Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Modification of tyrosine (TyrOH) is used as a marker of oxidative and nitrosative stress. 3,3′-Dityrosine formation, in particular, reflects oxidative damage and results from the combination of two tyrosyl phenoxyl radicals (TyrO). This reaction is in competition with reductive processes in the cell which ‘repair’ tyrosyl radicals: possible reductants include thiols and ascorbate. In this study, a rate constant of 2 × 10⁶ M⁻¹ s⁻¹ was estimated for the reaction between tyrosyl radicals and glutathione (GSH) at pH 7.15, generating the radicals by pulse radiolysis and monitoring the tyrosyl radical by kinetic spectrophotometry. Earlier measurements have suggested that this ‘repair’ reaction could be an equilibrium, and to investigate this possibility the reduction (electrode) potential of the (TyrO,H⁺/TyrOH) couple was reinvestigated by observing the fast redox equilibrium with the indicator 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate). Extrapolation of the reduction potential of TyrO measured at pH 9–11 indicated the mid-point reduction potential of the tyrosyl radical at pH 7, E_m7(TyrO,H⁺/TyrOH) = 0.93 ± 0.02 V. This is close to the reported reduction potential of the glutathione thiyl radical, E_m7 = 0.94 ± 0.03 V, confirming the ‘repair’ equilibrium constant is of the order of unity and suggesting that efficient reduction of TyrO by GSH might require removal of thiyl radicals to move the equilibrium in the direction of repair. Loss of thiyl radicals, facilitating repair of TyrO, can arise either via conjugation of thiyl with thiol/thiolate or oxygen, or unimolecular transformation, the latter important at low concentrations of thiols and oxygen.     

Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Erythropoietin (Epo) is crucial for promoting the survival, proliferation, and differentiation of mammalian erythroid progenitors. The central role played by tyrosine phosphorylation of erythropoietin receptor (EpoR) in Epo-cell activation has focused attention on protein tyrosine phosphatases (PTPs) as candidates implicated in the pathogenesis of the resistance to therapy with human recombinant Epo. Prototypic member of the PTP family is PTP1B, which has been implicated in the regulation of EpoR signaling pathways. In previous reports we have shown that PTP1B is reciprocally modulated by Epo in undifferentiated UT-7 cell line. However, no information is available with respect to the modulation of this phosphatase in non-Epo depending cells or at late stages of erythroid differentiation. In order to investigate these issues we induced UT-7 cells to differentiate and studied their PTP1B expression pattern. Simultaneous observations were performed in TF-1 cells which can be cultured either with GM-CSF, IL-3 or Epo. We found that Epo induced PTP1B cleaveage in TF-1 and differentiated UT-7 cells. This pattern of PTP1B modulation may be due to an increased TRPC3/TRPC6 expression ratio which could explain the larger and sustained calcium response to Epo and calpain activation in Epo treated TF-1 and differentiated UT-7 cells.     

Ascorbate interacts with sodium selenite to increase glutathione peroxidase activity in selenium-deficient chick duodena cultured in vitro

Duodena from Selenium (Se)/vitamin E-depleted 19 d chick embryos were cultured in vitro for 0-30 h. The addition of sodium selenite to the culture medium was associated with increased selenium-dependent glutathione peroxidase (SeGSHpx) activity after 24 h of incubation. In the absence of Se or in the presence of sodium ascorbate supplementation alone, SeGSHpx activity showed a gradual decline over the same time period. When ascorbate was added, along with sodium selenite, SeGSHpx activity was increased earlier and to a greater extent than in the presence of Se alone. These observations show that ascorbate can influence the metabolism of sodium selenite, resulting in increased SeGSHpx activity.     

Induction of phospholipid hydroperoxide glutathione peroxidase in human polymorphonuclear neutrophils and HL60 cells stimulated with TNF-alpha

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is characterized as an important enzyme for protecting cells from oxidative stress-induced apoptosis and regulating the production of leukotrienes and prostanoids in cells overexpressing PHGPx. We studied whether the expression level of PHGPx fluctuates in polymorphonuclear leukocytes (PMNs) which were exposed to reactive oxygen species (ROS) and inflammatory cytokines at an inflammation site. Human peripheral PMNs up-regulated the expression level of PHGPx following culture with TNF-alpha, but not with IL-1beta, IL-8, and GRO. The up-regulated PHGPx expression was also observed in neutrophil-like cells that differentiated from the human leukemia cell line HL60 only after stimulation with TNF-alpha. However, macrophage-like differentiated HL60 cells and other cell lines, A498, ECV304, HeLa, U937, and HEK293, showed no increase in the PHGPx expression. This up-regulation of PHGPx was inhibited by treatment with the anti-oxidants, pyrrolidine dithiocarbamate, and N-acetyl-L-cysteine, and by inhibitors of NFkappaB and Src kinases. The stimulation of neutrophil-like differentiated HL60 cells with TNF-alpha induced activation of NFkappaB and c-Src kinase, and the activation was attenuated by treatment with the anti-oxidants. Up-regulation in neutrophil-like HL60 cells was also observed following exposure to H(2)O(2). These results indicate that activation of NFkappaB and/or Src kinases through ROS signaling may be involved in the up-regulation of the PHGPx in human PMNs stimulated by TNF-alpha.     

Ascorbate and glutathione homeostasis in vascular smooth muscle cells: cooperation with endothelial cells

Human umbilical vein smooth muscle cells (HUVSMCs) utilizeextracellular cystine, glutathione (GSH), andN-acetylcysteine (NAC) to synthesizecellular GSH. Extracellular cystine was effective from 5 µM, whereasGSH and NAC were required at 100 µM for comparable effects. Theefficacy of extracellular GSH was dependent on de novo GSH synthesis,indicating a dependence on cellular -glutamyltransferase (glutamyltranspeptidase). Coculture of syngenetic HUVSMCs and corresponding human umbilical vein endothelial cells (HUVECs) on poroussupports restricted cystine- or GSH-stimulated synthesis of HUVSMC GSHwhen supplied on the "luminal" endothelial side. Thus HUVSMC GSHrapidly attained a steady-state level below that achieved in theabsence of interposed HUVECs. HUVSMCs also readily utilizeboth reduced ascorbate (AA) and oxidized dehydroascorbate (DHAA) overthe range 50-500 µM. Phloretin effectively blocked both AA- andDHAA-stimulated assimilation of intracellular AA, indicating a role fora glucose transporter in their transport. Uptake of extracellular AAwas also sensitive to extracellular, but not intracellular, thioldepletion. When AA was applied to the endothelial side of the coculturemodel, assimilation of intracellular AA in HUVSMCs was restricted to asteady-state level below that achieved by free access.

     

pH homeostasis in promyelocytic leukemic HL60 cells

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.     

Defective chemiluminescence response in differentiated HL60 cells due to impaired degranulation

In the presence of dimethyl sulfoxide, the promyelocytic leukemic cell line, HL60, differentiates into apparently mature polymorphonuclear leukocytes. When correlating the superoxide production from HL60 cells with the number of phagocytozing and NBT-positive cells, no difference was observed in comparison with normal peripheral blood leukocytes. In contrast, the luminol-dependent chemiluminescence was greatly impaired in the differentiated HL60 cells. Analysis of degranulation, i.e., release of myeloperoxidase and N-acetyl-beta-glucosaminidase- and myeloperoxidase-mediated iodination by HL60 cells, suggested that the defective chemiluminescence response observed in HL60 cells may be due to impaired release of myeloperoxidase from azurophilic granulae. This may lead to impaired microbicidal activity in these cells.     

Myeloperoxidase-catalyzed phenoxyl radicals of vitamin E homologue, 2,2,5,7,8-pentamethyl- 6-hydroxychromane, do not induce oxidative stress in live HL-60 cells

We used myeloperoxidase-containing HL-60 cells to generate phenoxyl radicals from nontoxic concentrations of a vitamin E homologue, 2,2, 5,7,8-pentamethyl-6-hydroxychromane (PMC) to test whether these radicals can induce oxidative stress in a physiological intracellular environment. In the presence of H(2)O(2), we were able to generate steady-state concentrations of PMC phenoxyl radicals readily detectable by EPR in viable HL-60 cells. In HL-60 cells pretreated with succinylacetone, an inhibitor of heme synthesis, a greater than 4-fold decrease in myeloperoxidase activity resulted in a dramatically decreased steady-state concentrations of PMC phenoxyl radicals hardly detectable in EPR spectra. We further conducted sensitive measurements of GSH oxidation and protein sulfhydryl oxidation as well as peroxidation in different classes of membrane phospholipids in HL-60 cells. We found that conditions compatible with the generation and detection of PMC phenoxyl radicals were not associated with either oxidation of GSH, protein SH-groups or phospholipid peroxidation. We conclude that PMC phenoxyl radicals do not induce oxidative stress under physiological conditions in contrast to their ability to cause lipid peroxidation in isolated lipoproteins in vitro.     

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5–1.000 mV/cm p-p) were applied for 1 h to cells (10⁷/ml) in Teflon chambers at 37 °C in the presence or absence of 2 μM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100–1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA. EFs (33–330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents. © 1996 Wiley-Liss, Inc.     

Synthesis of hydroxyferrocifen and its abilities to protect DNA and to scavenge radicals

Abstract  

The aim of this work was to clarify the effect of the position of the hydroxyl group on the antioxidant capacity of hydroxyferrocifen by means of a chemical kinetic method. Propionylferrocene and benzoylferrocene condensed with 4-hydroxydiphenylketone, 3,4-dihydroxydiphenylketone, and 4,4′-dihydroxydiphenylketone to form FP3, FP4, FB3, and FB4 with a single hydroxyl group and FP34, FP44, FB34, and FB44 with two hydroxyl groups. These hydroxyferrocifens were applied in Cu²⁺/glutathione (GSH)-induced, hydroxyl radical (·OH)-induced, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA, and in trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+·). It was found that these hydroxyferrocifens acted as prooxidants in Cu²⁺/GSH-induced oxidation of DNA and exhibited very weak effects on ·OH-induced oxidation of DNA. FP3, FP4, FB3, and FB4 can only retard the rate of AAPH-induced oxidation of DNA, whereas FP44, FB44, FB34, and FP34 can trap 11.9, 7.1, 6.2, and 4.9 radicals, respectively, in AAPH-induced oxidation of DNA. The ability to trap ABTS^+· followed the order FB4 > FP44 > FB34 > FB44 > FP34. It was concluded that two hydroxyl groups at the para position of two benzene rings rather than at the ortho position in the same benzene ring were beneficial for hydroxyferrocifen to increase the antioxidant capacity.     

Dual Mg2+ control of formyl-peptide-receptor--G-protein interaction in HL 60 cells. Evidence that the low-agonist-affinity receptor interacts with and activates the G-protein

In neutrophils and several other phagocytic cell types, a pertussis- and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) Gp couples receptors for N-formylmethionine-containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formyl-peptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and (b) the receptor-mediated activation of Gp. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet-Leu-[3H]Phe. At the same time, Mg2+ largely increased the potency of guanosine-5'-(3-O-thio)triphosphate, but not of GDP or guanosine-5'-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mg2+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor-Gp complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non-selective fashion. It is suggested that the former site is located on Gp and that interaction of Mg2+ with this site is necessary for the receptor-mediated G-protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl-peptide receptor binding properties by guanine nucleotides is independent of Gp activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high-affinity GTPase activity is stimulated by fMet-Leu-Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor--G-protein interaction and (b) high-affinity agonist binding is not a prerequisite for the receptor-mediated activation of the G-protein.     

Dendritic antioxidants with pyrazole as the core: ability to scavenge radicals and to protect DNA

Chalcones with or without a para-hydroxyl group were condensed with phenylhydrazine-related compounds to form 1,3,5-triphenyl-1H-pyrazole (TPP), 4-(1,5-diphenyl-1H-pyrazol-3-yl)phenol (APP), 4-(1,3-diphenyl-1H-pyrazol-5-yl)phenol (BPP), and 4-(3,5-diphenyl-1H-pyrazol-1-yl)phenol (CPP), in which the phenyl group formed a dendritic structure with pyrazole as the core. Thus, the aim of this work was to explore the antioxidant capacities of TPP, APP, BPP, and CPP in trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+?) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) and in inhibiting Cu^2 +/glutathione (GSH)-, ^?OH-, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. TPP can react with ABTS^+? and DPPH, indicating that the N atom in pyrazole possesses radical-scavenging ability. Moreover, APP, BPP, and CPP can trap 1.71, 1.81, and 1.58 radicals, respectively, in protecting DNA against AAPH-induced oxidation. Thus, the combination of pyrazole with a phenyl group exerted antioxidant ability although only one phenolic hydroxyl group was involved. However, these compounds showed weak protective effect against Cu^2 +/GSH-induced oxidation of DNA and even a pro-oxidant effect on ^?OH-induced oxidation of DNA.     

Cell cycle arrest induced by Pisosterol in HL60 cells with gene amplification

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 μg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 μg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 μg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 μg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.     

Cytotoxicity effect of algal polysaccharides on HL60 cells

The present study shows the cytotoxic effect of three different classes of algal polysaccharides on HL60 cells. Three galactofucans, fucoidan, and glucan were the polysaccharides utilized in this analysis. The parameters used for evaluating cell viability were [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) reduction, protein content, and phosphatase activity. We demonstrated stimulation of phosphatase activity, MTT reduction, and protein content in relation to three types of galactofucans (1, 2, and 3) with different molecular weights (1600, 1200, and 360 kD). However, when HL60 cells were treated with galactofucan type 3, the total protein remained unchanged. Under the same experimental conditions, an expressed increase in the phosphatase activity was detected when galactofucan 3 was utilized. In relation to the mitochondrial function, the stimulation was higher in cells treated with galactofucan type 1. Fucoidan did not have a significant effect on MTT reduction, but protein content was decreased (IC₅₀ around 30 μg/ml). Glucan also activated all the parameters that were analyzed, and this effect was more expressed in the phosphatase activity and in the protein content. This study provides new insights into the cytotoxic action of polysaccharides on HL60 cells and suggests for the first time the possible involvement of phosphatases in this process. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1613–1617.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.     

Analysis of P-glycoprotein phosphorylation in HL60 cells isolated for resistance to vincristine.

In the present study we have analyzed the involvement of phosphorylation in the function of P-glycoprotein and have also examined sites of phosphorylation along the P-glycoprotein polypeptide chain. The results show that in HL60 cells isolated for resistance to vincristine the protein kinase inhibitor staurosporine induces a major inhibition in the phosphorylation of P-glycoprotein. Further studies show that under the same conditions in which staurosporine inhibits P-glycoprotein phosphorylation there is a concomitant increase in cellular drug accumulation and a major inhibition in drug efflux. Additional studies using pulse-chase experiments show that the P-glycoprotein phosphate groups are metabolically active and that the protein undergoes rapid cycles of phosphorylation and dephosphorylation in the cell. Structural analyses demonstrate that cleavage of 32P-labeled P-glycoprotein at Asp-Pro linkages with formic acid results in the formation of a major phosphorylated peptide of 35 kDa and a minor peptide of 42 kDa. Western blot analysis using site-specific anti-sera against P-glycoprotein suggests that P35 represents a phosphorylated fragment containing P-glycoprotein amino acids 446-744. Analysis of tryptic peptides using site-specific antisera identifies a second major phosphorylated region of P-glycoprotein which contains amino acids 745-1088. These studies thus suggest that phosphorylation plays an important role in the biological activity of P-glycoprotein. The results also indicate that two adjacent internal regions are highly phosphorylated in the P-glycoprotein molecule.     

Antileukemic agent alkyllysophospholipid regulates phosphorylation of distinct proteins in HL60 and K562 cells and differentiation of HL60 cells promoted by phorbol ester

The tumor-promoting 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulated phosphorylation of several proteins in block I (including protein Ia) and protein 3 in HL60 cells. The antileukemic agent alkyllysophospholipid (ALP) inhibited the TPA-stimulated phosphorylation of these proteins and the TPA-induced differentiation of the cells. In comparison, TPA only stimulated phosphorylation of protein 3 in K562 cells which, in contrast, were not induced to differentiate by TPA and lacked protein Ia and had a very high basal phosphorylation of protein B. ALP inhibited phosphorylation of protein 3 as well as protein B in K562 cells. The data suggest that the presence of distinct phosphoproteins and regulation of their phosphorylation may be related to the selective susceptibility of the two leukemia cell lines to the maturating effect of TPA and cytotoxicity of ALP.     

Transglutaminase activity increases in HL60 cells induced to differentiate with retinoic acid and TPA but not with DMSO

HL60 cells induced to differentiate into myeloid cells by retinoic acid exhibited a 300-fold increase in transglutaminase (TGase) activity which peaked on day 5. HL60 cells induced to differentiate into monocytes by a phorbol ester tetradecanoylphorbol-12-myristate-13-acetate (TPA) had a greater than 840-fold increase in TGase activity on day 7. In contrast, cells induced to differentiate along the myeloid pathway by dimethyl sulfoxide (DMSO) exhibited no increase in TGase activity. Elevation of TGase activity appears to be characteristic of monocyte differentiation and retinoic acid-induced myeloid differentiation but not of myeloid differentiation in response to DMSO.