Experimental Hematology 30 (2002) 640–648

0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(02)00809-3

Extracellular nucleoside diphosphate kinaseNM23/NDPK modulates normal hematopoietic differentiation

Roel Willems

a,b

, Herman Slegers

b

, Inez Rodrigus

c

, Adriaan C. Moulijn

c

,Marc Lenjou

a

, Griet Nijs

a

, Zwi N. Berneman

a

, and Dirk R. Van Bockstaele

a

a

Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital (UIA/UZA), Edegem-Antwerp, Belgium;

b

Laboratory of Cellular Biochemistry, University of Antwerp (UIA), Wilrijk-Antwerp, Belgium;

c

Department of Cardiac Surgery, Antwerp University Hospital (UIA/UZA), Edegem-Antwerp, Belgium

(Received 24 July 2001; revised 28 January 2002; accepted 26 February 2002)

Objective.

We previously demonstrated the presence of nucleoside diphosphate kinase NDPK/NM23 in normal human plasma. It also was reported that extracellular NM23 could inhibitdifferentiation of certain hematopoietic cell lines. We further investigated the extracellular ef-fect of NM23 on hematopoiesis by adding recombinant NM23-H1, NM23-H2, and NM23-H3proteins to in vitro differentiation assays of normal human hematopoietic progenitors.

Materials and Methods.

To study the effect on the earlier stages of hematopoietic maturation,NM23 was added to serum-free pre–colony-forming unit (pre-CFU) assays starting from im-

mature CD34

��

CD38

�

bone marrow cells. Serum-free CFU assays starting from CD34

�

CD38

�

bone marrow cells were used as a model for terminal hematopoietic differentiation.

Results.

In pre-CFU assays, none of the NM23 isoforms used significantly changed the expan-

sion of CD34

��

CD38

�

cells, nor did NM23 alter the CD34

��

CD38

�

cell lineage commitment.In contrast, terminal differentiation of CD34

�

CD38

�

progenitor cells in CFU assays was sig-nificantly altered by addition of NM23 protein. More erythroid burst-forming units and fewermacrophage colonies were observed in cultures containing any of the NM23 isoforms exam-ined. Similar effects were observed using the enzymatically inactive H118N mutant of NM23-H1,strongly suggesting that the observed effect is independent of the nucleoside diphosphate ki-nase activity of NM23.

Conclusion.

We demonstrated a modulating effect of extracellular NM23 proteins on the ter-minal stages of normal hematopoietic differentiation. Therefore, the fairly high concentrationsof NM23 constitutively present in plasma could have a physiologic role in supporting erythro-poiesis and inhibiting excessive macrophage formation. © 2002 International Society for Exper-

imental Hematology. Published by Elsevier Science Inc.

Currently, the human

nm23

gene family consists of eightmembers:

nm23-H1

to

nm23-H8. Nm23-H1

,

nm23-H2

, and

nm23-H3

(also called

DR-nm23

) are highly expressed inhematopoietic progenitors and down-regulated during dif-ferentiation [1,2]. In addition, high expression levels of

nm23-H1

and

nm23-H2

have been observed in proliferatinglymphocytes [1,3]. Most of the gene products have nucleo-side diphosphate kinase (NDPK) activity; therefore, NM23has been proposed to be involved in cellular processes that

consume nucleotides other than ATP, such as polymeriza-tion of microtubules [4,5] or G-protein–mediated signaltransduction [6,7]. However, compelling evidence indicatesthat some NM23 isoforms have several other functions,such as transcriptional regulation of the c-

myc

oncogene [8]and protein kinase activity [9,10] (for review see [11]).Steeg et al. [12] identified NM23-H1 as an overexpressedgene in melanoma cells with low metastatic potential. Thisled to the hypothesis that NM23-H1 was a metastasis sup-pressor gene, which was confirmed by transfection of the

nm23-H1

cDNA in highly metastatic cell lines, resulting ina reduction of metastatic potential [13,14]. To date, morethan 200 research papers have described an inverse relation-ship between

nm23

expression and tumor metastasis in sev-

Offprint requests to: Zwi N. Berneman, M.D., Ph.D., Laboratory ofExperimental Hematology, Antwerp University Hospital (UZA), Wilrijk-straat 10, B-2650 Edegem, Belgium; E-mail: Zwi.Berneman@uza.be

R. Willems et al./Experimental Hematology 30 (2002) 640–648

641

eral types of carcinomas, including breast cancer, mela-noma, gastric cancer, and many others [15]. In contrast, inhematologic malignancies, high NM23 levels are indicativeof aggressive tumoral behavior. In acute myelogenous leu-kemias (AML), high NM23-H1 expression, and to a lesserextent high NM23-H2 expression, correlates with shortersurvival times [16]. High-grade malignancy lymphomashave significantly higher NM23-H1 levels than low-gradelymphomas [17]. Patients with myelodysplastic syndromeor chronic myelogenous leukemia up-regulate intracellularNM23-H1 upon transformation into leukemic phase andblast crisis, respectively [18].

We previously reported the presence of NM23 in humanplasma [1]. In normal peripheral blood plasma, bone mar-row plasma, and umbilical cord blood plasma, NDPK activ-ity correlated with the hemoglobin levels, indicating thepresence of NM23 in plasma as a consequence of red bloodcell lysis. Niitsu et al. [19–21] demonstrated that plasmaNM23-H1 concentration is a valuable prognostic factor inAML as well as in non-Hodgkin’s lymphoma. In thesepathologic conditions, NM23-H1 level did not correlatewith hemoglobin concentration, but high amounts of plasmaNM23-H1 were indicative of poor prognosis. In non-Hodgkin’s lymphoma, the plasma concentration of NM23-H1correlated significantly with the Ann Arbor stage and theWHO performance status; in AML, plasma NM23-H1 cor-related with white blood cell counts and LDH levels. More-over, of all prognostic factors examined in AML, theNM23-H1 plasma level (

�

80 ng/mL) was the most reliablefor predicting patient survival.

These data clearly demonstrate the constitutive presenceof NM23 in plasma, which is increased in certain pathologicconditions. However, the physiologic relevance of thesefindings is not yet known. In 1985, Okabe-Kado et al. [22]described a factor present in the conditioned medium of aclone of the mouse myeloid cell line M1 that inhibited mac-rophage differentiation of this clone. This “I-factor” wasisolated and identified as NM23-M2, the murine homologueof NM23-H2 [23]. In addition, it was demonstrated that dif-ferent NM23 isoforms were able to inhibit the induction ofdifferentiation in several other cell lines [24]. The effectswere most pronounced on erythroid differentiation of HEL,K562, and KU812 cells, and could also be observed withmutants of NM23 lacking NDPK activity [25]. These find-ings support the possibility that NM23 is present in plasmaas a regulator of hematopoiesis. Unfortunately, all of theseexperiments were performed in serum-containing condi-tions, because the presence of NM23 in plasma and also infetal calf serum (FCS) [1] was still unknown.

In view of the results that high concentrations of NM23can be present in plasma and the findings that this proteincan inhibit differentiation of certain cell lines, we investi-gated the effects of several recombinant NM23 isoforms ondifferent stages of normal hematopoietic differentiation.Here, we show for the first time on primary cells and in se-

rum-free conditions that extracellular NM23 can affect latestages of hematopoiesis, i.e., stimulation of erythropoiesisand inhibition of macrophage differentiation.

Materials and methods

Materials

Supernatant of the 43A1 hybridoma (IgG

3

murine monoclonalantibody, provided by Dr. H.J. Bühring, University of Tübingen,Tübingen, Germany) was used as the source of anti-CD34 anti-bodies [26]. Fluorescein isothiocyanate (FITC)-conjugated rabbitanti-mouse (RAM) immunoglobulins [F(ab)

�

2

fragments] werepurchased from Dako (Glostrup, Denmark). Phycoerythrin (PE)-conjugated anti-CD38 antibodies (IgG

1

) as well as isotype-specificcontrol antibodies were purchased from Becton-Dickinson (Erem-bodegem, Belgium). Mouse

�

-globulins were obtained from Jack-son ImmunoResearch Laboratories (West Baltimore Pike, PA,USA). Recombinant human (rh)IL-1 (specific activity

�

10

9

U/mg),rhIL-6 (

�

1

�

10

8

U/mg), and rh

kit

ligand (

�

1

�

10

5

U/mg) wereobtained from Boehringer Mannheim GmbH (Penzberg, Ger-many), and rhIL-3 (10

4

U/

�

g) and rh

Flt3/Flk2

ligand (FL; 2.10

5

U/mg) were obtained from Biosource Europe (Nivelles, Belgium).Erythropoietin (Epo;

�

10

5

U/mg) was obtained from Janssen-Cilag (Brussels, Belgium). Insulin, transferrin, cholesterol, and leci-thin were obtained from Sigma (Bornem, Belgium). FCS (batchno. 40G5182K; Life Sciences, Paisley, UK) was heat inactivatedfor 30 minutes at 56

�

C. Deionized bovine serum albumin (BSA;Intergen, Toronto, Canada) was tested for NDPK activity as de-scribed previously [1] and had an activity

1 mU/g. TherhNM23-H1, rhNM23-H2, and rhNM23-H3 (cut to the length ofNM23-H1 and NM23-H2, i.e., amino acid 18–170) were a kindgift of Dr. I. Lascu (Université de Bordeaux, Bordeaux, France),and the H118N mutant of NM23-H1 was a kind gift of Dr. M.L.Lacombe (INSERM U 402, Paris, France) [27,28].

Isolation of hematopoietic progenitor cells

After obtaining informed consent according to the regulations ofthe Ethics Committee of the University of Antwerp, bone marrowsamples were collected by sternal puncture from hematologicallynormal patients undergoing cardiac surgery, in tubes containing Is-cove’s modified Dulbecco’s medium (IMDM; Life Sciences), 10%FCS, and 100 U/mL preservative-free heparin (B. Braun, Melsun-gen, Germany). Mononuclear cells were isolated by lymphocyteseparation medium (LSM; ICN Biomedicals, Costa Mesa, CA,USA) density gradient centrifugation and washed twice withIMDM. Bone marrow cells were incubated with 43A1 supernatantfor 15 minutes at room temperature, washed twice in IMDM, andincubated with F(ab)

�

2

fragments of a fluoresceinated rabbit anti-mouse IgG antibody (1/40 dilution) for 15 minutes at room tem-perature. After two washes with IMDM, the cells were labeled for15 minutes with PE-conjugated anti-CD38 monoclonal antibody inthe presence of a 10-fold excess of normal mouse

�

-globulins,added 10 minutes in advance in order to saturate the remainingRAM binding sites. After two washes with IMDM, the cells weresorted on a FACStar Plus Cell Sorter (Becton-Dickinson) equippedwith an air-cooled argon ion laser (ILT model 5500 A; Ion LaserTechnology, Salt Lake City, UT, USA), tuned to 488 nm at 40 mWpower. Cells with a low-to-medium forward scatter, a low sidescatter, and a highly positive FITC fluorescence were retained.

642

R. Willems et al./Experimental Hematology 30 (2002) 640–648

This population was split into two fractions (right and left sorted).Cells with a PE fluorescence signal lower than the mean fluores-cence

2 SD of cells labeled with an irrelevant isotype-matchedcontrol antibody were retained as CD34

CD38

�

cells. Cells with ahigher PE fluorescence signal were retained as CD34

CD38

cells(Figure 1). Because the average CD34 expression of the CD34

CD38

�

cells was always higher than that of the CD34

CD38

fraction, theCD34

CD38

�

population is designated as CD34

CD38

�

. Cells werecounted in a hemocytometer, after which CD34

CD38

�

cells were in-oculated in serum-free pre–colony-forming unit (pre-CFU) assays,and the CD34

CD38

cells were plated in serum-free CFU assays.

Pre-CFU assays

To avoid interference of NDPK present in the culture media, weoptimized serum-free pre-CFU assays [29]. CD34

CD38

�

bonemarrow cells were cultured in flat-bottom 96-well plates (Costar,Corning, NY, USA) in serum-free medium (StemPro34; Life Sci-ences) containing 200 U/mL penicillin, 200

�

g/mL streptomycin,1

�

g/mL amphotericin B, rhIL-1 (100 U/mL), rhIL-6 (1000 U/mL),rhIL-3 (20 U/mL), rh

kit

ligand (100 ng/mL), and rhFL (100 ng/mL). To obtain an exact quantification of the number of inoculatedcells, the sorted CD34

CD38

�

bone marrow cells were countedin a hemocytometer. Depending on the sort yield, 500 to 1000 cellswere seeded in each well. Two hours after inoculation, the exactnumber of cells per well was determined by cell counting in thewell, under an inverted light microscope (magnification

�

400).The number of inoculated cells ranged from 300 to 1500 cells/well. Cultures were performed in the presence or absence of a low(50 ng/mL) or a high (1

�

g/mL) concentration of rhNM23-H1,rhNM23-H2, rhNM23-H3, and rhNM23-H1(H118N).

After 14 days of primary liquid culture at 37

�

C in 7.5% O

2

and5% CO

2

in a fully humidified incubator, expanded cells werecounted using a hemocytometer or, in case of a low cell number, di-rectly in the well, and CFU assays in semisolid medium were per-formed as a readout. In brief, 1000 cells were cultured in six-wellplates (Costar) in 2 mL of medium containing 0.9% methylcellu-lose supplemented with 20% FCS, 1% deionized BSA, 10% condi-tioned medium of the 5637 bladder carcinoma cell line containinggranulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) [30,31], 20 U/mL rhIL-3, 2 U/mL Epo, 10

�

5

M 2-mercaptoethanol, 200 U/mLpenicillin, 200

�

g/mL streptomycin, and 1

�

g/mL amphotericin B,which were optimal concentrations for colony formation as deter-mined in preliminary experiments [32]. The cultures were micro-scopically scored for colony formation after 14 days of culture at37

�

C in 7.5% O

2

and 5% CO

2

in a fully humidified incubator. Colo-nies were differentially counted and scored making the distinctionbetween macrophage colonies (CFU-M), granulocyte colonies(CFU-G), granulocyte-macrophage colonies (CFU-GM), erythroidcolonies (CFU-E), burst-forming units (BFU-E), and mixed colo-nies (CFU-GEMM). The number of colonies obtained is expressedas cloning efficiency (CE), which is the percentage of inoculatedcells that was capable of colony formation in semisolid culture.

CFU assays

One thousand CD34

CD38

bone marrow progenitor cells werecultured in a six-well plate (Costar) in 2 mL of serum-free semi-solid medium consisting of IMDM with 0.9% methylcellulose sup-plemented with 1% BSA, 10

�

g/mL human insulin, 400

�

g/mLfreshly iron-saturated bovine transferrin, 12

�

g/mL cholesterol, 36

�

g/mL lecithin, 20 U/mL rhIL-3, 100 ng/mL rh

kit

ligand, 2500 U/mLG-CSF, 3000 U/mL GM-CSF, 2 U/mL Epo, 10

�

5M 2-mercapto-ethanol, 200 U/mL penicillin, 200

�

g/mL streptomycin, and 1

�

g/mL amphotericin B. The assays were performed in the presence orabsence of a low (50 ng/mL) or a high (1

�

g/mL) concentration ofrhNM23-H1, rhNM23-H2, rhNM23-H3, and rhNM23-H1(H118N).After a 14-day culture at 37

�

C in 7.5% O

2

and 5% CO

2

in a fullyhumidified incubator, the cultures were microscopically scored forcolony formation, as explained earlier.

Statistical analysis

Comparison of cell expansions and colony types was performedwith a paired Student’s

t

-test.

p

0.05 was considered statisticallysignificant.

Results

To investigate the effects of extracellular NM23 on normalhematopoietic differentiation, recombinant human NM23-H1,NM23-H2, and NM23-H3 were added to in vitro hemato-poietic differentiation assays. As a model for early hemato-poietic differentiation, adult human bone marrow CD34

CD38

�

immature progenitor cells were used as a startingpopulation for serum-free pre-CFU assays. Effects of NM23on more mature bone marrow progenitor cells with theCD34

CD38

phenotype were investigated in semisolid

Figure 1. Sorting criteria for CD34CD38� and CD34CD38 hemato-poietic progenitors. A representative example of the sorting criteria for theisolation of CD34CD38� (R2) and CD34CD38 (R3) hematopoieticprogenitors from normal human bone marrow is shown. (A) Side scatter(SSC) vs forward scatter (FSC) dot plot of mononuclear adult bone marrowcells labeled with a primary anti-CD34 antibody, an FITC-conjugated sec-ondary antibody, and a PE-conjugated anti-CD38 antibody. (B) Dot plotshowing the fluorescence intensities of CD34-FITC and CD38-PE of thecells in region R1. (C) FSC vs SSC dot plot of the cells after flow cytomet-ric sorting of the population in gate R2. (D) Fluorescence intensities ofCD34-FITC and CD38-PE of the sorted CD34CD38� hematopoieticprogenitor cells (population in gate R2).

R. Willems et al./Experimental Hematology 30 (2002) 640–648 643

clonogenic assays (CFU assays). Because NM23 is presentin FCS [1], CFU assays (starting from CD34 CD38 cells)and the primary phase of the pre-CFU assays (starting fromCD34CD38� cells) were performed in serum-free con-ditions.

Effects of NM23 on immatureCD34CD38� hematopoietic progenitorsIn the pre-CFU assay, CD34CD38� adult bone marrowcells were cultured in a serum-free primary liquid culturecontaining a stimulatory combination of early-acting cyto-kines (IL-1, IL-3, IL-6, kit-ligand, and FL) to induce earlydifferentiation. These cultures were performed in the ab-sence or presence of 50 ng/mL or 1 �g/mL NM23-H1,NM23-H2, and NM23-H3. In order to evaluate if NM23had a modulating effect on early hematopoietic differentia-tion, cells were counted after 14 days of culture and platedin semisolid clonogenic assays. Cell counts provided infor-mation about the effect of NM23 on cell expansion, whilethe secondary CFU assay served as a readout for the differ-entiation state of the cells after 14 days of liquid culture.Therefore, this secondary readout CFU assay does not con-tain NM23 and contains FCS to ensure optimal growth. Inthis way, it was possible to identify the effects of NM23 onthe initial phase of proliferation and differentiation of prim-itive CD34CD38� hematopoietic progenitor cells, whichare otherwise difficult to grow in direct semisolid CFU as-says [32,33]. In the primary liquid phase of the pre-CFU as-says, the average cell expansion in control conditions was8.2 � 3.6-fold (mean � SEM). Expansion in the presence of50 ng/mL or 1 �g/mL NM23-H1, NM23-H2, and NM23-H3 (n 6) is depicted in Figure 2A. The mean CE (n 6)is shown in Figure 2B. These results are expressed in rela-tion to control conditions, which were taken as 100%. Incontrol conditions, CE was 11% � 3% (mean � SEM). Theactual, not normalized, CE for the other conditions were(mean � SEM): NM23-H1 50 ng/mL: 8% � 3%; NM23-H11 �g/mL: 8% � 4%; NM23-H2 50 ng/mL: 10% � 3%;NM23-H2 1 �g/mL: 9% � 3%; NM23-H3 50 ng/mL: 12% �3%; and NM23-H3 1 �g/mL: 15% � 3%. The number ofcolonies formed per initially inoculated CD34CD38�

cell (n 6) is shown in Figure 2C. These results also areexpressed in relation to control conditions, in which 1.1 �0.6 (mean � SEM) colonies were scored per initially inocu-lated CD34CD38� cell. Although there are two statisti-cally significant differences in comparison with controlconditions, no consistent and/or dose-dependent modulationof the proliferation of primitive hematopoietic progenitorsby extracellular NM23 could be demonstrated with this as-say. After differential scoring, the proportion of the variousdifferentiation lineages was determined for each condition.However, comparison of these data did not reveal any statis-tically significant effect caused by any of the NM23 iso-forms (n 5; data not shown).

Figure 2. Effects of extracellular NM23 in pre-CFU assays starting fromCD34CD38� cells. Human CD34CD38� bone marrow cells werecultured in serum-free medium in the absence or presence of 50 ng/mL or 1�g/mL NM23-H1, NM23-H2, and NM23-H3. After 14 days of culture,cells were counted to determine the expansion during the liquid phase andthen inoculated in standard CFU assays. (A) Expansion in the differentconditions in relation to control conditions (mean SEM; n 6). (B) CEof the expanded cells in the secondary CFU assay in relation to controlconditions (mean SEM; n 6). (C) Number of colonies generated perinitially inoculated CD34CD38� progenitor in relation to control condi-tions (mean SEM, n 6). Statistical comparisons were made using apaired Student’s t-test. Results were considered as statistically differentfrom the control condition if p 0.05 (star).

644 R. Willems et al./Experimental Hematology 30 (2002) 640–648

Effects of NM23 on matureCD34CD38 hematopoietic progenitorsA possible effect of NM23 on the later phases of hemato-poietic differentiation was studied using serum-free CFUassays as in vitro differentiation assays. Adult bone mar-row CD34CD38 cells were plated in a semisolid assay inthe presence or absence of 50 ng/mL or 1 �g/mL NM23-H1 (n 16), NM23-H2 (n 10), and NM23-H3 (n 6).Average CE after 2 weeks of culture in control conditionswas 12% � 2% (mean � SEM; n 17). Addition ofNM23 did not significantly alter this value. However, dif-ferential scoring revealed a clear shift in differentiationpathway from monocyte differentiation to erythroid differ-entiation (Fig. 3). Addition of any of the NM23 isoformssignificantly decreased the proportion of CFU-M and sig-nificantly increased the proportion of BFU-E. Remarkably,while the effect on BFU-E was significantly dose depen-dent for two isoforms (NM23-H1 and NM23-H3), the de-crease in percentage of CFU-M was already maximal at 50ng/mL for all the isoforms examined. The proportion ofother types of colonies was not consistently altered afteraddition of NM23.

NM23 not only induced a shift from macrophage toerythroid differentiation, but a significant decrease in mac-rophage colony formation and a significant increase inerythroid burst formation was observed in conditions con-taining NM23. This is apparent when the CE of each colonytype (i.e., the percentage of inoculated cells that induce theformation of a certain type of colony) in control conditionsis compared with the CE of each colony type in NM23-con-taining conditions (Fig. 4A and B). Further analysis of theCFU assay data showed that for samples characterized by ahigh colony formation, the decrease in CFU-M was morepronounced from a statistical viewpoint after NM23 addi-tion compared to samples with a poorer outcome in terms ofCE. On the other hand, samples with a lower total CE weremore susceptible to stimulation of BFU-E growth by NM23(up to almost 10-fold of control) compared to samples withhigh colony formation. This is apparent when the experi-ments were divided into two groups (based on the CE). Fig-ure 4 shows the CE of CFU-M and BFU-E for the sampleswith a high total CE (Fig. 4C and D) and for the sampleswith a low total CE (Fig. 4E and F).

To investigate whether the effect of NM23 is a conse-

Figure 3. Differential scoring of CFU assays starting from CD34CD38 cells, with and without NM23. Human CD34CD38 bone marrow cells wereinoculated in serum-free semisolid clonogenic assays and cultured for 2 weeks in the presence or absence of 50 ng/mL or 1 �g/mL NM23-H1 (n 16),NM23-H2 (n 10), and NM23-H3 (n 6). Subsequently, the formed colonies were counted microscopically, distinguishing (A) granulocyte colonies (CFU-G),(B) macrophage colonies (CFU-M), (C) granulocyte-macrophage colonies (CFU-GM), (D) erythroid colonies (CFU-E), (E) erythroid bursts (BFU-E), and(F) mixed colonies (CFU-GEMM). For each type of colony, the results are expressed as the percentage of the total number of colonies formed in each condi-tion. Results were statistically different if p 0.05 (star), using a paired Student’s t-test.

R. Willems et al./Experimental Hematology 30 (2002) 640–648 645

quence of its enzymatic NDPK activity, CFU assays wereperformed using the enzymatically inactive H118N NM23-H1mutant. In 10 experiments, mutant NM23-H1 was added toserum-free CFU assays at concentrations of 50 ng/mL or 1�g/mL and compared to wild-type NM23-H1 (Fig. 5).These experiments showed that the H118N mutant is able toalter hematopoietic differentiation, favoring the formationof BFU-E and inhibiting CFU-M formation to a similar ex-tent as wild-type NM23-H1.

DiscussionThe relation of NM23 proteins to hematopoiesis has beenreported in several studies [34]. Down-regulation of nm23expression (H1, H2, and H3) is observed during maturationof CD34 progenitor cells [1,2] and nm23-H1 and nm23-H2are strongly up-regulated after activation of peripheral bloodlymphocytes [1,3]. When present in the culture medium ofseveral hematopoietic cell lines, extracellular NM23-H1 andNM23-H2 are able to inhibit induction of differentiation.This inhibition was demonstrated for macrophage anderythrocyte differentiation and was independent of theNDPK activity of NM23 proteins [24,25].

In this study, we investigated the effects of extracellularNM23-H1, NM23-H2, and NM23-H3 on different stages ofnormal hematopoietic differentiation. Using the pre-CFU as-say starting from CD34CD38� cells as a model for earlydifferentiation, we could not detect a consistent effect on pro-liferation or on differentiation. In contrast, terminal differen-tiation of mature hematopoietic progenitors was influencedby addition of NM23 protein, as shown in CFU assays start-ing from CD34CD38 cells. We observed a consistent andsignificant inhibition of macrophage differentiation that wasmost pronounced at the level of CFU-M. The most pro-nounced was the effect of NM23 on erythroid differentiation.The presence of NM23 in the medium resulted in a consider-able, significant, and consistent increase in the number ofBFU-E, but not of CFU-E. This effect was prominent, even inthe presence of IL-3 and GM-CSF, which have been shownto possess “burst promoting activity” [35]. In accordancewith the previous studies on leukemia cell lines [25], these ef-fects were observed for all investigated isoforms. This isprobably a consequence of the high degree of homology intheir primary structure. NM23-H1 and NM23-H2 share 88%homology and NM23-H3 has approximately 70% sequencehomology with NM23-H1 and NM23-H2 (for a review of all

Figure 4. Differential effects of NM23 on erythroid and macrophage differentiation. Serum-free CFU assays performed in the presence or absence of 50 ng/mLor 1 �g/mL NM23-H1, NM23-H2, and NM23-H3. The scoring of (A,C,E) macrophage colonies (CFU-M) and (B,D,F) erythroid bursts (BFU-E) is presentedas cloning efficiency (CE), i.e., the percentage of inoculated CD34CD38 cells that was capable of colony formation in semisolid culture (mean SEM).Experiments with a high CE (total CE �10%) (C,D) were separated from experiments with a low CE (total CE 10%) (E,F). In panels A and B (all experi-ments): n 16 (control, NM23-H1), n 10 (NM23-H2), and n 6 (NM23-H3); in panels C and D: n 6 (control, NM23-H1), n 5 (NM23-H2), and n 4 (NM23-H3); in panels E and F: n 10 (control, NM23-H1), n 5 (NM23-H2), and n 2 (NM23-H3). Results were statistically different if p 0.05(star), using a paired Student’s t-test.

646 R. Willems et al./Experimental Hematology 30 (2002) 640–648

eight known isoforms, see Lacombe et al. [11]; an amino acidsequence comparison of NM23-H1, NM23-H2, and NM23-H3 is shown in references [2,36]). In addition, the tertiary andquaternary structures are conserved throughout the NM23family [37,38]. In our study, the same differentiation lineages(macrophage and red blood cell differentiation) were affectedcompared to the cell line studies [24], although in experi-ments with cell lines, red blood cell differentiation was inhib-ited while we observed a stimulation of bone marrow erythro-poiesis. Perhaps this is less contradictory than it seems,because differentiation of cell lines usually is concomitantwith inhibition of proliferation while the formation of BFU-Erequires a large cell expansion.

Several hypotheses can be made about the molecular

mechanism of this novel NM23 property. As most NM23isoforms can transfer the terminal phosphate of nucleosidetriphosphates to nucleoside diphosphates (NDPK activity)[39,40], NM23 could influence hematopoietic differentia-tion by modulation of the extracellular nucleotide pool.However, compared to wild-type NM23, the effect was nei-ther qualitatively nor quantitatively altered when an enzy-matically inactive mutant of NM23-H1 was used. This sug-gests that NM23 modulates hematopoiesis through amechanism independent of its phosphotransferase activity.Because NM23-H1 has the capacity to convert nucleotidesat a high rate (kcat of 580s�1 [27]) and nucleotides present inthe culture medium have to be secreted by the cells, it is ex-pected that the equilibrium of extracellular nucleotides ispredominantly regulated by the concentration of the nucle-otides itself rather than by the enzyme concentration. Thatthe stimulating effect of NM23 on BFU-E formation is notsaturated at low NM23 concentrations but was higher at aconcentration of 1 �g/mL underscores the hypothesis thatthis effect is not mediated by the NDPK activity of NM23.Taken together, these data suggest that NM23 mediates itsextracellular effect through protein interaction with either areceptor or a soluble molecule (e.g., a growth factor), result-ing in the stimulation or inhibition of certain signal trans-duction pathways. The action of NM23 through an (un-known) receptor could be challenged in view of the highactive concentration of NM23 proteins compared to otherreceptor-mediated responses. However, this must be viewedin light of the high molecular mass of native NM23 (hex-amer of 105 kDa [41]). Moreover, high saturating concen-trations have been described for several cytokines (e.g.,Flt3/Flk2 ligand, thrombopoietin [42]).

Previously, we reported that high NDPK activity ispresent in normal plasma and that the NDPK activity is cor-related with the plasma hemoglobin concentration [1]. Us-ing an enzyme-linked immunosorbent assay (ELISA), Ni-itsu et al. [20] quantified the level of NM23-H1 in normalplasma as 6.1 � 4.1 ng/mL. In this view, our data suggestthat, under physiologic conditions, there is not a pronouncedeffect of NM23 on erythropoiesis, but when the extracellularconcentration of NM23 is increased, hematopoietic differen-tiation is biased toward erythropoiesis. Therefore, NM23might be one of the factors that stimulates red blood cellsynthesis after hemolysis. On the other hand, the effect ob-served on macrophage differentiation was already maximalat 50 ng/mL. This suggests that plasma NM23 might consti-tutively inhibit terminal macrophage differentiation. As ithas been demonstrated that several molecules present inplasma promote macrophage differentiation (e.g., adenosineor platelet-derived factors [43,44]) and that serum biases he-matopoiesis toward the monocyte lineage [29], one couldhypothesize that the physiologic function of plasma NM23is to (partially) neutralize the monocyte-differentiating ef-fect of serum, thereby preventing massive macrophage dif-ferentiation.

Figure 5. Effect of mutant NM23-H1 on differentiation. Scoring of mac-rophage colonies (CFU-M, upper panel) and erythroid bursts (BFU-E,lower panel) in serum-free semisolid CFU assays, using CD34CD38

bone marrow cells as starting material. Cells were cultured in the presenceor absence of 50 ng/mL or 1 �g/mL of NM23-H1 or its enzymatically inac-tive H118N mutant (n 10). Bar charts show the percentage of the totalnumber of colonies formed in each culture condition (mean SEM). Thescoring is presented as a percentage of the control condition. Results werestatistically different if p 0.05 (star), using a paired Student’s t-test. NS statistically nonsignificant differences.

R. Willems et al./Experimental Hematology 30 (2002) 640–648 647

AcknowledgmentsThis work was supported by the Concerted Research Action 1996-2001 (GOA no. 21/1996) of the University of Antwerp “ControlMechanisms of Cell Proliferation in Eukaryotic Cells”. R.W. is afellow of the Concerted Research Action and the “Vlaamse Ligategen Kanker”. The authors thank Dr. I. Lascu (Institut de Bio-chimie et Génétique Cellulaire, Centre Nationale de la RechercheScientifique (CNRS), Université de Bordeaux, France) and Dr. M.L.Lacombe (INSERM U 402, Faculté de Médecine Saint Antoine,Paris, France) for the generous gift of recombinant NDPK. Dr. H.J.Bühring of the University of Tübingen (Germany) is acknowl-edged for kindly providing us with the 43A1 hybridoma.

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