromatin staining by TUNEL, and fluorescent cytometric analyses demonstrated that TF5 failed to elicit apoptosis, however, suggesting that this material instead drove treated cells into growth arrest and an unanticipated cytostasis. Qualitatively similar responses were noted in the human monoblastic leukemia cell line U937. Partial purification of TF5 by FPLC yielded a component containing an antiproliferative activity associated with the ∼1000-Da fraction. These results demonstrate that TF5 contains a sub-fraction possessing a growth-suppressive activity capable of restraining normal proliferation of human myeloid neoplasms via the apparent induction of true cytostasis.

Costimulatory molecule expression following exposure to orthopaedic implants wear debris.

Patients with long-term orthopedic implants may develop inflammatory reactions due to the accumulation of biomaterial particles both around the implant and in distant organs. The exact impact of these particles on the normal immune cell function still remain relatively unclear. Activation of T-cells following exposure to biomaterial particles is driven by macrophages and requires synergistic signals primed by both antigen presentation and costimulation. The pattern of costimulatory molecule expression (CD80,CD86) was primarily examined using immunohistochemistry on tissue specimens of bone/implant interface membranes taken from sites of bone erosion. Additionally, costimulatory molecule expression was also assessed in the monocytic leukemia cell line U937 following exposure to clinically relevant titanium aluminum vanadium (TiAlV) and stainless steel particles (FeCrNi) cultured in vitro. This study demonstrates the induction and prominent expression of CD86 on almost all macrophage subsets at the bone/implant interface, including fused forms and large multinucleated giant cells (MNGC). In vitro analysis also indicated phagocytosis of metal particles by differentiated U937 caused significant induction of both CD80 and CD86 (p < 0.01), although the expression of CD86 dominated following prolonged exposure. The data presented highlights that CD86 is the predominant costimulatory molecule ligating to the complementary CD28 molecule at the inflammatory lesion of the interface. We propose that the intracellular presence of indigestible implant material, in addition to elevated costimulatory molecule expression, may promote T-cell inflammatory reactions at sites close to and distant from the orthopedic implant.

Differential expression of glutaredoxin and thioredoxin during monocytic differentiation

Macrophages generate reactive oxygen intermediates (ROIs) as the effectors of anti-bacterial defense mechanism. Intracellular ROIs and reduction/oxidation (redox) status play crucial roles in signal transduction. We therefore investigated the expression of redox-regulating proteins such as glutaredoxin (GRX) and thioredoxin (TRX) during the differentiation of murine monocytic leukemia cell line M1 cells and human monocytic leukemia cell line U937 cells. When M1 cells were treated by IL-6, GRX mRNA markedly increased and TRX mRNA also increased slightly. In contrast, there was no increase of GRX mRNA in D-cell, which is a sub-cell line derived from M1 lacking in the capacity of differentiation. GRX mRNA also increased in U937 cells differentiated by phorbol 12-myristate 13-acetate (PMA). By immunohistochemistry, unstimulated M1 cells showed strong staining of TRX and marginal staining of GRX. In contrast, TRX expression in IL-6 treated M1 cells is as strong as in unstimulated M1 cells, whereas GRX expression is slightly enhanced in IL-6 treated M1 cells. Phagocytosis is markedly enhanced and hydrogen peroxide production is slightly enhanced in IL-6 treated M1 cells. These results showed that TRX is steadily expressed whereas GRX is induced in association with the differentiation in macrophage-like cell line cells, suggesting differential roles of these redox regulators in macrophage lineage.

Effects of two sex steroids (17 β estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937

We investigated the effects of two sex steroids (17 β estradiol and testosterone) on five human leukemia cell lines. We observed a statistically significant inhibition of proliferation, dose and time dependent, of the human monoblastic leukemia cell line U937. This inhibition was associated with a dose dependent decrease in the number of CFU-blasts in clonogenic cultures. Cytostatic effect was obtained with doses of 5 μM for estrogen and 10 μM for androgen and was not due to a non-specific cytotoxic effect, some cell viability remained high (>90%) even after 6 days of incubation. More accurately, we demonstrated that growth inhibition was associated with a cell cycle arrest, U937 cells accumulating in G2/M phase. This blockade was dose related with a maximum number of cells accumulating at day 4. Sensitivity of these cells to an S-phase specific agent (hydroxyurea) was not increased, suggesting that these cells were blocked in G2/M and did not undergo mitosis. Expression in U937 cells of high affinity nuclear receptors for estrogen and androgen was negative which was in favour of a type II estrogen binding site, mediated mechanism. Moreover, a small fraction of these cells underwent apoptosis or differentiation with about 12% apoptotic cells and a significant increase (more than 30%) of two myelomonocytic markers (CD13 and CD64). These results demonstrate that the proliferation of some leukemic cells may be inhibited by micromolar concentrations of sex steroids, independently of nuclear receptor expression. The main mechanism seems to be a block in cell cycle associated with modulation of apoptosis and differentiation. It provided additional evidence for the potential value of sex steroids and their analogues in the treatment of leukemias.

Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes.

To examine the potential of methotrexate (MTX) to act as a differentiation-stimulating factor for monocytes, which could explain the antiinflammatory properties of this agent in the treatment of rheumatoid arthritis (RA). Fluorescence-activated cell sorter analysis was used to measure the changes in antigen expression (CD11b/c, CD16, CD64, CD14, CD68, and CD95) in response to MTX, 1,25-OH-cholecalciferol (1,25-OH-CCF), and granulocyte-macrophage colony-stimulating factor in the human monoblastic leukemia cell line U937, bone marrow mononuclear cells (BMMC), and peripheral blood mononuclear cells (PBMC). Release of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor a, and soluble tumor necrosis factor receptors (sTNFR) p55 and p75 during the differentiation in vitro was assessed by immunoassay in the culture supernatants. MTX alone and in combination with 1,25-OH-CCF markedly stimulated the differentiation of the monocytic U937 cells and simultaneously increased Fas-antigen expression. Differentiation was associated with enhanced IL-1Ra and sTNFR p75 release from U937 cells. MTX had fewer effects on phenotypic differentiation of human BMMC and PBMC, but did stimulate IL-1Ra release and inhibit IL-1beta synthesis in BMMC. MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells. Differentiation in vitro is associated with an increase in natural cytokine inhibitor release and a simultaneous down-regulation of IL-1beta. These findings may explain the marked clinical antiinflammatory effects of MTX when used in the treatment of RA.

Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes

Objective To examine the potential of methotrexate (MTX) to acts as a differentiation-stimulating factor for monocytes, which could explain the antiinflammatory properties of this agent in the treatment of rheumatoid arthritis (RA). Methods Fluorescence-activated cell sorter analysis was used to measure the changes in antigen expression (CD11b/c, CD16, CD64, CD14, CD68, and CD95) in response to MTX, 1,25-OH-cholecalciferol (1,25-OH-CCF), and granulocyte-macrophage colony-stimulating factor in the human monoblastic leukemia cell line U937, bone marrow mononuclear cells (BMMC), and peripheral blood mononuclear cells (PBMC). Release of interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor α, and soluble tumor necrosis factor receptors (sTNFR) p55 and p75 during the differentiation in vitro was assessed by immunoassay in the culture supernatants. Results MTX alone and in combination with 1,25-OH-CCF markedly stimulated the differentiation of the monocytic U937 cells and simultaneously increased Fas-antigen expression. Differentiation was associated with enhanced IL-1Ra and sTNFR p75 release from U937 cells. MTX had fewer effects on phenotypic differentiation of human BMMC and PBMC, but did stimulate IL-1Ra release and inhibit IL-1β synthesis in BMMC. Conclusion MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells. Differentiation in vitro is associated with an increase in natural cytokine inhibitor release and a simultaneous down-regulation of IL-1β. These findings may explain the marked clinical antiinflammatory effects of MTX when used in the treatment of RA.

Effect of bryostatin 1 on taxol-induced apoptosis and cytotoxicity in human leukemia cells (U937)

We have examined the effects of the macrocyclic lactone protein kinase C (PKC) activator bryostatin 1 on taxol-induced apoptosis and inhibition of clonogenicity in the human monocytic leukemia cell line U937. Exposure of cells to bryostatin 1 (10 nM; 15 hr) after (but not before) a 6-hr incubation with 0.5 μM taxol significantly increased apoptosis and resulted in an approximately 3 log reduction in clonogenicity. Cell cycle analysis revealed that the increase in apoptotic cells following bryostatin 1 treatment occurred primarily in the population undergoing taxol-mediated G 2M arrest. The actions of bryostatin 1 were not attributable to potentiation of taxol-induced tubulin stabilization or to a reduction in the intracellular retention of taxol. Following exposure of cells to taxol, the Bcl-2 protein displayed an alteration in mobility that was not modified appreciably by bryostatin 1 treatment. The mobility shift in Bcl-2 protein from cells exposed to taxol followed by bryostatin 1 was eliminated by treatment of lysates with the protein phosphatase 2A (PP2A); the latter effect was blocked by okadaic acid. Treatment of cells with taxol followed by bryostatin 1 did not increase the amount of total Bax (compared with treatment with taxol alone), but did increase the amount of free Bax in the supernatant fraction. Finally, the ability of bryostatin 1 to potentiate taxol-induced apoptosis in U937 cells was mimicked closely by 2′-amino-3′-methoxyflavone (PD98059), a specific inhibitor of the mitogen-activated protein kinase (MAPK) kinase (MEK). Collectively, these findings indicate that bryostatin 1 increases the susceptibility of U937 cells to taxol-induced apoptosis and inhibition of clonogenicity. They also raise the possibility that this phenomenon may involve functional alterations in Bcl-2 and/or other proteins involved in regulation of the cell death pathway.