Role of Catalase in Monocytic Differentiation of U937 Cells by TPA: Hydrogen Peroxide As a Second Messenger

Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger

T Yamamoto1, N Sakaguchi1, M Hachiya1, F Nakayama1, M Yamakawa2 and M Akashi1

1Department of Radiation Emergency Medicine, The Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba-city, Chiba, Japan and 2Department of Pathology, Yamagata University Faculty of Medicine, Yamagata-city, Yamagata, Japan

Human promonocytic cell line U937 cells can be induced to atherosclerosis.4,5 Internalization of foreign substances by differentiate into macrophages by treatment with 12-O-tetra- macrophages is mediated through distinct surface receptors that decanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and recognize their targets, such as microorganisms, tumor cells and CD36, with concomitant morphological changes. Moreover, cellular debris. Following phagocytosis, macrophages synthe- TPA enhanced reactive oxygen species (ROS) generation in size and release reactive oxygen species (ROS), a process called these cells, and phagocytic ability was also stimulated during ‘respiratory burst.’ Thus, the generation of ROS is important differentiation. The antioxidant agent N-acetyl-L-cysteine inhib- for much of the microbicidal and antitumor activity of ited the TPA-induced differentiation of U937 cells. TPA treat- macrophages. ment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H O )to Since the discovery of ROS, primary focus has been directed 2 2 at the oxidative damage to biologic macromolecules including H2O and O2. In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of proteins as well as DNA and lipids. Potential sources of ROS in superoxide into H2O2 and O2 without affecting the levels of phagocytic cells are nicotinamide adenine dinucleotide phos- copper-zinc superoxide dismutase or glutathione peroxidase 1, phate (NADPH) oxidases and the mitochondrial electron which removes H2O2 using glutathione as substrate. Treatment transport chain.6–8 NADPH oxidases are plasma membrane- of U937 cells with catalase inhibited the enhancement of ROS associated enzymes found in a variety of cells. This enzyme generation induced by TPA, and blocked the TPA-induced À differentiation of U937 cells. Human promyelocytic cell line catalyzes the production of O2 by the one-electron reduction HL60 cells were also induced to differentiate into macrophages of oxygen using NADPH as electron donor. On the other hand, by TPA. However, HP100-1 cells, its variant cell line over- it has also been shown that ROS are generated in a variety expressing catalase, were resistant to TPA-induced differentia- of cells other than phagocytes, and that ROS are involved tion. Our results suggest that catalase inhibits monocytic in normal physiologic signaling including cell proliferation or differentiation by TPA; the decrease in catalase level and the survival.6,9,10 Previously, we showed that ROS regulate cell accumulation of H2O2 are significant events for monocyte/ 11 macrophage differentiation by TPA. growth in human promyelocytes HL60. Leukemia (2009) 23, 761–769; doi:10.1038/leu.2008.353; Recently, there is a growing body of evidence regarding the published online 18 December 2008 ROS signal playing an important role in hematopoietic Keywords: catalase; hydrogen peroxide; monocytic differentiation; systems. The regulation of oxidative stress by ROS has been TPA shown to be necessary for the maintenance of the capacity of hematopoietic stem cells to self-renew.12 Activation with granulocyte-macrophage colony-stimulating factor and other growth factors, including interleukin-3, stem cell factor and thrombopoietin, have been shown to be associated with 13,14 Introduction alterations in the levels of hydrogen peroxide (H2O2). Moreover, it has been shown that differentiation of monocytes Stem cells form hematopoietic progenitor cells with a more into macrophages leads to an increase in the baseline oxidant limited differentiation potential. These progenitors yield blood tone.15,16 Furthermore, studies have suggested that ROS may be precursors that cause unilineage differentiation and production involved in the regulation of neuronal or cardiomyocyte of mature blood cells, including red blood cells, megakaryo- differentiation.17,18 However, the roles of ROS remain unclear, cytes, monocytes/macrophages, granulocytes and lympho- and the type of ROS involved in the differentiation of 1–3 cytes. Monocytes enter the circulation and migrate into hematopoietic cells is not yet known. various tissues, where they terminally differentiate into macro- Leukemic cell lines, such as U937 and HL60, are blocked phages in response to stimuli. Macrophages are phagocytes that at different stages of differentiation.19 These cells can differ- play important roles in the immune system, especially in the entiate to more mature cells following exposure to various host defense against infection or malignancy, and they also agents in vitro. Therefore, these leukemic cell lines provide participate in the development of a variety of diseases including not only the opportunity to study the induction of cell differentiation but also the mechanisms for cell maturation. Correspondence: Dr M Akashi, Department of Radiation Emergency Using these cell lines, we studied the roles of ROS in phagocytic Medicine, The Research Center for Radiation Emergency Medicine, differentiation induced by 12-O-tetradecanoylphorbol-13-acet- National Institute of Radiological Sciences, Chiba-city, Chiba 263- ate (TPA), a member of phorbol esters. We found that the levels 8555, Japan. E-mail: [email protected] of catalase are reduced and H2O2 acts as a second messenger Received 26 July 2008; revised 18 September 2008; accepted 14 in TPA signaling essential for the differentiation of phaogocytic October 2008; published online 18 December 2008 cells. Role of catalase in monocytic differentiation T Yamamoto et al 762 Materials and methods cells were combined and were stained with either anti-CD11b or –CD36 antibody-FITC (Immnotech, Marseille, France) at 4 1C Reagents for 30 min. Fluorescent intensities were analyzed by FACSCa- 12-O-tetradecanoylphorbol-13-acetate was purchased from libur (Becton, Dickinson and Company, Franklin Lakes, NJ, Sigma-Aldrich Co. (St Louis, MO, USA). TPA was dissolved in USA). To determine nonspecific binding, FITC-conjugated IgG1 dimethyl sulfoxide. When TPA was added to cells, the isotype antibody was used, and its intensities were subtracted concentration of dimethyl sulfoxide was less than 0.1%. from those of cells with CD11b or CD36. N-acetyl-L-cysteine (NAC) and human catalase were purchased from Calbiochem (La Jolla, CA, USA). NAC was dissolved in water at a concentration of 1 M and stored at À20 1C until use. Analysis of phagocytic activity Superoxide dismutase (SOD) (Cu/Zn type) from bovine The cells were incubated at 37 1C for 10 min with luminol- erythrocytes was purchased from Wako Pure Chemical bound microbeads (Kamakura Techno-Science Inc., Kanagawa, 20 Industries Ltd (Osaka, Japan). Anti-catalase rabbit polyclonal Japan), as directed by the manufacturer’s protocol. Chemilu- antibody was from Calbiochem. Anti-manganese SOD minescence, which was generated by the reaction between (MnSOD) rabbit polyclonal antibody was a product of Stressgen internalized microbeads-bound luminol and intracellular ROS, (Ann Arbor, MI, USA). Anti-copper-zinc SOD (CuZnSOD) rabbit was measured for 1 min using a Lumat LB 9507 Luminometer polyclonal antibody was purchased from Santa Cruz Biotech- (Berthold Technologies, Bad Wildbad, Germany). nology Inc. (Santa Cruz, CA, USA). Anti-glutathione peroxidase 1 (GSH-Px1) sheep polyclonal antibody was from Abcam plc. (Cambridge, UK). Anti-glyceraldehyde-3-phosphate dehydrogen- Intracellular levels of ROS Intracellular ROS were measured according to the methods ase (G3PDH) rabbit polyclonal antibody was purchased from 21 described previously. Briefly, cells were incubated with 5 mM Trevigen (Gaithersburg, MD, USA). Horseradish peroxidase- CM-H DCFDA (5-6-chloromethyl-20,70-dichlorodihydrofluores- conjugated anti-rabbit immunoglobulin G (IgG) antibody was a 2 cein diacetate, acetyl ester) (Molecular Probes, Eugene, OR, product of Amersham Biosciences (Buckinghamshire, UK). USA) in phosphate-buffered saline for 15 min at 37 1C in the Horseradish peroxidase-conjugated anti-sheep/goat IgGs dark. During incubation, the acetate groups on CM-H DCFDA antibody was purchased from Binding Site LTD (Birmingham, 2 are cleaved by intracellular esterase, trapping the probe inside UK). cells. CM-H2DCFDA was chosen because it showed better retention in cells than other derivatives. Endogenous ROS levels Cells and cell culture were analyzed by FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with excitation and U937 cells, derived from a diffuse human histiocytic lymphoma emission settings of 488 and 530 nm, respectively. (American Type Tissue Culture Collection, Manassas, VA, USA), were cultured in minimum essential medium-a (Gibco Invitro- gen, Carlsbad, CA, USA) supplemented with 10% fetal calf Cytotoxicity assay serum (Biological Industries, Kibbutz Beit Haemek, Israel) at The cytotoxicity of cells was determined by a colorimetric 1 37 C in a humidified atmosphere containing 95% air/5% CO2. assay based on the cleavage of the tetrazolium salt WST-1 (4-[3- Human promyelocytic HL60 and HP100-1 cells (Japanese (4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene Cancer Research Resources Bank, Tokyo, Japan) were cultured disulfonate) by mitochondrial dehydrogenase in viable cells a 11 in minimum essential medium- supplemented with 7% fetal (Roche Diagnostics GmbH, Mannheim, Germany). Cells were calf serum. HP100-1 cells were maintained in the same medium seeded at an initial concentration of 5 Â 104 cells/well in a supplemented with 100 mM of H2O2. These cells were washed medium with or without various concentrations of H2O2 in a 96- and cultured in medium without H2O2 1 week before each 5 well microplate and cultured for 48 h. Then tetrazolium salt experiment. For differentiation, the cells were seeded at 2 Â 10 WST-1 was added to the cultures and incubated at 37 1C for 1 h. cells/ml and TPA was added. The production of formazan was measured by microplate reader (Bio-Rad Model 3550, Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 480 nm. Giemsa staining The floating U937 cells were fixed in methanol at À20 1C overnight. One drop of the fixed cell suspension was applied Northern blot analysis onto a glass slide and dried. For staining of the adherent cells, Northern blot analysis was performed as described previously.11 the micro cover glasses (Matsunami Glass IND. LTD, Osaka, Briefly, total RNA from cells was obtained by the guanidinium/ Japan) were placed on the bottom of culture dishes before hot phenol method.22 After denaturation at 65 1C, RNA (15 mg/ culture. After treatment with TPA, these cover glasses were lane) was electrophoresed in an agarose-formaldehyde gel (1%) removed and cells adherent to these cover glasses were also and transferred to a nylon-membrane filter (Amersham fixed in methanol. The fixed cells were stained with May- Biosciences). The filters were sequentially hybridized with Gruenwald (Wako Pure Chemical Industries Ltd, Osaka, Japan) 32P-labeled probe for 16 to 24 h at 42 1C. After washing, filters and Giemsa’s solution (Merck & Co. Inc., Whitehouse Station, were exposed to X-ray film (RX, Fuji Photo Film Co. Ltd, NJ, USA). Kanagawa, Japan). Autoradiograms were developed at different exposures. The DNA probes for catalase, MnSOD, CuZnSOD and GSH-Px1 were prepared as described previously.11 Analysis of CD11b and CD36 expression For measurement of the expression of CD11b and CD36, adherent cells were collected by scraping with a rubber Western blot analysis policeman, after removing non-adherent cells and washing with Cells were sonicated in ice-cold potassium phosphate buffer phosphate-buffered saline. Adherent cells and non-adherent (0.05 M, pH 7.8) with diethylenetriamine pentaacetic acid and

Leukemia Role of catalase in monocytic differentiation T Yamamoto et al 763 protease inhibitor cocktail Complete (Boehringer Mannheim, 30 Mannheim, Germany). After centrifugation, the protein concentration of the supernatant was determined by the method of 25 Bradford (Bio-Rad Laboratories). An equal amount of protein 20 (40 mg) was boiled for 5 min in SDS-polyacrylamide gel electrophoresis sample loading buffer, and separated on 10% 15 SDS-polyacrylamide gel. Proteins were transferred to nitro- 10 cellulose membranes, and then probed with the indicated antibodies. Membranes were subsequently incubated with CD11b expression 5 (Fluorescent intensity) secondary antibody conjugated to horseradish peroxidase, and 0 immunocomplexes were visualized by ECL Western Blotting Control 0.2 2 20 200 detection reagents (Amersham Biosciences) according to the TPA (nM) instructions of the manufacturer. 2500 TPA(-) Assay for catalase activity 2000 TPA(+) Catalase activity was assayed as described previously.11.Cells were sonicated in ice-cold potassium phosphate buffer (0.05 M, 1500 pH 7.8) with diethylenetriamine pentaacetic acid. After centrifugation, the protein concentration in each sample was determined by the method of Bradford. An equal amount of 1000 each sample, 30 mg of protein, was electrophoresed in a 10% non-denatured polyacrylamide gel. The gel was stained with Phagocytic activity 500 (Luminescent intensity) 50 mg/ml of horseradish peroxidase in potassium phosphate buffer for 45 min and then added with 5 mM H O for 10 min. 0 2 2 Control 3 6 12 24 Then the gel was soaked in 0.5 mg/ml of diaminobenzidine until staining was completed. Time (hr) Figure 1 Monocytic differentiation of U937 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). U937 cells were induced to Results differentiate into mature monocytes/macrophages by TPA. (a) Expres- sion of CD11b in U937 cells treated with TPA. Cells were cultured for Monocytic differentiation of U937 cells induced by TPA 24 h with TPA at various concentrations. Expression of CD11b was analyzed by flow cytometry. The results are presented as mean±s.d. U937 cells are known to differentiate along a monocyte/ of fluorescent intensities from three independent experiments. (b) macrophage lineage by TPA, and this cell line represents Phagocytic activity in U937 cells treated with TPA. Cells were cultured 19,23 a good model for cell differentiation. The expression of the with TPA at a concentration of 20 nM for various durations and then gene encoding CD11b (integrin a-M) is one of the characteristics luminol-bound microbeads were added. Phagocytic activity was for the differentiation of myeloid precursors into mature measured by luminometer. The results are presented as mean±s.d. monocytic cells.24 To investigate the effect of TPA on of luminescent values from three independent experiments. the expression of this monocytic differentiation marker, CD11b, U937 cells were treated with different concentrations of TPA (0–200 nM) for 24 h, and the expression of cell surface marker CD11b was monitored by flow cytometry cytes and in tissue-resident macrophages, and it is thought to (Figure 1a). U937 cells constitutively expressed the CD11b mediate the phagocytosis of apoptotic cells and the endocytic protein at a very low level (fluorescent intensity: 0.3±0.2), uptake of modified lipoproteins.27–29 When U937 cells were while culturing without TPA did not affect the expression of treated with 20 nM TPA, a significant induction of CD36 CD11b in these cells. On the other hand, the CD11b expression expression was observed at 6 h (P ¼ 0.0073), with the expression was induced almost dose dependently when these cells were levels then increasing in a time-dependent manner similar to the treated with TPA. Cells treated with 0.2 nM of TPA had a CD11b expression (data not shown). significant higher level of CD11b expression compared with Differentiation induces changes in cells that also make control cells (7.0±0.8, P ¼ 0.00039) and 2 nM of TPA addition- them more functional. One of these changes is an activation ally induced the CD11b expression (23.2±2.5, P ¼ 0.00022). of their phagocytic ability.30 The phagocytic ability of U937 However, there was no significant difference in CD11b cells was determined by an assay that uses chemiluminescence expression among cell groups receiving 2, 20 and 200 nM of caused by interaction between phagocytized microsphere- TPA. Kinetic studies found that a significant increase in the bound luminol and reactive oxygen in phagosomes.20 A time- expression of CD11b was observed in cells treated with 20 nM of course experiment was performed in cells treated with 20 nM of TPA for 3 h (P ¼ 0.018), with the fluorescent intensities TPA (Figure 1b). A significant increase in phagocytic activity then increasing in a time-dependent manner until 24 h (data was observed in cells treated with TPA for 3 h (P ¼ 0.0049), and not shown). its activity increased in a time-dependent manner. We also We also studied the expression of CD36 in differentiation studied the differentiation of U937 cells morphologically by induction by TPA. CD36 is an integral membrane glycoprotein Giemsa staining. U937 cells normally grow in suspension and and a member of the scavenger receptor type B family show a smooth surface. Exposure to 20 nM of TPA induced implicated in the binding of lipoproteins, phosphatidylserine, differentiation of U937 cells, as confirmed by changes in thrombospondin-1 and the uptake of long-chain fatty acids.25,26 morphology; cells became adherent to the surface of the culture In the monocytic lineage, CD36 is expressed during the late plate within 24 h and extended pseudopodia within 48 h (data stages of differentiation in bone marrow, in circulating mono- not shown).

Leukemia Role of catalase in monocytic differentiation T Yamamoto et al 764 Accumulation of ROS in TPA-treated U937 cells during accumulation of MnSOD protein in a time-dependent manner. monocytic differentiation However, TPA did not affect the levels of CuZnSOD or GSH- The generation of ROS is an important function for the defense Px1 in these cells. Similar results were obtained by an assay for mechanism against pathogens in phagocytes. To examine catalase activity, and studies by northern blot analysis showed whether the induction of U937 cells to differentiate into results parallel to those from western blot analysis (data not macrophages affected the generation of ROS, TPA-treated shown). U937 cells were stained with cell-permeable and oxidation- sensitive dye, CM-H2DCFDA, and the fluorescent intensity was analyzed by flow cytometry (Table 1). The intracellular ROS Effect of NAC on differentiation of U937 cells level was significantly higher at 3 h in cells treated with 20 nM of To further study the involvement of ROS generation in the TPA than in untreated cells (P ¼ 0.0057), and TPA increased the differentiation of U937 cells, the effect of an antioxidant agent, level of ROS in a time-dependent manner. NAC, on TPA-induced differentiation of U937 cells was determined. U937 cells were treated with 25 mM of NAC for 1 h, and then TPA was added to these cells. Morphologically, no macrophage-like cells were observed when U937 cells were Levels of antioxidant enzymes in U937 cells during cultured with 20 nM of TPA for 48 h in the presence of NAC (data differentiation not shown). Then, the induction of CD11b expression by TPA As shown above, the accumulation of ROS occurred during the was studied. Treatment with NAC inhibited the expression of differentiation of U937 cells by TPA. We also studied the levels CD11b by these cells treated with TPA by almost 70% (TPA: of antioxidant enzymes in these cells (Figure 2). Western blot 93±2%, TPA þ NAC: 28±4%, P ¼ 0.00001). Furthermore, analysis showed that U937 cells constitutively expressed stimulation of phagocytic activity by TPA was also blocked by catalase, MnSOD, CuZnSOD and GSH-Px1 at various levels. more than 60% upon NAC treatment (vehicle control: TPA treatment decreased the level of catalase at 24 h and even 111±12%, NAC: 41±0.2%, P ¼ 0.0006). Although it has been more at 48 h. In contrast, TPA markedly stimulated the reported that NAC treatment caused apoptosis during the differentiation of human monocytes,16 NAC treatment failed to show any effect on cell viability in this experiment (data not Table 1 Accumulation of ROS in U937 cells treated with TPA shown).

Control 3 h 6 h 12 h 24 h Effects of catalase or SOD on accumulation of ROS by Untreated 1430±18 1417±23* 1433±23** 1442±24*** 1445±25**** * ** *** **** TPA in U937 cells TPA (20 nM) 1551±27 1634±25 1798±33 1948±31 À Superoxide dismutases catalyze dismutation of superoxide (O2 ) Abbreviations: PBS, phosphate-buffered saline; ROS, reactive oxygen into oxygen (O2) and H2O2, and catalase catalyzes the species; TPA, 12-O-tetradecanoylphorbol-13-acetate. decomposition of H2O2 to water (H2O) and O2. The decrease Cells were cultured with 20 nM of TPA for various durations and in the cellular levels of catalase during differentiation suggests then stained with CM-H2DCFDA in PBS for 15 min at 37 1C. Generation of ROS was analyzed by flow cytometry. The results are that the accumulation of H2O2 might be involved in the expressed by fluorescent intensities and presented as mean±s.d. differentiation by TPA. To investigate the roles of H2O2 in the of three independent experiments. *P ¼ 0.00571, **P ¼ 0.00108, differentiation of U937 cells, these cells were pretreated with ***P ¼ 0.00024, ****P ¼ 0.00006. either catalase (500 U/ml) or SOD (300 U/ml) for 30 min, and then cultured for 24 h with or without TPA (20 nM) in the presence of each factor (Figure 3a). Treatment of cells with Control 12 24 48 (hr) catalase alone significantly reduced the level of ROS in cells ± ± TPA (20nM) – – +–+–+ (control: 1451 54, catalase alone: 1056 38, P ¼ 0.00104). However, SOD treatment alone did not affect the levels in these Catalase cells. On the other hand, catalase inhibited the accumulation of ROS by TPA completely (P ¼ 0.00022). SOD also blocked the accumulation of ROS by TPA (P ¼ 0.00230). MnSOD

CuZnSOD Effects of catalase or SOD on expression of CD11b and phagocytic activity by TPA In parallel to the ROS levels, we also studied the effects of GSH-Px1 catalase and SOD treatment on the CD11b expression proﬁles (Figure 3b) and phagocytic activities in cells treated with TPA G3PDH (Figure 3c). Treatment of these cells with either catalase or SOD blocked the CD11b expression by TPA signiﬁcantly (control Figure 2 Expression of antioxidant enzymes in 12-O-tetradecanoyl- and catalase: P ¼ 0.00025, control and SOD: P ¼ 0.01750) phorbol-13-acetate (TPA)-treated U937 cells. Cells were cultured with (Figure 3b). Thus, the results of CD11b expression were in or without 20 nM TPA for various durations. After cell lysis, 40 mgof accord with those of ROS accumulation in TPA-treated cells, protein was electrophoresed in a 10% SDS-polyacrylamide gel and whereas no changes in CD11b expression were observed in transferred to a nitrocellulose membrane. Western blot analysis was cells without TPA treatment. On the other hand, only catalase performed using anti-catalase, manganese superoxide dismutase treatment inhibited the phagocytic activity induced by TPA (MnSOD), copper-zinc SOD (CuZnSOD), anti-glutathione peroxidase 1 (GSH-Px1) or anti-glyceraldehyde-3-phosphate dehydrogenase (P ¼ 0.00045), and none of the other factors had any effect on (G3PDH) antibody. The levels of G3PDH were presented as loading these cells without TPA (Figure 3c). During a series of control. experiments, treatment with catalase or SOD had no effect on

Leukemia Role of catalase in monocytic differentiation T Yamamoto et al 765 11 3000 H2O2. Northern blot analysis showed that the level of catalase TPA(-) mRNA was more than six times higher in HP100-1 cells than in 2500 TPA(+) HL60 cells (Figure 4a). On the other hand, there were no differences in the levels of MnSOD, CuZnSOD or GSH-Px1 2000 between the two cell lines. We also studied the sensitivity of these cells to H O . Both cell lines were cultured for 48 h with 1500 2 2 or without H2O2 (0, 20, 100, 200 mM), and a colorimetric assay 1000 for cytotoxicity was performed using tetrazolium salt WST-1

ROS production (Figure 4b). Spectrometric analysis showed that the treatment of

(Fluorescent intensity) 500 HL60 cells with H2O2 decreased the absorbance in a dose- dependent manner; a significant decrease was observed at 0 100 mM of H2O2 in HL60 cells (P ¼ 0.0006), and 200 mM of H2O2 Control Catalase SOD decreased the absorbance to as low as 12±3% of the control level (P ¼ 0.0004). However, no significant changes in absor- 45 TPA (-) bance were observed even when HP100-1 cells were exposed 40 TPA (+) to 100 mM of H2O2 compared with control cells. Furthermore, 35 we studied the capability for the dismutation of H2O2. 30 Fluorescence analysis using DCFH-DA showed that culturing m 25 HL60 cells with 100 M of H2O2 for 5 min increased the mean and peak fluorescent intensities from 92 and 63 to as high as 524 20 and 133, respectively (Figure 4c). On the other hand, the mean 15 and peak fluorescent intensities were shifted from 103 and 42 to

CD11b expression 10 117 and 64, respectively, in HP100-1 cells cultured with H2O2, (Fluorescent intensity) 5 suggesting that HP100-1 cells had a significantly greater capability for the dismutation of H2O2 than HL60 cells. These 0 11 Control Catalase SOD results were in line with our previous findings. To further explore the roles of catalase in monocytic 3500 differentiation, we compared the differentiation induction by TPA(-) TPA between HL60 cells and its variant cell line, HP100-1 cells. 3000 TPA(+) HL60 and HP100-1 cells were cultured with TPA at various 2500 concentrations for 24 h, and then the expression of CD11b was examined by flow cytometry (Figure 4d). Both cell lines showed 2000 no expression of CD11b without TPA. However, a markedly increased level of fluorescent intensity was detected in HL60 1500 cells treated with TPA at a concentration of 2 nM (control and 1000 2nM TPA, P ¼ 0.001457), and a plateau was reached at 20 nM Phagocytic activity (control and 20 nM TPA, P ¼ 0.000139). Treatment with TPA (Luminescent intensity) 500 also increased the levels of fluorescent intensity in HP100-1 0 cells. However, the magnitude of the induction of CD11b Control Catalase SOD expression was much more prominent in HL60 than in HP100-1 cells, with the induced level in HP100-1 cells being less than Figure 3 Effects of antioxidants on 12-O-tetradecanoylphorbol-13- acetate (TPA)-induced differentiation in U937 cells. Cells were half of that in HL60 cells (HL60 and HP100-1, 2 nM: pretreated with catalase (500 U/ml) or superoxide dismutase (SOD) P ¼ 0.01723, 20 nM: P ¼ 0.01308, 200 nM: P ¼ 0.01073). (300 U/ml) for 30 min and then cultured with 20 nM TPA for 24 h in the Furthermore, we also compared the phagocytic activity induced presence of each. (a) Effects of catalase and SOD on reactive oxygen by TPA in these two cell lines (Figure 4e). Both cell lines had species (ROS) levels in U937 cells treated with TPA. Cells were stained almost no phagocytic capability when differentiation was not with CM-H2DCFDA and the generation of ROS was measured by flow ± induced. However, phagocytic activity was markedly stimulated cytometry. The results are shown as mean s.d. of three independent ± ± experiments. (b) Effect of TPA on CD11b expression in U937 cells in HL60 cells by TPA (untreated: 247 27, TPA: 11854 1346, pretreated with catalase and SOD. Expression of CD11b was measured P ¼ 0.0003). In contrast, TPA failed to induce phagocytic by flow cytometry. The results are shown as mean±s.d. of three activity in HP100-1 cells (untreated: 235±19, TPA: 269±4, independent experiments. (c) Effects of catalase and SOD on P ¼ 0.0645). phagocytic activity in U937 cells treated with TPA. Cells were cultured with 20 nM of TPA and catalase (500 U/ml) or SOD (300 U/ ml) for 24 h and then luminol-bound microbeads were added. Phagocytic activity was measured by luminometer. The results are Discussion shown as mean±s.d. of three independent experiments. Reactive oxygen species are constitutively generated from a partial reduction of molecular oxygen in cells. Since the cell viability and did not induce apoptosis at the concentrations discovery of ROS, attention has primarily focused on the used (data not shown). oxidative damage elicited by ROS within cells. Thus, ROS have traditionally been regarded as cytotoxic products of aerobic metabolism. However, recent studies have shown that ROS can Roles of catalase in monocytic differentiation act as a signaling molecule involved in various physiological The human promyelocytic cell line HL60 cells can be induced processes including cell growth, survival and cell differentia- to differentiate along a monocytic lineage by TPA.31 Its variant tion.6,9,10,18,21,32 On the other hand, phagocytes, such as cell line overexpressing catalase, HP100-1, is resistant to monocytes and macrophages, kill microorganisms through an

Leukemia Role of catalase in monocytic differentiation T Yamamoto et al 766 HL60 HP100

200 Catalase HL60 HP100 150

MnSOD 100

50 CuZnSOD

Cytotoxicity (% of control) 0 0 20 100 200 GSH-Px1 H2O2 (µM)

70 Untreated HL60 HL60 60 HP100 H2O2 50 40 30

Cell number 20

HP100 CD11b expression 10 (Fluorescent intensity) 0 0 100 101 102 103 104 Control 0.2 2 20 200 Fluorescence TPA (nM)

14000 Untreated 12000 TPA 10000 8000 6000 4000

Phagocytic activity 2000 (Luminescent intensity) 0 HL60 HP100

Figure 4 Differentiation induction of HL60 cells overexpressing catalase by 12-O-tetradecanoylphorbol-13-acetate (TPA). (a) The levels of catalase mRNA in HL60 and HP100-1 cells. Cells were harvested and total RNA was prepared. The basal levels of catalase, manganese superoxide dismutase (MnSOD), copper-zinc SOD (CuZnSOD) and anti-glutathione peroxidase 1 (GSH-Px1) mRNA were determined by northern blot 4 analysis. (b) Cytotoxicity of H2O2 in HL60 and HP100-1 cells. HL60 and HP100-1 cells (5 Â 10 per well) were cultured with or without H2O2 (20, 100, 200 mM) for 48 h in a 96-well microplate. Tetrazolium salt was added to cell cultures and incubated for 1 h at 37 1C. Absorbance was measured by microplate reader at 480 nm and normalized to the absorbance of untreated cells. Results are shown as mean±s.d. for triplicate assays. (c) Capacity for catalyzing H2O2 in HL60 and HP100-1 cells. Cells were incubated with 10 mM DCFH-DA for 15 min and then treated with or without 100 mM of H2O2 for 5 min. The levels of intracellular H2O2 were analyzed by ﬂow cytometry. (d) Expression of CD11b in differentiation- induced HL60 and HP100-1 cells with TPA. HL60 and HP100-1 cells were cultured for 24 h with TPA at various concentrations. Expression of CD11b was analyzed by ﬂow cytometry. The results are shown as mean±s.d. of three independent experiments. (e) Phagocytic activity in differentiation-induced HL60 and HP100-1 cells with TPA. HL60 and HP100-1 cells were cultured with TPA at a concentration of 20 nM for 48 h and then luminol-bound microbeads were added. Phagocytic activity was measured by luminometer. The results are presented as mean±s.d. of luminescent values from three independent experiments.

oxygen-dependent system; these cells produce ROS in response leukemia cell lines, including U937 and HL60.19 TPA is a to various stimuli, such as inflammation and infection,3 protein kinase C (PKC) activator, and the activation of PKC is suggesting that ROS are important especially in these phago- necessary for TPA-induced macrophage differentiation.33–36 In cytes. In this study, we showed that the level of catalase was our study, differentiation of U937 cells toward macrophages decreased along a monocytic differentiation induced by TPA, was documented by changes in cell morphology; floating cells and catalase inhibited its differentiation induction. The inhibi- form clumps and become adherent cells with protruding tory effect of catalase on monocytic differentiation was also pseudopodia. These morphological changes were accompanied confirmed by using cells overexpressing catalase, which had a by progressive increases in the levels of CD11b and CD36, 24,27,29 greater capability of dismutating H2O2. The data presented here surface markers of differentiated macrophages. More- show that catalase plays an important role in the regulation of over, the differentiated cells showed high phagocytic activity. the TPA-induced differentiation toward a macrophage/mono- Thus, we confirmed that TPA induced U937 cells to differentiate cyte lineage. into monocytic mature cells.19 The TPA is a well-known agent that signals monocyte/ Reactive oxygen species are generated mainly by NADPH macrophage differentiation of myeloid precursors and myeloid oxidase in monocytes and macrophages.6 There are many

Leukemia Role of catalase in monocytic differentiation T Yamamoto et al 767 reports on phagocytes, which were mainly based on studies of in monocytic differentiation induction by TPA, namely, that the the production of ROS in the context of bacterial killing. Studies accumulation of H2O2 is, at least in part, required for the have shown that the components of NADPH oxidase increased induction of differentiation by TPA. Our results suggest that during macrophage differentiation of HL60 and U937 cells.37–39 catalase is an important physiological regulator of differentiation It is well known that differentiation of monocytes into by TPA. macrophages leads to increased baseline level of ROS.15,16 In this study, SOD inhibited CD11b expression. However, TPA is known to induce ROS production in many different types SOD failed to block the stimulation of phagocytic activity by 37,40 of cells including U937 and HL60 cells. Moreover, NADPH TPA in U937 cells. H2O2 is a diffusible form of ROS that can oxidase is activated by TPA through the activation of PKC,7 and transverse from the outside in or the inside out of cells. PKC- TPA-induced ROS production is dependent on activation of stimulated mitochondrial-derived H2O2 is able to diffuse from PKCbII.41 Thus, there have been many studies that indicated that mitochondria, passing through the cytoplasm and crossing the ROS increase is mediated by the activation of PKC and NADPH plasma membrane.52–54 Therefore, changes in the concentration oxidase induced by TPA during macrophage differentiation. of extracellular H2O2 will in turn affect the intracellular redox However, the roles of ROS in monocytic differentiation of status; the addition of catalase to the cell culture leads to an hematopoietic cells are not yet well understood. In this study, increase in intracellular catalase activity. As catalase is an À treatment of cells with TPA resulted in an increase in the level of enzyme that dismutates H2O2 to H2O and SODs react with O2 intracellular ROS, consistent with the results by other investi- to form H2O2, the difference of membrane permeability 37,40–42 À gators. During differentiation, protein components re- between H2O2 and O2 may reflect the different effects of lated to ROS are induced in leukemic cells.43–45 SOD activity exogenously added catalase and SOD on the inhibition of cell has been shown to increase dramatically in a variety of tumor differentiation by TPA. 46–48 cell systems when they undergo differentiation, and it has We showed that H2O2 is actively involved in macrophage also been reported that non-differentiated cells usually have differentiation by TPA. In agreement with our finding, antiox- lower SOD activities than differentiated cells.49 In this study, the idants have been shown to inhibit the differentiation of myeloid levels of MnSOD were increased, whereas those of CuZnSOD cells. Catalase abolished but SOD promoted differentiation of 49 were not altered during differentiation, indicating that the total HL60 cells into granulocytes by L-ascorbic acid. Exogenously levels of SOD were increased during differentiation by TPA. In added SOD blocked the monocytic differentiation induced by contrast, the levels of catalase were decreased during differ- the combination of superoxide ions and platelet-activating entiation in these cells. These results were consistent with factor in rat monocytic leukemia c-WRT-LR cells.55 The previous studies reporting that the catalase activity decreased differentiation of bone marrow monocyte-macrophage lineage during macrophage differentiation50 and also during polymor- cells into osteoclasts was inhibited by the treatment with NAC phonuclear leukocyte differentiation.46 Catalase directly de- to remove OHÀ.56 However, our findings are also contrary to composes H2O2 into H2O and O2, and the glutathione cycle earlier studies showing that antioxidants promote the differ- metabolizes H2O2 by using GSH as a reductant for H2O2 in a entiation of cells. Plant-derived antioxidants have been shown reaction catalyzed by GSH-Px1. Thus, H2O2 is degraded by to potentiate differentiation of HL60 cells induced by 1a, 25- 57 catalases and peroxidases in cells. However, the levels of GSH- dihydroxyvitamin D3. Moreover, the oxidizing agent diethyl Px1 remained constant during the differentiation by TPA in maleate has been shown to prevent the differentiation of HL60 À 58 U937 cells. As SOD converts O2 into H2O2, these findings cells stimulated by TPA. Antioxidants have a cooperative suggest that accumulation of H2O2 possibly occurs in U937 effect with 1, 25-dihydroxyvitamin D3 in the differentiation of cells treated with TPA. leukemia HL60 cells.59 Further, hydroxyl radical scavenger In this study, scavenging ROS by NAC abrogated the inhibited TPA-stimulated differentiation in HL60 cells, whereas induction of CD11b expression by TPA in U937 cells. More- SOD or catalase did not affect TPA-induced HL60 cell over, treatment of U937 cells with either catalase or SOD differentiation.40 The mechanisms responsible for the discre- inhibited the accumulation of ROS by TPA. Furthermore, pancies between our and other studies are not clear. The catalase or SOD blocked the induction of CD11b expression functional role of ROS in macrophage differentiation might be by TPA, with the magnitude of the inhibitory effect by catalase dependent on the type of ROS and the nature of the being larger than that by SOD. Catalase also inhibited differentiation inducers. Therefore, it will be worthwhile for phagocytic activity in TPA-treated cells. These results suggest future studies to investigate how individual ROS are involved in that H2O2 may be, at least in part, involved in differentiation macrophage differentiation induced by different factors or along a monocytic lineage by TPA. To further study the role of signaling pathways. catalase on macrophage differentiation by TPA, we compared Hydrogen peroxide is one of the most frequently occurring À À the potential differentiation capability between HL60 promye- ROS in the biosphere. H2O2 is more stable than O2 and OH 51 locytes and their H2O2-resistant variant HP100-1 cells. HL60 and has been found to be more active in cell metabolism and cells are well known to differentiate toward macrophages in signaling than other forms of ROS. On the other hand, its rapid response to TPA. Previously, we have shown that exogenously and efficient removal is of primary importance, as excessive added H2O2 suppressed cell growth in HL60 cells, but not in H2O2 is harmful for almost all cell components. H2O2 is HP100-1 cells, and HP100-1 cells had an almost 10-fold higher degraded by peroxidases and catalases; catalase is able both to activity of catalase than HL60 cells without differences in the reduce H2O2 to water and to oxidize it to molecular oxygen. levels of GSH-Px1, MnSOD and CuZnSOD.11 We confirmed Thus, the activity of catalase is important not only in modulating that HP100-1 cells had a much greater capability to dismutate the redox status but also in directly mediating signals. The H2O2, with concomitantly much higher levels of catalase, than catalase gene has an overlapping Sp1/ early growth response-1 HL60 cells, and they were resistant to H2O2. When HL60 cells (Egr-1) recognition sequence within its core promoter, and the were treated with TPA, differentiation was induced into competition of Egr-1 with Sp1 for binding to the promoter results phagocytic monocytes in these cells. In contrast, HP100-1 cells in reduced promoter activity of the catalase gene.60 Egr-1 is the failed to differentiate into monocytic lineage in response to TPA. zinc-finger transcription factor, has been shown to be induced In this study, we for the first time clarified a certain role of H2O2 during macrophage differentiation, and is essential for macro-

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