Life Sciences 84 (2009) 458–467

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Nm23-H1 regulates the proliferation and differentiation of the human chronicmyeloid leukemia K562 cell line: A functional proteomics study

Lin Jin b,c, Ge Liu a, Chuan-hai Zhang a, Chun-hua Lu c, Sheng Xiong a, Mei-Ying Zhang a, Qiu-Ying Liu a,Feng Ge c, Qing-Yu He c, Kaio Kitazato d, Nobuyuki Kobayashi d, Yi-Fei Wang a,⁎a Guangzhoujinan Biomedicine Research & Development Center, Jinan University, No. 601, West Huangpu Road, Guangzhou, 510632 Chinab The Institute of Pharmacology Science, Jinan University, Guangzhou, 510632 Chinac The Institutes of Life and Health Engineering, Jinan University, Guangzhou, 510632 Chinad Division of Molecular Pharmacology of Infectious Agents, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki,852-8521 Japan

⁎ Corresponding author. Tel.: +86 20 85223426; fax:E-mail address: twangyf@jnu.edu.cn (Y.-F. Wang).

0024-3205/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.lfs.2009.01.010

a b s t r a c t

a r t i c l e i n f o

Article history:

Aims: Nm23-H1 is a suppre Received 26 August 2008Accepted 23 January 2009

Keywords:Nm23-H1K562 cellsMegakaryocytic differentiationProteomicsCdc42

ssor of metastasis that has been implicated in the regulation of proliferation anddifferentiation of hematopoietic cells, although specific mechanisms for Nm23-H1 have not been well-characterized. Our study is designed to further elucidate the role of Nm23-H1 in the human chronic myeloidleukemia K562 cell line.Main methods: In this study we generated and selected two cell clone pools of human chronic myeloidleukemia K562 cells with up-regulated and down-regulated Nm23-H1 expression.Key findings: Our data show that knockdown of Nm23-H1 decreased proliferation and increased thepercentage of cells arrested in the G0/G1 phase of the cell cycle. Correspondingly, K562 cells overexpressingNm23-H1 were more proliferative. After treatment of these two cell types with phorbol 12-myristate13-acetate (PMA) for 48 h, cells with reduced Nm23-H1 expression had a higher percentage of 8N ploidy andhigher expression of CD41 than K562 cells overexpressing Nm23-H1. A functional proteomics analysisidentified ten proteins, including ANP32A, Cdc42GAP, and the isoform 2 of SET, whose expression levels weresignificantly altered by down-regulation of Nm23-H1. In addition, cells with decreased levels of Nm23-H1 hadsignificantly reduced expression of Cdc42 independent of treatment with PMA. The interaction of theendogenous Nm23-H1 and Cdc42 proteins has been further validated by reciprocal immunoprecipitations.Significance: We provide data that complement functional studies of Nm23-H1 in regulating hematopoieticcells, and address action mechanisms of Nm23-H1 that have not previously been reported.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Nm23 is a metastasis suppressor protein that was first cloned fromthe murine malignant melanoma cell line K-1735 (Steeg et al. 1988).A total of nine isoforms of human Nm23 have been identified andall have been shown to have catalytic activity. Of the nine isoforms,Nm23-H1 encodes a nucleoside diphosphate kinase A (NDPKA) thathas been shown to be involved in awide variety of biological activities.Correspondingly, Nm23-H1 generates nucleoside triphosphates(NTPs) necessary for DNA and RNA synthesis, protein elongation,signal transduction, etc. (Biggs et al. 1990). In various cell lines, ex-pression of Nm23-H1 has been shown to positively or negatively affectcell behavior through signaling pathways or associations with co-factors (Lacombe et al. 2000). For example, decreased expression ofNm23-H1 was shown to block TGF-β1 signaling to inhibit growth ofcolon carcinoma cells (Hsu et al. 1994). Nm23-H1 has been shown to

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complex with the Ras-related protein associated with diabetes (Rad) toinfluence cell proliferation and differentiation (Otsuki et al. 2001; Zhuet al. 1999). Moreover, interaction of Nm23-H1 with Tiaml has beenshown to induce metastases (Otsuki et al. 2001; Habets et al. 1994). Inbreast carcinoma cells high-Nm23-H1 correlated well with reducedphospho-ERK expression (Salerno et al. 2005; Hartsough et al. 2002).In hematologic malignancies, high levels of Nm23 expression havebeen indicative of aggressive tumor behavior. In acute myelogenousleukemias (AML), increased Nm23-H1 expression correlates with apoor clinical course especially in AML-M5 (Yokoyama et al. 1998).Similarly, in myelodysplastic syndrome and chronic myelogenousleukemia, high intracellular Nm23-H1 levels can induce the leukemicphase of the disease and blast crisis (Yokoyama et al. 1998). High-grademalignant lymphomas have also been shown to higher expressionlevels of Nm23-H1 (Aryee et al. 1996).

It has been extensively demonstrated that K562 cells can beinduced to differentiate towards erythroid and megakaryocytelineages by various differentiation inducers (Sutherland et al. 1986;Alitalo 1990). With PMA treatment, K562 cells exhibit a well-known

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phenotype with the loss of erythroid markers and the appearance ofearly megakaryocytic markers. It includes acquisition of megakaryo-cytic surface marker CD41, production of megakaryocytic cytokines,development of polyploidy nuclei, cellular enlargement, clusterformation, cell cycle arrest (D'Angelo et al. 1996; Hocevar et al. 1992;Chih et al. 2007).

Althoughmany reports have characterized the role and expressionof Nm23-H1 in various differentiated cell lines, there is little in-formation regarding the function of Nm23-H1 in K562 cells. Ourstudy is designed to highlight changes in the differentiation capacityof K562 cells as a result of changes in Nm23-H1 protein expression.We investigate possible interaction between Nm23-H1 and Cdc42,and use proteomics analysis to identify ten proteins that have sig-nificant changes in expression following PMA-treatment of K562 cellsexpressing Nm23-H1-targeted siRNA.

Materials and methods

Chemical reagents

Phorbol 12-myristate 13-acetate (PMA) was purchased fromSigma-Aldrich (USA) and U0126 was purchased from Cell SignalingTechnology (USA).

Cell culture

Human chronic myeloid leukemia K562 cells were purchased fromATCC and, grown in RPMI 1640 (Gibco, UK), supplemented with 10%heat inactivated fetal bovine serum (Gibco, UK), containing 100 U/mlof penicillin and streptomycin.

Proliferation assay

Cells were plated at 1×105/ml and viable cells were counted dailyin a hemocytometer after staining with trypan blue. Each proliferationassay was performed in triplicate.

RNA isolation and real-time quantitative PCR

Total RNA was isolated from 5×105 cells using TRIzol reagent(Invitrogen, USA) according to the manufacturer's protocol. Purity andquantification of RNAwere assessed by OD260/280 ratios. One microgramof total RNA was reverse transcribed into cDNA using oligo(dT) primersand SuperScript II Reverse Transcriptase (Invitrogen, USA). Real-timequantitative PCR reaction was performed using SYBR Green I Real-timePCRMasterMix (TAKARA, Japan) andABI Prism7000 SequenceDetectionSystem (Applied Biosystem, UK). For amplification, Nm23-H1 sequencespecific primers [forward-5′-CAAGCCTGGGACCATCCGTGGAGACT-3′ andreverse-5′-TTCCTCAGGGTGAAACCACAAGCCGA-3′] and 18sRNA se-quence specific primers [forward-5′-CCTGGATACCGCAGCTAGGA-3′ andreverse-5′-GCGGCGCAATACGAATGCCCC-3′] were used. The reactionwasperformed for 40 cycles [95 °C for 1 min, 95 °C for 15 s, 58 °C for 15 s]followed by incubation at 72 °C for 40 s. The relativemRNA expression ofNm23-H1 was normalized to the housekeeping gene, 18sRNA, and wascalculated using the formula: Rel Exp=2(−ΔΔC

T) (Livak and Schmittgen

2001). Each real-time PCR reaction was performed in triplicate.

Generation of stable cell clone pools of K562

We cloned a previously selected siRNA sequence shown to beeffective in down-regulatingNm23-H1 (sense: 5′-CUGGAUCUAUGAAU-GACAGtt-3′, antisense: 5′-CUGUCAUUCAUAGAUCCAGtt-3′), into thepSilencer4.1-CMVneo vector (Ambion, USA) (Chen et al. 2006). K562cells (4×105)were produced by transfectingwith 8 μg of plasmidmixedwith Lipofectamine 2000 (Invitrogen, USA). After 48 h, the cells wereremoved from the wells, diluted 1:10, and transferred to 6-well plates

containing 800 μg/ml of G418 (Invitrogen, USA). After 2–4weeks, G418-resistant cell clone pool was collected and expanded. The same methodwas used to select a K562 cell clone pool with overexpression of Nm23-H1 using the pcDNA3.1-Nm23-H1 vector (constructed in our lab).

Soft agar colony formation assay

Transfected cells (1×103) in the exponential growth phase wereseeded in 35 mm2 dishes containing 0.7% and 1.2% layers of agar in 2×RPMI 1640. After 10 days at 37 °C, colonies of N50 cells were counted.All experiments were performed in triplicate, and the results are theaverage of three independent experiments.

Flow cytometry analysis cell cycle, DNA ploidy, and surface antigen CD41

Approximately 1×106 cells for each experimental conditionwere harvested by centrifugation, washed twice with PBS, fixed in70% cold ethanol, and kept at 4 °C overnight. Cells were resuspendedin 20mg/ml propidium iodide (PI) and 100mg/ml RNase for 30min atroom temperature in the dark. DNA content was measured on a FACSCaliber (Becton-Dickinson, USA). Each sample contained up to 10,000cells. Cell cycle and DNA ploidywere analyzed using CellQuest software.

CD41 was used as a megakaryocytic differentiation marker anddetected using flow cytometry. For this analysis, 1×106 cells weresuspended in PBS, stained with phycoerythrin (PE) conjugated anti-CD41 and fluorescein isothiocyanate (FITC) conjugated anti-glyco-phorin A monoclonal antibodies (Beckman Coulter, USA). All antibodyincubations were for 30 min on ice. Cells were stained with FITC-conjugated mouse IgG and PE alone as a negative control.

Two-dimensional gel electrophoresis (2-DE)

Cells induced with 20 nM PMA for 48 h were harvested, rinsed inwashingbuffer, and lysed (8MUrea, 4% CHAPS, 2% IPGbuffer, 0.2mg/mlPMSF) for 30 minutes at 4 °C with constant agitation. Cell lysates werecentrifuged (12,000 g at 4 °C) for 15minutes and protein concentrationswere determined by the Bradford method. Protein samples were se-parated by isoelectric focusing (IEF) using Immobilized pH Gradient(IPG) drystrips with a pH range of 3–10 on Ettan IPGphor 3 (GeneralElectric Company, USA)with a programmed voltage gradient. Resolutionin the second dimensionwas achieved using SDS-PAGE (12.5%) and gelswere stained with silver nitrate overnight. Gel images were scanned andanalyzed using Image Master 2D Platium 6.0.

Protein digestion

Silver-stained protein spots on polyacrylamide gels were excised,rinsed twice with ddH2O, then destained in a 1:1 solution of 30 mMpotassium ferricyanide and 100mMammonium bicarbonate (pH 8.0).After hydrating with acetonitrile and drying in a speedVac, gelsamples were rehydrated in a minimal volume of sequencing gradeporcine trypsin solution (20 μg/ml in 25 mMNH4HCO3) and incubatedat 37 °C overnight. Supernatants were extracted once with 67% ace-tonitrile containing 1% trifluoroacetic acid. The peptide extract and thesupernatant of the gel spot were combined and completely dried.

MALDI-TOF/TOF analysis

Digested protein extracts (tryptic peptides) were re-suspendedwith 5 μl of 0.1% trifluoroacetic acid. The peptide samples were mixed(1:1 ratio) with a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in 50% acetonitrile–1% trifluoroacetic acid. Aliquots(0.8 μl) were spotted onto stainless steel sample target plates. Peptidemass spectrawere obtained on anApplied Biosystem Sciex 4800MALDITOF/TOF mass spectrometer. Data were acquired in a positive MSreflector using a CalMix5 standard to calibrate the instrument (ABI4700

Fig. 1. Effect of PMA on K562 cell differentiation. A: RT-qPCR was used to detect levelsof Nm23-H1 mRNA from untreated and treated cells (20 nM PMA) at 0 h, 6 h, 12 h, 24 h,and 48 h timepoints. Data were normalized using the housekeeping gene, 18sRNA.B: Western blot analysis was used to detect Nm23-H1 protein levels. Detection of β-actinserved as a loading control and was used to normalize quantitation of Nm23-H1 proteinlevels (shown in bar graph). Results were representative of three independent experi-ments and values represent the mean±SD. ⁎⁎pb0.01, compared with untreated K562 cells.

Fig. 2. Expression of Nm23-H1 in stably transfected K562 cells. A: RT-qPCR detectedNm23-H1 mRNA levels in untransfected and selected K562 cell types. The results werenormalized to 18sRNA. ⁎⁎pb0.01, compared with K562 control. B: Changes in Nm23-H1protein levelsweredetectedusingWesternblot analysis andwere consistentwithRT-qPCRdata. The same blot was re-probed with an anti-β-actin antibody as a loading control andfor normalization of quantitated protein levels (shown in bar graph). Results wererepresentative of three independent experiments and values represent the mean±SD.⁎⁎pb0.01, compared with untransfected K562 cells.

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CalibrationMixture).Mass spectrawereobtained fromeach sample spotby accumulation of 600–800 laser shots in a 900–4000 mass range. ForMS/MS spectra, the fivemost abundant precursor ions per samplewereselected for subsequent fragmentation and 900–1200 laser shots wereaccumulated per precursor ion. The criteria for the precursor selectionwere a minimum S/N of 50. The MS and MS/MS spectra for each spotwere combined and submitted to the MASCOT search engine (V2.1,Matrix Science, U.K.) using GPS Explorer software (V3.6, AppliedBiosystems) and were searched with the following parameters: IPIHuman database (V3.36), taxonomy of Homo sapiens (human), trypsinof the digestion enzyme, one missed cleavage site, partial modificationof cysteine carboamidomethylated and methionine oxidized, no fixedmodifications, MS tolerance of 30–60 ppm, MS/MS tolerance of 0.2–0.3 Da. Known contaminant ions (keratin) were excluded. A total of69,012 sequences and 29,002,682 residues in the database were

searched. MASCOT protein scores N61 (based on combined MS andMS/MS spectra) were considered statistically significant (pb0.05).

Co-immunoprecipitation

For endogenous protein immunoprecipitation, K562 cells seedingfor 48 h were lysed with immunoprecipitation buffer (50 mM Hepes,pH 7.5, 50 mM NaCl, 0.1%Tween 20, 1% Triton X-100, 10% glycerol andprotease and phosphatase inhibitors). Clarified cell lysates were mixedwith goat monoclonal antibodies against Nm23-H1 (37.6, Santa Cruz,USA), Cdc42 (c-20, Santa Cruz, USA) or goat IgG, respectively, andincubated 90min at 4 °Cwith gentle shaking, followed by adsorption toprotein G plus-agarose beads (Santa Cruz, USA). Beads were extensivelywashed with immunoprecipitation buffer, the complex was re-suspended in SDS sample buffer, separated by 12.5% SDS-PAGE.

Western blot analysis

Cells were lysed in RIPA buffer and protein concentrations weredetermined using the Bradford method. Cell extracts were separatedby SDS-PAGE (12.5%) and transferred to polyvinylidene difluoride

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membranes (Millipore, USA) in Tris–glycine buffer containing 20%methanol. Membranes were blocked with TBST containing 5% non-fatmilk and washed 3× with TBST. Detection with primary antibodiesranging in dilution from 1:1000–1:2000 and included: anti-Nm23-H1,anti-Cdc42, anti-ANP32A, anti-EEF2, and anti-β-actin. Except for anti-Nm23-H1 (37.6, SantaCruz, USA), anti-Cdc42 (c-20, SantaCruz, USA) andanti-ANP32A (Chemicon, USA), all other antibodies were purchasedfrom Cell Signaling Technology (USA). Corresponding secondaryantibodieswere conjugated tohorseradishperoxidase (1:5000dilution)(Cell Signaling, USA). ECL chemiluminescent substrate solution (Pierce,USA) was used to visualize the antibody–antigen complexes, and bandswere imaged by autoradiography.

Fig. 3. Effects of up- and down-regulation of Nm23-H1 on K562 cells. A: Wrights–Giemsa staspecific siRNA and non-specific siRNA (magnification: 400×). Arrows indicate cells with 2–3Results were representative of three independent experiments and values represent themeanrepresentative of three independent experiments and are reported as the mean±SD. D: FlowPMA for 48 h. E: Expression of CD41 in transfected K562 cells induced with 20 nM PMA forthree independent experiments. ⁎⁎pb0.01 compared with K562 control.

Statistical analysis

Results are presented as themean±standard deviation (SD) deter-mined by t test. Results were considered significant if pb0.05.

Results

Expression of Nm23-H1 during PMA-induced megakaryocytedifferentiation of K562 cells

PMA concentrations of 5 nM and higher suppressed proliferation ofK562 cells relative to the concentration of PMA used. After 72 h, the

ining examined the morphological changes in K562 cells transfected with shNm23-H1-lobated nuclei. B: A growth curve is shown for untransfected and selected K562 cells.±SD. C: The number of colonies in each of the five cell groupswere counted. Values arecytometry analysis of cell ploidy distribution of K562 clones after treatment with 20 nM48 h analyzed by flow cytometry. Histograms for CD41 expression are representative of

Fig. 3 (continued ).

Table 1Cell cycle distribution for transfected K562 cell types

Cell type G0/G1 S G2/M

shNm23-H1 59.4±2.1 33.3±1.7 6.2±1.9shCon 43.4±1.7 28.7±3.9 27.9±2.4pcNm23-H1 32.0±1.2 60.2±1.5 7.7±2.8pcCon 46.8±3.9 45.5±6.4 7.7±2.5

Values represent the mean±SD for three experiments.

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proliferation rate decreased from71.8±2.3% to 40.2±5.4% (pb0.05). Toinvestigate changes in Nm23-H1 expression following PMA-induceddifferentiation of K562 cells without inducing cytotoxicity, 20 nM PMAwas selected for further experiments. K562 cells were treated with20 nM PMA for 6–48 h and Nm23-H1 mRNA levels were detected usingreal-timequantitative PCR (RT-qPCR). After 12hof PMAtreatment, K562cells showed a decrease of N82% in Nm23-H1 mRNA expression(pb0.01). By 48 h, levels of Nm23-H1 mRNA had nearly returned topretreatment levels (Fig. 1A). Western blot analysis was also used todetermine if there was a correlation between changes in Nm23-H1protein levels and stimulation of differentiation. The amount of Nm23-H1 protein significantly decreased ~60% after 12 h compared to un-treated controls, and further decreased to 71% by 48 h (Fig. 1B).

Confirmation of transfected K562 cells

Based on the above results, we evaluated the possible role of Nm23-H1 in K562 differentiation. We transfected and selected K562 cell clone

pools that overexpressed Nm23-H1 (referred to as pcNm23 cells) andhad down-regulated Nm23-H1 (referred to as shNm23 cells). Two addi-tional cell clone pools were used as controls (pcCon and shCon,respectively) that expressed Nm23-H1 at equivalent mRNA levels asuntransfected K562 cells (Fig. 2A). For the pcNm23 cells, mRNA ex-pressionwas increased 3.1-fold (Fig. 2A), while shNm23 cells hadmRNAlevels ofNm23-H1 reducedup to 88% comparedwithuntransfectedK562and shCon cells. Western blot analysis of the transfected cell lines foundsimilar changes in Nm23-H1 protein levels (Fig. 2B).

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The phenotype of shNm23 and pcNm23 K562 cells

The morphology of shNm23 cells included the presence of somecells with 2–3 lobated nuclei as detected by Wrights–Giemsa staining(Fig. 3A). In terms of proliferation, shNm23 cells had the lowest pro-

Fig. 4. Representative 2-D gel images for shNm23 and shCon cells treated with PMA for 48treatment are shown. Triplicate gels were prepared from protein extracts obtained in sepahighlight areas where significant differences in protein expression are present. B and C: Specieach image. Arrows label the specific protein spot of interest.

liferation rate, while pcNm23 cells exhibited the greatest growthcapacity and the proliferation rate is 19.3±0.8% and 43.3±2.9%, res-pectively (Fig. 3B). Similar results were obtained from colony formationassays. The number of colonies for each of thefive cells types is shown inFig. 3C. The data show that the shNm23 cells had an inhibition of colony

h. Separated proteins were visualized by silver staining. Representative gels from eachrate experiments. A: An overall view of a 2-D gel with separated cell extract. Circlesfic regions of the 2-D gel are highlighted to identify specific proteins labeled to the left of

Fig. 4 (continued ).

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formation capacity compared to pcNm23 and control cells. To furthercharacterize the cell proliferation phenotype, cell cycle analysis wasperformed and revealed that the percentage of cells in the G0/G1-phasewas59.4±2.1% for shN23 cells, 32.0±1.2% forpcNm23cells, 43.4±1.7%for shCon cells, and 46.8±3.9% for pcCon cells (Table 1). The higher thepercentage of cells in the G0/G1-phasemeans the greater the inductioninG0/G1 arrest. Therefore, cellswith down-regulatedNm23-H1 showedthe greatest induction of G0/G1 arrest.

We also used flowcytometry analysis to compare the DNA content ofour cell lines. There was no obvious difference in DNA content betweenknockdown and control cells, however, we found that low levels ofNm23-H1 accelerated the appearance of polyploidy in response to PMA.After 48 h of PMA stimulation, about 18.4%, and 6.5% of 2N and 4N cellsshifted to 8N and higher ploidy in shNm23 and shCon, respectively. Incontrast, K562 cells overexpressing Nm23-H1 showed no significantchange relative to the pcCon cells (Fig. 3D). Lastly, we analyzed theexpressionof surface antigen, CD41, after treatmentof cellswith PMA for48 h. Despite a low percentage of megakaryocytes in the population ofknockdown cells, ~23% were CD41-positive, which was significantlyhigher than that of the control groups (pb0.01). The opposite result wasobserved for pcNm23 cells (Fig. 3E). Increased expression of CD41

Table 2Up- and down-regulated proteins in response to PMA with decreased endogenous expressi

Protein name Spot no. Protein scorea Sequence

Up-regulatedElongation factor 2 50 189 34Isoform neutral α-glucosidase AB precursor 58 119 16Pantothemate kinase 4 87 95 11Zyxin 116 85 9Transketolase 217 444 48Cdc42 GAPase-activating protein 1 523 107 26Isoform 2 of succinyl CoA ligase 525 63 5

Down-regulatedIsoform 2 of protein SET 589 106 25Acidic leucine-rich nuclear phosphor-protein

32 family member A856 102 26

Phosphoglycerate 944 194 30

a MASCOT protein scores N61 (based on combined MS and MS/MS spectra) were considb Sequence coverage indicates the relative amount of protein sequence covered by the pep

abundance in the full scan by MALDI-TOF/TOF MS.c The fold change column corresponds to the expression of each protein in shNM23-H1 ce

independent experiments. ⁎pb0.05, ⁎⁎pb0.01, compared with the control.

indicates that cells with down-regulated Nm23-H1 were able toaccelerate the process of megakaryocytic differentiation of K562 cells.

Identification of the different proteins by functional proteomics

We used proteomics to identify specific proteins that differed inexpression in PMA-treated shNm23 cells compared to control cells.We detected 750–1120 protein spots in each gel (Fig. 4A). More than90% of overlapped protein spots were achieved in parallel gels fromthe same group, indicating that the spots used for differential analysiswas reproducible. When the two dimensional pattern of proteins fromshNM23 cells were compared with the control group, ten spots wereidentified as having a N2-fold change. As shown in Table 2, MALDI-TOF/TOF MS analysis identified each of the ten proteins. After PMAtreatment for 48 h, proteins EEF2, GANAB, PANK4, ZYX, Transketolase,isoform 2 of succiny1-CoA ligase, and Cdc42GAPwere significantly up-regulated (Fig. 4B). Alternatively, isoform 2 of protein SET, ANP32A,and PGK1 were significantly down-regulated (Fig. 4C).

Expression changes in ANP32A and EEF2 detected in the pro-teomics analysiswere confirmedusingWestern blot analysis. Consistentwith the proteomics results, Western blot analysis detected increased

on of Nm23-H1

coverageb PI/Mr (Da) Fold changec Function

6.41/95,277 2.34⁎⁎ Protein synthesis5.74/106,806 2.03⁎ Protein binding5.88/85,937 3.34⁎ Biosynthesis regulation6.92/67,242.3 2.09⁎ Protein binding; signal transduction7.58/67,834 6.09⁎ Calcium ion binding5.85/50,404.2 2.21⁎⁎ GTP binding6.63/48,009 3.96⁎ Metabolic regulation

4.09/31,170.3 2.22⁎ DNA replication4.75/24,053.2 6.99⁎⁎ Transcription regulation

8.3/44,586.1 2.73⁎⁎ ATP binding

ered statistically significant (p≤0.05).tides with matching masses, of which came from the top ten precursor ions peptides in

lls relative to its expression in control cells. Values represent the mean of three

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expression of EEF2 and decreased expression of ANP32A (Fig. 5). Thesedata confirm the reliability of the proteomics analysis.

Analysis of protein–protein interactions based on STRING database

In our study, two proteins of the ten identified by proteomicanalysis were further analyzed for protein–protein interactions basedon STRING database criteria. Protein–protein interactions wereevaluated using the STRING online database, developed at EMBL, SIB,and UniZH. Both direct (physical) and indirect (functional) associationswere analyzed. Selected proteins were uploaded by their SWISSPROTidentifier or SWISSPROT accession number. To determine the highestconfidence association, we set the parameters to include a networkdepth of 1 and nomore than 10 interactors. By uploading the Nm23-H1SWISSPROT accession data and choosing the protein form, a networkwas created. Analysis of protein–protein interactions ranked the pro-teins as SET, NME2, PRUNE APEX1, ANP32A, NME3, HMGB2, RRAD,STK6, and NME4. Strikingly, isoform 2 of SET and ANP32A were alsoidentified in our proteomics analysis.

Nm23-H1 directly effects expression of Cdc42

When shNm23 cells were treated with PMA, Cdc42 expressiondecreased further (Fig. 6A). Fig. 6B shows that expression of Cdc42 in

Fig. 5. Expression patterns of ANP32A and EEF2 determined byWestern blot. Expressionof ANP32A and EEF2 in shNm23-H1 cells after treatment with PMA for 48 h. Detectionof β-actin served as a loading control and was used to normalize quantitation ofANP32A and EEF2 protein levels (shown in bar graph). Results were representative ofthree independent experiments and values represent the mean±SD. ⁎pb0.05 and⁎⁎pb0.01, compared with shCon cells.

shNm23 cells declined to 0.4-fold, and increased 1.25-fold in pcNm23cells compared to control K562 cells. To validate whether there is theinteraction between endogenous Nm23-H1 and Cdc42, a reciprocalimmunoprecipitation experiment was further performed. As shown inFig. 6C, Cdc42 was detected in the Nm23-H1 immunoprecipitate, andNm23-H1 was also present in the Cdc42 immunoprecipitate. Thesedata suggest that endogenous Nm23-H1 can directly effect expressionof endogenous Cdc42 in K562 cells.

Discussion

The role of Nm23-H1 in hematopoietic cell differentiation has beenreported in several studies. When recombinant Nm23-H1 was con-stitutively present in plasma, it was shown to induce normal erythro-poiesis and inhibit macrophage formation (Willems et al. 2002).Alternatively, erythroid differentiation of HEL cells was inhibited byrecombinant Nm23-H1 (Okabe-Kado et al. 1995). Expression of surfaceNm23-H1 protein was found to decrease during both erythroid andgranulocyte differentiation in vitro (Okabe-Kado et al. 2002). Studies ofRNAi-mediated down-regulation of Nm23-H1 were conducted by ourlaboratory and showed that Nm23-H1-targeted siRNA could block K562cells fromentering S-phase bycausing them to arrest in theG0/G1phaseof the cell cycle.We also found that expression of Nm23-H1 significantlydeclined during PMA-induced megakaryocyte differentiation of K562cells, consistent with previous reports (Yamashiro et al. 1994). Since anarrest of the cell cycle correlates with the process of cell differentiation,we hypothesized that Nm23-H1 may regulate K562 proliferation anddifferentiation. To investigate this, we compared K562 cells withincreased versus decreased expression of Nm23-H1 by employingseveral clone pools.

Decreased expression of Nm23-H1mRNA increased the DNA ploidyof K562 cells, but not the process of normal cell division. Previous assaysof cell proliferation and cell cycle analysis of K562 cells also indicatedthat Nm23-H1 was a potential activator of K562 cell proliferation.Despite the appearance of 2–3 lobated nuclei in some of the shNm23cells, one of the characterization of the megakaryocyte (Coppola et al.2006), analysis of DNA content and expression of CD41 (data notshown) indicated that knockdown of Nm23-H1 mRNA was notsufficient to induce differentiation of K562 cells. Therefore, we inducedthe differentiation of K562 cells with PMA, and unexpectedly found anaccelerated appearance of polyploidy and expression of CD41 comparedto K562 cells that overexpressed Nm23-H1 with the same treatment.The combination of these data suggest that knockdown of Nm23-H1mRNA levels can accelerate the megakaryocyte differentiation of K562cells in the presence of PMA.

To further characterize the mechanism by which PMA-stimulatedshNm23-H1 cells, we analyzed changes in protein expression usingproteomics. Compared to control cells, 10 proteins had significantchanges in their expression levels. Transketolase was up-regulated 6-fold and ANP32A was down-regulated 7-fold. Based on protein–protein interactions predicted by STRING analysis, we chose to furtherstudy isoform 2 of SET, ANP32A, Cdc42, and Cdc42GAP.

SET is a 39-kDa phosphoprotein widely expressed in human andmouse tissues. SET has 2 isoforms, 1 and 2, the latter mediates anti-apoptosis activity by inhibition of Nm23-H1. This inhibition can bereversed by Gzma cleavage of SET which allows SET and Nm23-H1 tomove into the nucleuswhere SET is degraded. Now in anunbound form,the DNase activity of Nm23-H1 can damage DNA independent ofcaspase-mediated apoptosis (Zusen et al. 2003). Isoform 2 of SET canalso associate with other proteins as part of a SET complex whichincludes ANP32A, APEX1, HMGB2, and Nm23-H1. ANP32A is a memberof the ANP32 family and is found in self-renewing and stem cell-likepopulations. ANP32A expression is increased in largely undifferentiatedcells, whereas its expression is reduced in highly differentiated cells. Inour study, the expression of ANP32A was unchanged in shNm23-H1versus control cells (data not shown). However, after PMA treatment, its

Fig. 6. Nm23-H1 directly effects expression of Cdc42. A: Expression of Cdc42 in shNm23-H1 cells after treatment with PMA for 48 h. Detection of β-actin served as a loadingcontrol and was used to normalize quantitation of Cdc42 protein levels (shown in bar graph). Results were representative of three independent experiments and values represent themean±SD. ⁎pb0.05 and ⁎⁎pb0.01, comparedwith shCon cells. B: Detection of endogenous Cdc42 protein in untransfected and transfected K562 cells. Detection of β-actin served as aloading control and was used to normalize quantitation of Cdc42 protein levels (shown in bar graph). Results were representative of three independent experiments and valuesrepresent the mean±SD. ⁎pb0.05 and ⁎⁎pb0.01, compared with untransfected K562 cells. C: Analysis of interaction between endogenous Nm23-H1 and Cdc42. K562 cell lysate wasimmunoprecipitated (IP) with anti-Nm23-H1antibody, anti-Cdc42 antibody, or IgG control. The interaction between Nm23-H1 and Cdc42 was then examined by Western blotanalysis of the precipitate.

466 L. Jin et al. / Life Sciences 84 (2009) 458–467

expression was significantly lower than control cells. This observationwas verified by MALDI TOF/TOF mass spectrometry and Western blotanalyses. From these data, we can hypothesize that knockdown ofNm23-H1 mRNA does not directly down-regulate isoform 2 of SETand ANP32A without additional proteins, and decreased expressionof ANP32A in combination with decreased expression of Nm23-H1accelerates the differentiation of K562 cells to megakaryocytes.

Cdc42, a Rho-type GTPase, has been implicated in a variety ofcellular processes and has been shown to accelerate the G1-S phasetransition (Moon and Zheng 2003; Debidda et al. 2005). Cdc42 isintracellular binary molecular switches cycling between the activeGTP-bound state and the inactive GDP-bound state (Etienne-Manne-ville andHall 2002; Bar-Sagi andHall 2000). This active-inactive loop isregulated by three families of proteins: the GEFs (guanine nucleotide-exchange factors), the GAPs (GTPase-activating proteins) and the GDIs(guanine nucleotide-dissociation inhibitors) (Cerione and Zheng 1996;Jaffe and Hall 2005; Olofsson 1999). Cdc42GAP (Cdc42 GTPase-activating protein; also known as p50RhoGAP or ARHGAP1) is amajor member of the GAPs family and is a ubiquitously expressednegative regulator of Cdc42 (Moon and Zheng 2003). In this study, withor without the PMA treatment K562 cells expressing a Nm23-H1-targeted RNAi resulted in the down-regulation of Cdc42 as determined

byWestern blot. Furthermore, Cdc42GAP was found to be up-regulatedunder the same experimental conditions in proteomics analyses. Wealso performed the reciprocal immunoprecipitation experiment and theresults support that there may exit an interaction between endogenousNm23-H1 and Cdc42 in K562 cells, which is agreement with the recentresearch identification (Murakami et al. 2008a,b). In combination, ourdata show that Cdc42 was significantly reduced by knockdown ofNm23-H1 and up-regulated in K562 cells overexpressing Nm23-H1,which correlates with the arresting of cells in the G1/G0 phase to blockcell division in shNm23-H1 cells.

Conclusion

Our data indicate that Nm23-H1 plays a key role in mediating theproliferation of K562 cells by regulation of Cdc42. However, furtherstudies are needed to determine whether Nm23-H1 interacts withCdc42 directly, and whether this process requires NDP kinase activity.We also demonstrate that the role of Nm23-H1 in regulating thedifferentiation of K562 cells is enhanced by treatment of K562 cellswithPMA. Our data not only complement functional studies of themechanism of Nm23-H1 in regulating hematopoietic cells, but alsosupport thepotential for siRNA-mediated therapies to beapplied in vivo.

467L. Jin et al. / Life Sciences 84 (2009) 458–467

Acknowledgements

This work was supported by grants from the Nation “863” Programof China (No. 2007AA02Z142) and the National Natural ScienceFoundation of China (No. 30400071).

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