Induction of Differentiation and DNA Strand Breakage … · Induction of Differentiation and DNA Strand Breakage in Human HL-60 ... Mg/mlgenistein, was the induction of DNA damage

[CANCER RESEARCH 50. 2618-2624, May I. 1990)

Induction of Differentiation and DNA Strand Breakage in Human HL-60and K-562 Leukemia Cells by Genistein1

Andreas Constantinou, Kaoru Kiguchi, and Eliezer Huberman2

Biological and Medical Research Division, Argonne National Laboralor)-. Argonne, IL 60439

ABSTRACT

Genistein, an in vitro inhibitor of topoisomerase II and tyrosinekinasfs. suppressed growth and induced differentiation in HL-205 cells,a clonal population of the human promyelocytic HL-60 leukemia cells,and in K-562-J cells, a clonal population of the human erythroid K-562leukemia cells. Maturing I IL-205 cells acquired either granulocytic ormonocytic markers, namely, reactivity with the murine ORMI monoclonal antibody, expression of nitroblue tetrazolium dye reduction, andstaining for nonspecific esterase. The maturing K-562-J cells stainedwith benzidine, which indicates the presence of hemoglobin, an erythroidmaturation marker. Although the acquisition of the maturation markersin both HL-205 and K-562-J cells was time dependent up to 6 days, thekinetics of this induction differed between the two cell types. Despite thein vitro inhibitory effect of genistein, treatment of either HL-205 or K-562-J cells with 150 MR/ml genistein for up to 16 h did not altertopoisomerase II activity (as determined by the unknotting assay) in theirnuclear extracts. Analysis with the anti-phosphotyrosine PY-20 murinemonoclonal antibody indicated that treatment of K-562-J cells withgenistein decreased the reactivity of the antibody with two of the cellularproteins. However, no reactivity with the PY-20 antibody was detectedin untreated or genistein-treated HL-205 cells. An early event in the HL-205 and K-562-J cells, occurring after only l h of treatment with 30-200Mg/mlgenistein, was the induction of DNA damage as measured by analkaline elution assay. This damage may be a contributing factor in thegenistein-induced cell differentiation in the HL-205 and K-562-J cells.

INTRODUCTION

Decreased activity of either topoisomerases or tyrosine ki-nases has been implicated in the differentiation of a number ofcell types (1-8). With regard to the inhibition of topoisomerases, the activity of Topo II1 is decreased when mouse eryth-roleukemia cells acquire a mature phenotype (5) and when HL-60 cells differentiate following treatment with novobiocin, aTopo II inhibitor, or phorbol 12-myristate 13-acetate, a phorboldiester (7, 8). With respect to the inhibition of tyrosine kinase,a reduction in the amounts of protein phosphotyrosine residueshas been reported during granulocytic and monocytic maturation of the human promyelocytic HL-60 leukemia cells (1) anda reduction in the level of tyrosine-phosphorylated proteins hasbeen observed in the erythroid differentiation of K-562 leukemia cells (2).

These observations raised the possibility that genistein, because of its dual action as a topoisomerase inhibitor and atyrosine kinase inhibitor (9, 10), may induce cell differentiation.

Received 8/11/89; revised 12/20/89.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.

1This work was supported by the U.S. Department of Energy. Office of Healthand Environmental Research, under contract W-31-109-ENG-38.

2To whom requests for reprints should be addressed, at Biological and MedicalResearch Division. Argonne National Laboratory. 9700 South Cass Avenue.Argonne. IL 60439.

3The abbreviations used are: Topo II, type II topoisomerase; Topo I, type Itopoisomerase; EOF. epidermal growth factor; EGFTK. epidermal growth factorreceptor tyrosine kinase; m-AMSA. 4'-(9-acridinylamino)methanesulfon-m-an-

isidide; NBT. nitroblue tetrazolium: NSE. nonspecific esterase: SDS, sodiumdodecyl sulfate: TLCK. A'-<i-tosyl-L-lysyl chloromethyl ketone: VM-26. 4'-de-methylepipodophyllotoxin-4-(4,6-0-thenylidene-^-D-glycopyranoside) (tenipo-side); SSB, single-stranded break.

To test this possibility, we investigated the ability of genisteinto inhibit the activities of topoisomerases and tyrosine kinasesand to induce differentiation in the human HL-60 and K-562leukemia cells. We report here that genistein induces a maturephenotype in both cell types and that this induction is associatedwith its effect on DNA strand breakage, possibly mediated byTopo II.

MATERIALS AND METHODS

Chemicals and Reagents. Genistein (4',5,7-trihydroxyisoflavone) and

the murine PY-20 monoclonal antibody were purchased from ICNBiomedicals; novobiocin, proteinase K, TLCK from Sigma ChemicalCo.; and polycarbonate membrane filters from Nucleopore Corp. Themurine OKM1 monoclonal antibody was from Ortho PharmaceuticalCorp. Knotted bacteriophage P4 DNA was isolated from the taillesscapsids of the bacteriophage P4 viri I del IO (11). Topo II was purifiedfrom HL-525 as described by Drake et al. (12). Calf thymus Topo Iwas purchased from Bethesda Research Laboratories. Stock solutionsof genistein and TLCK were disolved in dimethyl sulfoxide at 30 mg/ml and stored at -70Â°C. Novobiocin was dissolved in double-distilledwater at 10 mg/ml and stored at â€”20Â°C.[/neiAy/-'H]Thymidine was

purchased from ICN Radiochemicals. The chemicals were added to theculture once at the beginning of the treatment.

Cells, Culture Conditions, and Differentiation Markers. The HL-205cell clone was isolated from HL-60 cells as described (13). The K-562-J cell clone was isolated in this laboratory by Dr. S. Maeda from theK-562 cells originally established from a chronic myelogenous leukemiapatient (14). Cells were inoculated into 100- or 150-mm tissue culturedishes at 1.5 x IO5 cells/ml of RPMI 1640 supplemented with 15%

fetal bovine serum, penicillin (100 units/ml), and streptomycin (100Mg/ml), and cultured at 37Â°Cin a humidified atmosphere of 8% CO2in

air. Immunofluorescence tests for reactivity with the antibodies, staining for NSE activity, and the NBT dye reduction assay were allperformed as previously described (15, 16). The presence of hemoglobinin the differentiating K-562-J cells was determined by the benzidine-staining assay (17).

Unknotting Assay for the Determination of Topo II Activity. Theenzyme source was either Topo II purified from HL-525 cells (13) orserial dilutions of nuclear extracts of either untreated cells or cellstreated with genistein. The preparation of the nuclear extracts, the P4DNA substrate, and the electrophoresis conditions were previouslydescribed (8). Reaction mixtures of 20 u\ contained 2 M' of enzymepreparation-50 mivi Tris-Cl, pH 8.0-100 mivi KCL-10 mM MgCl2-0.5mM dithiothreitol-0.5 mM EDTA-30 Mg/ml bovine serum albumin 1mM ATP. One unit of unknotting activity was defined as the amountof the enzyme that converts one-half of the knotted DNA to unknottedDNA during a 30-min incubation at 37Â°Cas previously described (8).

Relaxation Assay for the Determination of Topo I Activity. Reactionmixtures of 20 u\ contained 50 mM Tris-Cl. pH 7.4-50 mM KC1-10 mMMgCIj-l mM dithiothreitol-0.1 mM EDTA-30 Mg/m' bovine serumalbumin. The enzyme source was either purified Topo I (from calfthymus) or serial dilutions of nuclear extracts of either untreated cellsor cells treated with genistein; the substrate was pUC8 DNA (90%supercoiled). The electrophoresis conditions were as described previously (8). One unit of relaxation activity was defined as the amount ofthe enzyme that converts one-half of the supercoiled DNA to relaxedDNA during a 30-min incubation at 37Â°Cas previously described (8).

Topo H-mediated DNA Cleavage Assay. The conversion of super-coiled pUC8 DNA to its linear form was measured in the same reactionmixture as was used for the relaxation assay except that ATP was

2618

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GENISTEIN INDUCES DIFFERENTIATION IN HUMAN LEUKEMIA CELLS

added to a final concentration of 1 mM in some of the reactions andthe amount of Topo II was increased to 40 units. When applicable, theinhibitor was added prior to addition of Topo II to the reaction mixture.After 30 min incubation at 37'C, 1 mg/ml of proteinase K and \%

SDS were added to the reaction mixture. After a further 30-minincubation at 37Â°C,the reactions were stopped with sample buffer (5%

SDS, 50 mm EDTA, 25% Ficoll, and 0.05 mg/ml bromophenol blue)and the samples were electrophoresed at 3 V/cm for 4 h in Tris-borate-EDTA buffer containing 0.5 Â¿ig/mlof ethidium bromide.

Alkaline Elution Technique. The alkaline elution procedure used wasthat of Kohn et al. (18). Following incubation for 24 h with 0.2 Â¿iCi/ml[merA)7-'H]thymidine (56 Ci/mmol). the cells were washed twice with

fresh RPMI 1640 growth medium and incubated with genistein for 1h. (Controls were treated for l h with the appropriate concentration ofdimethyl sulfoxide. the genistein solvent.) Aliquots of cells containing4 x IO5cells were deposited onto polycarbonate membrane filters andlysed with 2% SDS-25 mM EDTA, pH 9.7, containing 0.5 mg/ml ofproteinase K. DNA was eluted with 34 ml of tetrapropylammoniumhydroxide-EDTA, pH 12.1, at a rate of 2.13 ml/h. The different DNAfractions were collected into scintillation vials at 90-min intervals. Atthe end of the elution. an alkaline solution (2 ml of 0.4 N NaOH) wasapplied to elute any remaining DNA from the walls of the holder. Thisfraction was considered as DNA remaining on the filter. SSB frequencywas calculated as SSB rad-equivalents according to the following equation:

SSB rad-equivalents = log (r,/r0)x 300 [Eq. 1]

where r,, r0, and R0 represent the retention of DNA from genistein-treated, control, and 300-rad 7-ray-treated ['Hjthymidine-labeled cells,respectively. Retention values for determining the rad-equivalents werebased on the 9-h fraction.

Neutral Elution Technique. Neutral elution was performed as described by Bradley and Kohn (19), with the following two exceptions.First, the lysis solution (0.05 M Tris-0.05 M glycine-0.025 M EDTA-2% SDS) was adjusted to pH 7.2, and, second, at the end of the neutralelution, an alkaline solution (2 ml of 0.4 N NaOH) was applied to eluteany remaining DNA from the walls of the holder. This fraction wasconsidered as DNA remaining on the filter.

Immunoblotting. Immunoblotting was performed as previously described (8) except that the primary antibody was the PY-20 antibodyand the secondary antibody was biotin-conjugate anti-mouse IgG followed by avidin-peroxidase conjugate (both from Sigma Chemical Co.).

Tyrosine Kinase Assay. The activity of tyrosine kinase was determined with Raytide (Oncogene Science) as the substrate. The solubi-lized membrane fractions of A-43I cells containing the EGFTK (20)were used as the source of the enzyme. These fractions were incubatedin a reaction volume of 0.03 ml containing 20 mM Tris-Cl, pH 7.4-1mM MnCl2-0.05 mM sodium vanadate-0.5 mM ethyleneglycol-bis(fi-aminoethyl ether) yV,W,W,JV'-tetraacetic acid-0.1 mM [7"P]ATP (spe

cific activity 200 cpm/pmol)-0.01 mg/ml leupeptin, with or withoutEGF. The reaction was initiated by the addition of the fraction containing EGFTK, and the reaction mixture was incubated at 23Â°Cfor 10

min. The reaction was terminated by spotting an aliquot of the reactionsolution on Whatman P8I paper and then washing the paper with ISOmM phosphoric acid.

RESULTS

Induction of Differentiation in HL-2O5 and K-562-J Cells byGenistein. Genistein, an isoflavone compound isolated from thefermentation broth of Pseudomonas sp., is an in vitro inhibitorof topoisomerases and tyrosine kinases (9, 10). This compoundwas tested for its ability to inhibit cell growth and to inducedifferentiation in the HL-205 and K-562-J cells. Treatment ofHL-205 cells for up to 6 days with different concentrations ofgenistein caused a time-dependent (Fig. 1) and dose-dependent(Fig. 2) decrease in cell numbers. A similar effect was also

observed in the K-562-J cells, but the degree of decrease wasless pronounced than in HL-205 cells (Figs. 1 and 2).

During a period of 6 days in culture, less than 7% of untreatedHL-2O5 cells reacted with the murine OKM1 monoclonal

antibody that detects a cell surface antigen common to maturemyeloid cells (21) and less than 10% of the cells stained positively for NSE activity, a characteristic of monocytes/macro-phages (22), or for NBT dye reduction, a marker of granulocyticmaturation in these cells (16). Treatment of these cells withgenistein resulted in not only a decrease in cell number but alsoan induction of cell differentiation (Fig. 3). In the HL-205 cells,genistein at 10 Mg/ml caused a time-dependent increase in thepercentage of cells reacting with the OKM1 antibody, stainingfor NSE activity, and exhibiting NBT dye reduction (Fig. 3).After 4 days of treatment, more than 95% of the cells reactedwith the OKM1 antibody, about 50% of the cells exhibitedNBT dye reduction, and 20% of the cells stained positively forNSE activity (Fig. 3). Under these conditions, less than 8% ofthe cells stained with trypan blue, a characteristic of dead cells.At a higher dose of genistein (20 Â¿/g/ml),the percentage of HL-205 cells staining for NSE activity rose to about 50% (Fig. 3).

Similarly, treatment of the K-562-J cells with genistein resulted in cell differentiation. At 10 Mg/ml, the genistein causeda time-dependent increase in the percentage of K-562-J cellsstaining positively with benzidine (Fig. 4), which indicates thepresence of hemoglobin, an erythroid maturation marker inthese cells (17). On the seventh day, more than 50% of cellsstained positively with benzidine, while during the 7 days ofculture less than 5% of control cells stained positively with thedye (Fig. 4). Under these conditions, less than 5% of the cellsstained with trypan blue. The increase in benzidine-stained cellswas also dose dependent up to 10 Mg/ml of genistein (Fig. 4).Higher doses of the agent were less effective. Genistein also did

HL-205 K-562-J

Fig. 1. Effect of 10 ,ig/nil of genistein on the growth of HL-205 and K-562-Jcells as a function of time. O, untreated HL-205 cells; A. untreated K-562-J cells:Â».treated HL-205 cells: A. treated K-562-J cells.

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5 10 15 20 25

GENISTEIN (M9/ml)

Fig. 2. Effect of different concentrations of gcnistein on the growth of HL-205 (â€¢)and K-562-J (A) cells after 4 days of treatment.

2619




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Fig. 3. Induction of differentiation of HL-205 cells treated with genistein.Left, time response: cells were treated with 10 ng/m\ of genistein and thepercentage of cells reacting with the OKMI monoclonal antibody (â€¢).reducingthe NBT dye (A), and expressing NSE activity (A) was determined. During the 6days in culture, less than 1% of the control cells reacted with OKMI antibody,and less than 5cr of the control cells reduced the NBT dye or expressed NSEactivity. Righi, dose response: cells were treated with increasing concentrationsof genistein and the percentage of cells reacting with the OKMI monoclonalantibody (â€¢)and expressing NSE activity (A) was determined after 4 days oftreatment. Each point represents the mean Â±SE of three independent determinations.

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Fig. 4. Induction of differentiation of K-562 cells with genistein. Left, timeresponse: cells were treated with 10 ^g/ml of genistein and the percentage ofbenzidine-stained positive cells was determined as a function of time. During the7 days in culture, less than 5cr of the control cells stained with benzidine. Right.dose response; cells were treated with increasing concentrations of genistein andthe percentage of benzidine positive cells was determined after 7 days of treatment.Each point represents the mean Â±SE of three independent determinations.

not cause an increase in the percentage of K-562-J cells reactingwith the OKMI antibody (data not shown).

These results indicate that genistein induces growth inhibition and differentiation in both the HL-205 and the K-562-Jcells. The kinetics of this induction, however, differ betweenthe two cell types.

Inhibition of Topoisomerase Activity and Stabilization of aTopo II-DNA Complex by Genistein. To confirm and extendprevious studies indicating that genistein can inhibit the activities of DNA topoisomerases and tyrosine kinases (9, 10), wemeasured the effect of genistein on these activities using purifiedTopo I and Topo II and a cell preparation that contains theEGFTK. For comparison we included novobiocin, a knownTopo II inhibitor, and TLCK, a known tyrosine kinase inhibitor. The activity of Topo II was determined by using theunknotting assay, and the activity of Topo I was determined byusing the relaxation assay. Our results indicated that genisteininhibited the activity of Topo II in a dose-dependent manner(Fig. 5). In the presence of 2 units of Topo II, genistein at 30Mg/ml inhibited about one-half of Topo II activity (Fig. 5, top,Lane 6), while a concentration of 100 Mg/ml resulted in a

complete inhibition (Fig. 5, top, Lane 7). In comparison, complete inhibition of Topo II activity required a concentration of200 Â¿ig/mlof novobiocin (Fig. 5, top. Lane 2). On a molar basis,360 /IMgenistein or 315 MMnovobiocin were required to inhibitthe Topo II activity. The inhibitory effect of genistein on TopoII activity was inversely related to the amount of the purifiedenzyme; in the presence of 20 units of Topo II, completeinhibition of enzyme activity required 10 times more genisteinthan in the presence of 2 units of the enzyme (data not shown).Genistein was less effective in inhibiting Topo I activity; aconcentration of 1000 ng/m\ inhibited less than one-half theactivity when 2 units of Topo I were present in the reactionmixture (Fig. 5, bottom. Lane 7). TLCK did not affect eitherTopo II (Fig. 5, top, Lanes 8-12) or Topo I activities (data notshown). These results confirm the ability of genistein to inhibitthe activity of purified Topo II.

Topo II interaction with DNA involves transient double-stranded breakage followed by rejoining of the two strands. TheTopo II-DNA complex causing the DNA strand breakage canbe stabilized by drugs such as m-AMSA or VM-26, formingwhat are termed "cleavable complexes" (23). These complexes

can be detected by the conversion of supercoiled plasmid DNAto its linear form following denaturation and proteolytic degradation of the DNA-bound Topo II. To determine whethergenistein can stabilize the Topo II-DNA complex, we analyzedits ability to produce linear DNA from supercoiled DNA in thepresence of Topo II (Fig. 6). Genistein at concentrations of 3-1000 Mg/m' produced double-stranded DNA breaks as shown

9 10 11 12

Fig. 5. Effect of genistein on the unknotting activity of purified HL-525 cell.Topo II (top), or the relaxation activity of purified Topo I (bottom). Top. 2 unitsof purified Topo II were incubated in the absence (Lane I) or presence of 200Mg/ml of novobiocin (Lane 2), or 1, 3, 10. 30, or 100 Mg/ml of genistein (Lanes3-7). or 1, 3, 10, 30, or 100 ug/ml TLCK (Lanes 8-12). Topo II activity wasdetermined by converting knotted P4 phage DNA (K) to its unknotted form (U).The phage DNA preparation contained also a small amount of linear form (L).Bottom, reaction mixtures contained 2 units of purified Topo I (Lane 2). plusgenistein at 10,30, 100. 300. and 1000 Mg/ml (Lanes 3-7). Lane 1. pUC8 plasmidDNA control (no enzyme added). Topo I activity was determined by convertingsupercoiled pUC8 plasmid DNA (.9) into its relaxed form (R).

2620




by the appearance of linear DNA (Fig. 6. Lanes 2-7). Linear

DNA was not detectable in the absence of genistein (Fig. 6,Lane Â¡)nor in the absence of Topo II (Fig. 6, Lane 9). In ourexperiment, ATP did not have a major effect on the productionof linear plasmid DNA (Fig. 6, compare Lanes 6 and 8). Thus,genistein, like m-AMSA and VM-26 (Fig. 6, Lanes 11-16).induces Topo Il-mediated double-stranded breaks. In contrast,novobiocin, which interacts directly with Topo II, does notstabilize the Topo II-DNA complex, as shown by its inabilityto induce Topo Il-mediated double-stranded breaks (Fig. 6,

Lanes //and 18). These results indicate that genistein can notonly inhibit Topo II activity but also stabilize the Topo II-DNA

complex.Enhancement of Protein-associated DNA Strand Breaks in

HL-60 and K-562-J Cells Treated with Genistein. In additionto determining the effect of genistein on purified topoisomer-ases, we measured Topo I and Topo II activities in nuclearextracts obtained from cells treated with this agent using therelaxation and unknotting assays, respectively. Treatment ofeither K-562-J or HL-205 cells with genistein at a concentrationas high as 150 Mg/ml for 16 h resulted in no detectable changesin either Topo I or Topo II activities in the nuclear extractsfrom these cells. Treatment of the cells with 10 ^g/m\ genisteinfor as long as 4 days caused less than a 2-fold decrease in thelevel of extractable Topo II activity (data not shown). Theseresults indicate that Topo I or Topo II activities in nuclearextracts from K-562-J and HL-205 cells are not effectivelyaltered by treating the cells with genistein. We cannot, however,rule out the possibility that the extraction process results in theelimination of genistein from the nuclear extracts and thusrestores the Topo II activity to control levels.

Since Topo Il-mediated cleavage in intact cells can be markedly decreased without an associated decline in extractableenzyme activity (7), we measured protein-associated DNAstrand breaks in control and genistein-treated K-562-J and HL-205 cells. In both cell types, treatment with 30-200 ng/m\ ofgenistein for only l h produced protein-associated DNA strandbreaks that increased in a dose-dependent manner, with theDNA from the K-562-J cells being somewhat less susceptible

1 2 3 4 56 7

Fig. 6. Effect of genistein on DNA cleavage mediated by purified Topo II.Purified Topo II was incubated with supercoiled plasmid pUC8 in the absence(Lane 1) or in the presence of Topo II inhibitors. Lanes 2-7: 3, 10, 30. 100. 300.and 1000 ^g/ml of genistein; Lane 8: 300 jjg/ml of genistein in the absence ofATP; Lane 9: 300 /ig/ml of genistein in the absence of Topo II. Lanes 11-13: 1,10, or 100 (ig/ml of m-AMSA: Lanes 14-16: 1. 10. or 100 Mg/ml of VM-26;Lanes 17 ana 18: 100 or 1000 fig/ml novobiocin; Lanes 10 and 20: linear pUC8DNA (positive control); and Lane 19: supercoiled pLJCS DNA (negative control).Following protcinase K-SDS treatment, samples were loaded on 0.8Ã‡ agarosegel and the linear DNA (/.) form, was separated from the supercoiled (S) and thenicked form (A') by electrophoresis in Tris-borate-EDTA buffer containing 0.5

>ig/ml of ethidium bromide. Results are represntative of three similar but independent experiments.

to the damaging effects of genistein (Fig. 7). Treatment of HL-205 cells for l h with 100 ng/ml of genistein resulted in DNAdamage equal to that induced by 105 rads of 7-rays. The samedose of genistein caused DNA damage in the K-562-J cells thatwas equivalent to that induced by 51 rads of 7-rays (Fig. 8).Treatment of HL-205 cells for 24 h with genistein doses rangingfrom 3 to 30 Mg/ml also resulted in protein-associated DNAstrand breaks (as determined by the alkaline elution assay) thatincreased in a dose-dependent manner (Fig. 9). Even thoughthese results are expressed as protein-associated single-stranded

DNA breaks under alkaline conditions (pH 12.1), a substantialfraction of genistein-induced DNA breaks are double strandedas determined from parallel neutral elution analysis (pH 7.2)of treated HL-205 and K-562-J cells (data not shown). However, DNA strand breaks were not detected in 100 ng/m\ ofgenistein-treated HL-205 cells when proteinase K was omittedfrom the lysis solution (under alkaline conditions), confirmingthat the observed DNA strand breaks were protein associated.

Effect of Genistein on Tyrosine Phosphorylation. To study theinhibitory effect of genistein on tyrosine kinase activity, weincubated solubili/od membrane fractions of A-431 cells, whichare enriched for the EGFTK, with genistein in the presence andabsence of its activator EGF. Our results indicated that genistein inhibited both the basal and EGF-activated EGFTK activity by about 90%. No such inhibition was observed with novobiocin, which is not known to be a tyrosine kinase inhibitor(Table 1).

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ELUTIONTIME(HOURS)Fig. 7. Alkaline elution of DNA from HL-205 and K-562-J cells incubated

with different concentrations of genistein. Cells containing |'H]DNA were treatedwith gcnistcin for l h and lysed on polycarbonate filters in the presence ofproteinase K. and the elutcd radioactivity was plotted against time of elulion.Treatments were as follows: no treatment (O), 30 /*g/ml (â€¢),100 pg/ml (A), and200 (jg/ml (A) of genistein. The variation of the data was within 8'r among twoindependent experiments, each performed in duplicate.

2621




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ELUTION TIME (HOURS)Fig. 9. Alkaline elution of DNA from HL-205 cells incubated with different

concentrations of genistein. Cells containing |'H]DNA were treated with genistein

for 24 h and then lysed as described in the legend of Fig. 7. and the amount ofeluted radioactivity was plotted against the elution time. Treatments were asfollows: no treatment (O), 3 ,.i: nil (â€¢).10 Â¡.u.nil (A), and 30 iug/ml (A) ofgenistein. The variation of the data was within 8?t among two independentexperiments, each performed in duplicate.

In addition to determining the effect of genistein on theactivity of EGFTK in a cellular preparation, we measuredchanges in the levels of phosphorylated tyrosine residues inHL-205 and K-562-J cells treated with genistein. The cellularproteins were separated following cell lysis and SDS gel elec-trophoresis, and the phosphorylated tyrosine residues weredetected by Western blotting with the murine anti-phosphoty-

Table 1 Effect of genistein and novobiocin on the tyrosine kinase activity in theabsence or the presence of EGF

The source of the enzyme was detergent solubili/ed material from the membrane fraction of A-431 cells. The substrate was Raytide.

TreatmentControl

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Fig. 10. The effect of genistein on the reactivity of proteins from K-562-J cellstreated with the anti-phosphotyrosine PY-20 antibody. Total lysates from aliquotscontaining 5 x 10s cells were prepared as described in "Materials and Methods."C'ell lysates containing 100 /jg of protein were loaded in each lane and were

separated by SDS-polyacrylamide gel electrophoresis and immunoblotted withthe PY-20 antibody. Lane I, untreated cells: Lane 2, cells treated with 20 ng/mlof genistein for 1 day: Lane 3. cells treated with 20 ><g/nil of genistein for 2 days:Lane 4. cells treated with 20 /ig/ml of genistein for 4 days. Ordinate, Mt of thebands detected with PV -20 expressed in thousands.

rosine PY-20 monoclonal anti-body. In the untreated K-562-Jcells, the PY-20 antibody detected protein bands of M, 210,000;155,000; 125,000; 105,000; 67,000; and 65,000 (Fig. 10). Theintensity of these bands changed very little during 4 days ofculture of the untreated cells. However, treatment of the K-562-J cells for more than 2 days with 20 ng/ml of genisteinresulted in a reduction in the reactivity of the PY-20 antibodywith the M, 210,000 protein to almost nondetectable levels.The level of the reactivity with the M, 105,000 protein increasedon the second and third day but fell sharply on the fourth. Onlyminor changes in the reactivity were observed following geni-stein treatment for the M, 67,000 and 65,000 proteins. Westernblots with PY-20 cell extracts from either control or genistein-treated HL-205 cells (as well as the parental HL-60 cells)showed no reaction, as also previously reported (24).

These results indicate that the PY-20 antibody detected ty-rosine residues in a number of proteins in untreated and ingenistein-treated K-562-J cells but not in control or treated

2622




HL-205 cells. Moreover, the extent of the reactivity of the anti-phosphotyrosine antibody with two of the proteins changedmarkedly following genistein treatment.

associated with the formation of DNA strand breaks, whichmay be a factor leading to the acquisition of the mature phe-

notype.

DISCUSSION

In the present study we report that genistein, an in vitroinhibitor of topoisomerases (10) and tyrosine kinases (9), induces a mature phenotype in the human myeloid HL-205 (13)and erythroid K-562-J (14) leukemia cells. Differentiation inthe HL-2O5 cells was characterized by an increase in thepercentage of cells reacting with the OKM1 monoclonal antibody directed to a myeloid cell surface antigen (21), positivestaining for NSE (22), a characteristic common to monocytes,and expression of NBT dye reduction (16), a marker of granu-locytic maturation in these cells. Although the reactivity withthe OKM1 antibody was observed in more than 95% of theHL-205 cells, the percentage of cells exhibiting lineage-specific(monocytic or granulocytic) markers did not exceed 50%. Thus,it is not clear whether these markers are expressed by the sameor different cell fractions in the HL-205 cells. The expressionof the lineage-specific markers may be related to the effect ofgenistein on a specific biochemical event; for example, inhibition of Topo II activity can result in one phenotype, andinhibition of tyrosine kinase can yield the other phenotype. Inthis context, the optimal level of genistein (10 ng/m\) induceda mature phenotype in only about 50% of the K-562-J cells.Higher doses of the agent caused a suppression of this induction.

The generation of DNA strand breaks is an early event in thegenistein-treated HL-205 and K-562-J cells because it is observed as early as l h after treatment. Moreover, in spite of thisshort treatment, these breaks were detected at doses that areeither similar or somewhat higher (3-fold) than those requiredto produce the mature phenotype. These genistein-evoked DNAstrand breaks may be linked to the induction of differentiationbecause they were also observed in other types of cells inducedto differentiate by a variety of chemical agents (17, 25-29).

Little or no decrease in Topo II activity was shown in nuclearextracts from cells treated with genistein, which inhibits thecatalytic activity of Topo II in vitro. This lack of effect may bedue to drug removal during the preparation of the nuclearextracts. In common with other Topo II-targeting drugs (eto-poside, teniposide, m-AMSA, and 9-hydroxy-eIlipticine), geni-stein may stabilize the complex between Topo II and DNA (thecleavable complex) (23, 30-32), which prevents the rejoiningof the two DNA strands following the Topo Il-mediated transient double-stranded breakage. Further support for this similarity in mode of action comes from an experiment in whichChinese hamster lung cells selected for resistance to 9-hydroxy-ellipticine also exhibited resistance to genistein (33). However,no definite conclusions can be drawn as to whether the double-stranded breakage in genistein-treated intact cells is indeedmediated through a Topo II mechanism. No matter how theDNA damage is caused, the resulting changes in chromatinstructure (34), perhaps combined with inhibition of some tyrosine kinases, may lead to changes in specific cellular programsthat cause the acquisition of the mature phenotype in the HL-205 and K-562-J cells. However, additional studies will berequired to establish the exact nature of the biological targetsfor genistein action that results in the acquisition of the maturephenotype in these cells.

In brief, our results indicate that genistein can induce maturation markers in HL-205 and K-562-J cells. This induction is

ACKNOWLEDGMENTS

We gratefully acknowledge the help of Drs. Jennifer Peak andMeyrick Peak, who made their equipment for the alkaline and neutralelution experiments available to us, as well as provided helpful suggestions in these experiments.

REFERENCES

1. Frank, D. A., and Sartorelli, A. C. Alterations in tyrosine phosphorylationduring the granulocytic maturation of HL-60 leukemia cells. Cancer Res.,48: 52-58. 1988.

2. Richardson. J. M.. Moria, A. O., and Wang, J. Y. J. Reduction of proteintyrosine phosphorylation during differentiation of human leukemia cell lineK-562. Cancer Res., 47: 4066-4070. 1987.

3. Nishimura. J., Takahira. H., Shibata. K.. Muta. K.. Vamamoto. M., Ideguchi.H., Umemura. T.. and Nawata, H. Regulation of biosynthesis and phosphorylation of P210b"/*blprotein during differentiation induction of K 562 cells.Leuk. Res., 12: 875-885. 1988.

4. Momma, Y., Okabe-Kado. J., Hozumi, M., Uehara, Y., and Mizuno, S.Induction of erythroid differentiation of K562 human leukemic cells byherbimycin A. an inhibitor of tyrosine kinase activity. Cancer Res.. 49: 331-334, 1989.

5. Bodley. A. L.. Wu. H-Y.. and Liu. L. F. Regulation of DNA topoisomerasesduring cellular differentiation. Nati. Cancer Inst. Monogr.. 4: 31-35. 1987.

6. Heck, M. M. S., and Earnshaw. W. C. Topoisomerase II: a specific markerfor cell proliferation. J. Cell Biol.. 103: 2569-2581, 1986.

7. Zwelling, L. A.. Chan. D.. Hinds. M.. Mayes. J.. Silberman, L. E.. and BlickM. Effect of phorbol ester treatment on drug-induced, topoisomerase II-mediated DNA cleavage in human leukemia cells. Cancer Res., 48: 6625-6633, 1988.

8. Constantinou. A.. Chubb, C. H.. and Huberman. E. Novobiocin- and phorbol-l2-myristate-13-acetate-induced differentiation of human leukemia cells associated with a reduction in topoisomerase II activity. Cancer Res.. 49:1110-1117. 1989.

9. Akiyama. T., Ishida. J., Nakagawa, S.. Ogawara, H., Watanabe, S., Itoh. N.,Shibuya. M.. and Ink.uni Y. Genistein, a spÃ©cifieinhibitor of tyrosine-specific protein kinases. J. Biol. Chem.. 262: 5592-5595. 1987.

10. Okura. A.. Arakawa. H.. Oka, H.. Yoshinari. T., and Monden, Y. Effect ofgenistein on topoisomerase activity and on the growth of [VAL 12]Ha-ras-transformed NIH 3T3 cells. Biochem. Biophys. Res. Commun., 157: 183-189. 1988.

11. Liu. L. F., Davis. J. L., and Calendar, R. Novel topologically knotted DNAfrom bacteriophage P4 capsids: studies with DNA topoisomerases. NucleicAcids Res.. 9: 3979-3989. 1981.

12. Drake, F. H.. Zimmerman. J. P., McCabe, F. L., Bartus, H. F.. Per. S. R.,Sullivan, D. M.. Ross, W. E., Mattern. M. R., Johnson. R. K., Crooke, S.T., and Mirabelli, C. K. Purification of topoisornerase II from amsacrine-resistant P388 leukemia cells: evidence for two forms of the enzyme. J. Biol.Chem.. 262: 16739-16747. 1988.

13. Homma, Y.. Henning-Chubb. C. B.. and Huberman. E. Translocation ofprotein kinase C in human leukemia cells susceptible or resistant to differentiation induced by phorbol 12-myristate 13-acetate. Proc. Nati. Acad. Sci.USA. S3: 7316-7319, 1986.

14. Lozzio, C. B., and Lozzio, B. B. Human chronic myclogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 45: 321-334, 1975.

15. Murison. G., Chubb. C. B.. Maeda. S., Gemmell, M. A., and Huberman, E.Cannabinoids induce incomplete maturation of cultured human leukemiacells. Proc. Nati. Aead. Sci. USA. 84: 5414-5418. 1987.

16. Collins, S. J.. Bonder, A., Ting, R., and Gallo, R. C. Induction of morphological and functional differentiation of human promyclocytic leukemia cells(HL-60) by compounds which induce differentiation of murine leukemiacells. Int. J. Cancer. 25: 213-218. 1980.

17. Scher, W., and Friend. C. Breakage of DNA and alterations in foldedgenomes by inducers of differentiation in friend erythroleukemic cells. CancerRes., 38: 841-849. 1978.

18. Kohn, K. W., Ewig, R. A. G., Erickson, L. C., and Zwelling, L. A. Measurement of DNA strand breaks and crosslinks by alkaline elution. In: E. C.Friedberg and P. C. Hanawalt (eds.), DNA Repair, a Laboratory Manual ofResearch Procedures, pp. 379-401. New York, NY Marcel Dekker. Inc.,1981.

19. Bradley, M. O., and Kohn, K. W. X-ray induced DNA double-strand breakproduction and repair in mammalian cells as measured by neutral filterelution. Nucleic Acids Res., 7: 793-804. 1979.

20. Cohen. S. Purification of the receptor for the epidermal growth factor fromA-431 cells: its function as a tyrosine kinase. Methods Enzymol., 99: 379-387. 1983.

21. Foon, K., Schroff, R. W., and Gale, R. P. Surface markers on leukemia andlymphoma cells: recent advances. Blood, 60: 1-19, 1982.

2623




22. Li, C. V., Lam. K. \V., and Yam, L. T. Esterases Â¡nhuman leukocytes. J.Histochem. Cytochcm.. 21: 1-12, 1973.

23. Ross. \V. E. DNA topoisomerases as targets for cancer therapy. Biochem.Pharmacol.. .?* 4191-4195. 1985.

24. Naldini. L.. Stacchini. A.. Cirillo. D. M., Aglietta. M.. Gavosto. F.. andComoglio. P. M. Phosphotyrosine antibodies identify the p210c"bl tyrosinekinase and proteins phosphorylated on tyrosine in human chronic myclogen-ous leukemia cells. Mol. Cell. Biol.. 6: 1Ã•803-I811.1986.

25. Modak. S. P., and Traurig. H. Appearance of strand breaks in the nuclearDNA of terminally differentiating vaginal epithelium. Cell Differ., /: 351-355, 1972.

26. Appleby. D. NV..and Modak. S. P. DNA degradation in terminally differentiating lens fiber cells from chick embryos. Proc. Nati. Acad. Sci. USA. 74:5579-5583. 1977.

27. Farzaneh, F.. Zalin, R.. Brill. D.. and Shall, S. DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation. Nature (Lond.). 300:362-366. 1982.

28. Weisinger. G.. Korn, A. P., and Sachs. L. Protein that induces cell differentiation causes nicks in double-stranded DNA. FEBS Lett.. 200: 107-110.1986.

29. Farzaneh. F.. Meldrum. R., and Shall. S. Transient formation of DNA strandbreaks during the induced differentiation of a human promyelocytic leukemiccell line. HL-60. Nucleic Acids Res., 15: 3493-3502, 1987.

30. Long, B. H.. Musial, S. T.. and Brattain. M. G. Single-and double-strandDNA breakage and repair in human lung adenocarcinoma cells exposed toetoposide and teniposide. Cancer Res.. 45: 3106-3112. 1985.

31. Pommier. Y., Schwartz, R. E., Zwclling, L. A., Kerrigan, D.. Mattern. M.R.. Charcosset. J. Y., Jacquemin-Sablon, A., and Kohn. K. W. Reducedformation of protein-associated DNA strand breaks in Chinese hamster cellsresistant to topoisomerase II inhibitors. Cancer Res., 46: 611-616, 1986.

32. Pierson, V., Pierre. A.. Pommier, Y., and Gros, P. Production of protein-associated DNA breaks by lO-ldiethylaminopropylaminol-o-methyl-S//^^ido[3'.4':4.5|pyrrolo[2.3-g]isoquinoline in cultured L1210 cells and in iso

lated nuclei: comparison with other topoisomerase II inhibitors. Cancer Res.,48: 1404-1409. 1988.

33. Markovits, J., Linassier, C., Fosse, P., Couprie. J.. Pierre, J., Jacquemin-Sablon, A., Saucier, J. M., Le Pecq. J. B., and Larsen, A. K. Inhibitory effectsof the tyrosine kinase inhibitor gcnistein on mammalian DNA topoisomeraseII. Cancer Res.. 49: 5111-5117. 1989.

34. Pienta. K. J.. Partin. A. W.. and Coffey, D. S. Cancer as a disease of DNAorganization and dynamic cell structure. Cancer Res.. 49: 2525-2532. 1989.

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1990;50:2618-2624. Cancer Res Andreas Constantinou, Kaoru Kiguchi and Eliezer Huberman HL-60 and K-562 Leukemia Cells by GenisteinInduction of Differentiation and DNA Strand Breakage in Human

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Induction of Differentiation and DNA Strand Breakage … · Induction of Differentiation and DNA Strand Breakage in Human HL-60 ... Mg/mlgenistein, was the induction of DNA damage