Decrease in Nucleoside Diphosphate Kinase (NDPK/nm23) Expression during Hematopoietic Maturation
1998; Elsevier BV; Volume: 273; Issue: 22 Linguagem: Inglês
10.1074/jbc.273.22.13663
ISSN1083-351X
AutoresRoel Willems, Dirk R. Van Bockstaele, Filip Lardon, Marc Lenjou, Griet Nijs, Hans‐Willem Snoeck, Zwi Berneman, Herman Slegers,
Tópico(s)Metastasis and carcinoma case studies
ResumoThe nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34+ progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells. The nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34+ progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells. Currently, the family of nucleoside diphosphate kinases (NDPK/nm23) 1The abbreviations used are: NDPK, nucleoside diphosphate kinase; PHA, phytohemagglutinin; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; IMDM, Iscove's modified Dulbecco's medium; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; FCS, fetal calf serum. consists of four members encoded by the genes nm23-H1, nm23-H2, DR-nm23, and nm23-H4 (1Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M. J. Natl. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1299) Google Scholar, 2Stahl J.A. Leone A. Rosengard A.M. Porter L. King C.R. Steeg P.S. Cancer Res. 1991; 48: 6550-6554Google Scholar, 3Venturelli D. Martinez R. Melotti P. Casella I. Peschle C. Cucco C. Spampinato G. Darzynkiewicz Z. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7435-7439Crossref PubMed Scopus (156) Google Scholar, 4Milon L. Rousseau-Merck M.-F. Munier A. Erent M. Lascu I. Capeau J. Lacombe M.L. Hum. Genet. 1997; 99: 550-557Crossref PubMed Scopus (112) Google Scholar). These enzymes share the ability to transfer the terminal phosphate of nucleoside triphosphates to nucleoside diphosphates. Beside their function to maintain the cellular GTP level (5Otero A.D.S. Biochem. Pharmacol. 1990; 39: 1399-1404Crossref PubMed Scopus (104) Google Scholar), several other functions and properties have been assigned to the NDPK family. These include the identity of nm23-H2 to PuF, a transcription factor of the proto-oncogene c-myc (6Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (483) Google Scholar), the ability of nm23-H1 to suppress the metastatic potential of several tumors and cell lines (1Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M. J. Natl. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1299) Google Scholar,7Leone A. Flatow U. King C.R. Sandeen M.A. Margulies I.M.K. Liotta L.A. Steeg P.S. Cell. 1991; 65: 25-35Abstract Full Text PDF PubMed Scopus (545) Google Scholar, 8Leone A. Flatow U. VanHoutte K. Steeg P.S. Oncogene. 1993; 8: 2325-2333PubMed Google Scholar), the association of NDPK with transcription regulating factors such as the retinoic acid receptor-related orphan receptor α and the retinoic Z receptor β, and with the heat shock protein hsc70 (9Paravicini G. Steinmayr M. André E. Becker-André M. Biochem. Biophys. Res. Commun. 1996; 227: 82-87Crossref PubMed Scopus (34) Google Scholar, 10Leung S.-M. Hightower E. J. Biol. Chem. 1997; 272: 2607-2614Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Recently, it was also reported that NDPK has protein kinase activity (11Wagner P.D. Vu N.-D. J. Biol. Chem. 1995; 270: 21758-21764Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 12Lu Q. Park H. Egger L.A. Inouye M. J. Biol. Chem. 1996; 271: 32886-32893Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The importance of nm23 expression in hematopoiesis has been demonstrated by Okabe-Kado et al. (13Okabe-Kado J. Kasukabe T. Honma Y. Hayashi M. Henzel W.J. Huzomi M. Biochem. Biophys. Res. Commun. 1992; 182: 987-994Crossref PubMed Scopus (111) Google Scholar), who identified NDPK B as a differentiation inhibiting factor (I-factor) in the conditioned medium of the differentiation-resistant mouse M1 cell line and demonstrated that both nm23-H1 and nm23-H2 can inhibit the in vitro induced differentiation of several hematopoietic cell lines. This inhibition was independent of the phosphotransferase activity as demonstrated with nm23 mutants lacking the enzymatic activity (13Okabe-Kado J. Kasukabe T. Honma Y. Hayashi M. Henzel W.J. Huzomi M. Biochem. Biophys. Res. Commun. 1992; 182: 987-994Crossref PubMed Scopus (111) Google Scholar, 14Okabe-Kado J. Kasukabe T. Hozumi M. Honma Y. Kimura N. Baba H. Urano T. Shiku H. FEBS Lett. 1995; 363: 311-315Crossref PubMed Scopus (68) Google Scholar, 15Okabe-Kado J. Kasukabe T. Baba H. Urano T. Shiku H. Honma Y. Biochim. Biophys. Acta. 1995; 1267: 101-106Crossref PubMed Scopus (42) Google Scholar). Moreover, reported data indicated a correlation between proliferation and nm23 expression in hematopoietic cells. In lymphoma and monocytic leukemia, overexpression of nm23-H1 was correlated with a higher tumor aggressiveness and resistance to chemotherapy, respectively (16Aryee D.N.T. Simonitsch I. Mosberger I. Kos K. Mann G. Schlo¨gl I. Po¨tschger U. Gadner H. Radaszkiewicz T. Kovar H. Br. J. Cancer. 1996; 74: 1693-1698Crossref PubMed Scopus (36) Google Scholar, 17Yokoyama A. Okabe-Kado J. Sakashita A. Maseki N. Kaneko Y. Hino K. Tomoyasu S. Tsuruoka N. Kasukabe T. Honma Y. Blood. 1996; 88: 3555-3561Crossref PubMed Google Scholar). Up-regulation of nm23-H1 and -H2 was observed in normal lymphocytes induced to proliferate with phytohemagglutinin (PHA) (18Keim D. Hailat N. Melhem R. Zhu X.X. Lascu I. Veron M. Strahler J. J. Clin. Invest. 1992; 89: 919-924Crossref PubMed Scopus (146) Google Scholar, 19Caligo M.A. Cipollini G. Fiore L. Calvo S. Basolo F. Collecchi P. Ciardiello F. Pepe S. Petrini M. Bevilacqua G. Int. J. Cancer. 1995; 60: 837-842Crossref PubMed Scopus (73) Google Scholar).In vitro, DR-nm23 mRNA is up-regulated in CD34+ hematopoietic progenitor cells during early stages of myeloid differentiation and is completely down-regulated upon terminal differentiation (3Venturelli D. Martinez R. Melotti P. Casella I. Peschle C. Cucco C. Spampinato G. Darzynkiewicz Z. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7435-7439Crossref PubMed Scopus (156) Google Scholar). Studies on the localization of nm23 mRNA in mouse embryos by “in situ” hybridization with nm23-H1 probes showed an overall staining and an increased expression in differentiating tissue (20Lakso M. Steeg P.S. Westphal H. Cell Growth & Differ. 1992; 3: 873-879PubMed Google Scholar). NDPK phosphotransferase activity has also been observed in the cytosol and membrane fraction of neutrophils (21Guignard F. Markert M. Biochem. J. 1996; 316: 233-238Crossref PubMed Scopus (10) Google Scholar). These studies support the hypothesis that nm23 is ubiquitously expressed in cells. Using enzymatic assays, surface-bound NDPK has been observed on platelets (22Adler J.R. Handin R.I. J. Biol. Chem. 1979; 254: 3866-3872Abstract Full Text PDF PubMed Google Scholar). In addition, several hematopoietic tumor cell lines stained positive for NDPK in flow cytometric analysis (23Urano T. Furukawa K. Shiku H. Oncogene. 1993; 8: 1371-1376PubMed Google Scholar). So far, the presence of NDPK in extracellular fluids has not been demonstrated. In this article we studied the nm23-H1 and nm23-H2 expression in cells of human hematopoietic origin and demonstrated that both isoforms are highly expressed in hematopoietic progenitors. A decrease in expression levels was observed during myeloid and lymphoid differentiation. Surface expression of nm23 was not detected on normal hematopoietic cells and was only observed on tumor cell lines of hematopoietic origin. We also provide evidence for the presence of NDPK in human plasma. Iscove's modified Dulbecco's medium (IMDM), phosphate-buffered saline (PBS), and Tris were obtained from Life Technologies, Inc. (Paisly, United Kingdom). Casein and paraformaldehyde were from Merck (Darmstadt, Germany). ATP, CDP, CHAPS, ethidium bromide, RNase A, and phytohemagglutinin-L (PHA-L) were obtained from Sigma (Bornem, Belgium). Benzidin was from Fluka (Bornem, Belgium). The fluorescein isothiocyanate (FITC)-labeled anti-nm23-H1 and anti-nm23-H2–2 antibodies were obtained from Seikagaku Corporation (Tokyo, Japan), the isotype control antibodies, mouse IgG2a-FITC and mouse IgG1-FITC, respectively, and the phycoerythrin-labeled anti-CD14 (LeuTM-M3) and anti-CD34 (anti-HPCA-2) antibodies were obtained from Becton Dickinson (Erembodegem, Belgium). The Hybond-C cellulose membranes and the enhanced chemoluminescence (ECL) kit were from Amersham (Slough, UK). Bovine serum albumin (BSA) was obtained from Merck (Darmstadt, Germany) and Sigma (Bornem, Belgium). Fetal calf serum (FCS, Myoclone Super Plus) was from Life Technologies, Inc. (Paisley, UK) and horse serum from Sera Lab (Crawley, W Sussex, UK). The hematopoietic cell lines K562, HL-60, U937, KG-1, Jurkat, Molt3, Molt4, SupT1, and HSB2 were obtained from ATCC (Rockville, MD) and cultured in IMDM containing 10% (v/v) FCS. Blood of healthy volunteers was collected in CTAD-tubes (Becton Dickinson, Erembodegem, Belgium), without using a tourniquet and placed on ice for at least 10 min in order to avoid platelet activation. Umbilical cord blood was collected from the placenta of healthy full-term infants. Bone marrow samples were aspirated by sternal puncture from hematologically normal patients undergoing cardiac surgery after informed consent. Umbilical cord blood and bone marrow samples were collected in tubes containing 2 ml of IMDM with 10% (v/v) FCS and 5 units/ml preservative-free heparin (Novo Industries, Bagsvaerd, Denmark). Subsequently, the samples were centrifuged at 400 × g for 15 min at 4 °C. The middle part of the plasma column was collected and centrifuged at 700 × g for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22-μm microfilter (Millipore, Molsheim, France). White blood cells were isolated from normal blood or bone marrow by a 1/100 dilution of the sample in hemolysis buffer (155 mm NH4Cl, 0.1 mm EDTA, 10 mm KHCO3, pH 7.3). After an incubation for 5–10 min at room temperature, cells were washed three times with PBS and labeled for flow cytometric analysis. Peripheral blood lymphocytes were isolated from normal blood by density gradient centrifugation on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and washed three times with IMDM. Purified lymphocytes were cultured in IMDM supplemented with 20% (v/v) FCS and 5 μg/ml PHA. Platelets were isolated from normal blood samples, collected in CTAD-tubes. The samples were centrifuged at 400 × gfor 15 min and the intermediate, platelet-containing fraction of the plasma collected. Platelets were washed three times with PBS containing 3.7 mm adenosine, 15 mm theophylline, and 0.198 mm dipyridamole and pelleted by a 10-min centrifugation at 700 × g (24Contant G. Gouault-Heilmann M. Martinoli J.L. Thromb. Res. 1983; 31: 365-374Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Erythrocytes were isolated by repeated (3 times) centrifugation of normal blood samples at 400 ×g for 15 min and removal of the buffy coat and supernatant. Cell lysates were obtained by four repetitive cycles of freeze-thawing and sonication for 5 min in the presence of 0.02% (w/v) CHAPS. Based on an exploratory screening, plasma samples and cell lysates were diluted with PBS containing 0.1% (w/v) casein to approximately equal NDPK phosphotransferase activities. The diluted samples were incubated at 37 °C in a buffer containing 50 mm Tris-HCl (pH 7.6), 100 mm NaCl, 10 mm MgSO4, 1 mm ATP, 2 mm CDP, and 0.1 mCi/ml [γ-32P]ATP (ICN Biochemicals Inc., Costa Mesa, CA). At relevant time intervals, samples were taken and the reaction was stopped by addition of an equal volume of 0.5 m EDTA. Radiolabeled ATP and CTP were separated by polyethyleneimine-cellulose thin layer chromatography in 750 mm KH2PO4 (pH 3.65). The spots were visualized by autoradiography, excised, and counted in a liquid scintillation counter. The protein content was measured by the biuret method in a Vitros 750XRC analyzer (Johnson & Johnson Clinical Diagnostics Inc., Rochester, NY). Free plasma hemoglobin was determined according to Crosby and Furth (25Crosby W.H. Furth F.W. Blood. 1956; 11: 380Crossref PubMed Google Scholar). In brief, 20 μl of plasma was added to a solution of 1% (w/v) benzidin in 90% (v/v) acetic acid. The reaction was initiated by addition of 1 ml of 1% (w/v) H2O2 and stopped after an incubation of 20 min at room temperature by addition of 10 ml of 10% (v/v) acetic acid. After 10 min, the optical density (OD) of the samples was measured at 595 nm. In this assay, 0.1% (w/v) hemoglobin has an OD of 1.5. Cells were washed three times with PBS. For the detection of membrane-bound NDPK, 5 × 105 nucleated cells or 107thrombocytes or erythrocytes, were suspended in a volume of 50–200 μl, blocked for aspecific staining with 10 μg of unlabeled mouse gamma globulins (Jackson ImmunoResearch Laboratories, West Grove, PA) for 10 min and incubated for an additional 15 min with 0.5 μg of antibody, washed with PBS, and analyzed on a FACScan flow cytometer (Becton Dickinson, Erembodegem, Belgium). For detection of intracellular NDPK, 5 × 105 cells were suspended in 50–200 μl, fixed, and permeabilized with Fix&Perm (An-der-Grub, Kaumberg, Austria) according to the manufacturer's instructions. The cells were labeled with 0.5 μg of antibody and washed once before analysis. FITC-labeled mouse IgG1 and IgG2awere used as isotype-matched controls for the anti-nm23-H2 and anti-nm23-H1 antibodies, respectively. All data were obtained with the same batches of antisera. Nm23 expression and DNA content were simultaneously measured according to the method of Clevenger et al. (26Clevenger C.V. Bauer K.D. Epstein A.L. Cytometry. 1985; 6: 208-214Crossref PubMed Scopus (117) Google Scholar). In brief, 4 × 106cells were fixed in 1 ml of 0.5% (w/v) paraformaldehyde for 15 min at 4 °C. After centrifugation at 400 × g for 5 min, the cells were resuspended in 1 ml of 0.1% (v/v) Triton X-100 (BDH Chemicals, Poole, UK) for 5 min at 4 °C. After centrifugation, the pellet was divided in four equal parts: one part was incubated with 0.5 μg of anti-nm23-H1-FITC antibody, one part with 0.5 μg anti-nm23-H2-FITC antibody, and the two remaining parts with 0.5 μg of the isotype-matched control antibodies, mouse-IgG2a-FITC, and mouse-IgG1-FITC, respectively. After an incubation for 1 h at 4 °C, all samples were washed with PBS and resuspended in 400 μl of PBS containing 10 μg/ml ethidium bromide and 0.1% (w/v) RNase A. After an incubation at room temperature for at least 20 min, cells were analyzed on a FACScan flow cytometer with standard optics except for the 515 ± 4.5 nm bandpass filter in front of the Fl-1 photomultiplier. Proteins in the lysates of 15 × 106 platelets or 106 erythrocytes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on a 20% (w/v) gel (27Anciaux K. Van Dommelen K. Willems R. Roymans D. Slegers H. FEBS Lett. 1997; 400: 75-79Crossref PubMed Scopus (22) Google Scholar). The gel was electroblotted onto a Hybond-C cellulose membrane and nm23 immunodetected with either anti-nm23-H1 (Novocastra, Newcastle, UK) or anti-NDPKα/nm23-H2 antibody (Dr. Kimura, Tokyo Metropolitan Institute of Gerontology, Japan). After an additional incubation with a horseradish peroxidase-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), visualization was performed by ECL. Comparisons of nm23 expression in different cell types was performed with a paired Student'st test using Statview 4.5 (Abacus Concepts, Berkely, CA).p Values smaller than 0.05 were considered as statistically significant. Regression analysis was performed in Excel 7.0 (Microsoft Corp., Redmond, WA). NDPK was measured in the physiological medium of hematopoietic cells. Platelet-free plasma was prepared from human peripheral blood, bone marrow, and umbilical cord blood and was assayed for NDPK phosphotransferase activity, protein content, and free plasma hemoglobin. NDPK assays were performed in conditions of linear kinetics. The activity in bone marrow plasma exceeded by far that in peripheral blood and umbilical cord blood plasma. All plasma samples contained approximately equal amounts of protein and a statistically significant correlation (p < 0.01) between NDPK activity and free hemoglobin was observed (Fig.1). However, peripheral blood samples containing very low levels of hemoglobin still contained NDPK activity. Extrapolation to zero hemoglobin revealed a basal NDPK phosphotransferase activity of 33 ± 5 milliunits/ml in peripheral blood plasma (p = 0.05). The NDPK activity was also quantified in several protein solutions routinely used in cell cultivation, i.e. FCS, horse serum, BSA, and casein. With the exception of casein, all assayed solutions contained NDPK activity (Table I).Table INDPK activity in plasma and culture mediaProtein solutionNDPK activityProtein contentNDPK activitymilliunits/mlmg/mlmilliunits/mg proteinPeripheral blood plasma1-aMean ± SD; n, number of samples.46 ± 862 ± 30.74 ± 0.15 (n = 10)Bone marrow plasma1-aMean ± SD; n, number of samples.1139 ± 54850 ± 323 ± 11 (n = 12)Umbilical cord blood plasma1-aMean ± SD; n, number of samples.58 ± 1238 ± 61.53 ± 0.24 (n = 9)Fetal calf serum1-bGibco BRL, catalog number 10081–164, lot 30A1263A.239386Horse serum1-cSera Lab, catalog number S-0004a, lot 104020.8562110% (w/v) BSA1-dSigma, catalog number A-4503, lot 66H0674.701000.710% (w/v) BSA1-eMerck, catalog number 12018, lot 231ZA1094118.31000.0310% (w/v) Casein1-fServa, catalog number 16260.Not detectable100Not detectable1-a Mean ± SD; n, number of samples.1-b Gibco BRL, catalog number 10081–164, lot 30A1263A.1-c Sera Lab, catalog number S-0004a, lot 104020.1-d Sigma, catalog number A-4503, lot 66H0674.1-e Merck, catalog number 12018, lot 231ZA1094118.1-f Serva, catalog number 16260. Open table in a new tab Intracellular nm23 in blood and bone marrow cells was monitored by flow cytometry after intracellular staining with FITC-conjugated anti-nm23-H1 and anti-nm23-H2 antibodies. In peripheral blood leukocytes, distinction between lymphocytes and granulocytes was made by the scatter profile of the cells. Monocytes were identified by labeling with a phycoerythrin-conjugated anti-CD14 antibody (Fig.2 A). In bone marrow, a distinction was made between mononuclear and myeloid precursors by their scatter profile. CD34+ progenitors were identified by labeling with a phycoerythrin-conjugated anti-CD34 antibody (Fig.2 B). All analyzed cells showed a positive intracellular immunofluorescence for nm23-H1 and nm23-H2. The results presented in Fig. 3 demonstrated that the expression of nm23 is high in bone marrow CD34+ cells and lower in more differentiated cell types. In addition, peripheral blood polymorphonuclear cells have a higher expression of nm23-H1 than lymphocytes, which in turn express more nm23-H2 than polymorphonuclear cells. Among the peripheral blood leukocytes, monocytes have the highest nm23 expression. Due to cell lysis of platelets and red blood cells during fixation and permeabilization, the intracellular nm23 expression of these cells could not be measured by flow cytometry. Therefore, the presence of NDPK was measured by enzymatic assays and Western blotting. In lysates of red blood cells and platelets, a NDPK phosphotransferase activity of 1.3 ± 0.6 nanounits/cell and 0.40 ± 0.08 nanounits/cell (mean ± S.E., n= 3) was measured, respectively. Both nm23-H1 and nm23-H2 were identified in these cells by Western blotting (data not shown).Figure 3Decrease in nm23 expression upon maturation of hematopoietic cells. Nm23 expression is determined from the mean fluorescence intensity (MFI) measured by flow cytometric analyis of cells, labeled with FITC-conjugated antibodies for intracellular nm23. The plotted MFI is the measured value substracted with the MFI of cells labeled with isotype-matched control antibodies. Nm23-H1 expression is indicated in the upper bar chart, nm23-H2 expression in the lower bar chart. Error bars represent the standard deviation. Note that nm23-H1 and nm23-H2 cannot be compared due to different affinities and fluorescence intensities of the used antibodies. Statistical analysis using the Student's t test revealed a significantly different expression of nm23-H1 and nm23-H2 in all cells, except when mononuclear precursors are compared with monocytes and granulocytes. pValues smaller than 0.05 were considered as statistically significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Nm23 was rapidly up-regulated in lymphocytes after induction of proliferation by PHA. Using single parameter flow cytometry on fixed and permeabilized lymphocytes, we observed a more than 10-fold increase in immunoreactivity of nm23-H1 and nm23-H2 72–96 h after PHA stimulation (Fig. 4). Cells were also simultaneously examined for nm23 and DNA content using a multiparameter flow cytometric approach. From the obtained data, we were able to correlate the nm23 content with the cell cycle phase. The results presented in Fig. 5, are representative of five independent experiments. The most important conclusions which can be drawn from these data are: (i) unstimulated lymphocytes in G0 phase have a basal level of nm23 expression; (ii) the onset of proliferation takes place between 24 and 48 h after stimulation; (iii) there is an up-regulation of nm23 expression after 24 h; (iv) cells that enter the S-phase have a nm23 content which exceeds that of unstimulated cells; (v) there is no further up-regulation of nm23 during the S-phase. The obtained results were similar for nm23-H1 and nm23-H2.Figure 5Cell cycle-dependent expression of nm23.Dot plots of multiparameter flow cytometric analysis of PHA-stimulated lymphocytes labeled for their DNA content (ethidium bromide fluorescence, FL2-A) and for nm23-H1 (upper panel) or nm23-H2 (middle panel) using FITC-conjugated antibodies. The expression level of nm23 in unstimulated lymphocytes is indicated by the horizontal lines. Lower panel, DNA histograms of lymphocytes after different periods of PHA treatment. Percentage of cells in the S or G2/M phase is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The surface expression of nm23-H1 and nm23-H2 on normal hematopoietic cells was investigated by flow cytometric analysis. In five independent experiments, we could not demonstrate nm23 on the surface of any normal hematopoietic cell,i.e. bone marrow CD34+ progenitors, mononuclear precursors (lymphoid and erythroid), myeloid precursors, lymphocytes, monocytes, granulocytes, and purified thrombocytes and erythrocytes. Apart from the latter two, cells were identified by flow cytometric analysis of the scatter profiles and the CD34 membrane marker (Fig. 2,C and D). PHA stimulation of lymphocyte proliferation did not induce surface expression of nm23. In contrast, of the hematopoietic cell lines examined for surface-bound nm23, the myeloid cell lines (K562, KG1, HL-60, and U937) showed a weak but reproducible positive staining for nm23-H1, whereas the lymphoid ones (HSB2, SupT1, Jurkat, Molt3, and Molt4) were negative for both isoforms. K562 also showed a comparable positive staining for nm23-H2. Four representative examples are shown in Fig.6. Flow cytometric and enzymatic analysis were used to study the NDPK/nm23 expression in several plasma types and in all cells of hematopoietic origin. This article is mainly focused on the nm23 isoforms nm23-H1 and nm23-H2. They have a molecular mass of 21 and 19 kDa, respectively, are encoded by separate genes, colocalized on chromosome 17q21.3, which are probably independently regulated (28Backer J.M. Mendola E. Kovesdi I. Fairhurst J.L. O'Hara B. Eddy Jr., R.L. Shows T.B. Mathew S. Murty V.V.V.S. Chaganti R.S.K. Oncogene. 1993; 8: 497-502PubMed Google Scholar,29Okada K. Urano T. Baba H. Furukawa K. Furukawa K. Shiku H. Oncogene. 1996; 13: 1937-1943PubMed Google Scholar). Recently, two new isoforms, DR-nm23 and nm23-H4, have been characterized and localized on chromosome 16q13 and 16p13, respectively (4Milon L. Rousseau-Merck M.-F. Munier A. Erent M. Lascu I. Capeau J. Lacombe M.L. Hum. Genet. 1997; 99: 550-557Crossref PubMed Scopus (112) Google Scholar, 30Martinez R. Venturelli D. Perrotti D. Veronese M.L. Kastury K. Druck T. Huebner K. Calabretta B. Cancer Res. 1997; 57: 1180-1187PubMed Google Scholar). An interesting extracellular property of nm23-H1 and nm23-H2 is their ability to inhibit the induced differentiation of hematopoietic cell lines, independently of their enzymatic activity (13Okabe-Kado J. Kasukabe T. Honma Y. Hayashi M. Henzel W.J. Huzomi M. Biochem. Biophys. Res. Commun. 1992; 182: 987-994Crossref PubMed Scopus (111) Google Scholar, 14Okabe-Kado J. Kasukabe T. Hozumi M. Honma Y. Kimura N. Baba H. Urano T. Shiku H. FEBS Lett. 1995; 363: 311-315Crossref PubMed Scopus (68) Google Scholar, 15Okabe-Kado J. Kasukabe T. Baba H. Urano T. Shiku H. Honma Y. Biochim. Biophys. Acta. 1995; 1267: 101-106Crossref PubMed Scopus (42) Google Scholar). In this study we demonstrated the presence of NDPK in plasma from peripheral blood, umbilical cord blood, and bone marrow. The highest activity was measured in bone marrow plasma. However, the observed correlation between the hemoglobin content and the NDPK phosphotransferase activity in the plasma samples indicated that the presence of nm23 in plasma is probably due to lysis of red blood cells. In the latter cells, a relatively high level of NDPK was detected. The high activity observed in bone marrow plasma is probably due to the harsher treatment of cells during bone marrow sampling. However, even in vivo, a limited lysis of senescent red blood cells occurs and causes release of hemoglobin and NDPK in the plasma. In addition, a basal level of 33 ± 5 milliunits/ml was estimated in human peripheral blood plasma. These data suggest that in vivo, NDPK is present in plasma, and may have a physiological significance. We also demonstrated the presence of NDPK in several protein solutions, often used in cell cultivation. These findings emphasize that studies on the effect of extracellular nucleotides or on the differentiation inhibitory effect of exogenous NPDK should be performed in serum-free conditions, and with NDPK-free serum substitutes. Previously, extracellular NDPK has been reported on the cell surface of several cell lines (23Urano T. Furukawa K. Shiku H. Oncogene. 1993; 8: 1371-1376PubMed Google Scholar). These results were confirmed in our study, although the observed cell surface immunoreactivity was always small and close to the flow cytometric detection limit (±1000 molecules/cell). In contrast, the presence of ecto-NDPK on platelets (22Adler J.R. Handin R.I. J. Biol. Chem. 1979; 254: 3866-3872Abstract Full Text PDF PubMed Google Scholar) was not confirmed in our study. Moreover, we could not detect any nm23 surface expression on normal hematopoietic cells. Apparently, malignant cells seem to express more surface NDPK than normal cells. Probably this expression is not due to the proliferating state of the cell, since PHA-stimulated lymphocytes or CD34+ progenitors did not express detectable surface-bound NDPK. In addition, nm23 surface expression seems to occur on tumor cells in a lineage specific way, since we detected this expression on all myeloid cell lines and could not demonstrate any on lymphoid cells. Whether nm23 surface expression can be used as a marker for certain hematopoietic malignancies remains to be determined. Our data demonstrated the presence of NDPK in all investigated cells of hematopoietic origin, indicating the constitutive expression of nm23-H1 and nm23-H2 in these cells in agreement with the hypothesis that nm23 is ubiquitously expressed in all tissues (20Lakso M. Steeg P.S. Westphal H. Cell Growth & Differ. 1992; 3: 873-879PubMed Google Scholar). However, the main finding of our study is the inverse correlation between nm23 expression and hematopoietic maturation. In fact, hematopoietic CD34+progenitors express high levels of both nm23-H1 and -H2, and their expression decreases in more differentiated cell types. Previously, high levels of DR-nm23 mRNA have also been reported in CD34+ progenitors (3Venturelli D. Martinez R. Melotti P. Casella I. Peschle C. Cucco C. Spampinato G. Darzynkiewicz Z. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7435-7439Crossref PubMed Scopus (156) Google Scholar). Taken together, these data point to an important role for nm23 in the early stages of hematopoiesis. The CD34+ compartment represents a population of proliferation inducible and early proliferating cells, therefore the expression of NDPK might reflect the proliferative potential of hematopoietic cells. The data generated with PHA-stimulated lymphocytes also supported this hypothesis and demonstrated that during the G0/G1 transition, peripheral blood lymphocytes up-regulate nm23-H1 and nm23-H2 approximately 10-fold, a level which is maintained during further cell cycle progression. Although our findings indicate a correlation between nm23 expression and the proliferation of nucleated hematopoietic cells, the specific function of nm23 in this process remains unknown. Several non-enzymatic functions have been proposed for NDPK. Nm23-H2 has been identified as PuF, a transcription factor which positively regulates c-mycexpression in Burkitt's lymphoma (6Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (483) Google Scholar, 31Ji L. Arcinas M. Boxer L.M. J. Biol. Chem. 1995; 270: 13392-13398Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This proto-oncogene is well known to be overexpressed in proliferating cells (32Marcu K.B. Bossone S.A. Patel A.J. Annu. Rev. Biochem. 1992; 61: 809-860Crossref PubMed Google Scholar), and in vitro and in vivo terminal differentiation of hematopoietic cells is accompanied by down-regulation of c-myc expression (33Liebermann D.A. Hoffman B. Stem Cells. 1994; 12: 352-369Crossref PubMed Scopus (62) Google Scholar, 34Nguyen H.Q. Selvakumaran M. Liebermann D.A. Hoffman B. Oncogene. 1995; 11: 2439-2444PubMed Google Scholar). Therefore, the high nm23 expression in proliferating hematopoietic cells could be responsible for the constitutive expression of c-myc. However, in contrast to nm23, c-myc is not ubiquitously expressed in all cells. This apparent contradiction indicates that the transcriptional activity of nm23 must be strictly regulated. Regulatory mechanisms may include their subcellular localization, post-translational modifications, or changes in the oligomeric structure (35MacDonald N.J. De La Rosa A. Benedict M. Freije J.M.P. Krutsch H. Steeg P.S. J. Biol. Chem. 1993; 268: 25780-25789Abstract Full Text PDF PubMed Google Scholar, 36Gilles A.-M. Presecan E. Vonica A. Lascu I. J. Biol. Chem. 1991; 266: 8784-8789Abstract Full Text PDF PubMed Google Scholar, 37Mesnildrey S. Agou F. Karlsson A. Bonne D.D. Véron M. J. Biol. Chem. 1998; 273: 4436-4442Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Another important hypothesis concerning the level of nm23 expression is the necessity for cells of guanosine 5′-triphosphate (GTP) or other triphosphate nucleotides. In the promyelocytic leukemia cell line HL-60, a down-regulation of the intracellular GTP level after vitamin D3-induced granulocyte differentiation was demonstrated (38Pilz R.B. Huvar I. Scheele J.S. Van den Berghe G. Boss G.R. Cell Growth & Differ. 1997; 8: 53-59PubMed Google Scholar). In this cell line, the down-regulation of nm23 expression upon granulocyte differentiation has also been reported (39Caligo M.A. Cipollini G. Petrini M. Valentini P. Bevilacqua G. Leukemia Res. 1996; 20: 161-167Crossref PubMed Scopus (38) Google Scholar). Similarly, the high expression of nm23 in normal proliferating hematopoietic cells may reflect their necessity for a high intracellular GTP level. As proposed previously, NDPK may be involved in providing GTP for the activation of G-proteins, for the activation of the translation apparatus or for the polymerization of microtubuli (5Otero A.D.S. Biochem. Pharmacol. 1990; 39: 1399-1404Crossref PubMed Scopus (104) Google Scholar, 40Sonnemann J. Mutzel R. Biochem. Biophys. Res. Commun. 1995; 209: 490-496Crossref PubMed Scopus (25) Google Scholar). As induction of proliferation requires a reorganization of the cytoskeleton, it is not unlikely that the nm23 up-regulation is correlated with the latter process. Research is in progress to elucidate the function of NDPK in the G0/G1 transition upon induction of proliferation in lymphocytes. We thank the Laboratory of Biochemistry of the Antwerp University Hospital (UZA) for the determination of the protein content of the plasma samples. Dr. N. Kimura (Tokyo Metropolitan Institute of Gerontology, Japan) is acknowledged for the anti-rat NDPK α and β antibodies and Dr. I. Lascu (University of Bordeaux, France) for the gift of recombinant human nm23. We also thank Dr. M. Véron (Institut Pasteur, Paris, France) for the polyclonal anti-NDPK antibody (CRS-B).
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