A critical role of redox state in determining HL-60 cellgranulocytic differentiation and apoptosis via involvementof PKC and NF-κB

Jurate Savickiene & Grazina Treigyte & Arunas Gineitis &

Ruta Navakauskiene

Received: 9 September 2009 /Accepted: 14 January 2010 /Published online: 12 March 2010 / Editor: J. Denry Sato# The Society for In Vitro Biology 2010

Abstract The modifications of intracellular redox balanceleads to important cellular changes in many cell types.Here, a causal relationship among redox state, granulocyticdifferentiation induced by all-trans retinoic acid (RA) ordibutyryl cAMP (dbcAMP) and apoptosis have beenstudied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygenspecies levels by D, L-buthionine-(S, R) sulfoximide (BSO),and N-acetyl-L-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growthinhibition, differentiation and subsequent apoptosis. Thepresence of BSO during the commitment stage suppressedRA—but not dbcAMP-mediated differentiation, whileNAC inhibited both. BSO alone and in combination withRA or dbcAMP-induced apoptosis, which was prevented byNAC in dbcAMP—but not in RA-treated cells. Usingprotein kinase C inhibitor, calphostin C, cross-talk effectsbetween the intracellular redox state and PKC signalling wasidentified by demonstrating inducer-dependent changes incell differentiation or apoptosis, which were associated withthe changes in DNA-NF-κB binding activity. These obser-vations suggest a critical role of redox state in determiningHL-60 cell behaviour and provide new insights into thecomplex effects of redox perturbations on the intracellularsignalling network via the involvement of PKC and NF-κB.

Keywords Granulocytic differentiation . Apoptosis . ROS .

PKC . NF-κB

Introduction

Recent evidence suggests that the redox state of the cell maybe important to control cell growth, differentiation andapoptosis (Allen and Tresini 2000; Shackelford et al. 2000;Sauer et al. 2001; Ueda et al. 2002). Superoxide anions arethe primary reactive oxygen species (ROS) generated bynormal cellular metabolism (Fridovitch 1978; Cross andJones 1991). Tumour cells with respect to normal counter-parts exhibit the increase in metabolic rate and elevatedintracellular ROS levels causing the maintenance of cellcycle activity and resistance to apoptosis (Szatrowski andNathan 1991; Clement and Stamenkovic 1996; Gorman et al.1997). The maturation of differentiating granulocytes leadsto the superoxide anion production as well (Cerutti 1985).Excessive accumulation of ROS is toxic but the productionof intracellular ROS is tightly regulated by antioxidantmolecules and ROS-scavenging enzymes (Fridovitch 1978;Jacobson 1996). Many studies demonstrated that oxidantsinduce cell death, whereas antioxidants can protect fromapoptosis (Verhaegen et al. 1995; Jacobson 1996; Simon etal. 2000; Martindale and Holbrook 2002). Some evidencepoints to the beneficial roles of oxidants or antioxidants aschemotherapeutic agents. In acute myeloid leukaemia blasts,the prooxidant agent L-buthionine-(S, R) sulfoximide (BSO)promotes high level of ROS generation by causing reducedglutathione (GSH) depletion (Hedley et al. 1998) andsignificantly enhances the toxicity of many anti-canceragents (Bailey 1998). N-Acetyl-L-cysteine (NAC), a precur-sor of cysteins, elevates the GSH level, scavenges intracel-lular ROS, and exerts protective effects from initiatingapoptosis (Aruoma et al. 1989; Verhaegen et al. 1995),cancer cell invasion, and metastasis (De Flora et al. 1996;Sen 1998). There are conflicting observations that ROS mayhave prosurvival effect through activation of antiapoptotic

In Memoriam: Arunas Gineitis (1939-2007)

J. Savickiene :G. Treigyte :A. Gineitis : R. Navakauskiene (*)Department of Developmental Biology, Institute of Biochemistry,Mokslininku st. 12,08662 Vilnius, Lithuaniae-mail: ruta.navakauskiene@bchi.lt

In Vitro Cell.Dev.Biol.—Animal (2010) 46:547–559DOI 10.1007/s11626-010-9296-0

redox-sensitive signalling pathways (Filomeni and Circolo2006) or the inhibition of ROS generation by antioxidantsmay result in apoptotic response (Maraldi et al. 2009).

Although ROS have been demonstrated to be involved inthe regulation of many signal-transduction pathways (Gabbitaet al. 2000; Sauer et al. 2001) the molecular targets are notprecisely defined. Besides the activation of different mem-bers of signalling cascades, ROS may directly regulate theactivity of transcription factors, such as NF-κB, AP-1, c-fos,c-myc, c-jun, Sp-1, p53 (Gabbita et al. 2000). The redox-sensitive transcription factor, NF-κB, known as a centralregulator of stress responses, is involved in the immune andinflammatory processes, cell-cell interactions, apoptosis, cellproliferation, etc. (Verma et al. 1996; Janssen-Heininger et al.2000). Findings that NF-κB activation or overexpression isassociated with tumourigenesis suggest the role for thistranscription factor in the regulation of cell cycle progressionand antiapoptotic defence (Pahl 1999). The redox regulationof NF-κB has been investigated extensively (Li and Karin1999; Janssen-Heininger et al. 2000). However, the origin ofthe dependency of NF-κB activation on ROS generation andthe molecular basis for this regulation is still largelyunknown.

Oxidative stress increases the activity of variety ofprotein kinases, including serine/threonine protein kinaseC (PKC). Several lines of evidence demonstrated thatPKC is an upstream target for both oxidants andantioxidants (Gopalakrishna and Jaken 2000). Prolongedoxidant exposure can lead to sustained PKC activation andtumor promotion. Antioxidants do not appear to directlyregulate PKC activity but may counteract the effects ofoxidants on stimulating PKC activity (Gopalakrishna andJaken 2000). Redox modification of PKC contributes toredox-mediated signalling events (Suzuki et al. 1997). Thedirect activation of PKC by ROS induces the activation ofdownstream signalling pathways (e.g., MAPK, ERK 1/2,JNK, p38 MAPK) that leads to increased transcriptionalactivity and the changes in gene expression (Sauer et al.2001). Obviously, exogenous ROS elicit complex of cellularresponses and regulation of redox state remains as apromising therapeutic approach.

The aim of this study was to investigate the role ofintracellular redox state modulated by oxidant BSO andantioxidant NAC on relationship among ROS levels,apoptosis and cell commitment to differentiate towardgranulocytes in the human promyelocytic leukaemia HL-60 cells, and possible beneficial involvement of PKCinhibitor calphostin C (Clp) in these events. This is basedon previous data that PKC inhibitors induce apoptosis inproliferating HL-60 cells (Jarvis et al. 1994; Ferraris et al.1997) and potentiate retinoic acid (RA)-mediated HL-60cell differentiation (Hui and Yung 1992; Savickiene et al.1995). The task of this study was to reveal whether the

modulation of cellular redox state may be useful inleukaemia therapy.

We report here that the modulation of intracellular ROSlevels causes different effects on HL-60 cell growth andsurvival, and leads to inducer-dependent or stage-specificchanges in HL-60 cell differentiation and apoptosis via aredox-sensitive mechanisms that involves PKC and NF-κB.

Materials and Methods

Materials. [γ-32P-ATP] and X-ray films were from Amer-sham Biosciences UK, (Buckinghamshire, England); T4polynucleotide kinase was from MBI Fermentas, Vilnius,Lithuania. All other reagents used, including Clp, BSO,dbcAMP, RA, nitro blue tetrazolium (NBT), NAC, cyto-chrome C, superoxide dismutase, phorbol myristate acetate(PMA), propidium iodide (PI), RNAse, trypan blue, acridineorange (AO), ethidium bromide (EB) were purchased fromSigma (St. Louis, MO).

Cell culture. The HL-60 human promyelocytic cell line wasobtained from German Collection of Microorganisms andCultures (GmbH, Braunschweig, Germany) and cultured inRPMI 1640 medium supplemented with 10% fetal calfserum, 100 U/ml penicillin and 100 μg/ml streptomycin(Gibco, Grand Island, NY) in 5% CO2 and humidified in a37°C incubator. Cells were tested negative for mycoplasmainfection using the MycoProbe ™. Mycoplasma detection kit(R&D Systems Europe, Ldt, Abingdon, UK). For eachexperiment, logarithmically growing HL-60 cells were usedat a density 4×105 cells/ml. In pretreatment experiments,cells were incubated with BSO, NAC or Clp for 24 h, thecompound was removed by centrifugation at 300×g for5 min. The pelleted cells were rinsed once with freshmedium and resuspended in the medium containing arequired compound alone or in combination with anothercompound. Treated cells were cultured and harvested at thetime points indicated. Three independent experiments wereperformed with control and treated flasks. The stocksolutions used were: PMA (1 mg/ml), Clp (25 μM) inDMSO; RA (1 mM) in ethanol, dbcAMP (20 mM) indouble-distilled water, BSO (1 mg/ml or 100 mM) in RPMImedium. NAC (500 mM) was dissolved in RPMI mediumand taken to neutrality by adding 0.1 N NaOH. The requiredvolume of stock solutions was added to cell culture mediumto achieve the desired concentration.

Cell viability, proliferation and differentiation. Viable cellswere evaluated by the trypan blue exclusion test. Of the cellsuspension, 0.1 ml was mixed with 0.1 ml of 0.4% trypanblue and viable and dead cell number was determined bycounting in a haemocytometer. The growth inhibition of

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treated cells was expressed as the percentage of total cellnumber from untreated control (100%). The degree ofdifferentiation was assayed by the ability of cells to reduceNBT to insoluble blue-black formazan on stimulation byPMA (Collins et al. 1977). Cell suspension (100 μl) wasmixed with an equal volume of 0.2% NBT dissolved inphosphate-buffered saline (PBS) containing 40 ng/ml PMA,and incubated at 37°C for 30 min. NBT-positive cells werecounted in a haemocytometer. At least 400 cells werescored for each determination. NBT-positive cell numberwas expressed as a percentage of the viable cell number.

Apoptosis. Apoptotic cells were determined after stainingwith 0.01% AO-0.01% EB mixture (1:1, v/v) by morpho-logical evidence of surface blebs as apoptotic bodies, usingfluorescence microscopy (Mercille and Massie 1994). Atleast 300 cells were scored for each determination, and thenumber of apoptotic cells in the test was expressed as thepercentage of the total cell number. In differentiation andapoptosis experiments of combined treatments, results areexpressed as the percentage of mean number of NBT-positive (or apoptotic) cells in treated culture relative tocontrol (without drugs). For evaluation of apoptosis by flowcytometry, cells were collected by centrifugation, sus-pended in PBS and fixed in 70% ethanol (ratio 1:10) for24 h at −20°C. Then cells were centrifuged at 800×g for5 min and cell pellet (1-2×106 cells/ml) were resuspendedin 40 μl of phosphate-citrate buffer consisting of 192 partsof 0.2 M Na2HPO4 and eight parts of 0.1 M citric acid (pH7.8) at room temperature for at least 30 min (Gong et al.1994). After centrifugation at 1,000×g for 5 min, cells weresuspended in 1 ml PBS containing PI (50 mg/ml) andRNAse (0.2 mg/ml) in polypropylene tubes and incubatedat room temperature for 30 min. The tubes were then placedat 4°C in the dark before the flow cytometric analysis inFACscan flow cytometer (Becton-Dickinson, FranklinLakes, NJ). The data were registered on a logarithmic scale.

To assess apoptotic DNA fragments, cells were washedwith PBS and pelleted by centrifugation. Cell pellets werethen treated for 10 s with lysis buffer (1% Nonidet P-40 in20 mM EDTA, 50 mM Tris-HCl, pH 7.5; 10 μl/106 cells,minimum 50 μl). The supernatant was collected bycentrifugation for 5 min at 1,600×g, and the extractionwas repeated with the same amount of lysis buffer. Thesupernatants were brought to 1% SDS and treated for 2 hwith RNAse A (final concentration 2.5 μg/μl) at 56°Cfollowed by digestion with proteinase K (final concentra-tion 2.5 μg/μl) for at least 2 h at 37°C. After addition of 0.5vol. of 10 M ammonium acetate, DNA was precipitatedwith 2.5 vol. ethanol and dissolved in TE buffer (10 mMTris-HCl, 1 mM EDTA, pH 8.0; Herman et al. 1994).

DNA (15 μg) in 10 μl of TAE buffer containing 0.2 vol.30% glycerol: 0.25% bromphenol blue was loaded into

each well and electrophoresed on a 1.5% agarose gelcontaining TAE running buffer (40 mM Tris-acetate,20 mM sodium acetate, 2 mM EDTA, pH 7.8) at 100 Vfor 2-3 h. The agarose gel was stained with EB for 10 minand destained in H2O. The DNA marker of 123 base pairswas used as a molecular weight standard (Sigma).

Superoxide anion production. Superoxide anion productionwas assayed by superoxide dismutase-inhibitable cytochromec reduction (Azzi et al. 1975). Cells (1×106 cells/ml) werewashed two times in PBS and suspended in 0.5 ml PBScontaining 0.07 mM CaCl2, 1 mM MgCl2, then 75 μl of80 μM cytochrome c was added and the cells werestimulated with PMA (final concentration 100 mg/ml) at37°C for 20 min in the presence or absence of 50 μg/mlsuperoxide dismutase. The absorbance at 550 nm wasmeasured against a blank of cytochrome c incubated forthe indicated time at 37°C in the absence of cells. Theamount of superoxide anion produced was calculated byFabian et al. (1992). Results were expressed as nanomoles ofsuperoxide anion per 106 cells/20 min.

Preparation of nuclear extracts. Cells were harvested andpelleted at 300×g for 6 min, then washed twice in ice coldPBS and lysed in Nuclei EZ lysis buffer (Sigma) for 5 minon ice. After centrifugation at 500×g for 5 min at 4°C,nuclei were washed in the same cold buffer, vortexedbriefly and set on ice for 5 min. Nuclei were pelleted at500×g for 5 min, then completely suspended in Nuclei EZstorage buffer and frozen at −70°C. Nuclear protein extractsfor electrophoretic mobility shift assay (EMSA) wereprepared by lysis of nuclei in a buffer (20 mM Tris-HCl,pH 8.0, 200 μM EDTA, 2 mM EGTA, 20% glycerol,400 mM NaCl) containing 3 mM DTT, 1 mM PMSF and1× protease inhibitor cocktail P2714. After incubation for1 h on ice, extracts were centrifuged at 18,000×g for20 min, and used immediately. Protein concentrations weredetermined by RCDC protein assay as recommended by themanufacturer (Bio-Rad Lab. GmbH, Munchen, Germany).

Electrophoretic mobility shift assay. The probes used weresynthetic oligonucleotides (MWG-Biotech MG, Ebersberg,Germany) representing binding sites: (5′-AGTTGAGGGGACTTTCCCAGGC-3′) consensus NFκB; (AAGCCTGGGCAACATAGAAAGTCCCCATCTGTACAAAAA-3′)NFκB from the FasL promoter. Complementary oligonu-cleotides were annealed and labelled at their 5′ ends using[γ-32P-ATP] and T4 polynucleotide kinase. Standard DNAreactions were performed with 12-15 μg nuclear extracts ina 20 μl of reaction buffer (10 mM HEPES pH 7.9, 3 mMMgCl2, 0.1 mM EDTA, 40 mM NaCl, 10% glycerol)containing 2 μg BSA, 1 μg poly(dI-dC) and 1 pM labelledoligonucleotide for 30 min at room temperature. When

ROLE OF REDOX STATE IN HL-60 CELLS 549

desired, unlabeled competitor oligonucleotide (cold) wasadded to protein extracts at 100-fold molar excess for15 min preincubation. DNA-protein complexes wereresolved on 6% polyacrylamide gel containing 1× Tris-borate buffer. After electrophoresis, gels were dried andthen exposed to X-ray films.

Statistical analysis. Data are expressed as the representa-tive result or as the mean of at least three independentexperiments. Error bars represent ±SD. The student t testwas used to perform statistical analysis and values of p<0.0002 (**) and p≤0.05 (*) were stated as statistically highsignificant and statistically significant, respectively.

Results

Effects of BSO and NAC on growth of proliferating anddifferentiating HL-60 cells. The modulation of the intracel-lular level of ROS was performed by the use of oxidantBSO, which selectively inhibits GSH synthesis andincreases ROS levels, and antioxidant NAC, whichincreases intracellular levels of GSH and directly scavengesoxygen radicals. In the initial experiments, we have studieddose- and time-dependency of human promyelocyticleukaemia HL-60 cell growth under stress conditions byusing various concentrations of BSO (250-750 μM) andNAC (5-15 mM). The curves presented in Fig. 1 illustratethe time course of HL-60 cell growth inhibition. The drugscaused differences in their potency to inhibit cell growthduring 4 d of incubation, with NAC being most efficientthan BSO. All concentrations of NAC used induced dose-dependent cell growth inhibition to about 60% on Day 2,while all doses of BSO caused maximal cell growthinhibition (about 65%) even on Day 4 (Fig. 1A). Thecotreatment with both drugs at optimal concentrations andtwo different inducers (RA and dbcAMP) enhanced cellgrowth suppression (Fig. 1B). The moderate molar concen-trations of BSO (500 μM) and NAC (10 mM) were chosenas optimal for following experiments.

Effects of BSO and NAC on superoxide production and HL-60 cell differentiation. HL-60 cell granulocytic differentia-tion was induced by 700 nM RA and 350 μM dbcAMP.Various concentrations of BSO reduced HL-60 cell differ-entiation induced by RA or dbcAMP in similar manner,although the effect in dbcAMP-treated cells was lower. Onthe contrary, all concentrations of NAC inhibited bothdbcAMP- and RA-mediated differentiation (about 2- and1.4-fold, respectively; Fig. 2A). The present data demon-strated that modulation of redox state by BSO- or NAC-affected HL-60 cell differentiation in inducer-specificmanner.

To understand whether the changes in the intracellularredox state were important determinants for cell differenti-ation, we attempt to correlate the level of produced ROSwith the extent of the event. The kinetics of superoxideproduction and the response of differentiating HL-60 cellsin the presence of 500 μM BSO or 10 mM NAC are shownin Fig. 2. In HL-60 cell culture induced by RA or dbcAMP,the level of superoxide production (Fig. 2C) was in parallelwith the degree of differentiation (Fig. 2B). The combinedtreatment with BSO-elevated superoxide production at thefirst day and continued to increase up to the third d, whileNAC caused low superoxide level. Our data demonstratedthat the concentrations of BSO and NAC used wereprecisely those that modulate the release of superoxideanion in treated cultures.

BSO and NAC have distinct effects on apoptosis ofproliferating and differentiating HL-60 cells. We thenstudied the effects of BSO and NAC on survival ofproliferating and differentiating HL-60 cells. Staining withAO/EB indicates the drug-induced changes in cell mor-phology consistent with apoptosis. As shown in Fig. 3A,continuous exposure to increasing concentrations of BSO(100-1,000 μM) during 6 d resulted in apoptosis increase ina dose-dependent manner. Lower doses of NAC (5-10 mM)prevented apoptosis, while higher (15-20 mM) increased it(Fig. 1A). By contrast, the presence of various concen-trations of BSO in differentiating cell population induced

Figure 1. Dose- and time-dependent effect of modulation of ROSlevels by BSO or NAC on growth inhibition of HL-60 cells. A, Cellswere exposed to increasing concentrations of BSO (250-750 μM) orNAC (5-15 mM) for 4 d. B, Cells were treated with 700 nM RA or350 μM dbcAMP in the presence of 500 μM BSO or 10 mM NAC for4 d. Cells were counted daily and growth inhibition was determined asthe percentage of the total number of treated cells relative to the totalnumber of untreated control (100%). Results are expressed as means ±SD of four independent experiments.

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by RA or dbcAMP did not show marked changes inapoptosis level. NAC increased apoptosis in a dose-dependent manner in RA- (but not dbcAMP) treated HL-60 cells (Fig. 3A).

Apoptosis was judged by morphology of cells, DNAfragmentation detected by agarose electrophoresis (at 72 h),and subdiploid DNA content determined by flow cytometry(at 48 h). Using both latter methods, we detected that500 μM BSO induced apoptosis in proliferating and RA- ordbcAMP- induced HL-60 cells (Fig. 3B, C). 10 mM NACcompletely inhibited DNA fragmentation in control anddbcAMP-treated cells but effectively induced the ladderformation due to DNA degradation and caused substantialincrease in the sub-G1 region in RA-induced cell culture(Fig. 3B, C). As was determined by FACS, on average, thedegree of early apoptosis corresponded 11-13% in HL-60cells treated with NAC alone or cotreated with dbcAMP,and proapoptotic effect was obtained after treatment withBSO alone (44%) and in combinations with RA (30%) or

dbcAMP (35%; Fig. 3C). These results further confirmedthat modulation of intracellular level of ROS causedinducer-dependent effects on apoptosis of differentiatingHL-60 cells.

Redox state modulation by BSO and NAC during HL-60cell differentiation causes stage- and inducer-dependenteffects. In order to evaluate the influence of redoxperturbations during commitment and maturation stages ofHL-60 cell differentiation, the cells were cultured with700 nM RA or 350 μM dbcAMP in the presence of500 μM BSO for different time (24, 48, 72 and 96 h),rendering cells more susceptible to oxidative stress. Afterremoving of BSO at indicated time-points, cells werecultivated with differentiation inducers alone for 6 d, thenumber of differentiated or apoptotic cells were determinedat Day 4 or Day 6, respectively. All data presented in Fig. 4was normalised to control (without treatment with BSO orNAC). The presence of BSO for different time during RA-induced differentiation linearly decreased the level ofdifferentiation from 76%±1.52 to 38±3.51% of control(Fig. 4A) and had no effect on apoptosis. On the contrary,the presence of BSO during the commitment stage (48 h)increased dbcAMP-induced cell differentiation to 123±1.54% of control, while prolonged exposure to BSOdecreased it to 86±2.10% of control (Fig. 5B). A linear,time-dependent loss of viability, which was associated withcell growth inhibition (Fig. 1), was observed during allcotreatments with BSO and RA or dbcAMP (Fig. 4A, B).However, the antiproliferative effect of BSO used incombination with RA or dbcAMP was not associated withthe induction of apoptosis by BSO and was independent onduration of the presence of BSO in cell culture (Fig. 4A, B).BSO appeared to be mostly responsible for no apoptoticdeath because it affected cell viability by forcing ROS tostay within the cells during cell differentiation.

In order to test the prediction that ROS depletionprovided by NAC might inhibit apoptosis in similarexperiment, HL-60 cells were treated with 10 mM NACfor different time (24-96 h) simultaneously with RA ordbcAMP during cell differentiation. NAC in coculture withRA decreased cell differentiation and apoptosis approxi-mately by 20% of control (Fig. 4C). The suppressing effecton cell differentiation was remarkable, when NAC wasmaintained more than 48 h in dbcAMP-induced cell culture(Fig. 4D). This effect was associated with high viability andapparent decrease in the number of cells undergoingapoptosis. Thus, the efficacy of NAC on the prevention ofapoptosis was inducer-dependent.

To extend the study of redox state modification beforethe induction of HL-60 cell differentiation, we pretreatedHL-60 cells with optimal concentrations of BSO or NACfor 24 h, then the drugs were washed out, and cells

Figure 2. Dose- and time-dependent effect of BSO and NAC oninduced HL-60 cell differentiation to granulocytes and superoxideanion production. A, Cells were cultured with 700 nM RA or 350 μMdbcAMP in the presence of various concentrations of BSO (100-1,000 μM) or NAC (1-20 mM) for 4 d. B–C, Cells were treated with700 nM RA or 350 μM dbcAMP alone or in the presence of 500 μMBSO or 10 mM NAC for 3 d. A, Cell differentiation was determineddaily and expressed as the percentage of NBT-positive cells numberrelative to viable cell number (100%). C, Superoxide production wasmeasured daily as described under Materials and methods. Results areexpressed as means ± SD of three independent experiments induplicate.

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incubated with RA or dbcAMP for 6 d. The data in Fig. 5Ademonstrated that preincubation with both agents sup-pressed RA- and dbcAMP-induced granulocytic differenti-ation compare to control (without preincubation). BSOshowed the greatest ability to inhibit dbcAMP-mediateddifferentiation and enhance apoptosis, while NAC causedsuppressing effect on RA-mediated differentiation andfailed to inhibit apoptosis. The ability of both drugs toblock partially HL-60 cell differentiation led to suggest thatbasal redox state is necessary for HL-60 cell commitment todifferentiate toward granulocytes.

Taken together, the changes in redox balance before theinduction of differentiation suppressed granulocytic differ-entiation in inducer-independent manner, while the increaseof ROS level by BSO caused inducer-dependent effects onapoptosis. The data implies that antileukemic activity ofdifferentiation inducers, RA and dbcAMP, depends onaltered oxidative conditions within the cell.

Redox state modulation effects on granulocytic differentia-tion and apoptosis of PKC inhibitor-treated HL-60 cells.Next, we determined whether ROS might be involved in

specific signalling pathways that trigger cellular events,such as differentiation and apoptosis. In order to verify thecausal relationship between oxidative stress and PKC-dependent signalling, we used a highly specific PKCinhibitor, Clp. Clp was used at a dose (100 nM) thateffectively inhibited PKC kinase activity and did not affectcell viability.

HL-60 cells were pretreated with Clp for 24 h, theninhibitor was removed and the cells were cotreated withBSO (500 μM) or NAC (10 mM) and differentiationinducers for 6 d. Clp pretreatment (Fig. 5B) resulted in thesuppression of RA- and dbcAMP-induced cell differentia-tion by about 30% and elevation of apoptosis by 30-40% ofcontrol (RA- or dbcAMP-treated cells), suggesting theinvolvement of PKC activity in both cellular events. Theaddition of BSO or NAC to Clp-pretreated and RA-inducedcells did not change Clp-dependent reduced level of celldifferentiation and elevated level of apoptosis with theexception that the addition of BSO with dbcAMP to Clp-pretreated cells significantly increased granulocytic differ-entiation with smaller changes in apoptosis (Fig. 5B). NACas an antioxidant was able to reverse apoptotic effects to

Figure 3. Dose- and time-dependent effect of BSO or NAC onapoptosis of control HL-60 cells and induced to differentiate by RA ordbcAMP. A, Cells were treated with 700 nM RA or 350 μM dbcAMPin the presence of different concentrations of BSO (100-1,000 μM) orNAC (1-20 mM) for 6 d. Apoptotic cells were determined inpreparations stained with AO/EB on Day 6. Data is presented as thepercentage of apoptotic cell number relative to total cell number (100%)and expressed as means ± SD of four independent experiments. B–C,

Cells were treated with 500 μM BSO or 10 mM NAC alone or in thepresence of 700 nM RA or 350 μM dbcAMP: B, DNA was isolated at72 h of treatment and electrophoresed in 1.5% agarose gel using 123 bpDNA ladder as a marker; C, apoptosis (AP) was determined as ahypodiploid peak from flow cytometric analysis of PI stained cells at48 h and presented as the percentage of the total events collected. Datais representative of three independent experiments.

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control and lower levels in examined cell cultures. Thus,only in dbcAMP-treated cells, where PKA but not PKCactivity is required for the induction of differentiation(Savickiene et al. 1995), the rise of intracellular ROS levelby BSO after depletion of PKC activity by Clp wasbeneficial stimulus for HL-60 cell differentiation.

Next, we have examined the effects of BSO and NACused during the commitment stage of induced HL-60 cellswith depleted PKC on the cell maturation and apoptosis.HL-60 cells were incubated with redox modulators (BSO orNAC) and RA or dbcAMP for 24 h (the commitmentstage); then, agents were removed and cells exposed to RAor dbcAMP in the presence of Clp. In control experiment,prolonged incubation with Clp caused a significant increasein RA-induced differentiation (by 40% of control withoutClp), a decrease in dbcAMP-mediated differentiation (by30% of control without Clp) and enhanced apoptosis inboth cell cultures (by 50-40% of control, respectively;Fig. 5D). Figure 5C presents the data expressed relative to

control (inducer with Clp, 100%). The presence of BSOduring commitment stage diminished both RA- anddbcAMP-mediated cell differentiations in coordination withdecreased apoptosis level. In similar experiment, NAC hadstimulating effect on differentiation induced by RA but notby dbcAMP with any effect on apoptosis. These observa-

Figure 5. Effects of the modulation of redox state by BSO or NACon the induction of HL-60 cell differentiation, differentiation itself andapoptosis after treatments with PKC inhibitor. A, Cells were pretreatedwith 500 μM BSO or 10 mM NAC for 24 h, then agents were washedout and cells were cultured with differentiation inducers (700 nM RAor 350 μM dbcAMP). B, Cells were pretreated with 100 nM Clp for24 h, then Clp was washed out and cells treated with RA or dbcAMPalone or in the presence of 500 μM BSO or 10 mM NAC. C, Cellswere treated with BSO or NAC concomitantly with RA or dbcAMPfor 24 h, then agents were washed out and cells incubated with RA ordbcAMP in the presence of 100 nM Clp. C, Cells were cotreated withRA or dbcAMP and Clp without or with BSO or NAC. Celldifferentiation was determined as the percentage of NBT-positive cellnumber on Day 4 after induction of differentiation. Apoptosis wasevaluated after staining with AO/EB on Day 6. The results areexpressed as the percentage of NBT-positive cells or apoptotic cellsrespect to controls carried out in the presence of RA or dbcAMP alone(A, B, D), or in the presence of Clp with RA or dbcAMP (B). Resultsare expressed as means ± SD of three independent experiments. *p≤0.05 and **p<0.0002, significantly different from control samples (C,underline) treated with RA or dbcAMP alone (A), with RA ordbcAMP following pretreatment with Clp (B), with RA or dbcAMPwith Clp (C, D).

Figure 4. BSO or NAC causes the inducer- and stage-dependenteffects on HL-60 cell differentiation, viability and apoptosis. Cellswere treated with 700 nM RA or 350 μM dbcAMP alone or in thepresence of (A, B) 500 μM BSO or (C, D) 10 mM NAC for differenttime (24, 48, 72 and 96 h). After exposure at the indicated time, agents(BSO or NAC) were washed out and cells were cultured with RA ordbcAMP alone for 6 d. Cell differentiation was determined by NBT+test on Day 4. Apoptosis was evaluated after staining with AO/EB onDay 6. Results are expressed as the percentage of the controls carriedout in the absence of BSO or NAC (100%). Full line curve representcell viability assessed by the trypan blue exclusion test. Results areexpressed as means ± SD of three independent experiments. *p≤0.05and **p<0.0002 is significantly different from controls without BSOor NAC.

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tions showed the significance of redox state during thecommitment stage on cellular events which are dependenton PKC activity.

Next, we examined the influence of prolonged incuba-tion for 6 d with BSO or NAC simultaneously with Clp anddifferentiation inducers on HL-60 cell maturation andapoptosis (Fig. 5D). We found that BSO and NAC hadlow effects on cellular events caused by Clp in RA-treatedcells. The combined treatment with BSO or NAC and Clpresulted in significant decrease of dbcAMP-induced differ-entiation, and low level of differentiation caused by BSObut not by NAC was paralleled with decrease in apoptosis.Confirming the data, we suggest that intracellular ROSlevels may be responsible for the modulation of PKC orPKA activity and subsequently for the events of differen-tiation or apoptosis.

Modulation of redox state by BSO or NAC results inchanges of NF-κB binding activity in differentiation-induced HL-60 cells. In our experimental model, weproceeded to verify the effects BSO and NAC asintracellular ROS modulators on a redox sensitive tran-scription factor, NF-κB. Nuclear extracts were preparedfrom HL-60 cells after treatment with BSO or NAC fordifferent time, and analyzed by EMSA with 32P-labelledNF-κB oligonucleotide probe for NF-κB DNA bindingactivity. Both BSO and NAC resulted in a rapid (at 0.5-2 h)increase in NF-κB DNA binding, although the effect ofNAC was slightly faster (at 6 h; Fig. 6A). The lineardecrease in NF-κB DNA binding activity occurred with theduration of the exposition (1-4 d) to both agents. Alterna-tively, cotreatment with BSO and differentiation inducers,RA or dbcAMP, as expected resulted in a marked increasein NF-κB DNA binding at the commitment stage (1-2 d)and a decrease during the cell maturation (3-4 d; Fig. 6B).NAC exposed with RA exhibited no obvious effect on NF-κB DNA binding activity during 2 d with subsequentdecrease on Day 4, while the combined treatment withdbcAMP caused initially the suppression but later a time-dependent increase in NF-κB DNA-binding in correlationwith high survival (Fig. 4D).

Next, we have examined the relationship between NF-κB DNA-binding activity to the FasL promoter and thepromotion of apoptosis in HL-60 cells treated with BSO orNAC and RA. The results in Fig. 6C demonstrated that NF-κB binding to consensus sequences and to the FasLpromoter was induced initially by BSO or NAC treatmentalone or in combination with RA at Day 1 but remained inrelatively steady state in coculture with BSO and RA in theaccordance with enhancement of apoptosis (Fig. 3C).

Since NF-κB activation occurs during granulocyticdifferentiation of HL-60 cells, a link among intracellularredox state, NF-κB activity and PKC-dependent signalling

was investigated. To examine this, HL-60 cells werepretreated with Clp for 24 h, PKC inhibitor was removed,the cells treated with RA or dbcAMP alone or in thepresence of BSO or NAC for 24 h (Fig. 6D). Pretreatmentwith Clp alone inhibited NF-κB DNA binding activity inRA- and dbcAMP-treated cells, suggesting that Clp-sensitive protein kinases are involved in the signallingpathway. Raising ROS levels by BSO in Clp-pretreatedcells remarkably increased NF-κB binding activity indbcAMP-treated cells compare to RA-treated cells, whileNAC substantially enhanced it in both cell cultures.Simultaneous exposition to BSO or NAC and Clp increasedNF-κB DNA binding in RA-induced cells but decreased itin dbcAMP-treated cells (Fig. 6E). The data suggest thatdefects in PKC-dependent pathway and pathway sensitiveto redox reagents may mediate the NF-κB levels andcontribute HL-60 cell responses.

Another support for the involvement of oxidative stress inNF-kB signalling was provided by 24-h pretreatement withBSO or NAC before the treatment with RA or dbcAMP in thepresence of Clp (Fig. 6F). NF-κB DNA binding activityincreased under treatment with RA alone or in combinationwith Clp at 24 h, and decreased when the cells werepretreated with BSO but not with NAC. By contrast,preincubation with NAC abolished NF-κB activation causedby treatment with dbcAMP and Clp, and BSO pretreatmentfailed to block it. Thus, the modulation of ROS level beforethe induction of HL-60 cell differentiation in the presence ofPKC inhibitor was able to regulate NF-κB signalling. Theextent of NF-kB activation appears to vary depending onstimulus that triggers differentiation process.

Discussion

Redox regulation is an important mechanism foroxidant-induced changes in gene expression that influ-ence overall cellular responses. The results of our studyshowed that redox balance perturbations cause unexpectedand stimulus-specific effects on promyelocytic leukaemiaHL-60 cell differentiation and apoptosis related to thetiming and degree of ROS modulation. First, the redoxstate disturbances in proliferating HL-60 cells by bothprooxidant BSO and antioxidant NAC (being mostefficient) caused dose- and time-dependent cell growthinhibition that was synergistically enhanced by differenti-ation inducers, RA or dbcAMP (Fig. 1). Other observationsalso provide evidence that redox signalling is involved in cellcycle regulation, checkpoint control and activation ofMAPK, ERK and SAPK signalling to stimulate cell growthor cell death dependently on ROS-signal intensity andduration (Esposito et al. 1997; Qiu et al. 1998; Gabbita etal. 2000).

554 SAVICKIENE ET AL.

Second, our study clarified the influence of redoxperturbations on promyelocytic leukaemia HL-60 celldifferentiation and subsequent apoptosis when ROS mod-ulators were added before the induction of differentiation,during the commitment stage and full time of RA- anddbcAMP-mediated differentiation. Results obtained afterprolonged exposition to BSO revealed that ROS werecritical mediator to determine proliferating cell apoptosiswhich was abrogated by antioxidant NAC (Fig. 3). This isconsistent with data that activation of apoptosis is usuallyassociated with ROS generation by oxidants (Fernandesand Cotter 1994; Sandstrom et al. 1994; Beaver and Waring1995; Jacobson et al. 1996) and prevention of apoptosis byantioxidants (Ho et al. 1997; Vahrmeijer et al. 2000). Thesurprising observations were that the presence of BSOduring RA-mediated differentiation inhibited cell matura-tion and did not enhance apoptosis but temporary stimulat-ed cell differentiation and apoptosis in dbcAMP-treatedcells (Fig. 4A, B); NAC prevented apoptotic cell death indbcAMP—but not RA-induced cells and decreased celldifferentiation in both cell cultures (Fig. 4C, D). It is notclear why the same agent provokes the opposite effects oncell fate. It is possible that intracellular ROS can playstimulatory or inhibitory roles depending on type ofinducer, which triggers cellular responses using differentsignalling pathways. This prediction is in agreement withpresented data, demonstrating inducer- and stage-specificeffects obtained under redox perturbation conditions in HL-

60 cells that utilise different, PKC- or PKA-dependentsignalling pathways. Previously, our work demonstratedthat the induction of RA-mediated HL-60 cell differentia-tion requires a substantial decrease in PKC activity andbasal PKA activity during the commitment stage, whileboth PKC and PKA activity are important for dbcAMP-induced differentiation and maturation (Savickiene et al.1995). Also, the inhibition of PKC activity during thecommitment stage markedly increases HL-60 cell differen-tiation and enhances induced apoptotic programme (Jarviset al. 1994; Savickiene et al. 1999). PKC is sensitive forredox modification (Gopalakrishna et al. 1995) and may beactivated following oxidative stress by oxidants andinhibited by antioxidants (Boscoboinik et al. 1991; Whislerat al. 1994; Brawn et al. 1995). According these and ourobservations, BSO produced ROS excess in RA-treatedcells may activate PKC and inhibit the cell commitmentto differentiate to granulocytes. The enhancement ofdbcAMP-mediated differentiation by BSO (Fig. 4B) maybe due to the ability of ROS to increase PKC activity and toalter PKA activity (Raynaud et al. 1997; Dimon-Gadal et al.1998). It is known that HL-60 cells in vitro differentiated togranulocytes, like mature neutrophils die via apoptosis(Jarvis et al. 1994, Tosi et al. 1994). In our experiments,apoptotic events were partially dissociated from differenti-ation (Fig. 4A, C and Fig. 5), supporting the evidence thatdifferentiation and apoptosis may be separately regulatedentities (Park et al. 1994; de Vente et al. 1995). This is

Figure 6. Modulation of redox state by BSO or NAC results inchanges of transcription factor NF-κB binding activity in proliferatingand differentiating HL-60 cells. A, Cells were treated with 500 µMBSO or 10 mM NAC. B–C, Cells were treated with 700 nM RA or350 µM dbcAMP alone or cotreated with BSO or NAC for 4 d. D,HL-60 cells were pretreated with 100 nM Clp for 24 h, Clp waswashed out, and cells were treated with 700 nM RA or 350 µMdbcAMP alone or in combination with 500 µM BSO or 10 mM NACfor 24 h. E, Cells were treated with RA or dbcAMP or cotreated with

BSO or NAC for 24 h. F, Cells were pretreated with BSO or NAC for24 h, both agents were washed out, and cells were treated with RA ordbcAMP alone or in combination with 100 nM Clp for 24 h. EMSAwas performed using a total 12-15 μg protein from each nuclearextract and NF-κB probe harbouring a consensus NF-κB motif (NF-κB cons) or the FasL binding site (NF-κB/FasL). Arrows indicatespecific band, the decrease of which is shown by competition analysis(cold). Results are from a representative experiment of three.

ROLE OF REDOX STATE IN HL-60 CELLS 555

consistent with reports that actively proliferating cells canbe triggered to apoptosis by modulation of PKC or PKAactivities (Lanotte et al. 1991; de Vente et al. 1995). Thereare evidences that basal activity of PKC prevents theoccurrence of apoptosis in HL-60 cells (Gorczyca et al.1993) and this prevention may be supported by prolongedactivation of PKC by ROS (Konishi et al. 1997; Korchak etal. 1998) or by the ability of RA to suppress the productionof ROS (Ahlemeyer and Krieglstein 1998). NAC mayexert survival effects by direct inactivation of redoxsensitive cysteine-containing enzymes, caspases and maymodulate also the activity of different kinases (Robinsonet al. 1999).

Third, we demonstrated the causal relationship betweenROS and PKC-dependent signalling, modulating the intra-cellular ROS levels and PKC activity before and duringHL-60 cell differentiation. We found that any disturbanceof redox state by BSO or NAC as like as the depletion ofPKC by Clp before the induction of differentiation causedthe decrease in both RA- and dbcAMP-mediated differen-tiation (Fig. 5A, B), suggesting the importance of basalredox state and PKC activity for HL-60 cell commitment todifferentiate. The presence of PKC inhibitor during the pre-commitment and the commitment stages promoted thosecells to initiate apoptosis. It is known that PKC inhibitorssuppress cell growth irreversibly (Begemann et al. 1998;Zhu et al. 1998) and induce apoptosis in many cell lines(Jarvis et al. 1994; Begemann et al. 1998; Zhu et al. 1998)via activation of caspase pathway in the early stage ofapoptosis (Drew et al. 1998). This may be a reason that anymode of treatment with BSO or NAC did not change Clp-mediated high level of apoptosis when PKC inhibitor wasused before the induction of differentiation (Fig. 5).However, Clp added together with differentiation inducershad distinct effect, enhancing RA-mediated and suppressingdbcAMP-mediated differentiation (Fig. 5D). The presenceof BSO for short- or long-time in cell cultures with depletedPKC abolished these effects and led to marked decrease incell differentiation, while short-time exposure to NACmarkedly increased the level of RA-mediated differentia-tion without changes in apoptosis (Fig. 5C), implying ROSas an agent changing PKC or PKA activity which are maintargets for appropriate type of differentiation.

The results obtained in this study allow us to propose aschematic model describing causal relationship betweenROS and PKC- or PKA-dependent signalling, determiningcellular responses (Fig. 7). It is necessary to point out thatother factors should be also considered, including cross-talkbetween ROS and activation of signalling cascades such asRas-dependent MAPK, p38, JNK/SAPK (Goldstone andNicholas 1997; Muller et al. 1997; Suzuki et al. 1997).Furthermore, cellular redox mechanisms may regulate theexpression of genes of early responses (c-fos, c-myc, c-jun,

NF-κB, AP-1, etc.) that may also contribute to themodulation of differentiation or apoptosis (Jabbar et al.1994; Keogh et al. 1998).

Redox-sensitive NF-κB is implicated in many cellularprocesses as a central regulator to determine cell death andcell survival (Barkett and Gilmore 1999). A constitutiveNF-κB activity is required for survival of several cancercells (Baldvin 2001). Activation of NF-κB results ininhibition of apoptotic signalling cascades, includingcaspases, JNK and ROS. Under conditions when NF-κBactivation is blocked, those signalling cascades are activat-ed, resulting in apoptosis or necrosis (Barkett and Gilmore1999; Piao et al. 2004). Numerous studies have demonstrat-ed that NF-κB is activated by oxidants and inhibited byantioxidants (Barkett and Gilmore 1999; Epinat and Gilmore1999; Li and Karin 1999; Pahl 1999) but other reports havenot (Li and Karin 1999).

A characteristic of NF-κB is that many different agentscan induce its DNA-binding activity (Janssen-Heininger etal. 2000). The suggestion that oxidants provide co-stimulatory signals towards the activation of NF-κB(Janssen-Heininger et al. 2000) was confirmed by ourresults of EMSA, demonstrating a marked increase in NF-κB DNA-binding activity under treatments with BSO aloneor BSO combined with RA or dbcAMP during the first 2 dand dramatic decrease on Day 4 (Fig. 6B) paralleled withdecreased cell viability (Fig. 4). The observation that NAC

Figure 7. Schematic representation of cellular responses to themodulation of intracellular redox state by BSO or NAC and inhibitionof PKC activity by calphostin C in HL-60 cells induced to differentiateby RA or dbcAMP.

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alone also induce NF-κB DNA-binding is consistent with areport, indicating that NAC may induce NF-κB by DNA-binding activity independently of its antioxidant function(Liu et al. 2008). By contrast, NAC used with dbcAMP,caused initially reduced NF-κB DNA-binding activitycharacteristic for antioxidants which linearly increasedduring 4 d (Fig. 6B) in association with high cell viability(Fig. 3).

The activity of NF-κB is controlled by the transcriptionalupregulation of its inhibitor, IκB or NF-κB phosphorylationby protein kinases. For instance, activation of NF-κBinvolves its phosphorylation by PKA pathway; a directphosphorylation of IκB through PKC release IκB andthereby activate NF-κB (Gopalakrishna and Jaken 2000;Janssen-Heininger et al. 2000). In our present study, we haveexamined NF-κB DNA-binding activity under conditionsthat increase or decrease intracellular ROS level in concertwith PKC inhibition. We found that 24-h pretreatment withClp before the induction of differentiation by RA ordbcAMP caused reduced NF-κB DNA-binding activity(Fig. 6D) as like as in a case when RA or BSO with RAwere added to Clp-pretreated cells that was associated withearly apoptosis induction by Clp (Fig. 5B). Inversely, othertreatments significantly increased NF-κB DNA-binding(Fig. 6D). The inducer-specific reduction of NF-κB DNA-binding activity was detected under combined treatment withboth ROS modulators, Clp and dbcAMP (but not RA;Fig. 5E), and also when cells were pretreated with BSO orNAC before the addition of RA and Clp or dbcAMP andClp, respectively, (Fig. 6F) that correlated with the decreasedlevel of differentiation following such treatments (Fig. 5C, D).The enhanced NF-κB binding to the FasL promoter during2 d of treatment with RA and BSO (Fig. 6C) demonstratedthe involvement of NF-κB in Fas/FasL death-inducingsignalling as well. The results of our investigation confirmedthat redox signalling can interfere with PKC or PKA-dependent signalling pathways and modulate NF-κB path-way which is likely dependent on the nature of the NF-κBactivator, ROS extent and the selectivity of signallingpathways activated.

In conclusion, we have demonstrated in this report thatthe change in intracellular ROS levels resulted in different,a context-specific cellular response, such as differentiationand apoptosis. ROS can have adverse or beneficial effectsdepending on cellular redox environment, concentrationand duration of ROS exposition. From a therapeutic pointof view, oxidant BSO might serve as an inducer ofapoptosis in proliferating leukaemia cells; antioxidantNAC that prevents cell growth but protects from cell deathis much less efficacious; the selective combinatorial treat-ments that potentiate cell differentiation and apoptosis andconcerted stimulatory action of RAwith PKC inhibitor, Clpwould be useful for anti-cancer therapy.

Acknowledgments The study was supported by the LithuanianState Science and Studies Foundation (project No. V-12/2009).

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