Genistein Protects Hematopoietic Stem Cells against G-CSF … · Research Article Genistein Protects Hematopoietic Stem Cells against G-CSF–Induced DNA Damage Liliana R. Souza1,

Research Article

Genistein Protects Hematopoietic Stem Cells againstG-CSF–Induced DNA Damage

Liliana R. Souza1, Erica Silva1, Elissa Calloway1, Omer Kucuk1, Michael Rossi2, and Morgan L. McLemore1

AbstractGranulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in various clinical

settings. Although clearly beneficial, there are concerns that the chronic use of G-CSF in certain conditions

increases the risk of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The most

striking example is in severe congenital neutropenia (SCN). Patients with SCN developMDS/AML at a high

rate that is directly correlated to the cumulative lifetime dosage of G-CSF. Myelodysplastic syndrome and

AML that arise in these settings are commonly associated with chromosomal deletions. We have demon-

strated in this study that chronicG-CSF treatment inmice results in expansionof the hematopoietic stemcell

(HSC) population. In addition, primitive hematopoietic progenitors from G-CSF–treated mice show

evidence of DNA damage as demonstrated by an increase in double-strand breaks and recurrent chromo-

somal deletions. Concurrent treatment with genistein, a natural soy isoflavone, limits DNA damage in this

population. The protective effect of genistein seems to be related to its preferential inhibition of G-CSF–

induced proliferation of HSCs. Importantly, genistein does not impair G-CSF–induced proliferation of

committed hematopoietic progenitors, nor diminishes neutrophil production. The protective effect of

genistein was accomplished with plasma levels that are attainable through dietary supplementation.Cancer

Prev Res; 7(5); 534–44. �2014 AACR.

IntroductionSevere congenital neutropenia (SCN) is a rare, heritable

disorder characterized by isolated neutropenia from birth(1). Before the clinical use of granulocyte colony-stimulat-ing factor (G-CSF), individuals typically died before the ageof 2 from overwhelming infections. With G-CSF treatment,patients with SCN now routinely survive until adolescenceor even adulthood. Unfortunately, a substantial number ofpatients with SCN now develop myelodysplastic syndrome(MDS) and/or acute myeloid leukemia (AML; refs. 1, 2).After 10 years of G-CSF treatment, the rate of MDS/AML inpatients with SCN is estimated to be 2% to 3% per year (2).The etiology of AML in SCN is not well defined. The initialhypothesis was that a molecular defect responsible for SCNpredisposed individuals to AML. Because accumulatingevidence has demonstrated that MDS/AML arises fromhematopoietic stem cells (HSC), this hypothesis seems lesslikely as the most frequently mutated gene in SCN, ELA2, is

not expressed in HSCs. Another hypothesis is that chronicG-CSF treatment promotes expansion of a malignant mye-loid clone. This remains a concern as the risk of myelodys-plastic syndrome and/or AML roughly correlates with life-time cumulative dosage of G-CSF (1, 3). Other researchershave suggested that G-CSF usage in certain settings maypromote leukemic transformation (4–9). These studies arefar from conclusive, as others have shown no increase in therisk of leukemic transformation and/or relapse with G-CSFtherapy (10). In particular, a prospective study has shownthat G-CSF usage did not increase the rate of relapse ordecrease complete remission rates in AML (11). However,SCN is unique in its cumulative lifetime dosage of G-CSF.Patients with SCN typically receive G-CSF multiple timesper week for life, as opposed to patients undergoing che-motherapy who may receive a short-term treatment.

G-CSF signals through the granulocyte colony-stimulat-ing factor receptor (G-CSFR). The G-CSFR is a non-tyrosinekinase receptor that is present at low levels onHSCs (12). Inmice, G-CSF treatment results in an increase inHSCs (13). Itis possible that chronic G-CSF treatment results in HSCproliferation and acquisition of mutations. Several lines ofevidence lend support to this hypothesis. First, acquisitionof hyperproliferative mutations of the G-CSFR increases therisk of leukemic transformation (14). Second, it has beendemonstrated that HSCs preferentially use error-proneDNA repair pathways when entering cell cycle, and chro-mosomal deletions are often seen not only in patients withSCN and AML, but also in patients with aplastic anemiatreated with G-CSF (7, 9, 14, 15).

Authors' Affiliations: Departments of 1Hematology and Oncology and2Radiology and Oncology, Winship Cancer Institute, Emory University,Atlanta, Georgia

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Liliana R. Souza, Department of Hematology andOncology,WinshipCancer Institute, EmoryUniversity, 1365CCliftonRoad,Room C3078, Atlanta, GA 30322. Phone: 404-778-1829; Fax: 404-778-5520; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-13-0295

�2014 American Association for Cancer Research.

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Published OnlineFirst March 10, 2014; DOI: 10.1158/1940-6207.CAPR-13-0295

http://cancerpreventionresearch.aacrjournals.org/

In this article, we provide evidence that prolonged G-CSFtreatment results in genomic instability in murine HSCs. Inaddition, we demonstrate that treatment with the soy iso-flavone genistein lessens DNA damage. This effect isachieved by using genistein at a dosage that can be easilyattainable by dietary supplementation, suggesting thatgenistein may be an effective preventive agent for thosepatients who require prolonged G-CSF treatment.

Materials and MethodsMouse strainC57BL/6J mice were obtained from The Jackson Labora-

tory. All mice were housed in a specific pathogen-freeenvironment. We used 6- to 10-week-old mice in all studiesand the experiments were approved by the Emory Univer-sity and Institutional Animal Care and Use Committee(IACUC protocol number, 2000678).

In vivo G-CSF treatmentC57BL/6J mice were treated subcutaneously 5-times a

week with G-CSF (10 mg/kg; Neupogen; Amgen) or diluentalone (control mice) for different amounts of time varyingup to 1 year.

Array comparative genomic hybridization analysisBonemarrow cells were harvested frommice treated with

G-CSFor diluent for 4months. Lin�Scaþ cellswere isolatedusing the instructions provided by the manufacturer (EasySep 18756, 19756A; STEMCELL Technologies). GenomicDNA was extracted from Lin�Scaþ cells using the QiagenDNeasy kit, and quantification and quality assessment wereperformed with Picogreen, NanoDrop, and a standardagarose gel. DNA was analyzed by array comparative geno-mic hybridization (aCGH) at the Florida State UniversityNimbleGenMicroarray Facility using NimbleGen 3� 720Kmouse whole-genome tiling arrays. Copy-number abnor-malities were identified using NimbleScan and BioDiscov-ery Nexus softwares. Aberrant segments were queried in theUCSC Genome Browser (GRCm38/mm10) for overlappingBAC alignments to be used for FISH validation of copy-number loss and gain. Data from the aCGHare available onthe Gene Expression Omnibus (GEO) repository underaccession number GSE54737.

Immunofluorescence microscopyBone marrow cells from mice were harvested and LSK

cells (lineage-negative, cKitþ, Scaþ) were sorted on a BDFACSAria (BD Biosciences) and spotted on a slide. The cellswere fixed using 10% methanol and 10% formalin in PBSand washed twice with PBS. Cells were then permeabilized,blocked, and incubated with antibodies overnight at 4�C.The antibodies used individually were: pH2AX (07–164;Millipore), pGSK3 A555 (bs-5367R-A555; Bioss), cyclinD1A555 (bs-0623R-A555; Bioss), and cyclin D3 A555 (bs-0660R-A555; Bioss). The following day, the sample waswashed with PBS, incubated with a fluorescent secondaryantibody when necessary (cat#A21429, Invitrogen), andstained with DAPI (40,6-diamidino-2-phenylindole;

cat#D3571; Invitrogen). For quantification of immunohis-tochemistry, images from more than 100 cells were cap-tured using a Carl Zeiss LSM 510 META confocal micro-scope (Zeiss) with a Plan-Apo 63� 11.4 oil immersion lens.The maximum intensity per nucleus was determined andbackground was subtracted using Metamorph software(Molecular Devices).

Reactive oxygen species analysesLSK cells were sorted, stained with dihydroergotoxine

(DHET) (final concentration 50 mg/mL; cat# 7008; Sigma-Aldrich) at 37�C for 20 minutes, and analyzed by FACS-Canto (BD Biosciences).

FISHLineage-negative Scaþ (Lin�Scaþ) bone marrow cells

were isolated, fixed in a 1:3mixture ofmethanol:acetic acid,and spotted onto a glass slide. For FISH analysis, thechromosome region-specific Bac clone for Abl1 (RP23-156H9) on mouse chromosome 2, and Tsc2 (RP23-438P15) on mouse chromosome 17, were labeled with5-ROX dUTP by nick translation (Empire Genomics). Thedenaturation, hybridization, and signal detection proce-dures were carried out as described by the Oncology Cyto-genetics Facility at Emory University. Hybridization wasvisualized onaLSM510METAconfocalmicroscope (Zeiss).

In vivo genistein treatmentC57BL/6J mice were treated subcutaneously 3-times a

week for 6 weeks as follows: genistein (10 mg/kg; CaymanChemical Company; genistein þ 25 mL Peg400 þ 75 mL0.1% BSA in PBS), G-CSF (10 mg/kg; Neupogen; Amgen;G-CSFþ 25 mL Peg400þ 75 mL 0.1% BSA in PBS), genisteinþG-SCF (genisteinþ 25 mL Peg400þG-CSFþ 75 mL 0.1%BSA in PBS), diluent [DMSO (dimethyl sulfoxide) þ 25 mLPeg400þ 75 mL 0.1% BSA in PBS; refs. 16, 17].

Cell sorting and flow cytometric analysisWeusedaBDFACSAria andFACSCanto (BDBiosciences)

for cell sorting and flow cytometric analysis, respectively,followed by analysis using FlowJo Software (TreeStar Inc).The following antibodies were used for cell sorting and flowcytometric analysis: MAC1 (cat#0112), GR1 (cat#5931),Ter119 (cat#5921), B220 (cat#0452), CD3 (cat#0031),c-Kit (cat#1171), Sca1 (cat#5981), CD48 (cat#0481), andCD150 (cat#1501); all were obtained from eBiosciences.

BrdUrd incorporationTo examine 5-bromo-20-deoxyuridine (BrdUrd) incorpo-

ration, we used the protocol previously described (18).Briefly, mice were given three daily intraperitoneal injec-tions of BrdUrd in 0.1%BSA in PBS (Sigma; 3mg/24 hours)and maintained on 0.2 mg/mL of BrdUrd in the drinkingwater for 72 hours.Mice were euthanized and bonemarrowcells were stained with antibodies against lineage markers,cKit, Sca, cd48, and cd150. Cells were fixed, permeabilized,and stained with anti-BrdUrd-PE (all from eBiosciences)and analyzed by FACSCanto.

Genistein Limits DNA Damage

www.aacrjournals.org Cancer Prev Res; 7(5) May 2014 535




Statistical analysisData were analyzed with Microsoft Excel. Paired t tests

were used to test for evidence of differences in groups.Values were considered statistically significant at P less than0.05.

ResultsG-CSF treatment induces DNA damage inhematopoietic progenitors

Mice were treated with G-CSF (10 mg/kg 5 � week) for 4months and bone marrow cells were analyzed by flowcytometry. The results demonstrated a 3-fold increase inthe lineage-negative, Sca-positive, and cKit-positive (LSK)population of G-CSF–treated mice (Fig. 1A). As phosphor-ylation of histone H2AX (p-H2AX) is an indicator of DNAdamage (19), we quantified the amount of nuclear pH2AXin individual LSKs using immunostaining. Our resultsdemonstrate that there is a 2-fold increase in double-strand

breaks (DSB) in LSK cells of treated animals in comparisonwith control animals (Fig. 1B and C). The LSK populationwas also analyzed for the presence of reactive oxygen species(ROS). LSK cells from G-CSF–treated animals displayed asignificant increase in ROS levels (Fig. 1D).

G-CSF induces chromosomal instabilityTo determine whether the increased proliferation and

DNA damage induced by G-CSF treatment lead to chromo-somal alterations, we performed aCGH. Bone marrow cellswere harvested from 3 mice treated with G-CSF and 3 micetreated with diluent for 4 months. Genomic DNA fromLin�Scaþ cells of G-CSF–treated mice and respective con-trols were analyzed in triplicate using NimbleGen 3� 720Kmouse whole-genome tiling arrays. Copy-number abnor-malities were identified using NimbleScan and BioDiscov-ery Nexus softwares. Aberrant segments were queried in theUCSCGenomeBrowser (GRCm38/mm10) for overlappingBAC alignments to be used for FISH. Significant deletions in

Figure 1. G-CSF induces proliferation, DNA damage, andROSproduction in LSK cells. C57BL/6Jmice (n¼ 4 per group) were given 5 doses/week of G-CSF ordiluent for 4 months. Bone marrow LSK cells were isolated and analyzed by FACSAria. A, fold change in bone marrow LSK cells between both groups.Data represent themean�SDof 4mice per group; a,P < 0.05. B, fold change in DNAdamage in sorted LSK cells quantified by intracellular staining of pH2AX;a, P < 0.05. For quantification, images from more than 100 cells were captured using a Carl Zeiss LSM 510 META confocal microscope (Zeiss) with aPlan-Apo 63 � 11.4 oil immersion lens. The maximum intensity per nucleus was determined and background was subtracted using Metamorph software(Molecular Devices). C, immunofluorescence stainingof pH2AX (red) in sorted LSKcells ofG-CSF–treatedmice. Imageswere capturedusing aCarl Zeiss LSM510 META confocal microscope with a Plan-Apo 63 � 11.4 oil immersion lens. D, fold change in ROS levels measured by staining sorted bone marrow LSKcells with DHET; data represent the mean � SD of 4 mice per group; a, P < 0.05.

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Cancer Prev Res; 7(5) May 2014 Cancer Prevention Research536




chromosome 2 and 17 were present in all G-CSF samplesand were chosen for validation (Fig. 2A and B; GEO acces-sion number, GSE54737).Validation of the copy-number abnormalities detected by

CGHwere examined using FISH analysis in Lin�Scaþ bonemarrow cells from animals treatedwithG-CSF for 4monthsusing BAC clones RP23-156H9 (chromosome 2, Abl1) andRP23-438P15 (chromosome 17, Tsc2) within the regions ofinterest. Two sets of 100 cells per mouse (n ¼ 44) werecounted and the average was calculated.FISH analysis confirmed alterations in chromosome 2 in

17% of cells, compared with 2% in equivalent control mice(Fig. 2C). Hybridizations also confirmed deletions in chro-mosome17 in 31%of cells comparedwith 3% in equivalentcontrol mice (Fig. 2D). Greater than 90% of abnormal cells

showed loss of one or two probe signals. In less than 10%ofabnormal cells, a gain of signal was noted.

Genistein decreases G-CSF–induced DNA damagePrevious studies have demonstrated that genistein pro-

tects hematopoietic progenitor cells from ionizing radiationand cytotoxic chemotherapy; therefore, the following pro-tocol was designed to examine whether genistein couldprotect HSCs against the deleterious effects of chronic G-CSF treatment.

Mice were concomitantly treated with G-CSF (10 mg/kg5 � week) and genistein (10 mg/kg) subcutaneously 3-times a week for a total of 6 weeks. At 6 weeks of G-CSFtreatment, the maximum expansion of HSCs was observed,with statistically significant evidence of genomic instability.

Figure 2. G-CSF induceschromosomal abnormalities inprogenitor cells. A and B, C57BL/6Jmice (n¼43)were given 5dosesper week of G-CSF or diluents(n ¼ 43) for 4 months. Lin�Scaþcells were isolated from the bonemarrow. DNA from 3 G-CSF–treated mice and respectivecontrols were analyzed by aCGHatthe Florida State UniversityNimbleGen microarray facilityusing NimbleGen 3 � 720K mousewhole-genome tiling arrays. Copy-number abnormalities wereidentified using NimbleScan andBioDiscovery Nexus softwares.Aberrant segments presentedin G-CSF–treated mice inchromosomes 2 and 17 werequeried in the UCSC GenomeBrowser (GRCm38/mm10) foroverlapping BAC alignments to beused for FISH validation of copy-number loss (GEO accessionnumber, GSE54737). C and D,C57BL/6Jmice (n¼ 44) were given5 doses per week of G-CSF ordiluents (n ¼ 44) for 4 months.Lin�Scaþ cells from bone marrowwere analyzed using FISH probesfor mouse chromosomes 2 and 17.Data represent the mean � SD ofabnormal cells per mouse; a, P <0.05.






The mean concentration of genistein in plasma reached1.70 mmol/mL 24 hours after the last treatment. Mice wereeuthanized and bone marrow cells were evaluated by flowcytometry. The LSK cell population in the bone marrowincreased 3.4-fold in mice treated with G-CSF; however,concurrent treatment with genistein lessened the expansionof LSK cells by 2.3-fold (Fig. 3A and Supplementary Fig. S1).The production of ROS increased after G-CSF treatment(Fig. 1D); however, we found that the levels of intracellularROSwere significantly lower inmice treated simultaneouslywith genistein and G-CSF (Fig. 3B). These cells were sortedand then analyzed for the amount of DNA DSBs via thepresence of nuclear pH2AX. We found that LSKs frommicetreated concurrently with genistein and G-CSF exhibitedless DNA damage than LSKs from G-CSF–treated mice(Fig. 3C).

Genistein decreases chromosomal instabilityBecause we determined that LSKs from mice treated

concurrently with genistein and G-CSF had less DNA dam-age, we verified the copy number of aberrant genomicsegments.

Lin�Scaþ cells were analyzed by FISH using BAC clonesfor chromosome 2 and chromosome 17. Two sets of 100

cells per mouse (n¼ 44) were counted and the average wascalculated.Our results demonstrated that after 6weeks ofG-CSF treatment, Lin�Scaþ bone marrow cells have altera-tions in chromosome 2 (6%) and chromosome 17 (20%),whereas cells fromanimals treatedwith genistein combinedwith G-CSF had fewer chromosomal abnormalities basedon alterations in chromosome 2 (2%; Fig. 3D) and chro-mosome 17 (9%; Fig. 3E).

Bone marrow differentials showed that G-CSF increasesthe percentage of granulocyte and concomitant treatmentwith genisteindidnot interferewith granulocytes expansion(Fig. 4A). Animals treated simultaneously with genisteinand G-CSF had a similar level of total neutrophils as theanimals treated only with G-CSF (Fig. 4B). Therefore, ourdata suggest that genistein protects against the deleteriouseffects ofG-CSF–induced excessiveHSCproliferation and atthe same time permits the desired increase in the neutrophilpopulation.

Genistein limits DNA damage through inhibition ofproliferation

To elucidate the mechanism of action of genistein, wecompared its effect to that of a known antioxidant, N-acetyl-cysteine (NAC; ref. 20). Animalswere treatedwithG-CSF for

Figure 3. Genistein protects LSK cells against G-CSF–induced damage. C57BL/6J mice (n¼ 44 per group) were concomitantly treated with G-CSF (10 mg/kg5�week) and genistein (10mg/kg) subcutaneously 3-times aweek for a total of 6weeks. A, bonemarrow LSK cells were isolated. Data represent themean�SD in LSK fold change in each treatment group. B, fold change in ROS levels measured by staining sorted bone marrow LSK cells with DHET. Datarepresent themean�SD.C, fold change inDNAdamage in sortedLSKcells quantifiedby intracellular stainingof pH2AX.Data represent themean�SD.DandE, bonemarrow Lin�Scaþ cells were isolated. Hybridization patternswere analyzed using FISHprobes formouse chromosomes2 and 17. Data represent themean � SD of abnormal cells per mouse; a, P < 0.05 versus diluent; b, P < 0.05 versus G-CSF.

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5 days and with genistein or NAC (50 mg/kg) on the last 2days of G-CSF treatment (Fig. 5A–C). The expansion in theLSKpopulation inmice treatedwithG-CSForNACþG-CSFwas comparable. However, genistein inhibited the prolif-erative effect of G-CSF (Fig. 5A). Our results also showedthat although NACþG-CSF–treated mice had significantlylower ROS levels than G-CSF–treated mice, short-termtreatment with genistein (2 doses) had a modest effect onROS concentration (Fig. 5B). We observed, however, thatLSK cells treated with genistein and G-CSF had significantlyless DNA damage, whereas NAC showed no significantprotective effect against DSBs induced by G-CSF treatment(Fig. 5C).Next, we compared the proliferation of different bone

marrow cell populations by analyzing BrdUrd incorpo-ration in newly synthesized DNA (Fig. 6A). Myeloid pro-genitors are found in the Lin�cKitþSca� fraction, whereasthe LSK (Lin�ckitþScaþ) cell surfacemarkers identify a cellpopulation highly enriched forHSCs (21, 22). Therewas nosignificant difference between BrdUrdþ incorporation inthe myeloid progenitor population (Lin�cKitþSca�) inmice treated with G-CSF or simultaneously treated with

genistein and G-CSF (Fig. 6B). However, the percentage ofBrdUrdþ cells in the LSK population was 70% in G-CSF–treated mice and 56% in mice concurrently treated withgenistein and G-CSF (Fig. 6C and Supplementary Fig. S2).These data suggest that genistein preferentially inhibits HSCproliferationwhile allowing proliferation/differentiation ofmyeloid progenitors.

To further elucidate the mechanism underlying thisphenomenon, we investigated the effects of G-CSF–trig-gered myeloid differentiation on GSK3 phosphorylationand cyclin D1 and D3 expression in the presence ofgenistein.

GSK-3 regulates HSC self-renewal and lineage commit-ment (23, 24) and controls genes that are important forproliferation such as cyclin D1 (25) and D3 (26). Myeloidprogenitor bonemarrow cells frommice treatedwithG-CSFfor 5 days and with genistein on the last 2 days of G-CSFtreatmentwere sorted, lysed, and theproteinswere analyzedby Western blot. Our results demonstrate that G-CSF treat-ment induces GSK3 phosphorylation and concomitantlycyclin D1 and D3 expression in comparison with untreatedcells (Fig. 6D). Genistein, however, blocked GSK3 phos-phorylation and cyclin D1 and D3 induction in G-CSF–treated mice. To evaluate the degree of G-CSF signal acti-vation in LSK cells, the levels of pGSK3 (Fig. 6E), cyclin D1(Fig. 6F), and D3 (Fig. 6G) were measured. LSK cells weresorted by flow cytometry directly on glass slides and ana-lyzed by immunofluorescence. G-CSF treatment inducesGSK3 phosphorylation and increases cyclin D1 and D3levels (Fig. 6E–G). Genistein treatment inhibited G-CSF–inducedphosphorylationofGSK3and cyclinsD1andD3 inLSK cells.

Taken together, these results suggest that genistein mod-ulates G-CSF induction of the GSK3-cyclinD1/D3 pathwayin LSK and myeloid progenitors; nonetheless, it does notinterfere with myeloid proliferation and differentiation ofneutrophils (Fig. 4A and B).

DiscussionThe effects of prolonged G-CSF treatment on HSCs are

not well understood. We hypothesized that excessive HSCproliferation induced by G-CSF could lead to deleteriousconsequences. Genistein is a soybean-derived isoflavonewith antioxidant effects (27). It also has tyrosine kinaseinhibitory properties that attenuate proliferation of bothnormal and cancerous cells (28). On the basis of theseproperties, we hypothesized that genistein could counteractthe deleterious effects of excessive HSC proliferationinduced by G-CSF.

G-CSF is widely used inmultiple clinical settings to lessenthe effects of neutropenia. Although clearly beneficial, thereare concerns about the long-term effects of G-CSF. A par-ticular concern is thatG-CSF therapymay increase the riskofmyelodysplastic syndrome and/or AML. G-CSF utilizationin both aplastic anemia and Fanconi’s anemia has beenassociated with clonal evolution to AML (7, 9). Further-more, usage of G-CSF has been associated with an increased

Figure 4. Genistein allows granulocytic differentiation. A, C57BL/6Jmice (n ¼ 44 per group) were concomitantly treated with G-CSF (10mg/kg 5 � week) and genistein (10 mg/kg) subcutaneously 3-times aweek for a total of 6 weeks. Bone marrow cells were isolated andanalyzed. Bone marrow cells were stained with Giemsa Wright Stain.Approximately 200 cells were counted and assessed for distribution ofmorphology. Myel, myelocytes; Meta, metamyelocytes; Pro,promyelocytes. B, average number of neutrophils per treatmentgroup. Neutrophil number was acquired during the bone marrowdifferential cell count and multiplied by the total number of bonemarrow cells; data represent the mean � SD.






risk of developing MDS/AML in women who undergochemotherapy for breast cancer (29). However, use of G-CSF during treatment of AML in one large prospective studyhad no impact on complete response rate or relapse rate(11).

The most compelling evidence for the increased risk ofMDS/AML through G-CSF therapy comes from SCN.Although G-CSF clearly improves survival, there are severallines of evidence to suggest that G-CSF treatment contri-butes to the development of leukemia in these patients.First, the risk of leukemia seems to correlate with thecumulative dose of G-CSF (3). Second, of all the congenitalmarrow failure syndromes predisposed to AML, SCN alonedoes not seem to be anHSCdisorder. Because AML seems torise from sequential mutations in HSC, this would suggestthat therapy, not the intrinsic cell defect, is causal (30). It hasbeen demonstrated that G-CSF does initiate signaling path-ways in HSCs (31). In addition, the presence of hyperpro-liferative truncation mutations and an activating mutationof the G-CSFR have been associated with the developmentof AML in SCN (1).

This article provides evidence that prolonged G-CSFexposure results in genomic instability in HSCs. Afterextended treatment with G-CSF, there is a significantincrease in DNA damage in LSK cells. HSCs from micetreated in vivo with G-CSF displayed consistent loss ofregions of chromosome 2 and 17. Interestingly, the chro-mosome 2 deletions align with ABL1, which has previouslybeen demonstrated to be involved in several rearrange-ments and chromosome translocations in various types ofhuman leukemia (32). A mouse model of acute promye-locytic leukemia and a radiation-induced model of AMLshowdeletionson chromosome2 in a region containing thegene PU.1, a transcription factor critical for myeloid devel-opment (33). Previous reports have implicated heterozy-gous loss of PU.1 as contributing to the development ofleukemia in the aforementioned settings and in othermouse models (34). Although similar deletions in PU.1are rare in human AML,mutations inRUNX1, an importantregulator of PU.1 expression, are commonly seen.

Consistent deletions on chromosome 17 that includethe tumor suppressor gene TSC2 were also detected. TSC2

Figure 5. Comparison of the effects of genistein and NAC on LSK cells treated with G-CSF. C57BL/6J mice (n ¼ 43 per group) were treated with 5 dosesof G-CSF followed by 2 doses of genistein or NAC (50 mg/kg). A single group was treated with diluent as a control. Bone marrow LSK cells were isolated byFACSAria. Data represent the mean � SD. A, fold change in LSK cells in the four treatment groups. B, fold change in ROS levels measured by stainingsorted bone marrow LSK cells with DHET; a, P < 0.05 versus diluent; b, P < 0.05 versus G-CSF. C, fold change in DNA damage in sorted LSK cells quantifiedby intracellular staining of pH2AX. a, P < 0.05 versus diluent; b, P < 0.05 versus G-CSF.

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is a negative regulator of mTOR and recent studies havedemonstrated that the expression of TSC2 is downregu-lated in patients with acute leukemia (35). The mTORpathway is frequently activated in blasts from patientswith AML (36) and high-risk myelodysplastic syndrome(37). Furthermore, constitutive activation of the AKT/mTOR pathway has been shown to induce acute leukemiain mice (38). Collectively, the results suggest thatprolonged G-CSF treatment induces DNA damage inHSCs and genistein acts as a genoprotective agent in thissetting.Despite evidence of genomic instability in HSCs, none of

the mice treated with G-CSF developed leukemia. Trans-genic mice overexpressing G-CSF also do not develop leu-

kemia (39). This is not unexpected, as patients with SCNonly develop AML after years of G-CSF treatment and theprevalence even after 10 years of treatment is less than 50%.As expected, the number of HSCs at risk for developing aleukemogenic mutation is significantly higher in a humanthan in a mouse.

Although prolonged G-CSF exposure promotes genomicinstability in HSCs and is associated with the developmentof AML, it remains the only effective treatment for SCN,besides a HSC transplant. An ideal treatment would pro-mote late myeloid differentiation without affecting HSCs.An alternative strategy would be to coadminister a complexthat selectively blocks the effect of G-CSF on HSCs. Onthe basis of previous studies, genistein is an attractive

Figure 6. Effect of genistein onHSCproliferation inmice treatedwithG-CSF.C57BL/6Jmice (n¼43per group; 4groups)weregiven 3 intraperitoneal injectionsof BrdUrd (3 mg/24 hours) in PBS andmaintained on 0.2 mg/mL of BrdUrd in the drinking water for 72 hours with concomitant injection of diluents, genistein,G-CSF, or genisteinþG-CSF. A, gating strategy to analyze BrdUrd incorporation inmouse bonemarrow. B, percentage of BrdUrd incorporation intomyeloidprogenitor Lin�Sca�cKitþ cells. Data represent the mean � SD. C, percentage of BrdUrd incorporation into LSK cells. Data represent the mean � SD. A,P < 0.05 versus diluents; b, P < 0.05 versus G-CSF. D, C57BL/6J mice (n ¼ 43 per group) were treated with 5 doses of G-CSF followed by 2 doses ofgenistein. Progenitor cells were isolated and extracted proteins were analyzed by Western blot with antibodies against pGSK3b/a, cyclin D1, cyclin D3, andb-actin. Shownare representative results of one of three experiments. E andG,C57BL/6Jmice (n¼43per group)were treatedwith 5doses ofG-CSF followedby 2 doses of genistein; LSK cells were FACS sorted in glass slides and stained against pGSK3b/a, cyclin D1, cyclin D3. Shown are representative results ofone of two experiments. For quantification, images from more than 100 cells were captured using a Carl Zeiss LSM 510 META confocal microscope (Zeiss)with a Plan-Apo 63 � 11.4 oil immersion lens. The maximum intensity was determined and background was subtracted using Metamorph software(Molecular Devices). Fold change in mean � SD of intensity.






compound. Genistein is a natural soy isoflavone withexcellent bioavailability that has both antioxidant andantiproliferative properties (16, 40, 41). Both oxidativestress and excessive proliferation have been postulated toresult in genomic instability in HSCs. In reality, previousstudies have demonstrated that genistein protects HSCsfrom radiation and chemotherapy (17, 42, 43).

G-CSF treatment leads to a modest increase in ROSin LSK cells, which is reduced by genistein (Fig. 3B).Although oxidative stress has been implicated in genomicinstability in HSCs, NAC treatment, while reducing ROS,did not reduce DNA damage. In addition, we did notobserve increased 8-oxo-guanine levels in LSK cells trea-ted with G-CSF (data not shown). Interestingly, ROS andcell-cycle progression seem to be linked in HSCs and thereduction in ROS may merely reflect decreased prolifer-ation (44).

In the current study, we used a dose of genistein thatresults in serum levels that can easily be obtained throughoral supplementation (16, 45). At this dosage, genisteinpartially blocked theG-CSF–induced expansion of LSK cellsand significantly reduced pH2AX levels in this population.This was also accompanied by a reduction in the level ofcells with an abnormal FISH signal. Importantly, genisteindid not block the G-CSF–driven expansion of mature neu-trophils, as the total number of neutrophils in mice treatedwith G-CSF and genistein were the same as in mice treatedwithG-CSF alone (Fig. 4AandB).Genistein seems to inhibitG-CSF–driven expansion of LSKcd48-cd150þ cells (Sup-plementary Fig. S2). This population, termed LSK-SLAM, ishighly enriched for HSCs.

Genistein did not block G-CSF–induced expansion of apopulation enriched for myeloid progenitors (Lin�Sca�cKitþ; Fig. 6B). Collectively, these results would suggestthat the effects of genistein are mediated primarilythrough preferential inhibition of HSC proliferation,while not impairing myeloid progenitor proliferation anddifferentiation. Genistein inhibits G-CSF–induced GSK3phosphorylation and cyclin D1 and D3 induction in LSKand myeloid progenitors (Fig. 6D–G). Our results suggestthat there is a distinct requirement for GSK3/cyclinD1/D3 in G-CSF–modulated pathways in myeloid pro-genitors and HSCs.

It has been shown that the absence of GSK3b impairslong-term self-renewal capacity of HSCs, although it is notessential for myeloid development (23). While HSCs fromthe triple cyclin knockout mouse (cyclins D1, D2, and D3)display delayed cell-cycle entry and multilineage hemato-poietic failure (46, 47), single knockouts for D1 or D2display normal granulocyte counts. Mice lacking cyclinD3 show impaired neutrophil development, however, allD1�/�, D2�/�, D3�/� single knockoutmice present normalmyelopoiesis (48).

The data imply that genistein treatment prevents G-CSF–induced GSK3 phosphorylation, thus activating GSK3.GSK3 has being shown to regulate cyclin D1 and D3through various mechanisms including mRNA transcrip-tion, protein localization, and ubiquitin-dependent prote-

olysis (49); however, the mechanism involved in genisteinmodulation of GSK3/cyclin D1/D3 in G-CSF–treated micerequires further elucidation.

Collectively, our results indicate that prolonged G-CSFtreatment induces DNA damage in HSCs by initiatingcell-cycle progression. HSCs are long-lived, quiescent cellsthat preferentially use nonhomologous end joining(NHEJ) for DNA repair when progressing from G0 toG1 (19, 50). NHEJ is a relatively error-prone DNA repairmechanism, and its preferential use by HSCs has beenpostulated as a reason that chromosomal deletions andtranslocations are often seen and are frequently causal inthe development of acute leukemia (19). Further evidenceis provided by recent whole genome wide array sequenc-ing that has shown that HSCs accumulate mutations overtime. Importantly, we demonstrate that genistein, atlevels obtainable through dietary supplementation, isable to reduce DNA damage by attenuating G-CSF–induced HSC proliferation without compromising theability of G-CSF to accelerate terminal neutrophilic dif-ferentiation. These results suggest that genistein may bean effective preventive agent in patients with SCN whorequire prolonged G-CSF support.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L.R. Souza, O. Kucuk, M.L. McLemoreDevelopment of methodology: L.R. Souza, O. Kucuk, M. Rossi,M.L. McLemoreAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): L.R. Souza, E. Silva, E. CallowayAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): L.R. Souza, E. Silva, M. RossiWriting, review, and/or revision of the manuscript: L.R. Souza, E. Silva,O. Kucuk, M. Rossi, M.L. McLemoreAdministrative, technical, or material support (i.e., reporting ororganizing data, constructing databases): L.R. Souza, M. Rossi,M.L. McLemoreStudy supervision: L.R. Souza, M.L. McLemore

AcknowledgmentsOmer Kucuk is a Georgia Cancer Coalition Distinguished Scholar. The

authors thank Dr. Debra F. Saxe and Faith Sheff (Emory University) fortheir assistance with the FISH assays; Dr. Daniel R. Doerge (U.S. Food andDrug Administration, Jefferson, AR) for analyzing the total isoflavonelevels on mice serumþ; Drs. Hanna J. Khoury and Leon Bernal-Mizrachi(Emory University) for their critical reading of this article; Anthea Ham-mond (Emory University) for the proofing and editing of this article; Dr.Adam Marcus and Deborah Eltzroth Martinson at the Winship CellImaging Core, for their expertise on imaging acquisition; and Aaron Raeat the Flow Cytometry Core at Emory Children’s Pediatric ResearchCenter, for his assistance in cell sorting and analysis. The authors alsothank Vinicius Miessler de Andrade Carvalho, PhD student at UNICAMP-Brazil, for his assistance on some of the assays using G-CSF–treated anduntreated mice during the summer of 2011.

Grant SupportThis work was supported by the "Charles Harris Leukemia" & the "Kelly

Wilhite Aplastic Anemia" Research Fund. No governmental grants fundedthis research.

The costs of publication of this articlewere defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 15, 2013; revised February 17, 2014; accepted February18, 2014; published OnlineFirst March 10, 2014.

Souza et al.





References1. Welte K, Zeidler C. Severe congenital neutropenia. Hematol Oncol Clin

North Am 2009;23:307–20.2. Rosenberg PS, Alter BP, Link DC, Stein S, Rodger E, Bolyard AA, et al.

Neutrophil elastase mutations and risk of leukaemia in severe con-genital neutropenia. Br J Haematol 2008;140:210–3.

3. Rosenberg PS, Alter BP, Bolyard AA, Bonilla MA, Boxer LA, Cham B,et al. The incidence of leukemia and mortality from sepsis in patientswith severe congenital neutropenia receiving long-term G-CSF ther-apy. Blood 2006;107:4628–35.

4. Avalos BR, Lazaryan A, Copelan EA. Can G-CSF cause leukemia inhematopoietic stem cell donors? Biol Blood Marrow Transplant 2011;17:1739–46.

5. Bennett CL, Evens AM, Andritsos LA, Balasubramanian L, Mai M,FisherMJ, et al. Haematologicalmalignanciesdeveloping in previouslyhealthy individuals who received haematopoietic growth factors:report from the Research on Adverse Drug Events and Reports(RADAR) project. Br J Haematol 2006;135:642–50.

6. Makita K, Ohta K, Mugitani A, Hagihara K, Ohta T, Yamane T, et al.Acute myelogenous leukemia in a donor after granulocyte colony-stimulating factor-primed peripheral blood stem cell harvest. BoneMarrow Transplant 2004;33:661–5.

7. Socie G, Mary JY, Schrezenmeier H, Marsh J, Bacigalupo A, Locas-ciulli A, et al. Granulocyte-stimulating factor and severe aplasticanemia: a survey by the European Group for Blood and MarrowTransplantation (EBMT). Blood 2007;109:2794–6.

8. Lyman GH, Dale DC, Wolff DA, Culakova E, Poniewierski MS, KudererNM, et al. Acute myeloid leukemia or myelodysplastic syndrome inrandomized controlled clinical trials of cancer chemotherapy withgranulocyte colony-stimulating factor: a systematic review. J ClinOncol 2010;28:2914–24.

9. Kaito K, Kobayashi M, Katayama T, Masuoka H, Shimada T, NishiwakiK, et al. Long-term administration of G-CSF for aplastic anaemia isclosely related to the early evolution of monosomy 7 MDS in adults.Br J Haematol 1998;103:297–303.

10. Khoury HJ, Loberiza FR Jr, Ringden O, Barrett AJ, Bolwell BJ, CahnJY, et al. Impact of posttransplantation G-CSF on outcomes ofallogeneic hematopoietic stem cell transplantation. Blood 2006;107:1712–6.

11. Wheatley K, Brookes CL, Howman AJ, Goldstone AH, Milligan DW,Prentice AG, et al. Prognostic factor analysis of the survival of elderlypatients with AML in the MRC AML11 and LRF AML14 trials. Br JHaematol 2009;145:598–605.

12. McKinstry WJ, Li CL, Rasko JE, Nicola NA, Johnson GR, Metcalf D.Cytokine receptor expression on hematopoietic stem and progenitorcells. Blood 1997;89:65–71.

13. Zandstra PW, Conneally E, Petzer AL, Piret JM, Eaves CJ. Cytokinemanipulation of primitive human hematopoietic cell self-renewal. ProcNatl Acad Sci U S A 1997;94:4698–703.

14. Zeidler C, Germeshausen M, Klein C, Welte K. Clinical implications ofELA2-, HAX1-, and G-CSF-receptor (CSF3R) mutations in severecongenital neutropenia. Br J Haematol 2009;144:459–67.

15. Li Y, Li X, Ge M, Shi J, Qian L, Zheng Y, et al. Long-term follow-up ofclonal evolutions in 802 aplastic anemia patients: a single-centerexperience. Ann Hematol 2011;90:529–37.

16. Klein CB, King AA. Genistein genotoxicity: critical considerations of invitro exposure dose. Toxicol Appl Pharmacol 2007;224:1–11.

17. Davis TA, Mungunsukh O, Zins S, Day RM, Landauer MR. Genisteininduces radioprotection by hematopoietic stem cell quiescence. Int JRadiat Biol 2008;84:713–26.

18. Zheng J,HuynhH,UmikawaM,SilvanyR, ZhangCC.Angiopoietin-likeprotein 3 supports the activity of hematopoietic stem cells in the bonemarrow niche. Blood 2011;117:470–9.

19. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J,WeissmanIL. Deficiencies in DNA damage repair limit the function of haemato-poietic stem cells with age. Nature 2007;447:725–9.

20. TesioM, Golan K, Corso S, Giordano S, Schajnovitz A, Vagima Y, et al.Enhanced c-Met activity promotes G-CSF-induced mobilization ofhematopoietic progenitor cells via ROS signaling. Blood 2011;117:419–28.

21. Barbier V, NowlanB, Levesque JP,Winkler IG. Flowcytometry analysisof cell cycling and proliferation in mouse hematopoietic stem andprogenitor cells. Methods Mol Biol 2012;844:31–43.

22. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches.Nat Rev Immunol 2006;6:93–106.

23. Huang J, Zhang Y, Bersenev A, O'Brien WT, Tong W, Emerson SG,et al. Pivotal role for glycogen synthase kinase-3 in hematopoietic stemcell homeostasis in mice. J Clin Invest 2009;119:3519–29.

24. Trowbridge JJ, Xenocostas A,Moon RT, BhatiaM. Glycogen synthasekinase-3 is an in vivo regulator of hematopoietic stemcell repopulation.Nat Med 2006;12:89–98.

25. D'Amico M, Hulit J, Amanatullah DF, Zafonte BT, Albanese C, Bou-zahzah B, et al. The integrin-linked kinase regulates the cyclin D1 genethrough glycogen synthase kinase 3beta and cAMP-responsive ele-ment-binding protein-dependent pathways. J Biol Chem 2000;275:32649–57.

26. Jin J, Wang GL, Shi X, Darlington GJ, Timchenko NA. The age-associated decline of glycogen synthase kinase 3beta plays a criticalrole in the inhibition of liver regeneration. Mol Cell Biol 2009;29:3867–80.

27. Wei H, Bowen R, Cai Q, Barnes S, Wang Y. Antioxidant and anti-promotional effects of the soybean isoflavone genistein. Proc Soc ExpBiol Med 1995;208:124–30.

28. Carlo-Stella C, Dotti G, Mangoni L, Regazzi E, Garau D, Bonati A, et al.Selection of myeloid progenitors lacking BCR/ABL mRNA in chronicmyelogenous leukemia patients after in vitro treatment with the tyro-sine kinase inhibitor genistein. Blood 1996;88:3091–100.

29. Kaplan HG, Malmgren JA, Atwood MK. Increased incidence of myelo-dysplastic syndrome and acute myeloid leukemia following breastcancer treatmentwith radiation aloneor combinedwith chemotherapy:a registry cohort analysis 1990–2005. BMC Cancer 2011;11:260.

30. Jan M, Majeti R. Clonal evolution of acute leukemia genomes. Onco-gene 2013;32:135–40.

31. GibbsKDJr,Gilbert PM, SachsK, Zhao F, BlauHM,Weissman IL, et al.Single-cell phospho-specific flow cytometric analysis demonstratesbiochemical and functional heterogeneity in human hematopoieticstem and progenitor compartments. Blood 2011;117:4226–33.

32. Greuber EK, Smith-Pearson P, Wang J, Pendergast AM. Role of ABLfamily kinases in cancer: from leukaemia to solid tumours. Nat RevCancer 2013;13:559–71.

33. Finnon R, Moody J, Meijne E, Haines J, Clark D, Edwards A, et al.A major breakpoint cluster domain in murine radiation-induced acutemyeloid leukemia. Mol Carcinog 2002;34:64–71.

34. Rosenbauer F, Wagner K, Kutok JL, Iwasaki H, Le BeauMM, Okuno Y,et al. Acute myeloid leukemia induced by graded reduction of alineage-specific transcription factor, PU.1.NatGenet 2004;36:624–30.

35. Xu Z, Wang M, Wang L, Wang Y, Zhao X, Rao Q, et al. Aberrantexpression of TSC2 gene in the newly diagnosed acute leukemia. LeukRes 2009;33:891–7.

36. Martelli AM, Evangelisti C, Chiarini F, McCubrey JA. The phosphati-dylinositol 3-kinase/Akt/mTOR signaling network as a therapeutictarget in acute myelogenous leukemia patients. Oncotarget 2010;1:89–103.

37. Follo MY, Mongiorgi S, Bosi C, Cappellini A, Finelli C, Chiarini F, et al.The Akt/mammalian target of rapamycin signal transduction pathwayis activated in high-riskmyelodysplastic syndromesand influences cellsurvival and proliferation. Cancer Res 2007;67:4287–94.

38. Kharas MG, Okabe R, Ganis JJ, Gozo M, Khandan T, Paktinat M, et al.Constitutively active AKT depletes hematopoietic stem cells andinduces leukemia in mice. Blood 2010;115:1406–15.

39. Serizawa I, Amano K, Ishii H, Ichikawa T, Kusaka M, Taguchi T, et al.Long-term overexpression of human granulocyte colony-stimulatingfactor in transgenic mice: persistent neutrophilia with no increasedmortality for more than one year. Cytokine 2000;12:630–5.

40. Yang Z, Kulkarni K, ZhuW,HuM.Bioavailability and pharmacokineticsof genistein: mechanistic studies on its ADME. Anticancer AgentsMedChem 2012;12:1264–80.

41. Banerjee S, Li Y, Wang Z, Sarkar FH. Multi-targeted therapy of cancerby genistein. Cancer Lett 2008;269:226–42.






42. Zhou Y, Mi MT. Genistein stimulates hematopoiesis and increasessurvival in irradiated mice. J Radiat Res 2005;46:425–33.

43. Tacyildiz N, Ozyoruk D, Yavuz G, Unal E, Dincaslan H, Dogu F, et al.Soy isoflavones ameliorate the adverse effects of chemotherapy inchildren. Nutr Cancer 2010;62:1001–5.

44. Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon DH, Cullen DE,et al. FoxOs are critical mediators of hematopoietic stem cell resis-tance to physiologic oxidative stress. Cell 2007;128:325–39.

45. Kaludjerovic J, Franke AA, Ward WE. Circulating isoflavonoid levels inCD-1 mice: effect of oral versus subcutaneous delivery and frequencyof administration. J Nutr Biochem 2012;23:437–42.

46. Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A,Sicinska E, et al. Mouse development and cell proliferation in theabsence of D-cyclins. Cell 2004;118:477–91.

47. Malhotra S, Kincade PW. Wnt-related molecules and signaling path-way equilibrium in hematopoiesis. Cell Stem Cell 2009;4:27–36.

48. Sicinska E, Lee YM, Gits J, Shigematsu H, Yu Q, Rebel VI, et al.Essential role for cyclin D3 in granulocyte colony-stimulating factor-driven expansion of neutrophil granulocytes. Mol Cell Biol 2006;26:8052–60.

49. McCubrey JA, Steelman LS, Bertrand FE, Davis NM, Abrams SL,Montalto G, et al. Multifaceted roles of GSK-3 and Wnt/beta-cateninin hematopoiesis and leukemogenesis: opportunities for therapeuticintervention. Leukemia 2014;28:15–33.

50. Shao L, FengW, LeeKJ, ChenBP, ZhouD. A sensitive and quantitativepolymerase chain reaction-based cell free in vitro non-homologousend joining assay for hematopoietic stem cells. PLoS ONE 2012;7:e33499.


Souza et al.




2014;7:534-544. Published OnlineFirst March 10, 2014.Cancer Prev Res Liliana R. Souza, Erica Silva, Elissa Calloway, et al. Induced DNA Damage

−Genistein Protects Hematopoietic Stem Cells against G-CSF

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Genistein Protects Hematopoietic Stem Cells against G-CSF … · Research Article Genistein Protects Hematopoietic Stem Cells against G-CSF–Induced DNA Damage Liliana R. Souza1,