(CANCER RESEARCH 52. 6200-6208. November 15. 1992]

Effects of Genistein on the Growth and Cell Cycle Progression of Normal HumanLymphocytes and Human Leukemic MOLT-4 and HL-60 Cells!

Frank Tráganos,2 Barbara Ardelt, Nadine Halko, Silvia Bruno, and Zbigniew Darzynkiewicz

The Cancer Research Institute, New York Medical College, Valhalla, New York 10595

ABSTRACT

Genistein (GEN) is an ¡soflavone known to inhibit both tyrosineprotein kinases and DNA topoisomerase II. The effects of GEN on cellproliferation and cell cycle kinetics of human myelogenous leukemiaHI.-60 and lymphocytic leukemia MOLT-4 cell cultures were studied,

and the data were compared to results obtained with normal humanlymphocytes stimulated to proliferate with phytohemagglutinin. GENconcentrations greater than 50 Mg/m' (185 MM)were cytotoxic to HL-60and MOLT-4 cells following exposure for 24 h; in HL-60 cell cultures,

a population of cells with decreased DNA content and nuclear fragmentation characteristic of apoptosis was observed within 8 h. The 50%inhibition concentration after 24 h of exposure for HL-60 and MOLT-4

cells was 8.5 and 13.0 Mg/ml, respectively. Normal proliferating lymphocytes survived a 24-h exposure of up to 200 Mg/ml GEN. Short-term(4-8 h) exposures of MOLT-4 or HL-60 cells to 5-20 ^g/ml GEN

resulted in a suppression of cell progression through S or through bothS and <•:phases, respectively, while equivalent treatment had no effecton proliferating lymphocytes. A stathmokinetic experiment usingMOLT-4 cells revealed that as little as 5 Mg/ml GEN suppressed cell

exit from S to G2 phase by 40%, with a terminal point of action at or nearthe S-( i, border. Cell progression through the very early portion of G,

phase (Glv, characterized by postmitotic chromatin decondensation)was also suppressed by approximately 40%, whereas cell advancementthrough the remainder of the GÃŒphase was not markedly affected.Longer (24 h) exposure of proliferating lymphocytes to 20 Mg/ml GENled to an S-phase arrest, while similar treatment of leukemic cells

caused cell arrest in G2 phase and an increase in the number of cellsentering the cycle at higher DNA ploidy. The mitogen-induced transi

tion of lymphocytes from (.,, to G! phase was extremely sensitive toinhibition by GEN; the 50% inhibition concentration was 1.6 Mg/ml.The chemotherapeutic value of GEN may be due to the fact that, interms of cytotoxicity, this agent is more active against proliferatingleukemic cells than against normal proliferating lymphocytes. The sensitivity of the G,, to GÃŒtransition in normal lymphocyte cultures and thesuppressive effect of GEN on the G!A exit in MOLT-4 cells both sug

gest that protein kinases involved in chromatin decondensation may bea target of this drug. In light of the observation that lymphocyte stimulation is sensitive to the presence of GEN, the drug is expected to be astrong immunosuppressant.

INTRODUCTIONThe isoflavone GEN3 is isolated from the fermentation broth

of Pseudomonas sp.(\) and is a naturally occurring phytoestro-gen present in a variety of plant foods, including soybeans (2).Akiyama et al. (1) were the first to describe the ability of GENto specifically inhibit PTKs, presumably at the level of theATP binding site. PTKs seem to play a key role in tumorigen-esis and are known to be associated with growth control receptors such as epidermal growth factor (3), platelet-derived

Received 4/24/92; accepted 9/11/92.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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by USPHS Grants R37 CA23296 and CA28704, as well as the CarlInserra and "This Close" Fund for Cancer Research.

2 To whom requests for reprints should be addressed, at The Cancer ResearchInstitute. New York Medical College. 100 Grasslands Road, Elmsford, NY 10523.

3 The abbreviations used are: GEN, genistein (4',5,7-trihydroxyisoflavone):lD;o- drug concentration which inhibited growth by 50% following a 24-h exposure:PTK, protein tyrosine kinase; PHA, phytohemagglutinin; HBSS, Hanks' balancedsalt solution; AO. acridine orange; DAPI, diamidino-2-phenylindole.

growth factor (4), insulin (5) and insulin-like growth factors (6),as well as with several oncogene products including pp60v~src(7), ppI108a8fcs (7), and the T-lymphocyte-specific PTKpp56lck (8). As a result of the ability of GEN to modulate PTKs,

the drug is expected to affect cell proliferation. Indeed, GEN iscapable of suppressing stimulation of both B- and T-lympho-cytes, preventing expression of growth factor receptors (i.e.,interleukin 2) or increases in free intracellular Ca2+ in thesecells (8-10). GEN has also been observed to induce the differentiation of human myelogenous leukemia K562 cells (11),mouse erythroleukemia cells (12), and a mouse megakaryoblas-tic(Cl) cell line (13).

In addition to its ability to inhibit PTKs, recent evidencesuggests that GEN may affect cell proliferation either via itsability to interact with phospholipase C, phosphatidylinositolkinases, or MAP kinases downstream from growth factor receptors (14) or, interestingly, by inhibition of DNA topoisomerase II (15). However, this diversity in cellular targets maybe more apparent than real. Markovits et al. (15) demonstratedthat PTKs (e.g., c-erb-B2), protein kinase C, cyclic AMP protein kinases, and human DNA topoisomerase II all share acommon sequence at or near the ATP binding site. The additional observation that both PTKs and topoisomerase II actthrough a common mechanism (phosphate ester formation between a nucleotide and the hydroxy group of tyrosine) could,when taken with the possibility of shared binding sites, accountfor the existence of inhibitors common to this diverse group ofenzymes (15).

The growth rates of human breast cancer lines MCF-7 andMDA-468, which are estrogen receptor positive and negative,respectively, were equally sensitive to GEN, as was the estrogenreceptor-positive, multidrug-resistant breast cancer cell lineMCF-7-D-40 (2). In another cell system, Chinese hamster lungcells (DC-3F) were sensitive to growth inhibition by GEN although the DC-3F/9-OH-E derivative, with an altered DNAtopoisomerase II, was more resistant (15). To date, however,relatively little is known about the mechanism of action of GENin inhibiting the proliferation of various tumor cell systems,especially with respect to its cell cycle specificity.

In the present study, GEN activity was assayed in severalhuman cell systems. Thus, GEN was tested for its ability toinhibit the stimulation of normal human lymphocytes exposedto the mitogen PHA. The drug's action on normal lymphocyte

proliferation was compared with its affect on two humanleukemic cell lines of lymphocytic (MOLT-4) and myelocytic(HL-60) origin. In addition, since DNA topoisomerase I or IIinhibitors induce apoptosis in the S-phase cells of HL-60, butnot MOLT-4 cell lines (16), the appearance of apoptotic HL-60cells in GEN-treated cultures could provide some indicationthat topoisomerases may be a target of this drug in leukemiccells.

MATERIALS AND METHODS

Cell Culture

MOLT-4 human lymphocytic leukemia cells, obtained from theAmerican Type Culture Collection (Bethesda, MD), were maintained in

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EFFECT OF GENISTEIN ON CELL PROLIFERATION

RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetalbovine serum, 100 units/ml of penicillin, 100 Mg/ml streptomycin, and2 HIML-glutamine (Gibco), as previously described (16). The cells growexponentially over the concentration range of 1 x IO5 to 2 x IO6

cells/ml, with an apparent doubling time of approximately 12 h.HL-60 human myelogenous leukemia cells were provided by Dr.

Harry A. Crissman of the Los Alamos National Laboratory (Los Alamos, NM). The cells were grown under the same culture conditions asMOLT-4 cells, which resulted in an apparent doubling time of 22-24 h

for these cultures.Human peripheral blood lymphocytes, obtained from healthy volun

teers by venipuncture, were isolated by lymphocyte separation medium(Histopaque 1077; Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. The mononuclear cells from the interface werewashed twice with buffered saline and resuspended in RPMI 1640containing 10% fetal bovine serum, antibiotics, and L-glutamine, at adensity of IO6 cells/ml. PHA (Sigma Chemical Co.) was added to ap

propriate cultures at a final concentration of 10 Mg/ml.

Drug Treatment

Exponentially Growing Cell Lines. GEN was purchased from Ko-miya BiomédicalCo. (Thousand Oaks, CA). Stock solutions (1 mg/ml)of the drug were prepared in dimethyl sulfoxide and stored at -20°C.

Intermediate dilutions were made, when necessary, in complete tissueculture medium.

Long-term drug stability was tested by first culturing MOLT-4 cellsfor 24 h in medium with varying concentrations of GEN, removing thecells by centrifugation, and utilizing this medium to resuspend fresh,previously untreated cells for an additional 24 h of incubation. The cellcycle distribution of cells from cultures treated with the preused drugsolutions were compared with those exposed to the same concentrations of fresh GEN in medium for the equivalent 24-h time period.

In separate experiments, fresh GEN was added to exponentiallygrowing MOLT-4 or HL-60 cells at varying concentrations. Cell countsof trypan blue-excluding cells were made by hemacytometer count oncell aliquots removed from culture at the designated times; results werecalculated as viable cells/ml in drug-treated cultures relative to control.

IDso values were calculated for the two leukemic cell lines.Unstimulated Human Lymphocytes. Lymphocytes were placed in

culture and received PHA and 0, 1, 3, or 10 Mg/mlGEN. Aliquots werethen removed from the respective cultures at 24 and 48 h, and the extentof lymphocyte stimulation (G0 to G, transition) was determined by flowcytometry as described below.

PHA-stimulated Lymphocytes. GEN was added at varying concentrations to lymphocyte cultures 48 h after the addition of PHA(i.e., during the peak of lymphocyte proliferation). Aliquots were removed 6 and 24 h after drug addition, and the cell cycle phase distribution was assayed by flow cytometry as described below.

Stathmokinetic Experiment. Exponentially growing MOLT-4 cells(100 ml) were split into two aliquots, each of which received theStathmokinetic agent vinblastine sulfate (Sigma) at a concentration of0.05 Mg/ml. One culture also received 5.0 Mg/ml GEN. After the firsthour of exposure, aliquots were removed from control and GEN-treatedcultures at 30-min intervals, centrifuged at 900 rpm for 5 min, resuspended in 1.0 ml HBSS, and fixed in 9.0 ml of 80% ethanol at 4°C.

Cell Cycle Analysis

Aliquots of cells were removed from control and drug-treatedMOLT-4 and HL-60 cultures at specified times and concentrated bycentrifugation at 900 rpm for 5 min, the pellet resuspended in 1 ml ofHBSS, and the cells were fixed in 9.0 ml of ice-cold 80% ethanol. Thecells were removed from fixative by centrifugation and resuspension in1.0 ml HBSS. Cellular DNA and the protein content of individual cellswere obtained by flow cytometry following staining with 1.0 Mg/mlDAPI (kindly provided by Dr. J. Kapuscinski of this Institute) and10 Mg/ml sulforhodamine 101 (Eastman Kodak, Rochester, NY)dissolved in 10 m\i piperazine-Ar,Af'-bis-(2-ethanesulfonic acid buffer

(Calbiochem, La Jolla, CA) containing 100 mm NaCl, 2 m\i MgCl2,and 0.1% Triton X-100 (Sigma) (pH 6.8) at 0-4°C,as previously de

scribed (16). The fluorescence of individual cells was measured with an

ICP-22 flow cytometer (Ortho Diagnostics, Westwood, MA) using appropriate dichroic mirror and emission filter combinations (17). Thedata were stored and analyzed using Acqcyte (Phoenix Flow Systems,San Diego, CA) on a Compaq 386 personal computer. The Multicycleprogram (Phoenix Flow Systems) was used for the analysis of cell cycledistributions.

MOLT-4 cells exposed to vinblastine with and without the additionof GEN (Stathmokinetic experiment) were centrifuged, rehydrated inHBSS, and incubated for 30 min at 37"C with 2 x IO3 units of RNase

(RASE; Worthington Biochemical Corp., Freehold, NJ). A 0.2-ml aliquot of cell suspension was then mixed with 0.5 ml of 0.1 M HC1solution (pH 1.5) followed 30 s later by the addition of 2.0 ml of a 4Mg/ml AO (chromatographically purified; Polysciences, Inc., War-rington, PA) solution in 0.1 Mcitric acid-0.2 MNa2HPO4 buffer at pH2.6 (18). Staining was done at room temperature. The green fluorescence resulting from AO binding to double-stranded DNA and redluminescence from AO bound to single-stranded (denatured) DNAwere recorded on an Ortho System 30 flow cytometer. Using Acqcytesoftware, as described above, the green and red luminescence for eachcell was combined to give total luminescence reflective of total DNAcontent (double- and single-stranded DNA), while the relative sensitivity of DNA in situ to acid-induced denaturation was expressed as theratio («,)of red luminescence to total luminescence (19).

Lymphocyte stimulation by PHA in the absence and presence ofGEN was monitored by flow cytometry following staining for cellularDNA and RNA content with the metachromatic dye AO as previouslydescribed (20). Briefly, 0.2 ml of lymphocytes in suspension were admixed with 0.4 ml of a detergent solution containing 0.08 N HC1, 0.15MNaCl, and 0.1 % Triton X-100. After 30 s at room temperature, 1.2mlof AO, at 6 Mg/ml, in a 0.2 MNa2HPO4-0.1 Mcitric acid buffer (pH 6.0)containing 1 miviEDTA, was added. The green fluorescence indicativeof AO binding to DNA and the red luminescence, in this case resultingfrom AO binding to RNA, were determined for individual lymphocyteson a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Datawere acquired and processed using the Consort 32 system (BectonDickinson).

RESULTS

Effects of GEN on HL-60 Cells. Cell proliferation was assayed in cultures exposed to 1-100 Mg/ml (3.7-370 MM)GEN.ID50, as measured by the number of viable cells in cultures 24 hafter the addition of GEN, was seen at a concentration of8.5 Mg/ml (Fig. 1). Cell viability remained high, with fewer than10% trypan blue-positive cells in cultures treated with GENconcentrations of up to 20 Mg/ml for 24 h. Treatment with 50

100

GEN

Fig. 1. The effect of GEN on cell viability. GEN was added at the appropriateconcentrations to exponentially growing cultures of MOLT-4 (•)and HL-60 (•)cells. Aliquots were removed from drug-treated and untreated control culturesafter 24 h and stained with trypan blue, and the number of viable cells/ml wasdetermined by hemacytometer count. Cell viability was expressed as the mean andSE of the ratio of trypan blue excluding cells in drug-treated cultures relative tocontrol cultures. The ID50 for HL-60 and MOLT-4 cultures was 8.5 and 13.0

l. respectively.

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and 100 Mg/ml GEN did not lead to cell death within the first 8h of culture, although by 24 h, fewer than 5% of the cells, ineither culture, excluded trypan blue.

The effects of GEN on the cell cycle distribution of HL-60

cells were dose and time dependent. The immediate effects(4 h), observed at drug concentrations of 5-20 Mg/ml, appearedprimarily as a 40-65% increase in the proportion of cells in theG2M phase of the cell cycle accompanied by a compensatorydecrease in G, phase cells (Fig. 2). Longer exposure (8 h) toGEN concentrations of 5 and 10 Mg/ml led to a further decreasein the proportion of G, cells, while the percentage of cells inboth S and G2M phase increased markedly (Figs. 2 and 3).Raising the drug concentration to 20 Mg/ml resulted in an S-phase rather than a G2M-phase accumulation (Fig. 2).

Prolonged (24 h) exposure of HL-60 cells to 5 or 10 Mg/ml ofGEN appeared not to completely inhibit proliferation of HL-60cells, since a higher proportion of G, cells remained in culturesexposed to those concentrations of GEN for 24 h compared tothat observed in identically treated cultures following only 8 hof exposure to the drug (Fig. 2). A loss of S-phase cells and amarked increase in the proportion of G2M phase cells were alsoapparent following 24 h of treatment with GEN concentrationsof 10 and 20 Mg/ml. At these higher drug concentrations, cellswith an increased DNA content (above that of G2M phase cells)also appeared in the cultures (Fig. 2).

A relatively small fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequencyhistograms as a distinct peak below that of the G| population,was evident following 24 h of exposure to 20 Mg/ml GEN. Thispopulation was more prominent in HL-60 cultures followingshorter (8 h) exposure to GEN concentrations of 50 or 100Mg/ml (Fig. 4); cell viability was still high at this point (10% orfewer trypan blue-positive cells), although it dropped to less

o>o.

GEN (jig/ml) GEN (ng/ml)

Fig. 2. The cell cycle phase distribution of MOLT-4 and HL-60 cell culturesfollowing 4. 8, and 24 h of exposure to various concentrations of GEN. Aliquotsfrom control and drug-treated cultures were removed at the indicated times, fixedin 80% cthanol overnight, rehydratcd in HBSS, and stained with the fluorescentdye DAPI for DNA content. Estimates of the cell cycle phase distribution werecalculated using the Multicycle program. The mean and SE are presented for G,(•),S (•).and G2M (A) phase cells; in some instances, cells at a higher ploidylevel (T) were detected.

than 5% by 24 h. Morphological examination of DAPI-stainedcytocentrifuge preparations of HL-60 cells treated with 20Mg/ml GEN for 24 h confirmed the presence of cells with nuclear fragmentation characteristic of apoptosis (Fig. 5).

Effects of GEN on MOLT-4 Cells. Proliferation of MOLT-4

cells was suppressed by 50% following 24 h of exposure to 13.0Mg/ml of GEN (Fig. 1); cell viability remained 90% or higherover the drug concentration range of 2-20 Mg/ml. Drug concentrations of 50 and 100 Mg/ml were toxic at exposure times of24 h, resulting in >95% cell death by trypan blue exclusion.

In MOLT-4 cell cultures, the cell cycle effects became evidentfollowing short-term exposure to GEN concentrations of 5

Mg/ml and higher (Fig. 2). In contrast to the increase of G2Mphase cells observed in HL-60 cultures, GEN caused a dramaticincrease in the proportion of S-phase cells, following 4 h ofincubation of MOLT-4 cultures with 5 and 10 Mg/ml GEN.Four h of exposure to 20 Mg/ml GEN resulted predominately inan S-phase accumulation, although fewer cells appear to haveexited GÃŒphase than at the lower drug concentrations (Fig. 2).At least part of the difference in the effect of GEN on the cellcycle phase distribution between HL-60 and MOLT-4 cells maybe due to the higher cell proliferation rate of the latter (10-12-happarent doubling time) compared to the former (22-24-happarent doubling time). As can be seen in Fig. 2, the effect ofthe drug on MOLT-4 cells at 4 h was similar to that observed inHL-60 cells following an 8-h exposure.

Longer (8 h) exposure to moderate (5 and 10 Mg/ml) GENconcentrations resulted in both S-phase and G2M-phase accumulation in MOLT-4 cultures (Fig. 3). At 20 Mg/ml, however,GEN continued to cause an S-phase accumulation following 8h of exposure (Fig. 3), as the percentage of cells in G( continuedto fall (Fig. 2).

Consistent with the observation in HL-60 cell cultures, 24-hcontinuous exposure of MOLT-4 cells to GEN resulted in theappearance of cells with DNA content above that of G2M cellsand a decrease in the proportion of S-phase cells, compared tothe respective cultures treated with GEN for 8 h (Fig. 2). Therewas no evidence, however, of the presence of the cells with adecreased DNA content, as observed with HL-60 cells.

Continuous (24 h) treatment with intermediate (5 and 10Mg/ml) concentrations of GEN did not completely inhibit celldivision, since the percentage of G, cells remained high in thesecultures compared to results observed following 8 h of treatment (Figs. 1 and 2). MOLT-4 cells differed from HL-60 cellsin that a relatively large percentage (12%) of GÃŒcells remainedin these cultures following 24 h of exposure to 20 Mg/ml (Fig. 2).Drug instability in tissue culture medium is one possible explanation for this phenomenon. This hypothesis was tested byincubating cells for 24 h with varying concentrations of GEN,discarding the cell pellet, resuspending previously untreatedMOLT-4 cells in the same medium for an additional 24 h, andcomparing the effects on the cell cycle distribution with celltreated with freshly prepared drug. The results in Table 1 demonstrated that preincubation had little effect on the ability ofthe drug to alter the cell cycle distribution.

Stathmokinetic Experiment. To reveal the early effects ofGEN on cell cycle progression in more detail, a Stathmokineticexperiment was designed in which vinblastine was added toexponentially growing MOLT-4 cell cultures, to arrest cells inmitosis. Parallel cultures were treated with GEN plus vinblastine. The concentration of GEN was chosen (5 Mg/ml) whichwas relatively low and yet caused distinct effects on the cellcycle distribution (Fig. 2). At different time intervals, the cellswere removed from these cultures, and their distribution in the

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HL-60 MOLT-4

DNA Content DMA Content

Fig. 3. DNA frequency histograms of HL-60 and MOLT-4 cells exposed to various concentrations of GEN for 8 h. Exponentially growing cells were treated with0-20 Mg/ml GEN for 8 h. The aliquots were stained with DAPI and sulphorhodamine as described in "Materials and Methods." The frequency histograms of DAPI

fluorescence relating to DNA content were recorded and analyzed using the Multicycle program. The various cell cycle phases are indicated in the histogram at thebottom left of the figure.

cell cycle was analyzed. By applying a staining technique thatallows one to identify mitotic (M) cells, cells in early G, phase(GiA cells), as well as the entire G! population, S-phase andG2-phase cells (Fig. 6) (19), it was possible to estimate thekinetics of cell progression through these phases and to measure the effect of GEN on cell kinetics. The predominant effectof GEN was a perturbation in cell progression through S phase,although the drug also had an effect on cell exit from the earlyportion of G] (G1A) (Fig. 6). Thus, the rate of cell entrance toM (M slope) was affected by GEN 2 h after addition of the drug,which indicated that the "terminal point" of GEN action, i.e.,

the latest point in the cycle prior to mitosis, at which cell cycleprogression is halted or slowed by the drug (21), was 2 h. Afterthat time, the rate of cell entrance to M, estimated from the Mslope, was suppressed by GEN by approximately 40% compared to control. In contrast, the rate of cell entrance to G2Mwas suppressed immediately after the addition of the drug, indicating an immediate effect of the drug on S-phase traverse(Fig. 6). By comparing the G2M slopes in the absence andpresence of GEN, it could be determined that the rate of cellentrance to G2M was also suppressed by 40%.

In the stathmokinetic experiment, since cell reentry into GÌwas prevented by the addition of the mitotic inhibitor, the rateof emptying of the GÌcompartment could be estimated (21).The data (Fig. 6) show that whereas during the initial 3 h oftreatment, GEN had no measurable effect on the overall rate ofcell exit from GÌ,it did slow down the exit from the earlyportion of GÌ(GIÀ).In the present experiment, the half-time ofMOLT-4 cell residence in GIA was increased from 0.75 h (incontrol) to 1.1 h (i.e., by more than 45%) in the presence ofGEN.

Effect of GEN on Normal Lymphocytes. The effect of GENon lymphocytes proliferation was assayed under two different

sets of experimental conditions, designed (a) to evaluate thepossible inhibition, by this drug, of the G0 to Gr transitioninduced by PHA and (¿>)to test the effect of GEN on the cellcycle distribution of actively proliferating lymphocytes.

GEN had a dose-dependent inhibitory effect on the ability ofnormal human lymphocytes to respond to stimulation by PHA.The drug was added simultaneously with the mitogen, and stimulation was assayed as the increase in RNA content (i.e., cellsundergoing the G0 to GÃŒtransition) (20). In cultures receivingno GEN, 30% of the lymphocytes were stimulated by PHA at24 h. The presence of 3 Mg/ml GEN inhibited stimulation by50% (not shown). When assayed at 48 h, at a time when stimulation would normally be nearing its peak, 70% of the lymphocytes had entered the cycle in control cultures (Fig. 7). Aconcentration of 1.6 Mg/ml GEN reduced the stimulation byone-half in parallel cultures sampled at the same time point.

The effects of GEN on the cell cycle of proliferating lymphocytes were estimated in a separate experiment, in which thedrug was added to cultures 48 h after stimulation with PHA.Following 6 h of incubation with GEN concentrations up to200 Mg/ml, there was no appreciable change in the cell cycledistribution of stimulated lymphocytes (not shown). When drugexposure was increased to 24 h, GEN, within the concentrationrange of 5-10 Mg/ml, caused an increase in the proportion ofboth S- and G2M-phase cells (Fig. 8). As the drug concentrationwas increased, the percentage of S-phase cells reached a maximum at 20 Mg/ml, while the percentage of G2M-phase cellsdecreased to a minimum at 50 Mg/ml GEN (Fig. 8).

DISCUSSION

The present study demonstrates that GEN modulates twoimportant cell cycle-related mechanisms: (a) the progression of

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Fig. 4. Cell cycle phase distribution ofHL-60 cells treated with 0-20 fig/ml GEN for24 h or 50 and 100 fig/ml GEN for 8 h. Arrow,a population to the left of the Gìpeak (see Fig..1) represents cells with decreased DNA stain-ability, typical of cells that undergo apoptosis.This population was apparent at 24 h in cultures exposed to 20 ,.u ml GEN or at 8 h incultures exposed to higher (50 and 100 eg/ml)GEN concentrations which, by 24 h, were cy-totoxic by trypan blue dye exclusion.

|

OO

IGO 13E

DNA Content

proliferating cells through S and G2 phases; and (b) the transition of cells from G0 to G, and from GIA to G)B- The formereffect was cell type- and drug concentration-specific, while thelatter effect, especially the transition of normal lymphocytesfrom quiescent G,, to actively proliferating G, phase cells, wasparticularly sensitive to the action of the drug.

Cytostatic concentrations of GEN (1-20 Mg/ml) had the im

mediate (4 h) effect of arresting human promyelocytic leukemicHL-60 cells in G2 phase. In the case of MOLT-4 lymphocyticleukemia cells, short-term exposure led to a perturbation in cellprogression through S phase with virtually no change or a slightdiminution in the percentage of G2M-phase cells (Fig. 2). Incontrast to the two leukemic cell lines, the drug had virtually noeffect on PHA-stimulated human lymphocytes treated at theheight of stimulation when assayed within 6 h of addition ofGEN.

At intermediate incubation times (8 h) and moderate (5 and10 Mg/ml) drug concentrations, the arrest of cells in G2 phasepredominated in both HL-60 and MOLT-4 cultures, althoughthe percentage of S-phase cells increased as well, indicating thesuppression of DNA replication (Fig. 2). However, the responseof the two cell lines to 8 h of treatment with 20 Mg/ml differedin that MOLT-4 cells were blocked almost exclusively in Sphase, whereas HL-60 cells, while also blocked in S phase,exhibited a higher proportion of cells which failed to exit G,phase than did HL-60 cultures exposed to lower GEN concentrations (Fig. 3).

Long-term (24 h) incubation did not completely inhibit celldivision at 5 and 10 Mg/ml GEN in either of the human leukemic cell lines (Fig. 2). However, in the presence of 20

GEN, a significant proportion of both HL-60 and MOLT-4cells appeared capable of growth at higher ploidy (i.e., an increase of cells with a DNA content >G2M-phase cells) (Fig. 2).Long-term (24 h) exposure to 10 Mg/ml GEN resulted in thearrest of cells in both S and G2 phase in cultures of PHA-stimulated lymphocytes (Fig. 8). Higher drug concentrations(>10 Mg/ml) resulted in an S-phase rather than the G2-phasearrest seen in both HL-60 and MOLT-4 cells (Figs. 2 and 8).Long-term (24 h) incubation with GEN concentrations in excess of 50 Mg/ml resulted in little cytotoxicity in PHA-stimulated cultures, although fewer than 5% of the cells in MOLT-4or HL-60 cultures remained viable under similar culture conditions. Thus, it would appear that proliferating normal humanlymphocytes were considerably more resistant to the action ofthe drug than were the human leukemic cell lines, in terms ofboth toxicity and the drug concentration required to inhibit celltransit through the cycle.

The data obtained from the stathmokinetic experiment confirmed that the principal action of the drug was to slow downcell transit through S, which was observed as the suppression ofcell entrance to G2 phase (Fig. 6). The "terminal point" of drug

action (21) in MOLT-4 cells, whose doubling time is about 12h, was calculated to be about 2 h prior to mitosis, which placedit near the border between S and G2 phase.

There are several possible mechanisms by which GEN couldcause the observed effects. GEN may (a) interact with DNAtopoisomerase II, stabilizing DNA-DNA topoisomerase IIcomplexes; (b) interact directly or indirectly to affect the phos-phorylation state of the p34cdc2/cyclin B complex which is re

sponsible for controlling cell entrance into mitosis; or (c) alter6204

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Fig. 5. Morphology of HL-60 cell nuclei, stained with the DNA-specific dye DAPI, from untreated cultures (A) or cultures exposed to 20 Mg/ml GEN (A) for 24h. Arrow, a typical apoptotic cell in the GEN-treated sample, with characteristic peripherally condensed chromatin. Note that the remaining nuclei are somewhatenlarged, consistent with the accumulation of cells in G2 and increased DNA ploidy observed by flow cytometry in these cultures (Fig. 2). Mitoses are also observed,indicating that under these culture conditions, sonic cells continue to divide. - 40.

Table I Long-term drug stability

GEN(/¿g/ml)Preincubation"0

+1010 +2020

+Cell

cycledistribution''G,36

202012

11S58

66627572G2M6 14181317

" GEN was added fresh to cultures in the absence of preincubation (—)or following preincubation (+) for 24 h in the presence of MOLT-4 cells. In the latterinstance, the original cells were removed, and the supernatant containing the drugwas used to resuspend previously untreated MOLT-4 cells. In each case, the totalexposure time was 24 h.

* Percentage cells in the various cell cycle compartments as calculated using

Multicycle.

the phosphorylation of other cellular proteins such as S6 kinasewhich modulate protein and/or RNA synthesis.

Inhibition of S-phase (and G2-phase) transit is often observedin response to the action of a variety of DNA topoisomerase IIinhibitors. Evidence exists that GEN, although not a DNAintercalator like most DNA topoisomerase II inhibitors, mayinteract with the enzyme (15). Thus, GEN (50 and 100 MM)prevents DNA topoisomerase II activity and stimulates unique,although fewer double-strand breaks in pBR322 DNA than dosimilar concentrations of classical inhibitors such as etoposide(100 MM)and 4'-(9-acridinylamino)-3-methanesulfon-m-anisid-

ide (20 MM)(15). However, it is not clear how the drug concentrations chosen in this study ( 15) compared with cytotoxicity invitro or in vivo. Nevertheless, the activity is specific to GEN,since six chemically related compounds do not induce enzymatic DNA cleavage over a similar range of concentration (15).

Finally, GEN causes protein-linked DNA strand breaks inDC-3F Chinese hamster lung cells, although a DC-3F variantline, containing an altered DNA topoisomerase II activity, issignificantly more resistant (15).

As noted, GEN treatment of HL-60 cells, at higher drugconcentrations and/or longer exposure times, led to the appearance of a cell population with diminished DNA stainability(Fig. 4) similar to that observed with other, classical DNAtopoisomerase II inhibitors (16). This population is typical ofapoptotic cells in which linker DNA is nicked, leading to partialDNA extraction from such cells (following fixation) and todecreased staining intensity with DNA specific stains (22).Morphological examination confirmed the presence of suchcells (Fig. 5). No apoptosis was evident in MOLT-4 cell cultures, which is in line with earlier observations (16) thatMOLT-4 cells do not respond to classical DNA topoisomeraseII inhibitors by undergoing apoptosis. It should be noted thatGEN-DNA topoisomerase II interactions were observed (15) atconcentrations which coincide with the appearance of apoptoticcells in HL-60 cultures in the present study.

Normally, DNA damage during S phase is sufficient to slowtransit through S or cause a block in G2 to allow repair ofpotentially lethal damage (23, 24). In fact, there appear to be"check points" (i.e., control mechanisms) which monitor S

phase and inhibit chromosome condensation until S phase hasbeen completed (25, 26). One of these check points seems tomonitor DNA damage which must be repaired prior to mitosisfor cell survival. Recently, Lock (27), using the DNA topoisomerase II inhibitor etoposide, and Tsao et al. (28), using theDNA topoisomerase I inhibitor camptothecin, describe a link

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EFFECT OF GENISTEIN ON CELL PROLIFERATION

nj 208

0 02 04 06 08

IDC 10

Hours

Fig. 6. The results of a stathmokinetic experiment in which MOLT-4 cellswere treated with 0 or 5 /jg/ml GEN and 0.05 »jg/mlvinblastine sulfate. Top,contour map representing the 0-h control distribution of MOLT-4 cells, the DNAof which was denatured by acid. The cells were stained with AO as described in"Materials and Methods." The a, ratio represents the degree of DNA denatur-

ation in situ as measured by the intensity of red luminescence of AO interactingwith single-stranded DNA per total DNA; the latter is represented by the sum ofred plus green fluorescence intensities (18, 19). The specific cell cycle compartments as shown (M, G,, G)A, S, and G2 phase) can be identified following thistype of cell staining and, in the stathmokinetic experiment, the rates of cellprogression through those compartments can be measured (19). Bottom, analysisof the percentage of cells in the individual compartments as a function of timesince addition of the stathmokinetic agent. Cells were treated with either 0 (•)or5 (•)..ij ml GEN. Left, the kinetics of cell accumulation in M and G2 + Mexpressed as the log of 1 + the fraction of cells in A' (fx), where fx is the

percentage of cells in either M or 62 + M. Its variation between 0 and 30.1represents the full scale (xlOO) of the possible increase of cells in each compartment. Right, the kinetics of emptying of the GÃŒand G,A compartments expressedas the initial percentage of cells in the total (., population.

between the DNA damage caused by these agents, presumablythrough their interaction with DNA topoisomerases, and theinhibition of p34edc2 kinase dephosphorylation, which prevents

cells from proceeding from G2 phase to mitosis.The fact that, at lower concentrations, GEN did not com

pletely inhibit cells from dividing and reentering G| phase (Fig.2) or resulted in cells which failed to divide but cycled at ahigher ploidy level, indicated that, if the drug causes DNAdamage, at least in some instances, that damage could be partially or completely repaired. This was not the case if the drugconcentration was 50 Mg/ml or higher where cell proliferationwas inhibited, leading to eventual cell death by 24 h. Failure ofGEN, at moderate (2-20 Mg/ml) concentrations, to completelyinhibit cell division was apparently not a result of decreaseddrug stability in culture (Table 1).

Alternatively, the effect of GEN on cell cycle progressionmay involve its direct action as an inhibitor of tyrosine kinase.Phosphorylation of the tyrosine of p34cdc2 occurs during late S

and early G2 phase in mammalian cells (29). Studies in vitro(30) suggest that the p34cdc2/cyclin B complex is tyrosine phos-

phorylated in the cytosol, which affects the association of the

complex with other, presumably regulatory, proteins and maybe necessary for the translation of the complex to the nucleus.While it is clear that the inhibition of tyrosine dephosphorylation during G2 phase inhibits cell entrance to mitosis, inhibitionof tyrosine phosphorylation during late S phase may directly orindirectly slow or block S-phase cell transit into G2.

Other evidence (14) suggests that GEN inhibition of S6 kinase activity may be important in its role in inhibiting cellproliferation. For instance, binding of epidermal growth factorto NIH-3T3 cells increases intracellular [Ca2"1"]and pH and

enhances the transcription ofc-fos and c-myc and the phosphorylation of ribosomal S6 protein. However, GEN does not inhibit epidermal growth factor induction of c-myc mRNA synthesis or the phosphorylation of several cytosolic proteins,suggesting that its cytostatic action is not mediated through the

<

Q

0 u,g/ml u,g/ml

3 tig/ml

G

10 jig/ml

0 200 400 600 8OO 1000 0 200 400 600 800 1000

RNAFig. 7. The effect of GEN on lymphocyte stimulation analyzed 48 h after

addition of PHA. Normal human lymphocytes were exposed to 0, 1, 3, or 10fig/ml GEN and PHA simultaneously. Aliquots were removed after 48 h ofculture. Stimulation was assessed by determining the percentage of cells whichresponded to PHA by increased RNA content (Gìcells) and increased DNAcontent (S + G2 + M cells). Arrows, maximum RNA content values for control,unstimulated (Go) lymphocytes (not shown).

<n"5o

40 T

20 50 200

Genistein (ng/ml)Fig. 8. The percentage of cells in S or G2M phase in 72-h cultures of normal

lymphocytes which received varying concentrations of GEN at 48 h, during theheight of PHA stimulation. Lymphocyte cultures were stimulated with PHA andat 48 h received varying concentrations of GEN and were returned to culture foran additional 24 h. The percentage of cells in the various cell cycle compartmentswere determined as shown in Fig. 7.

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EFFECT OF GENISTEIN ON CELL PROLIFERATION

direct inhibition of the epidermal growth factor receptor ty-

rosine kinase. GEN does prevent the stimulation of S6 kinaseactivity, probably via MAP kinase inhibition (31). Phosphory-

lation of S6 ribosomal protein modulates protein synthesis and,as such, represents a common pathway in the cellular responseto a variety of hormones, growth factors, transforming viruses,tumor promotors, etc. (14). As a result, GEN inhibition of S6kinase activity may lead to inhibition of the translation of mR-NAs necessary for DNA synthesis and/or cell division (14).

A second important observation of the present study was thatmitogen stimulation of human lymphocytes was relatively sensitive to the action of GEN inasmuch as the G0 to GÌtransition,measured by the accumulation of RNA and reflecting the activation of rRNA genes, was inhibited by 50% following 24 h ofcontinuous exposure to 3 ^g/ml or 48 h of exposure to 1.6Mg/ml GEN, while it was virtually abolished by treatment witha GEN concentration of 10 Mg/ml. These results were in agreement with the observations of others (8, 32) that GEN inhibitsPHA-induced T-cell receptor ¿"chainphosphorylation, interleu-

kin-2 receptor a subunit (p55) induction, the increase in orni-thine decarboxylase activity, blast transformation and T-cell

proliferation, within the concentration range found to be effective in this study. These data indicate that GEN may be a potentimmunosuppressor.

When GEN is added simultaneously with platelet-derivedgrowth factor to mouse fibroblasts, it inhibits platelet-derivedgrowth factor-induced increases in mRNA levels of c-fos, c-jun,

and jun B, preventing cell progression into and through G|phase (33). These data are in agreement with our finding of aslowdown in the rate of cell exit from the early portion of G,(G]A) phase induced by this agent (Fig. 6). This portion of G,is generally characterized by an exponential distribution of cellresidence times (21), and cells that reside in G)A have morecondensed chromatin than cells in late G, (G,B cells) (34).Because the G0 to G, transition of PHA-stimulated lymphocytes and cell exit from G,A both involve the decondensation ofnuclear chromatin (34, 35), it would appear that GEN may alsotarget enzymes involved in chromatin decondensation (e.g., de-

phosphorylation of histone Hl; Ref. 36).In summary, the effect of GEN on the proliferation of normal

and leukemic lymphocytes appears to be unique. The presentresults differ considerably not only from the data on classicaltopoisomerase inhibitors but also from that observed with theprotein kinase C inhibitor staurosporine. Staurosporine specifically blocks normal cells such as PHA-stimulated lymphocytesin G] phase at concentrations as low as 10 ng/ml while itinvariably blocks transformed and tumor cell lines exclusivelyin G2 phase at higher (50 ng/ml) concentrations (37, 38). Although the present results do not reveal what mechanism(s) isresponsible for the cell cycle phase-specific effects of GEN, thecomplexity of its effects suggests that this agent may have morethan one target in the cell.

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1992;52:6200-6208. Cancer Res   Frank Traganos, Barbara Ardelt, Nadine Halko, et al.   and HL-60 Cellsof Normal Human Lymphocytes and Human Leukemic MOLT-4 Effects of Genistein on the Growth and Cell Cycle Progression

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