(CANCER RESEARCH 49. 1110-1117. March 1. 1989]

Novobiocin- and Phorbol-12-myristate-13-acetate-induced Differentiation of HumanLeukemia Cells Associated with a Reduction in Topoisomerase II Activity1

Andreas Constantinou,2 Cynthia Henning-Chubb, and Eliezer Huberman

Biological, Environmental, and Medical Research Division, Argonne National Laboratory, Argonne, Illinois 60439

ABSTRACT

Studies were conducted to determine the possible involvement of DNAtopoisomerase II (Topo II) in the induction of differentiation in twohuman promyeloc)tic 111 (ill leukemia cell variants that are either susceptible or resistant to differentiation induced by phorbol-12-myristate-13-acetate (I'M \). a protein kinase C activator. The acquisition ofmaturation markers and changes in the activity, level, and phosphoryla-tion of Topo II were determined after treatment with either novobiocin,a Topo II inhibitor, or I'MA. Novobiocin at 50-150 MMinduced differentiation in the 111-20? cells but not in the HL-525 cells, although bothcell types were equally susceptible to novobiocin-evoked cytotoxicity. Inboth cell types, novobiocin induced similar reductions in topoisomerase Iactivity but different reductions in Topo II activity. Treatment withnovobiocin at 150 n\\ for 6 h or at 2 HIMfor 30 min resulted in a 4-foldor higher reduction in Topo II activity in the differentiation-susceptibleIII -2(1? cells but not in the differentiation-resistant HL-525 cells. Adifferential response in Topo II activity was also observed after treatmentwith I'M \. The novobiocin-evoked decrease in Topo II activity seems tobe due to an enhanced enzyme proteolysis, whereas the PMA-eliciteddecrease in Topo II activity is associated with an increase in Topo IIphosphor) lai ion. l-(5-Isoquinolinesulfonyl)-2-methylpiperazine, which isan inhibitor of protein kinases, including protein kinase C, diminishedthe novobiocin-elicited proteolysis of Topo II and the PMA-induced TopoII phosphorylation, as well as the decrease in Topo II activity and theacquisition of differentiation markers induced by either novobiocin orPMA. These results suggest that induction of differentiation in HL-60cells by novobiocin or PMA is associated with a reduction in Topo IIactivity, mediated directly or indirectly by a protein kinase(s), perhapsprotein kinase C.

INTRODUCTION

Topoisomerases are ubiquitous enzymes that change the conformation of DNA molecules. Their mode of action involves atransient breakage of one DNA strand (type I topoisomerase)or two DNA strands (type II topoisomerase) followed by rotation and rejoining of the strands (1, 2). In eukaryotes, theseenzymes have been implicated in the replication and processingof DNA, as well as in the transcription of specific genes (3-9).Studies with mammalian cells have shown that the activity ofTopo II3 is higher in replicating cells than in cells exhibiting amature phenotype (10-16), thus suggesting that a reduction inTopo II activity may be associated with either inhibition ofgrowth or the induction or maintenance of the differentiatedstate.

The present studies were initiated to distinguish betweenthese possibilities and to determine specifically if a reductionin Topo II activity is a prerequisite for the induction of cell

Received 8/23/88: revised 11/28/88: accepted 12/1/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by the United States Department of Energy, underContract W-31-109-ENG-38.

2Recipient of National Research Service Award CM 10662 from the NIH.3The abbreviations used are: Topo II, type II topoisomerase: m-AMSA, 4'-(9-

acridinylamino)methanesulfon-/n-anisidide, amsacrine: DDW, double-distilledwater; H-7, l-(5-isoquinolinesulfonyl)-2-methylpiperazine; NBT, nitroblue tetra-zolium; NSE, nonspecific esterase; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; SDS, sodium dodecyl sulfate; Topo I. type 1topoisomerase.

differentiation in the human promyelocytic leukemia HL-60cells (17). In one approach, we tested the ability of novobiocinand related agents that inhibit Topo II activity (1,2, 18-21) toinduce differentiation in HL-60 cells. For comparison, we included camptothecin, an inhibitor of Topo I (22). In anotherapproach, we tested the ability of PMA, a potent differentiationinducer (23-25), to reduce the activity of Topo II in the HL-60cells shortly (within 30 min) after treatment. Because the biological activity of PMA is believed to involve specific proteinphosphorylation induced by PKC (26), we examined the alterations in the activity, level, and phosphorylation of Topo IIafter treatment of the cells with either novobiocin or PMA inthe presence and absence of H-7, an inhibitor of protein kinases,including PKC (27). Induction of cell maturation and changesin Topo II level, activity, and phosphorylation were tested incell-line HL-205, a differentiation-susceptible HL-60 cell variant, and in cell-line HL-525, an HL-60 cell variant that is

resistant to induction of differentiation by PMA but not byother types of inducers (28).

Our results indicate that novobiocin caused the HL-205 cellsbut not the HL-525 cells to acquire a mature myeloid phenotype. Furthermore, in the HL-205 cells but not the HL-525cells, novobiocin and PMA were able to reduce the activity ofTopo II and to alter the level of phosphorylated Topo II within30 min after treatment. All of these changes were diminishedby pretreatment of the cells with H-7. On the basis of theseresults, we suggest that induction of differentiation in HL-60cells by some chemical agents is associated with a reduction inTopo II activity, which is facilitated by a protein kinase(s),perhaps PKC.

MATERIALS AND METHODS

Chemicals and Reagents. m-AMSA was obtained from the NationalCancer Institute, and novobiocin from Boehringer Mannheim. Cou-mermycin was purchased from Sigma Chemical Co., PMA from Chemicals for Cancer Research; and H-7 from Seikagaku America, Inc.These chemicals, stored as stock solutions at —¿�20°C,were used as

follows. Novobiocin at 10 mM in sterile DDW, m-AMSA at 2.5 mM in50% dimethyl sulfoxide in DDW, PMA at 1.6 mM in dimethyl sulfox-ide, and H-7 at 10 mM in DDW. When a combination of H-7 andanother drug was used, H-7 was added l h prior to the second drug.Bacteriophage P4 Vir\ del 10 was provided by R. Calendar (Universityof California, Berkeley, CA). The MoP-9 monoclonal antibody wasprovided by A. Dimitriou-Bona (Mt. Sinai School of Medicine, NewYork, NY), the B52.1 monoclonal antibody was provided by G. Trin-chieri (Wistar Institute of Anatomy and Biology, Philadelphia, PA),and two different rabbit IgG antibodies against mouse cell Topo II wereprovided by F. Drake (Smith Kline Beckman Co.). All other antibodieswere purchased from either Ortho Pharmaceutical Corp., Coulter Immunology, or Becton Dickinson. A Topo II control sample was theactive fraction of the Mono Q column partially purified from HL-60cells as described by Drake et al. (29). Protein kinase C from rat brainwas purified in our laboratory by J. Hardwick.

Cells, Culture Conditions, and Differentiation Markers. The HL-205cell variant was isolated from HL-60 cells, while the HL-525 cell variantwas obtained after cloning HL-60 cells that had been subcultured 102times in the presence of PMA (28). Both cell variants were stable for

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DIFFERENTIATION OF LEUKEMIA CELLS AND Topo II ACTIVITY REDUCTION

at least 50-60 subcultures with regard to their susceptibility or resistance to PMA-induced cell differentiation. Cells were inoculated into100-mmor 150-mm tissue culture dishes at 1.5x 10s cells/ml of RPMI

1640 supplemented with 20% fetal bovine serum, penicillin (100 units/ml), and streptomycin ( 100 Mg/ml), and cultured at 37°Cin a humidified

atmosphere of 8% CO2 in air.Immunofluorescence tests for reactivity with the antibodies, staining

for NSE activity (30), and the NBT reduction assay (17) were allperformed as previously described.

Preparation of Nuclear Extracts. Exponentially growing HL-205 orHL-525 cells in 150-mm culture dishes were treated with either theappropriate drugs or solvents only (controls). Following treatment, thedishes were placed on ice and a volume (equal to that of the growthmedium) of cold 50 IHMTris-HCl (pH 7.5) containing 25 mM KC1, 2IHM CaCl2, 3 mM MgCl2, and 0.25 M sucrose was added. The cells(about IO8) were centrifuged, then homogenized; the nuclei were iso

lated as described by Taudou et al. (10) and extracted as described byChampoux and McConaughy (11), except that immediately after thecell washing, we added 1 mM phenylmethylsulfonyl fluoride, 1 mMben/a miti ine. 10 Mg/ml soybean trypsin inhibitor, 50 Mg/ml leupeptin,l /¿g/mlpepstatin, and 20 Mg/ml aprotinin. These nuclear extractscontained more than 90% of the total Topo II levels as determined byimmunoblotting with the anti-Topo II antibodies (16, 18). The proteincontent of the nuclear extracts was determined by the Bradford method(31). Glycerol was added to a final concentration of 30%, and theextracts were stored at -20°C.These preparations served as the source

for Topo I and II activity, as well as for Topo II immunoblotting.Unknotting Assay for the Determination of Topoisomerase II Activity.

The substrate used in the assay was knotted DNA isolated from thetailless capsids of the bacteriophage P4 Vir\ del\0 as described by Liuet al. (32). Reaction mixtures of 20 M' contained 50 mM Tris-CI, pH

8.0,100 mM KC1, 10 mM MgCl2,0.5 mM dithiothreitol, 0.5 mM EDTA,30 Mg/ml bovine serum albumin (nuclease free), and 1 mM ATP. Serialdilutions of the nuclear extracts served as the enzyme source. Thereactions were started by the addition of 0.6 Mg knotted DNA andterminated by the addition of 5 n\ of a stop solution containing 5%SDS, 50 mM EDTA, 25% Ficoll, and 0.05 mg/ml bromophenol blue.Samples were loaded on 0.8% agarose gels. Electrophoresis was at 1.5V/cm for 15 h in Tris-borate-EDTA buffer. Gels were stained in 1 Mg/ml ethidium bromide, destained, and photographed over a UV lightsource. Densitometric scanning of the photographic negatives allowedthe quantitative determination of the two DNA forms. One unit ofunknotting activity was defined as the amount of enzyme that converts50% of the substrate (knotted DNA) into the reaction product (un-knotted DNA). The Topo II-DNA unknotting activity in the nuclearextracts was blocked by the omission of the enzyme cofactor ATP orby the addition of 0.5 mM novobiocin (Topo II inhibitor) but not bythe addition of 0.1 mM camptothecin (Topo I inhibitor). Under theseconditions, the Topo II activity is free from interference by Topo Iactivity and agents that may promote DNA aggregation (33).

Relaxation Assay for the Determination of Topoisomerase I Activity.The substrate used in the assay was pUC8 plasmid DNA (90% super-coiled). A reaction volume of 20 M!contained 50 mM Tris-CI, pH 7.4,50 mM KC1, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, and30 Mg/ml bovine serum albumin (nuclease free). Serial dilutions of thenuclear extracts served as the source of the enzyme. The reactions werestarted by the addition of 0.6 Me of the supercoiled plasmid DNA.Following a 30-min incubation at 37°C,the reactions were terminated

by the addition of 5 M'of a stop solution containing 50% sucrose, 2%SDS, 0.2 M EDTA, and 0.05% bromophenol blue. One-half of thesamples were loaded on 0.8% gels, electrophoresed in Tris-Borate-EDTA buffer, stained, and photographed as described previously. Densitometric determination of the supercoiled form provided the meansfor quantitation. One unit of relaxation activity was defined as theamount of enzyme that converts 50% of the substrate (supercoiledDNA) into the reaction product (relaxed DNA). Topo I DNA relaxationactivity in these extracts was blocked by 0.1 mM camptothecin (Topo Iinhibitor) but not by 0.5 mM novobiocin (Topo II inhibitor). Theomission of ATP from the assay eliminated the interference of Topo IIactivity (33).

Immunoblotting and Immunoprecipitation of Phosphoryiated Topoisomerase II. Immunoblotting was performed by the method of Towbinet al. (34) with a rabbit anti-Topo II IgG used as the primary antibody.The secondary antibody was peroxidase-conjugated anti-rabbit IgG

(purchased from Sigma). Prior to their use, the antibodies were diluted1:1000. For each preparation. 2 x IO7cells in their exponential growthphase were washed twice in serum-free, phosphate-free RPMI 1640and then resuspended in 1 ml of this medium. After the addition ofcarrier-free [12P1P¡(0.2 mCi/ml), the cells were incubated for 2 h at37°C,pelleted, and resuspended in serum-free RPMI 1640 containing

phosphate and the appropriate inducer. After a 30-min incubation at37°C,the cells were pelleted, lysed, and immunoprecipitated as de

scribed by Curran and Teich (35), except that the lysis buffer alsocontained I mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10Mg/ml soybean trypsin inhibitor, 50 Mg/ml leupeptin, 1Mg/ml pepstatin,and 20 Mg/ml aprotinin. Following pelleting and washing, the proteinA-Topo II complex was dissociated by boiling in 100 M' of Laemmlibuffer (36) and electrophoresed on 8% SDS-polyacrylamide slab gels.The gels were stained, dried, and autoradiographed. Topo II phospho-rylation was quantitated by densitometric determination of its M,170,000 band.

RESULTS

Cell Differentiation Induction by Inhibitors of TopoisomeraseII. Treatment of HL-205 cells for 7 days with up to 200 MMnovobiocin, a Topo II inhibitor, resulted in a time- and dose-dependent cell growth inhibition (Fig. 1). This treatment alsocaused a fraction of the HL-205 cells to acquire a variety ofmyeloid maturation markers, which included reactivity with theOKM1, MoP-9 (Fig. 1), Mo2, My4, and B52.1 monoclonalantibodies (37-40) (Table 1). Other markers included NSEactivity (41) and NBT reduction (17) (Table 1). However, thistreatment did not cause the cells to attach to the surface oftissue culture plates as did PMA treatment (23, 24). Thenovobiocin-induced differentiation in the HL-205 cells was bothtime and dose dependent; e.g., a 7-day treatment with 50 UMnovobiocin caused 25-40% of the cells to react positively withOKM1 and MoP-9 antibodies, while at 200 MM,the highestnovobiocin concentration tested, more than 80% of the cellsexhibited these markers (Fig. 1).

Treatment of the HL-525 cells (which are resistant to PMA-induced cell differentiation) with 50-200 pM novobiocin alsocaused cell growth inhibition. Furthermore, the degree of thiseffect was similar to that observed in the susceptible HL-205cells after novobiocin treatment (Fig. 1). However, in the HL-525 cells, 50-150 MMnovobiocin did not induce reactivity witheither the OKM1, Mo2, My4, MoP-9, or B52.1 antibodies, nordid it cause an increase in NSE activity or an enhancement ofNBT reduction (Fig. 1; Table 1), in contrast to the situation inthe HL-205 cells. At 200 MMnovobiocin, the most cytotoxicconcentration tested, a modest increase in reactivity with theOKM1 antibody was observed (in less than 25% of the cells)(Fig. 1). However, novobiocin at this concentration did notinduce any of the monocyte/macrophage markers.

HL-205 cells were also treated with the Topo II inhibitorscoumermycin and m-AMSA, and the Topo I inhibitor camptothecin. On a dose basis, m-AMSA and camptothecin wereabout three orders of magnitude higher than novobiocin in cellgrowth inhibition and cytotoxicity induction while coumermycin was only about one order of magnitude higher. Of these,coumermycin and, to a lesser degree, m-AMSA, were also ableto induce maturation markers in the HL-205 cells; however,the level of these markers did not reach that induced by novobiocin (Table 2).

Because of the similarity in the response of HL-205 and HL-

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DIFFERENTIATION OF LEUKEMIA CELLS AND Topo II ACTIVITY REDUCTION

HL-206 HL-625

01234667Time (Dajfi)

1234667Tine (Day«)

Fig. 1. Induction of differentiation and inhibition of growth, as a function ofdose and time, after treatment of HL-205 and HL-525 cells with novobiocin.Inhibition of cell growth (.-I. /.)): acquisition of reactivity with OK Ml (B, E);MoP-9 monoclonal antibody (C, F). Noboviocin at 0 jiM, T; 50 jiM, •¿�:100 t¡M,»:150 JIM.•¿�;and 200 ii\i. A was added to the medium 1 day after cell seeding.Cell number, number of viable cells after exclusion of cells stained with trypanblue. Results are representative of one of three similar but independent experiments.

Table 1 Induction of differentiation markers in HL-205 and HL-525 after 6 daysof treatment with 150 nM novobiocin

The percentages are the mean ±SE from at least three experiments.

HL-205 HL-525

MarkerControlNovobiocinControlNovobiocinCells

reacting with antibody(%)OKM1Mo2My4MoP-9B52.18±4<5<5<5<591±850±975

±1378±1792

±6<5<5<5<5<59±5<5<5<5<5Cellsexhibiting NBT reduction or NSE activity(%)NBTNSE8±313

±371±869

±14<5<57±4<5

525 cells to induction of differentiation by both PMA andnovobiocin, and because PMA-induced differentiation is believed to involve activation of PKC (26, 28), we tested theability of H-7, an inhibitor of protein kinases, including PKC(27), to suppress the expression of maturation markers evokedby these two inducers. Our results indicate that H-7 was indeedcapable of reducing the novobiocin- and PMA-induced reactivity with the OKM1 antibody and the activity of NSE (Table 3),as well as the induced reactivity with the MoP-9 antibody andreduction of NBT (novobiocin only) by about 60% (data notshown). However, H-7 was not able to prevent the novobiocin-evoked growth inhibition and cytotoxicity in either HL-205 orHL-525 cells. In addition, our results also indicate that the

Table 2 Induction of differentiation markers and growth inhibition in HL-205cells after 4 days of continuous treatment with topoisomerase inhibitors

The percentages are the mean ±SE from at least three experiments. Theconcentrations were chosen on the basis that they resulted in less than 30% cellkilling as determined by the trypan blue exclusion test. Coumermycin at 1 «¿M,m-AMSA at 0.05 nM, and camptothesin at 0.08 pM, which were less cytotoxic,did not induce these differentiation markers. The cell number is the number ofviable cells after exclusion of the cells stained with trypan blue.

TreatmentNone

NovobiocinCoumermycinmAMSACamptothesinConcen

tration(jM.)ISO5

0.100.15Cell

no.(10' cells/ml)1.4

1.20.40.40.5Cells

reactingwith

monoclonalantibody(%)OKM1

MoP-9<5

<568±9 36 ±10

25 ±5 26 ±715 ±2 <5

<5 <5Cells

exhibitingTONET

NSEreductionactivity<5

<549±11 57 ±12

19 ±3 21 ±49±2 10±3

<5 <5

Table 3 Inhibition of novobiocin- and PMA-induced differentiation markers byH-7 in HL-205 cells

The percentages are the mean ±SE from at least three experiments. H-7 at20 liM was added l h prior to the 4-day treatment with either l UMPMA or 150iiM novobiocin.

Cells reacting withOKM1 antibody(%)TreatmentControl

PMANovobiocinControl<582±851 ±4H-7<533

±920 ±4Cells

exhibiting NSEactivity(%)Control<533

±346 ±6H-7<514

±520 ±5

Table 4 Induction of differentiation markers after 4 days of treatment with eitherPMA or novobiocin in HL-205 cells

The percentages are the mean ±SE from at least three experiments. Novobiocin at 150 /<Mwas added l h prior to 0.3 nM PMA. ND, not determined.

Cells reacting withOKM1 antibody(%)fnnrentratmnTreatment

(nM)Control

PMAPMA0.31.0Control<522

±5>95Novobiocin64

±490 ±6

>95Cells

exhibiting NSEactivity(%)Control<59±2

NDNovobiocin46

±361 ±5

ND

induction of maturation markers (reactivity with OKM1 andNSE activity) by 150 ¿¿Mnovobiocin and 0.3 nM PMA at 4 daysafter treatment was additive. This novobiocin concentration didnot prevent the induction of differentiation by a higher dose ofPMA (1.0 nM), which by itself caused more than 95% of thecells to react positively with the OKM1 antibody (Table 4).

These studies implicate a protein kinase, most likely PKC,in the induction of cell differentiation by both PMA and novobiocin. However, this induction probably does not involve adirect effect of novobiocin on PKC because, unlike PMA,novobiocin did not compete for [3H]phorbol-12,13-dibutyratebinding to its HL-205 cellular receptors, nor did novobiocinactivate purified PKC when this kinase was tested for histoneH1 phosphory hition (28).

Activity of Topoisomerase I and II in HL-205 and HL-525Cells. Because of the possibility that a common step in novobiocin- and PMA-induced differentiation is a reduction in eitherTopo II or Topo I activity, we tested these activities in untreatedand inducer-treated HL-205 and HL-525 cells.

Analysis of the nuclear extracts from the untreated cellsrevealed that the extract from the differentiation-susceptibleHL-205 cells exhibited a specific activity for Topo II of 5 x IO3units/mg protein, whereas the extract from the resistant HL-525 cells contained a 4-fold higher activity of 20 x IO3 units/

mg protein (Fig. 2). Unlike these differences in Topo II activity,the activity of Topo I was similar in the extracts from both cell

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DIFFERENTIATION OF LEUKEMIA CELLS AND Topo II ACTIVITY REDUCTION

types, yielding an activity of 3.3 x IO5 units/mg protein (Fig.

2).Treatment of the HL-60 cell variants with 150 nM novobiocin

caused a reduction in Topo II activity in the extracts from bothHL-205 and HL-525 cells, with the reduction in the extractfrom the HL-205 cells being most prominant. After 6 h oftreatment, the Topo II activity in the extracts from the HL-205cells, but not the HL-525 cells, was reduced by 4-fold. A 24-htreatment resulted in an 8-fold reduction in Topo II activity inthe extracts from the HL-205 cells but less than one-half ofthis reduction in the extract from the HL-525 cells (Fig. 3). Incontrast, the response of Topo I activities in these extracts wassimilar in both cell types; these activities were reduced after 6h of treatment by 2-fold and after 24 h by 6-fold (Fig. 4).

We examined the possible role of protein kinases in theseevents by analyzing the reduction in topoisomerase activities inthe nuclear extracts from HL-205 cells treated with eithernovobiocin or PMA in the presence and absence of H-7. Toensure that the reduction in Topo II activity is an early eventand is thus associated with the induction process, we tested theeffects of novobiocin and PMA shortly after their incubationwith the cells. This shorter duration of treatments requiredhigher drug concentrations. Treatment of differentiation-susceptible HL-205 cells for only 30 min with either 500 fiM or 2mM novobiocin in the absence of H-7 resulted in a 2-fold anda 6-fold reduction of Topo II activity, respectively. A 2-fold

reduction in Topo II activity was also observed after a 30-mintreatment with 0.2 ^M PMA (Fig. 5). Little or no reduction inTopo II activity was observed after such treatments of theresistant HL-525 cells. The reductions in Topo II activity inthe treated HL-205 cells, however, were prevented when thecells were preincubated with H-7 (Fig. 5). In contrast, H-7 didnot markedly alter the Topo I activities in the nuclear extractsfrom HL-205 or HL-525 cells treated for 30 min with 2 mMnovobiocin.

Phosphorylation of Topoisomerase II in HL-205 and HL-525Cells. To study the involvement of protein kinases in modulating Topo II activity in the HL-205 and HL-525 cells, weincubated the cells with 32Pand immunoprecipitated the phos-

phorylated Topo II with a specific antibody and further separated the enzyme by SDS-polyacrylamide gel electrophoresis.The major fraction of the radioactivity was detected in the TopoII (A/, 170,000) band. Densitometric scanning revealed that thelevel of phosphorylated Topo II from the HL-205 cells was fourtimes lower than that from the HL-525 cells (Fig. 6). Treatmentof the HL-205 cells with 0.2 ^M PMA for 30 min resulted in a2-fold increase in the level of phosphorylated Topo II, while atreatment with 2 mM novobiocin caused an 8-fold decrease inthe level of the phosphorylated enzyme (Fig. 6). These PMA-and novobiocin-evoked changes in the level of phosphorylatedTopo II were prevented by pretreatment of the HL-205 cellswith H-7 (Fig. 6). Treatment of HL-525 cells with either PMA

A. TOPO I

Fig. 2. Topo I and Topo II activities innuclear extracts of HL-205 or HL-525 cells.Serial dilutions of the nuclear extracts servedas the enzyme source. A, Topo I activity wasdetermined by converting supercoiled pUC 8plasmid DNA (/) into its relaxed form (/ rei).The plasmid contained also a small amount ofthe nicked form (//). The negative control (-)contained the reaction mixtures without thenuclear extract. The positive control (+) contained the reaction mixture with a sample ofpurified Topo I from calf thymus. The reactionmixtures included the following amounts ofextract protein: Lane 1, 100 ng; Lane 2, 30 ng;Lane 3, 10 ng; Lane 4, ÃŒng; Lane 5, 1 ng. B,Topo II activity was determined by convertingknotted P4 phage DNA (K) to its unknottedform (f). The phage DNA preparation alsocontained a small amount of linear form (/.).The negative control (-) contained the reaction mixture without the nuclear extract. Thepositive control (+) contained the reactionmixture with a sample of knotted P4 DNA.The reaction mixtures included the followingamounts of extract protein: I.an? I, 800 ng:Lane 2. 400 ng; Lane 3, 200 ng; Lane 4, 100ng; Lane 5, 50 ng. Results are representativeof one of three similar but independent experiments.

I rei

HL-205

B. TOPO II- + 12345

HL-525

12345

K

HL-2051113

HL-525

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DIFFERENTIATION OF LEUKEMIA CELLS AND Topo II ACTIVITY REDUCTION

HL-205- + A. Control B. NOVO-6H C. NOVO-24H

K

Fig. 3. Topo II activity in nuclear extractsfrom untreated HL-205 and HL-525 cells (A)and from such cells treated with ISO /<Mno-vobiocin for 6 h (B) or 24 h (C). Nuclearextracts were serially diluted to contain thefollowing amounts of extract protein: Lane I,800 ng; Lane 2, 400 ng; Lane 3, 200 ng; Lane4, 100 ng; Lane 5. 50 ng. The negative (-) andpositive controls (+) are as described in thelegend of Fig. 2B.

Fig. 4. Topo I activity in nuclear extractsfrom untreated HL-205 and HL-525 cells (A)and from such cells treated with 150 pM no-vobiocin for 6 h (B) or 24 h (C). Nuclearextracts were serially diluted to contain thefollowing amounts of extract protein: Lane I,100 ng; Lane 2, 50 ng; Lane 3, 25 ng; Lane 4.12.5 ng; Lane 5, 6.2 ng; Lane 6, 3.1 ng. Thenegative (—)and positive controls (+) are asdescribed in the legend of Fig. 2A.

HL-525- + A. Control B. NOVO-6H C. NOVO-24H

U

12345 12345 12345

HL-205A. Control B. NOVO-6H C. NOVO-24H

or novobiocin did not result in a marked alteration in the levelof phosphorylated Topo II.

To determine whether the differences in the levels of phosphorylated Topo II are due to either changes in the amount ofthis enzyme or a different degree of its phosphorylation, we

1 23456 1234 56 123456

estimated the amount of the enzyme following its immunoblot-ting with an anti-Topo II antibody. The results indicated thepresence of similar Topo II levels in both HL-205 and HL-525cells, as well as in HL-205 cells treated with PMA (Fig. 7). Incontrast, treatment of the HL-205 cells with novobiocin re-

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K

DIFFERENTIATION OF LEUKEMIA CELLS AND Topo II ACTIVITY REDUCTION

- -f Control 1-H7 A. B.

12 123456

A Control

12345 12345

345 2345

B. NOVO

C. PMA

4 5

Fig. 5. Topo II activity in nuclear extracts from untreated HL-205 cells (A)and from HL-205 cells treated for 30 min with either 2 mM novobiocin (B), or0.2 >iMPMA (C) in the absence or presence of 0.2 mM H-7. The cells wereincubated with H-7 60 min prior to and during the treatment with eithernovobiocin or PMA. The negative (-) and the positive control (+) are as describedin the legend of Fig. IB. The reaction mixtures included the following amountsof extract protein: Lane I, 800 ng; Lane 2, 400 ng; Lane 3, 200 ng; Lane 4. 100ng; Lane 5, 50 ng. The results are representative of one of three similar butindependent experiments.

suited in a decrease in the amount of the M, 170,000 Topo IIband, most likely because of its degradation through a M,150,000 band (Fig. 7). This decrease in the amount of Topo IIwas also prevented by preincubation of the HL-205 cells withH-7 (Fig. 7).

These results suggest that the higher level of phosphorylatedTopo II in HL-525 cells as compared to that in HL-205 cellsand the increased level of phosphorylated enzyme in PMA-treated HL-205 cells results from an increase in Topo II phos-phorylation. In contrast, the decrease in the level of phosphorylated enzyme in the novobiocin-treated HL-205 cells resultsfrom enzyme degradation, most likely mediated by a proteinkinase-dependent protease.

DISCUSSION

In the present studies, we investigated the roles of Topo IIand Topo I in regulating the growth and maturation of twohuman HL-60 leukemia cell variants (HL-205 and HL-525)that differ in their susceptibility to differentiation induced byPMA but not other chemical inducers (28). Our results indicated that HL-205 cells that are susceptible to differentiationinduced by PMA are also susceptible to induction of differentiation by novobiocin. These results, and those from our otherstudies (which indicate that novobiocin does not inhibit PMA-induced differentiation but may rather act additively withPMA), are in contrast to those of Sahyoun et al. (42), who

•¿�t--

Fig. 6. Phosphorylated Topo II from HL-205 and HL-525 cells. The labeledTopo II [M, 170,000 (170 kOa)] band was obtained after the cells were incubatedwith 32P and Topo II immunoprecipitated with a specific antibody and furtherseparated by SDS-polyacrylamide gel electrophoresis. A, Topo II from untreatedHL-205 (Lane I) and HL-525 (Lane 2) cells. B, Topo II from untreated HL-205cells (Lane 1) and from HL-205 cells treated for 30 min with either 0.2 /iM PMA(Lane 2) or 2 itiM novobiocin (Lane 3), as well as Topo II from HL-205 cellstreated with 0.2 mM H-7 for 90 min (Lane 4), pretreated with 0.2 min H-7 for60 min and treated with 0.2 >JMPMA for 30 min in the presence of H-7 (Lane5), or pretreated with 0.2 mM H-7 for 60 min and treated with 2 nM novobiocinfor 30 min in the presence of H-7 (Lane 6).

116

84

Fig. 7. Topo II proteolysis after treatment of HL-205 cells with novobiocinand its inhibition by H-7. After cell lysis. Topo II was separated by polyacrylamidegel electrophoresis, transferred to a nitrocellulose membrane, and immunoblottedwith a specific antibody. The molecular weights shown on the right ordinate arethose of Topo II (M, 170,000) and its proteolytic products. Prestained standardswith their molecular weights in thousands are shown on the left ordinate (Lane1). The lanes represent Topo II from untreated HL-205 cells (Lane 2) and fromHL-205 cells treated with either 0.2 mM H-7 for 90 min (Lane 3), 2 mMnovobiocin for 30 min (Lane 4), or pretreated with 0.2 mM H-7 for 60 min andthen with 2 mM novobiocin for 30 min in the presence of H-7 (Lane 5).

found that novobiocin inhibited the differentiation induced byphorbol-12,13-dibutyrate. This apparent contradiction may bebecause Sahyoun et al. (42) determined cell differentiation afteronly 11.5 h of treatment with the phorbol diester, an insufficienttime period to detect commonly used maturation markers (17,24, 25, 28, 30). Moreover, cell attachment, the only markerused in their study, is a common response of different bloodcell types to treatment with phorbol diesters and may notnecessarily be indicative of cell maturation (43, 44).

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DIFFERENTIATION OF LEUKEMIA CELLS AND Topo II ACTIVITY REDUCTION

Topo II inhibitors such as coumermycin and m-AMSA wereless effective than novobiocin in inducing differentiation in HL-205 cells. This reduced ability may be attributed to their excessive cytotoxicity; unlike novobiocin, coumermycin is highlycytotoxic to the HL-60 cells, and its cytotoxicity is detected atconcentrations that are lower than those required to inhibitTopo II activity (45). The antitumor drug m-AMSA is a potentcytotoxic drug because of its ability to stabilize the double-strand DNA breakage that results from the interaction of TopoII with DNA (15, 46, 47), which may prevent the expression ofgenes critical for cell differentiation.

In addition to inducing differentiation in HL-205 cells, novobiocin and PMA were able to cause a reduction in the TopoII activities of the HL-205 cells within a short time (30 min);little or no reduction in this enzyme activity was found aftertreatment of the resistant HL-525 cells. This observation maybe attributed to the dissimilar levels of Topo II activities foundin the two cell types; Topo II activity in the nuclear extractfrom HL-205 cells is 4-fold lower than that in the nuclearextract from the HL-525 cells. However, in contrast, novobiocinreduced the Topo I activities to a similar degree in both HL-205 and HL-525 cells. Novobiocin also caused a similar reduction in the cell growth in both cell types. On the other hand,PMA, which does not inhibit the growth of HL-525 cells (28),also did not alter the Topo I level in these cells.

On the basis of these results, we suggest that the reductionof Topo II activity in HL-60 cells is associated with inductionof differentiation, while the reduction in Topo I seems to belinked to inhibition of cell growth. This suggestion is furthersubstantiated by results from our experiments with H-7, aninhibitor of protein kinases (27). In these studies, H-7 diminished both the induction of differentiation and the reduction inTopo II activities evoked by either novobiocin or PMA in theHL-205 cells. However, this inhibitor did not effectively preventthe novobiocin-elicited inhibition of cell growth or reduction inTopo I activities in either HL-205 or HL-525 cells.

The reduction in Topo II activity shortly (30 min) aftertreatment of the HL-205 cells with either novobiocin or PMAprobably occurs through different modes of action. The novo-biocin-evoked reduction in Topo II activity cannot be explainedby a possible reduction in the intracellular level of ATP (48),because the assay used in our study to measure Topo II activitycalls for an addition of exogenous ATP to the enzyme reaction(32). The novobiocin-evoked reduction in Topo II activity ismost likely due to an enhanced enzyme hydrolysis. Our resultshave shown that 30-min treatment of the HL-205 cells withnovobiocin resulted in a degradation of Topo II. The mechanism underlying this event may involve the interaction of novobiocin with Topo II, which causes the enzyme to change itsconformation, thus rendering it more susceptible to a proteo-lytic attack. Fluctuations in Topo II levels during the cell cycleof chicken lymphoblastoid cells are also attributed in part toproteolysis of the enzyme (49). The activity of the hydrolyticenzyme(s) in the novobiocin-treated cells is most likely dependent on protein kinase activities because Topo II degradation inthe HL-205 cells was prevented by preincubation with H-7. ThePMA-induced reduction in Topo II involves a different modeof action because treatment of HL-205 cells with this inducerdid not enhance degradation of Topo II. Recent studies haveshown that Drosophila and Geodia (sponge) Topo II can bemanipulated in vitro by phosphorylation with PKC (42, 50).Thus, PMA, a PKC activator (26), may inhibit Topo II activitythrough its effect on phosphorylation of the topoisomerase.Indeed, our studies have shown that PMA treatment of HL-

205 cells results in an increase in Topo II phosphorylation,which is abrogated by pretreatment with H-7, a PKC inhibitor(27). The PKC activities found in HL-205 and HL-525 cellsdiffer in a number of characteristics, including substrate specificities (51). It is thus possible that the types of PKCs that arefound and activated by PMA in HL-205 cells may phosphoryl-ate Topo II at sites that render this enzyme less active, whileother types of PKC, such as that found in HL-525 cells, mayphosphorylate Topo II at sites that make it more active. Another possibility is that HL-205 cells contain a Topo II inhibitorthat is dependent on PKC activation. Thus, the increase inTopo II phosphorylation and the resulting increase in enzymeactivity may be neutralized by activation of the Topo II inhibitor. A third possibility is that HL-205 and HL-525 cells eachhave a different type of Topo II, as described for m-AMSA-susceptible and -resistant cells (29), which may respond differently after phosphorylation.

In brief, our results suggest that a reduction in Topo II activityis an early event that is associated with induction of differentiation in HL-60 cells and perhaps other cell types. Furthermore,our studies raise the possibility that the antibiotic drug novobiocin, and perhaps some of its analogues, may become usefulin the control of some types of malignant cells by inducingthem to differentiate into nondividing mature cells.

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1989;49:1110-1117. Cancer Res   Andreas Constantinou, Cynthia Henning-Chubb and Eliezer Huberman  Reduction in Topoisomerase II ActivityDifferentiation of Human Leukemia Cells Associated with a Novobiocin- and Phorbol-12-myristate-13-acetate-induced

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