Influence of antioxidants on NO-dependent induction of heme oxygenase-1 in U937 monocytes

Thiol antioxidants are known to inhibit the nitric oxide-dependent induction of the hemoxygenase-1 gene (HOX-1). To estimate the degree to which the inhibitory effect of thiol antioxidants is accounted for by them scavenging oxidized NO derivatives or their precursors, the reactive oxygen and nitrogen species (ROS and RNS), we studied the inhibitory effect of nonthiol antioxidants: dimethyl sulfoxide, dimethylthiourea, sodium salicylate, sodium formate, uric acid, catalase, and superoxide dismutase. Partial inhibition of NO-dependent HOX-1 induction was observed in the presence of the nonpolar HO scavengers dimethyl sulfoxide and dimethylthiourea. The antioxidants which selectively bind other ROS had no effect on HOX-1 expression. To reveal the role of RNS in NO-dependent HOX-1 induction, cells were treated with the NO-generating compound DPTA-NO in the presence of 2-phenyl-4,4,5,5,-tetramethylimidazole-1-oxyl 3 oxide (PTIO), which oxidizes NO to NO₂. PTIO proved to significantly enhance NO-dependent HOX-1 induction. Thiol antioxidants completely inhibited the stimulating effect of PTIO, which is evidence that their inhibitory effect is explained by RNS scavenging. The results of this study indicate that antioxidants can be used to modulate the cell response to NO.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 89–95.Original Russian Text Copyright © 2005 by Litvinov, Prasolov, Bouton, Drapier, Turpaev.     

Chloride and potassium channels in U937 human monocytes

Summary Ionic channels in a human monocyte cell line (U937) were studied with the inside-out patch-clamp technique. A Ca²⁺-activated K⁺ channel and three Cl^–-selective channels were observed. The Ca²⁺-activated K⁺ channel had an inward-rectifying current-voltage relationship with slope conductance of 28 pS, and was not dependent on membrane potential. Among the three Cl^– channels, and outward-rectifying 28-pS channel was most frequently observed. The permeability ratio (Cl^–/Na⁺) was 4–5 and CH₃SO ₄ ^– was also permeant. The channel became less active with increasing polarizations in either direction, and was inactive beyond ±120 mV. The channel, observed as bursts, occasionally had rapid events within the bursts, suggesting the presence of another mode of kinetics. Diisothiocyanatostilbene-disulfonic acid (DIDS) blocked the channel reversibly in a dose-dependent manner. The second 328-pS Cl^– channel had a linear currentvoltage relationship and permeability ratio (Cl^–/Na⁺) of 5–6. This channel became less active with increasing polarizations and inactive beyond ±50 mV. DIDS blocked the channel irreversibly. The channel had multiple subconductance states. The third 15-pS Cl^– channel was least frequently observed and least voltage sensitive among the Cl^– channels. Intracellular Ca²⁺ or pH affected none of the three Cl^– channels. All three Cl^– channels had a latent period before being observed, suggesting inhibitory factor(s) presentin situ. Activation of the cells with interferon-, interferon-A or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused no change in the properties on any of the channels.     

Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells

Hydroxyl radicals (.OH) and superoxide anion radicals (O2.-) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44 degrees C, 30 min) treatment. Rate constants of the reactions between antioxidants and .OH or O2.- were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for .OH or O2.- was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for .OH or O2.-. The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia- induced apoptosis.     

Differential signaling of sphingosine derivatives in U937 human monocytes depends on the degree of N-methylation

Previously, we studied N,N-dimethyl-D-erythro-sphingosine (DMS)-induced cell death and signaling in U937 human monocytes; we found that DMS-induced sphingosine kinase- and PKC-independent apoptosis. In the present study, we studied apoptotic responses by three N-methyl derivatives of sphingosine: N-monomethyl-D-erythro-sphingosine (MMS), N,N,N-trimethyl-D-erythro-sphingosine (TMS), and D-erythro-sphingosine (SPH). The potency order in the apoptotic response was DMS>or=MMS>TMS>SPH. We compared cellular responses to the derivatives in terms of activities of MAPK signaling molecules, mitochondrial membrane potential (DeltaPsi(m)), and reactive oxygen species (ROS) generation. Our results suggest that the degree of N-methylation affects the apoptosis-inducing capacity and other related responses including MAPK modulation, DeltaPsi(m), and ROS generation. Dimethylation and monomethylation on the C2 amine of sphingosine enhance the apoptotic response; however, trimethylation induces differential modulation of signaling molecules and less cytotoxicity. Our investigation will be useful for understanding the actions of sphingolipids in apoptosis and for developing chemotherapeutics based on DMS structure.     

Comparison of biosynthesis and subcellular distribution of lysozyme and lysosomal enzymes in U937 monocytes

Using metabolic labelling and sucrose density fractionation we compared the synthesis of lysozyme and lysosomal enzymes in human monocytic U937 cells. In pulse-chase experiments in sucrose density gradients, the intracellular radioactively labelled lysozyme distributed similarly to cathepsin D and β-hexosaminidase. With the aid of immunochemical detection in Western blots, the steady-state distribution of lysozyme was found to be slightly different from that of β-hexosaminidase; relatively more lysozyme was present in fractions sedimenting between lysosomes and the Golgi apparatus. The observed distribution of the lysozyme antigen with a prominent peak in the lysosomal fraction was in striking contrast to the broad distribution of the lysozyme activity. The difference was explained by a bias in the determination of the activity of lysozyme by the ‘lysoplate’ diffusion assay.     

Progesterone, but not 17beta-estradiol, increases TNF-alpha secretion in U937 monocytes

The Women Health Initiative Clinical trial results suggest that post-menopausal women receiving estrogen + progesterone are at risk for heart disease compared with estrogen alone supplemented women. We examined the hypothesis that progesterone but not 17beta-estradiol (E) increases the secretion of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. U937 human monocytes were cultured with normal or high glucose in the presence and absence of estrogen or progesterone at 37 degrees C for 24 h. Results show that estrogen inhibits IL-6 but not TNF-alpha secretion (p < 0.05) in monocytes activated by lipopolysaccharide (LPS) or high glucose. In addition, progesterone increased the TNF-alpha secretion in activated monocytes. Thus, progesterone supplementation along with estrogen may increase blood levels of pro-inflammatory cytokine TNF-alpha and thus risk of heart disease in post-menopausal women.     

Structural diversity of the fibroblast-activating factors generated by human blood monocytes and U937 cells

The incubation of purified human blood monocytes with phytohemagglutinin (PHA) and of cultured U937 human monocyte-like cells with phorbol myristate acetate (PMA) evoked the generation of fibroblast-activating activity, as assessed by stimulation of the uptake of [3H]thymidine by human dermal fibroblasts. Filtration of the supernates from monocytes and U937 cells on Sephadex G-75 resolved fibroblast-activating factors of m.w. 25,000 to 40,000, designated FAF-M and FAF-U937, respectively, from smaller factors of an apparent m.w. of approximately 10,000. FAF-M and FAF-U937 were acidic by isoelectric focusing with respective pI values of 4.0 to 5.2 and 5.4 to 5.6. The smaller factors from both sources filtered on Sephadex G-25 in phosphate-buffered saline with an apparent m.w. of 10,000. However, filtration of the same factors on Sephadex G-25 in 0.1 M acetic acid revealed one predominant fibroblast-activating activity for each cell source of an apparent m.w. of 500 to 1000. The 500 to 1000 dalton factors were inactivated by treatment with trypsin and subtilisin, suggesting that the activity was attributable to fibroblast-activating peptides, termed FAP-M and FAP-U937. FAP-M and FAP-U937 each appeared to be composed of a predominant hydrophilic activity by reverse-phase high-performance liquid chromatography. Human blood monocytes and U937 monocytes both produce structurally diverse fibroblast-activating proteins and peptides, which may contribute to the immunologic regulation of wound healing and fibrosis.     

Characterization of Ca2+ influx induced by dimethylphytosphingosine and lysophosphatidylcholine in U937 monocytes

Calcium is a ubiquitous second messenger controlling a broad range of cellular functions. We previously observed that N,N-dimethyl-D-ribo-phytosphingosine (DMPH) and lysophosphatidylcholine (LPC) induced Ca2+ influx across the plasma membrane in U937 monocytes. In this study, we characterized the Ca2+ influx induced by DMPH and LPC. L-type voltage-gated Ca2+ channel blockers, verapamil and nifedipine, significantly reduced LPC-induced Ca2+ influx, but not DMPH-induced one. On the other hand, non-specific Ca2+ channel blockers, Ga3+ and La3+, considerably reduced DMPH- and LPC-induced Ca2+ influx. Preincubation of the cells with forskolin enhanced DMPH-induced Ca2+ influx, however, LPC-induced Ca2+ influx was not affected by the treatment. The enhancement by forskolin was blocked by KT5720, a PKA inhibitor. We also confirmed the presence of TRPM7 and absence of TRPM3 in U937 cells. Therefore, our characterization of Ca2+ influx in U937 human monocytes shows the presence of two different types of Ca2+ channels modulated by lysolipid molecules, DMPH and LPC. LPC may induce Ca2+ influx via L-type Ca2+ channels and DMPH seems to induce Ca2+ influx through TRPM7 in U937 human monocytes.     

Apoptosis induction by lectin isolated from the mushroom Boletopsis leucomelas in U937 cells

A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N'-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.     

The two mannose 6-phosphate receptors have almost identical subcellular distributions in U937 monocytes

Using a semiquantitative immunogold technique on ultrathin cryosections, the in situ subcellular distributions of the cation-dependent, 46-kDa mannose 6-phosphate receptor (small MPR) and of the cation-independent, 270-kDa mannose 6-phosphate receptor (large MPR) were for the first time compared. U937 cells were chosen because of their relatively high content of both receptor species. Of each receptor, about 12% occurred at the cell surface, 2% in the Golgi stack, and about 25% in vacuoles resembling endosomal vacuoles. About half of both receptors was found in tubules, presumably belonging to endosomes and trans-Golgi reticulum. It was concluded that the distribution of the small and large MPR were roughly similar. The only exception was formed by electron-dense vesicles occurring in the trans-Golgi region and surrounding endosomes. Dense vesicles contained significantly less small MPR (7%) than large MPR (12%).     

U937细胞中GATA1调控c-myb基因表达

c-myb是血细胞生成过程中的一个重要转录因子,与造血干细胞的增殖、分化、凋亡有关.在白血病、结肠癌、乳腺癌和黑色素瘤等恶性肿瘤中,c-myb异常表达,但是在白血病细胞中c-myb调控的机制尚不清楚.本研究探究了U937细胞中GATA1与c-myb的调控关系,可能对白血病研究和治疗提供帮助.用12-氧-十四烷酰佛波醇-13-乙酸酯(TPA)诱导U937细胞分化,并检测分化前后GATA1与c-myb蛋白的变化.Western blot结果显示U937细胞经TPA诱导分化后c-myb与GATA1蛋白均明显下降.利用慢病毒包装GATAl shRNA质粒和GATA1过表达质粒并感染到U937细胞中实现转录因子GATA1的敲降及过表达后,检测c-myb的mRNA和蛋白的表达水平.结果 显示:敲降GATA1后,c-myb的mRNA和蛋白质水平明显下降;过表达GATA1后,c-myb的mRNA和蛋白质水平明显上调.本研究揭示了U937细胞中GATA1对c-myb的正向调控关系,为白血病研究和治疗方案提供了新的思路.     

Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.