a novel human myeloid leukemia cell line, nkm-1 ...growth factors, leukemic cells proliferate in...

(CANCER RESEARCH 50. 7703-7709. December 1. I990|

A Novel Human Myeloid Leukemia Cell Line, NKM-1, Coexpressing Granulocyte

Colony-stimulating Factor Receptors and Macrophage Colony-stimulatingFactor Receptors'

Takae Kataoka,2 Yoshihisa Morishita, Michinori Ogura, Yasuo Morishima, Masayuki Towatari, Yukio Kalo,

Hideo Inoue, and Hidehiko SaitoThe First Department of Internal Medicine, Nagoya University School of Medicine fT. K., Y. M., Y. M., M. T., H. SJ. Showa-ku, Nagoya 466; Department ofllemalologyand Chemotherapy, Aichi Cancer Center Hospital [M. O.], Chikusa-ku. Nagoya 464; Department of Internal Medicine, Showa Hospital [Y. A'./. Konan 483; and Kirin

Brewery Co. [H. I./, Shihuya-ku, Tokyo Â¡50,Japan

ABSTRACT

A novel human myeloid leukemia cell line, NKM-1, was establishedfrom a patient with acute myeloid leukemia (FAB classification \12).The cells were positive for myeloperoxidase staining and cluster ofdifferentiation 15 cell surface antigen. Radiolabeled recombinant humangranulocyte (G) colony-stimulating factor (CSF) was used, and 60 specificbinding sites/cell with a Kd 100 pmol/liter were demonstrated on the cellsurface. '"I-G-CSF binding was not inhibited by interleukin-3, granulo-cyte-macrophage CSF, or macrophage (M) CSF. NKM-1 cells alsoexpressed M-CSF" receptors detected by c-fms mRNA expression. In

concordance with the receptor expression, NKM-1 cells proliferated inresponse to exogenous G-CSF or M-CSF in a dose-dependent manner(0.1-100 ng/ml), while interleukin-3 or granulocyte-macrophage CSFhad no effect. Colony-forming capacity of NKM-1 cells in semisolid agarwas also enhanced with the addition of 10 ng/ml of G-CSF or M-CSFbut decreased at higher concentrations. During CSF stimulation, noremarkable changes were observed morphologically and phenotypically.The stimulatory effect of G-CSF" and M-CSF on the cell growth was

additive. Neither G-CSF-binding capacity nor c-fms mRNA expressionwas altered by pretreatment with M-CSF or G-CSF, respectively. Thiscell line may provide a useful in vitro model for the study of CSF rolesin myeloid leukemia cell proliferation.

INTRODUCTION

AML' is characterized by excessive proliferation of myeloid

precursors and maturation arrest resulting in accumulation ofimmature hematopoietic cells. In the presence of appropriategrowth factors, leukemic cells proliferate in suspension cultureor form colonies in semisolid media. Recent studies describingthe effects of recombinant human hematopoietic growth factorson AML blasts have revealed that cells from most patientsproliferate in response to IL-3 (1-4), GM-CSF (1-9), G-CSF(1-5, 9, 10), or M-CSF (4, 11). The ability of a growth factorto exert its biological effect is linked to the expression ofreceptors specific for that factor on the surface of the cells,although there is no direct correlation between the ability to

Received 5/29/90; accepted 8/31/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science and Culture: by a Grant-in-Aid from theMinistry of Health and Welfare: and by a Grant-in-Aid from the Aichi BloodDisease Research Juridical Foundation in Japan.

2To whom requests for reprints should be addressed, at First Department ofInternal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho.Showa-ku, Nagoya 466. Japan.

3The abbreviations used are: AML, acute myeloid leukemia; IL-3, interleukin-3; CSF, colony-stimulating factor: GM-CSF, granulocyte-macrophage C'SF: G-CSF, granulocyte CSF; M-CSF. macrophage CSF: FCS, fetal calf serum; MGG,May-Grunwald-Giemsa: MPO, myeloperoxidase; CAE, naphlhol AS-Dchloroac-etate esterase; NBE, a-naphthyl butyrate esterase: CD, cluster of differentialon;NBT. nitroblue tetrazolium; TPA, 12-O-tetradecanoylphorbol-13-acetate; SB,sodium butyrate; MTT, 3-(4,5-dimethylthia/ol-2-yl)-2,5-diphenyltetra7.oliuni bromide; Kd. dissociation constant.

respond to a CSF and the absolute number of receptors expressed for that factor (12, 13). This lack of correlation may bedue to the heterogeneity of the leukemic cell population in apatient or to the fact that receptor levels are measured on wholeleukemic cell populations, which may not accurately reflectreceptor levels for each CSF on relatively rare clonogenic cells.Therefore, it would be advantageous to use a homogeneous cellpopulation when we study how CSFs regulate proliferation ofmyeloid leukemic cells.

Permanent cell lines of myeloid leukemia cells are consideredto be the clonal expansion of hematopoietic stem cells at somestage of maturation. Among established myeloid cell lines,however, proliferative responses to exogenous CSFs were rarelyobserved. This may be accounted for by the rarity of theexpression of CSF receptors on cell lines (13) or by the diversesignal transductions through the binding to its receptors. Herewe present a novel human myeloid leukemia cell line, NKM-1,which expresses both G-CSF receptors and M-CSF receptors.The cells proliferate remarkably in response to exogenous G-CSF and M-CSF.

MATERIALS AND METHODS

Case History. The cell line described in this report was derived froma peripheral blood sample of a 33-year-old male with AML. On October19, 1981, he was admitted to Daido Hospital (Nagoya, Japan) becauseof gingival bleeding and epigastralgia. Physical examination revealedmild splenomegaly and numerous ecchymotic spots. Peripheral bloodcell counts at the time of admission were as follows: RBCs, 3.65 xlO'V'iter; platelets, 17 x lO'/lUer; and WBCs, 108 x 10'/liter with

74% leukemic blasts. The number of iliac bone marrow nucleated cellswas 450 x 10g/liter with 88.4% blasts. Both prothrombin time and

partial thromboplastin time were markedly prolonged, the plasmafibrinogen level was 95 mg/dl, and the serum fibrinogen degenerationproducts value was 80 pg/ml. AML [FAB classification M2 (14)] withdisseminated intravascular coagulation was diagnosed. On the secondhospital day, he suddenly went into a coma and computed tomographyscanning showed a massive cerebroventricular hemorrhage. He died thesame day.

Cell Culture. On October 19, 1981, a heparinized peripheral bloodsample was obtained with the patient's consent and brought to the First

Department of Internal Medicine. Nagoya University. The mononu-clear cells were separated by Ficoll-Conray gradient centrifugaron andseeded in Falcon 3013 plastic tissue culture flasks (Falcon, Division ofBecton Dickinson, Oxnard, CA) at 10" cells/ml in RPMI 1640 medium

(GIBCO Laboratories, Grand Island, NY) supplemented with 20%FCS (Flow Laboratories, Stanmore, New South Wales, Australia),aqueous penicillin G (100 units/ml), and streptomycin (50 ^g/ml) andwere incubated at 37Â°Cwith a humidified atmosphere of 5% CO2.

Cultures were fed once weekly by partial replacement of spent mediumwith the fresh medium. No conditioned media or feeder cells were used.

Morphological Studies. The cells were stained with MGG, MPO,and dual esterase staining for CAE and NBE according to the standard

7703

Research. on February 23, 2021. © 1990 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

G-CSF-, M-CSF- RESPONSIVE CELL LINE

procedures. Ultrastructural morphology (conventional thin electronmicroscopy) and MPO were analyzed as described previously (15).

Cell Surface Markers. Cell surface antigens were determined by anindirect immunofluorescent method using monoclonal antibodies andanalyzed by flow cytometry (EPICS PROFILE, Coulter Electronics,Inc., Hialeah, FL) as described previously (15). The following monoclonal antibodies were used for the analysis. CD numbers were used fromthe designation of the Fourth International Workshop and Conferenceon Human Leukocyte Differentiation Antigens. 9.6 (CD2), 64.1 (CD3),10.2 (CDS) and 7.2 (HLA-DR) were provided by Dr. J. A. Hansen(Fred Hutchinson Cancer Research Center, Seattle, WA). ICIO (CD15)was the generous gift of Dr. I. Burnstein, Fred Hutchinson CancerResearch Center. 4A (16) (CD7), NL-1 (17) (CD10), and HPL3 (18)(CD41) were produced locally. B4 (CD 19), Bl (CD20), MOI (CD1 Ib),MY7 (CD13), MY4 (CD14), and MY9 (CD33) were purchased fromCoulter Co. Glycophorin A (erythrocytes) (Immunotech, Marseille,France), Leu 1Ib (CD16) (Becton Dickinson Co.), anti-CD18 (CD18),and anti-low affinity Fey receptor (CDw32) (Cosmo Bio Co., Tokyo,Japan) were also used.

Chromosome Analysis. The cultured cells were incubated in thepresence of Colcemid (GIBCO Laboratories) at 0.05 Mg/ml, for l h at37'C, then treated with 75 mmol/liter KC1 hypotonie solution for 30min at 37Â°C,and fixed by methanol:acetic acid (3:1). Chromosomes

were banded by the trypsin-Giemsa methods. Karyotypes were constructed from photographic enlargements according to the Paris Conference classification scheme.

NBT Reduction Test and Phagocytosis. The NBT (Sigma ChemicalCo., St. Louis, MO) reduction test was performed according to themethod of Park et al. (19). Phagocytosis was tested using immunobeads(Fluoresbrite Plain Microspheres; Polysciences Inc., Warrington, PA)or opsonized zymosan (Sigma Chemical Co.) (15).

Biological Reagents. Recombinant human G-CSF (KRN8601) wasobtained at Kirin Brewery Co. (Shibuya-ku, Tokyo, Japan) by transforming Escherichia coli with cloned G-CSF complementary DNA (20).Recombinant human M-CSF (21) was obtained from Morinaga MilkIndustry Co. (Minato-ku, Tokyo, Japan). Recombinant human GM-CSF (BI 71.018) (22) was provided by Hoechst Japan (Minato-ku,Tokyo, Japan) and recombinant human 1L-3 (23) was purchased fromGenzyme Corp. (Boston, MA). TPA (Paesel GmbH and Co., Frankfurt,West Germany), sodium butyrate (Wako Pure Chemical Co., Tokyo,Japan), dimethyl sulfoxide (Katayama Chemical Co., Tokyo, Japan),and retinoic acid (Sigma Chemical Co.) were used for in vitro stimulation.

Proliferate Response to CSFs. Cells (3 x 10*)were cultured in flat-bottomed 96-microwell plates (Nunc, Poskilde, Denmark) in serum-free medium (COSMEDIUM-001; Cosmo Bio Co.) which contains 5nK/ml insulin, 1.5 ng/ml sodium selenite, and 5 Mg/ml human transferrin with or without 0.1, 1, 10, and 100 ng/ml CSFs. The viable cellswere numerated by a trypan blue exclusion method or quantitated by acolorimetrie assay using tetrazolium salt, MTT, as described previously(24). Because the results with the MTT assay were consistently equivalent with the viable cell counts, data in this article represent resultsusing the MTT assay. The plates were read on a scanning multiwellspectrophotometer (SLT 210; SLT-LAB Instruments, Salzburg, Austria) using a test wavelength of 545 nm and a reference wavelength of650 nm. The results were expressed as absorbance.

Clonogenic Assay. Cells (5 x IO3)were plated in 96-microwell plates

(Nunc)in 100 Â¿Â«1of RPMI 1640 supplemented with 0.3% agar and 10%PCS, with or without various concentrations of G-CSF or M-CSF.After 7 days of incubation at 37"C in a humidified atmosphere of 5%

CO2 in air, colonies consisting of >20 cells were counted under aninverted microscope. The cultures were dried onto glass slides and thenexamined microscopically after staining with MGG and MPO. Forevaluating clonogenic cell recovery of NKM-1 cells after the exposureto G-CSF or M-CSF in suspension cultures, NKM-1 cells at a concentration of 3 x 105/ml were cultivated in COSMEDIUM-001 with orwithout G-CSF or M-CSF in 3047 multiwell tissue culture plates(Falcon) for the indicated days. After the cells were washed, colony-forming capacity was measured in the absence of CSF as described

above. The recovery of clonogenic cells/2 ml in suspension was calculated by multiplying the plating efficiency in agar by the number ofcells harvested from the suspension culture (25).

Cell Cycle Analysis. DNA histogram analysis with propidium iodidewas performed (26) by staining 1 x IO6leukemic blasts/sample with 50

Mg/ml propidium iodide (Sigma Chemical Co.) in 0.1 % sodium citratecontaining 0.2% Nonidet P-40 (Particle Data Inc., Elmhurst, IL) and250 ^g/ml RNase (Boehringer-Mannheim, Mannheim, West Germany)for 30 min at 0Â°Cand incubated for another 15 min at 37"C. The nuclei

were analyzed by flow cytometry (EPICS PROFILE). The percentageof the cells having S and ( ;.. + M content of DNA was estimated by aprogram developed by Ortho Diagnostics.

Tritiated Thymidine Incorporation. Cells (6 x 10') were cultured in200 Â»ilof COSMEDIUM-001 with or without G-CSF in 96-microtiterplates (Nunc). After 20 h of incubation at 37Â°C,7.4 kilobecquerel (kBq)tritiated thymidine (2.48 x 10* kBq; NEN Research Products, Boston,

MA) was added, and the cells were harvested 4 h later using a Titertekharvester 550 (Flow Laboratories) (4). Radioactivity was determinedwith a Beckmann II scintillation counter (Becton Instruments Inc.,Fullerton, CA).

'"I-G-CSF-Binding Analysis. The radiolabeling of G-CSF was greatly

enhanced by the presence of tyrosine residues at the NH2 terminus andthe specific activity was not altered by this engineering. Cells (5 x IO6)were incubated with various concentrations of '"I-labeled 'Tyr-3Tyr-recombinant human G-CSF for 3 h at room temperature in RPMI1640 containing 10% PCS and 20 HIM 4-(2-hydroxyethyl)-l-pipera-zineethanesulfonic acid (Sigma). Cells were spun over a 200-/J FCScushion in a 400-/J centrifuge tube. The bottom of the tube containing

the cell pellet was cut and counted (27). Nonspecific binding wasestimated in incubations with a 100-fold excess of unlabeled G-CSF.Effects of other CSFs on G-CSF binding to NKM-1 cells were testedby incubating cells with a fixed concentration of '"I-G-CSF in thepresence of unlabeled CSFs. In order to analyze the effect of M-CSFon G-CSF receptor expression, the radioligand assay was performedon NKM-1 cells pretreated with 2 ng/ml M-CSF for 20 h.

Northern Blot Hybridization. Total cellular RNA was extracted byguanidine isocyanate method (28). Ten m>of RNA was electrophoresedin 1% agarose/formaldehyde gel and transferred onto nitrocellulosemembranes (Nitroplus 2000; Micron Separation Inc., Westbord, MA).Hybridization was carried out at 42Â°Cusing "P-labeled probes. The

membrane was washed 3 times in 2x standard saline citrate and 0.1%sodium dodecyl sulfate for 30 min and in 0.2x standard saline citrateand 0.1 % sodium dodecyl sulfate for 30 min at 42Â°C.The probes used

in this study were a 1.4-kilobase human v-fms, pSM3 (provided by theJapanese Cancer Research Resources Bank with the developer's con

sent), and a 1.4-kilobase HLA-B7 probe, pDPOOl (a generous gift ofDr. Lloyd J. Old, Memorial Sloan-Kettering Cancer Center, NY).

Statistics. Data were analyzed by Student's t test.

RESULTS

Establishment of NKM-1. The cells began to proliferate after2 weeks of culture and consistently proliferated in single suspension without forming cell clumps or adhesion to the flask.At present, the cells have been in culture in RPMI 1640 mediumsupplemented with 10% heat-inactivated FCS with a doublingtime of 36-48 hours. This cell line was designated NKM-1.Cultures are free from Epstein-Barr virus and Mycoplasmacontamination. NKM-1 cells are able to proliferate in serum-free growth media, COSMEDIUM-001, for at least 5 monthswithout changing cell morphology or cell surface phenotypes.

Characterization of Fresh Leukemia Cells and NKM-1 LineCells. Fresh leukemic cells in the patient's bone marrow ranged

from 20-30 nm in diameter and had round or oval nuclei withfine chromatin structure and a few nucleoli. Cytoplasm wasbasophilic and contained many azurophilic granules and a fewvacuoles. No Auer rods were observed. Cytochemical stainings

7704



G-CSF-. M-CSF- RESPONSIVE CELL LINE

showed that almost all blasts were MPO, CAE, and NBEpositive. NKM-1 cells were mostly round to ovoid with almostthe same modal diameter and had round to ovoid nuclei withfine chromatin and one or more prominent nucleoli. Cytoplasmwas slightly basophilic and contained many vacuoles and azur-ophilic granules (Fig. 1). MPO was strongly positive in almostall the cells. About 20% of the cells were positive for CAE butnegative for NBE. Ultrastructural morphological study showedthat NKM-1 cells had one round to ovoid nucleus with a smoothto slightly irregular outline, moderately condensed chromatin,and one or more large nucleoli. The cytoplasm contained numerous mitochondria, moderately long segments of rough en-

doplasmic reticulum, a small Golgi apparatus, and granules(Fig. 2). MPO positivity was noted in granules. Cell surfaceantigens on the peripheral blood cells of the patient and NKM-1 cells are summarized in Table 1. Both cells were positive forCD 15, CD 18, and HLA-DR. CD 14, CDw32, and CD33 werepositive on some of the patient's cells but weak on NKM-1

cells. T-cell differentiation antigens such as CD2, CD3, CDS,and CD7, B-cell-related antigens such as CD 10, CD 19, andCD20, antigens for platelets (CD41), and erythrocytes (glyco-phorin A) were not demonstrated. Chromosomal analysis of 13NKM-1 cells showed that the karyotype of the modal numberwas 47XY,-2,+der(2)t(2;?)(q37;?),-6,+der(6)t(6;?)(p23;?),+8.NBT reduction and phagocytic activities were not detected.

Effects of Chemical Agents. To test whether chemical induc-ers could initiate differentiation, NKM-1 cells were incubatedfor 3-7 days with 100 nM TPA, 1 ^M retinoic acid, 0.3 mMsodium butyrate, or 1% dimethyl sulfoxide. None of the reagents but TPA had effects of differentiation on NKM-1 cellsby morphological and cytochemical studies and cell surface

antigen analysis (data not shown). During the 3-day exposureto 100 nM TPA, cells displayed macrophage-like appearancewith decreased staining for CAE.

Response to CSFs in Suspension Culture. The capacity of therecombinant human G-CSF, M-CSF, GM-CSF, and IL-3 tostimulate NKM-1 cells was titrated with cells cultured in COS-MEDIUM-001. Viable cells were measured after 1-5 days ofcultures using the MTT assay. The cells were in the growthphase at days 1-5. Fig. 3 illustrates growth of CSF-treated cellscompared with control cultures after 3 days of cultures. NKM-1 cells showed prominent proliferation in response to G-CSFand M-CSF in a dose-related manner. Neither GM-CSF norIL-3 affected the cell growth. The cells also proliferated withG-CSF or M-CSF in a dose-dependent manner in RPMI 1640with 10% PCS, although the proliferation was less apparent;after 3 days of cultures with 100 ng/ml of G-CSF, the cellsincreased approximately 1.3-fold in absorbance compared withcontrol cultures. The ability of G-CSF and M-CSF to inducecell differentiation was tested. Table 1 presents a phenotypicanalysis of NKM-1 cells after stimulation with 10 ng/ml G-CSF for 3 days. Although cells positive for CD 18 and HLA-DR diminished in number, few changes were observed in morphological appearance, NBT reduction, or phagocytic capacity.Identical results were obtained using cells stimulated for 7 days.

Effects of CSFs on Colony Formation. Fig. 4 shows the effectsof G-CSF and M-CSF on primary colonies of NKM-1 cells inagar. Without addition of CSFs, 31 Â±1 colonies/5 x IO3cells

were observed. Morphologically, the colonies were composedof myeloblasts. A greater number of colonies were formed inthe presence of G-CSF or M-CSF. The colony sizes also increased with CSFs. The effects of CSFs peaked at 10 ng/ml but

Fig. 1. Smear of NKM-1 cells stained withMGG. Cyloplasmic granules and vacuoles aredemonstrated. Original magnification, x 2000.

7705




Fig. 2. Electron microscopic photograph of NKM-I cells. A round to ovoidnucleus and mitochondria, rough endoplasmic rcticulum. and granules are noted.Original magnification, x 3200; bar, 2 ttm.

Table 1 Cell surface antigens on NKM-I cells

Cells were stained by an indirect immunofluoresccnt method and analyzedwith flow cytometer. The negative control showed 5C<of positive background.Furthermore, after incubation in COSMEDIUM-001 with or without 10 ng/mlof G-CSF of M-CSF for 3 days, cell surface antigens were examined.

Positive cells (%)

NKM-1

After 3-day incubation

CDCDlIbCD

13CD14CD

15CD16CD18CDw32CD33HLA-DRPBÂ°14.03.324.388.02.466.938.618.459.4TimeO6.20.64.599.22.458.611.96.922.9NoCSF0.81.96.694.82.120.48.36.132.6G-CSF0.31.95.992.71.15.37.51.37.1M-CSF7.32.23.883.01.53.87.37.83.3

' PB. peripheral blood mononuelear cells from the patient at time of diagnosis.

1.0-1

Control G-CSF M-CSF

0.1 ng/m1

1 ng/ml

10 ng/ml

fxl 100 ng/m!

IL-3GM-CSFFig. 3. Effects of CSFs on cell growth of N KM-1. Cells (3 x IO4)were cultured

in COSMEDIUM-001 with or without CSFs in 96-well microtiter plates. After3 days of culture, viable cells were measured using the MTT assay. Columns,means of triplicate cultures; bars, Â±SD.The absorbance was increased with G-CSF or M-CSF (with 1 ng/ml of G-CSF. P < 0.05: with 10 or 100 ng/ml of G-CSF, P < 0.01; with 1. 10, or 100 ng/ml of M-CSF. P < 0.01; compared withthe control).

declined at higher concentrations. The recovery of clonogeniccells in suspension culture was also measured using cells whichhad been incubated with G-CSF or M-CSF for 2-4 days (Table2). The clonogenic cell recovery on day 2 was well correlatedwith the dose of CSFs in suspension cultures. Conversely, thoseon days 3 and 4 were decreased at 10 or 100 ng/ml of CSFs(data on day 4 not shown).

601 A

:

01 5 10 20 40 60 80 100

G-CSF ( ng ml )

I

3

0 1 5 10 20 40 60 80 100M-CSF ( ng ml )

Fig. 4. Effects of CSFs on NKM-I colony growth. Cells were cultivated for 7days in scmisolid agar containing G-CSF (A) or M-CSF (B). Columns, means ofcolonies/5 x 10' cells in triplicate cultures; han, Â±SD.The colony number wasincreased with CSFs (with I or 5 ng/ml of G-CSF, P< 0.05; with 10 ng/ml ofG-CSF, P < 0.01 ; with 1, 5, 10, or 20 ng/ml of M-CSF, P < 0.01 ; compared withthe number without CSFs).

Table 2 Effects of G-CSF anil M-Ã•SI-' on xrowlh of clonogenic cells in suspension

NKM-I cells at a concentration of 3 x lO'/ml were cultivated in COSMEDIUM-001 with or without G-CSF or M-CSF. The recovery of clonogenic cells/2 ml in suspension was calculated by multiplying the plating efficiency in agar bythe number of cells harvested from the suspension culture.

No. of clonogenic cell recovery(xlO3)CSF

concentration (ng/

ml)0

1Id

100Day

2G-CSF8.9

10.4 Â±1.513.6 Â±2.5'18.2 Â±3.0"M-CSFÂ±0.7Â°

14.1 Â±0.4*I6.5Â± 1.6*20.1 Â±2.0*Day

3G-CSFM

CSF20.8

Â±4.926.5 Â±2.9 25.3 Â±5.5I3.6Â± 2.3 I9.3Â± 1.910.7 Â±1.3' 19.3 Â±1.6

" Mean Â±SD of three experiments.* Significantly different from the number without CSFs on the same day (P <

0.01).' Significantly different from the number without C'SFs on the same day (P <

0.05).

Effects of CSFs on Cell Cycle and |'H]Thymidine Uptake. Incontrol cultures in COSMEDIUM-001, the percentages of cellsentering the S/G2-M phase of the cell cycle were 24.5/5.5 attime 0 and 25.2/7.7 after 24 h without G-CSF. In contrast,37.1/7.0% and 37.5/8.6% of cells were in S/G2-M phase after24-h stimulation with 1 ng/ml and 10 ng/ml of G-CSF, respectively. This effect of G-CSF was also demonstrated by ['H]thymidine incorporation. The uptake was increased after 24-hexposure to G-CSF, and the increase was detected in cellsstimulated with as little as 0.1 ng/ml G-CSF (Fig. 5).

Interaction of G-CSF and M-CSF on Growth of NKM-1 Cells.Because both G-CSF and M-CSF actively influenced cellgrowth of NKM-1, proliferative potential was measured withthe combination of two factors. In a dose range from 0.1-10ng/ml of M-CSF, the additive increase with G-CSF was evidentin suspension cultures (Fig. 6). The additive effects were alsoobserved in colony sizes and numbers and most apparent at 10ng/ml of G-CSF and M-CSF. The additive effects in suspensioncultures were only demonstrated when the two CSFs were addedsimultaneously. Pretreatment of one of the two CSFs did not

7706




20-1

10G-CSF ( ng / ml )

Fig. 5. Dose-response analysis of [3H]thymidine uptake. Cells (6 x IO4)werecultured for 20 h in medium containing various doses of G-CSF and pulse labeledwith ['Hjthymidine for 4 h. Columns, means of triplicate cultures; bars, Â±SD.The uptake was increased with G-CSF (with 0.1, I, or 10 ng/ml of G-CSFcompared with G-CSF-untreated cells, P< 0.01).

1.0 n

0.9-

O)om.ooM 0.7-

0.6-

0.50.1 1 10

G-CSF (ng/ml)100

Fig. 6. Interactions of G-CSF and M-CSF on the proliferation of NKM-1cells. Cells were plated in microwell plates at 3 x 10"/well in COSMEDIUM-001 in the presence of various concentrations of G-CSF (0-100 ng/ml) and 0 (â€¢),O.I (O), 1 (â€¢),and 10 (D) ng/ml of M-CSF. The MTT assay was performed after3 days. Points, means of three experiments; bars, Â±SD.

alter the responsiveness of NKM-1 cells to the other CSF (datanot shown).

G-CSF Receptors on NKM-1 Cells. The 125I-labeled 'Tyr-3Tyr-G-CSF was used, and a binding analysis was performedon NKM-1 cells. The specific binding of '"I-G-CSF to NKM-1 cells increased depending on the cell number. NKM-1 cellsexpressed 60 specific binding sites with a calculated Kd of 100pM (Fig. 7). Scatchard analysis (29) of the data yielded a straightline, indicating a single class of binding sites. No competitionwas observed with 1L-3, GM-CSF, or M-CSF, while unlabeledG-CSF specifically inhibited I25I-G-CSF binding. When 125I-G-CSF-binding capacity was examined after incubation with M-CSF, the capacity was no different compared with the untreatedcells. These results suggest that NKM-1 cells expressed specificreceptors for G-CSF which were independent of M-CSF binding and not regulated by M-CSF.

c-fms Expression in NKM-1 Cells. The effects of M-CSF aremediated through binding to surface receptors. The response ofNKM-1 cells prompted us to examine c-fms mRNA expression

11*1-1

o

0.03-

0.02-

0.01-

100 200(pM)

300

O 10 20 30 10 50 60(molecules/cell)

Fig. 7. Top, binding of radiolabcled G-CSF to NKM-1 cells. 5x10' cells wereincubated with increasing amounts of radiolabeled G-CSF for 3 h at roomtemperature. Data are corrected for nonspecific binding measured in the presenceof excess unlabeled G-CSF. Bottom, Scatchard plots of these data. BF. bound/free ratio.

1 2 3

285-

18S-

^â€¢c-fms

Â«-HLA

Fig. 8. Expression of c-fms mRNA. Northern blot analysis was performedusing \-fms and HLA-B7 probes as described in "Materials and Methods." LaneI, NKM-1 cells in culture in RPMI with 10% PCS; lane 2, NKM-1 cells inCOSMEDIUM-OOI for 24 h; lane 3, NKM-1 cells in the same medium containing10 ng/ml of G-CSF for 24 h.

(30). By Northern blot hybridization, a 4.3-kilobase band wasdetected, indicating that NKM-1 cells expressed M-CSF receptor. The expression was also tested for RNAs from NKM-1cells which had been incubated with 10 ng/ml G-CSF for 24 h.No differences were observed between G-CSF-treated cells anduntreated cells (Fig. 8).

DISCUSSION

We established a myeloid leukemia cell line, NKM-1, from apatient with AML (FAB M2). In addition to the typical myeloidcharacteristics of positive staining for MPO and CAE withstrong CD 15 expression, this cell line has unique features:expression of G-CSF receptors and M-CSF receptors and pro-liferative responses to the two factors.

The stimulatory effects were demonstrated by viable cellcount, [3H]thymidine uptake, cell cycle analysis, colony forma-

7707




tion, and clonogenic cell recovery. G-CSF and M-CSF had noability to induce cell differentiation in NKM-1 cells, which wasdefined by morphological appearance or analysis of surfaceantigens. This lack of differentiation might be related to theweak differentiation potential of NKM-1 cells. Colony-formingcapacity was stimulated with 1-10 ng/ml of G-CSF or M-CSFbut suppressed with higher concentrations (>20 ng/ml) ofCSFs, which was reproducible in all of the experiments performed. This pattern of responsiveness was also demonstratedin some AML cells (31), which may represent optimal concentrations of CSFs for cell growth. No contaminating inhibitorymaterials were found in the solutions of colony assay. Theclonogenic cell recovery which reflects self-renewal capacity ofleukemia stem cells was rapidly increased on day 2 with G-CSFor M-CSF in a dose-dependent manner and decreased on day3 at higher concentrations of CSFs (10 or 100 ng/ml).

Several myeloid cell lines can be stimulated to differentiateor proliferate in the presence of G-CSF or M-CSF. Purified G-CSF is able to induce murine WEHI-3B D+ (20), human HL-

60 (32) cells to mature, followed by ultimate suppression ofclonogenic cells and terminal differentiation. In general, exogenous CSFs have little effect on long-term proliferation ofestablished cell lines. Some GM-CSF-dependent leukemia celllines retained their ability to respond to exogenous CSFs (33).Among three GM-CSF-dependent leukemia cell lines, G-CSFcan independently support the continuous growth of AML-193(34) cells but not of M V4-11 (34) cells or TALL-101 (35) cells.M-CSF also potentiated the proliferation of AML-193 cellsonly in the presence of suboptimal doses of GM-CSF. Recently,Matsuda et al. (36) reported a murine myeloid leukemia cellline, NFS-60, which is stimulated by murine and human G-

CSF to proliferate but not to differentiate. Considering theabove findings, one may regard NKM-1 as the first permanenthuman myeloid leukemia cell line that grows in serum-freemedium without adding any CSFs and shows proliferativeresponses to exogenous G-CSF and M-CSF.

The effects of CSFs are presumably mediated through binding to surface receptors. The '"I-G-CSF used retained fullbiological activity and receptor-binding capacity. By this labeledG-CSF, normal human neutrophils showed a single receptortype (230 receptors/cell; Kj 30 PM). The number of G-CSF-binding sites/cell was low on NKM-1 cells but the Kd wasrelatively low compared with that of previously reported myeloid leukemia cells. The prominent proliferative responses ofNKM-1 cells may be attributable to abnormalities in G-CSFreceptors or postreceptor pathways in NKM-1 cells.

It has been reported that of several human cell lines examinedonly the Raji cell line expressed c-fms (37, 38). NKM-1 is, toour knowledge, the first human myeloid leukemia cell line withc-fms mRNA expression. Dose-dependent proliferative responses to M-CSF indicate that NKM-1 cells express functionalM-CSF receptors, although M-CSF-binding analysis was notperformed in the current study. Therefore, the NKM-1 cell linepossesses both G-CSF and M-CSF receptors. Only the mousemacrophage cell line J774 (39) was shown to possess both G-CSF and M-CSF receptors (27, 40).

Myeloid leukemia cells are known to express multiple CSFreceptors (41), and interaction of CSFs on biological responseshas been reported. In NKM-1 cells, M-CSF showed additiveeffects with G-CSF in potentiating cell growth when two factorswere used simultaneously. This could occur in several ways. Itmay be that NKM-1 cells consist of two populations whichexpress one of the receptors for the two factors or the two

receptors appear on a single cell. In either case the cells couldbe responsive to both CSFs through separate, independentactivation pathways or the additive effects might depend on theup modulation of the growth factor receptor, thus making formore susceptibility to the other growth factor. The latter maynot be the case for NKM-1 cells, since the effects were notobvious when G-CSF and M-CSF were added sequentially.Moreover, c-fms mRNA expression was not up-regulated by G-CSF, and pretreatment of M-CSF did not alter 12SI-G-CSF-

binding capacity. Further studies would be required to elucidatethe molecular events underlying cellular responses to CSFs.This cell line may serve as a useful model for these studies.

ACKNOWLEDGMENTS

We would like to thank Dr. Shinsuke Saga and Dr. Shigeo Nakamurafor their assistance with the ultrastructural studies, Vlakoto Kondo foraid in the chromosome analysis, and Sayoko Sugiura for skillful technical assistance.

REFERENCES

1. Vellenga, E., Ostapovicz, D., O'Rourkc, B., and Griffin, J. D. Effects ofrecombinanl IL-3, GM-CSF, and G-CSF on proliferation of leukemic clonogenic cells in short-term and long-term cultures. Leukemia (Baltimore), I:584-589, 1987.

2. Delwel, R.. Dorssers, L., Touw, I., Wagemaker, G.. and LÃ¶wenberg, B.Human recombinant multilineage colony stimulating factor (interleukin-3):stimulator of acute myelocytic leukemia progenitor cells in vitro. Blood, 70:333-336, 1987.

3. Miyauchi, J., Kelleher, C. A., Yang, Y., Wong, G. G., Clark. S. C, Minden,M. D., Minkin, S., and McCulloch, E. A. The effects of three recombinantgrowth factors. IL-3, GM-CSF, and G-CSF, on the blast cells of acutemyeloblastic leukemia maintained in short-term suspension culture. Blood.70:657-663, 1987.

4. Delwel. R., Salem, M., Pellens, C., Dorssers, L., Wagemaker, G.. Clark, S.,and LÃ¶wenberg, B. Growth regulation of human acute myeloid leukemia:effects of five recombinant hematopoietic factors in a serum-free culturesystem. Blood, 72: 1944-1949. 1988.

5. Kelleher, C., Miyauchi, J., Wong, G., Clark, S., Minden, M. D., and McCulloch, E. A. Synergism between recombinanl growth factors, GM-CSFand G-CSF. acting on the blast cells of acute myeloblastic leukemia. Blood.69: 1498-1503. 1987.

6. Griffin, J. D., Young, D., Herrmann, F., Wiper, D.. Wagner, K., and Sabbath,K. D. Effects of recombinant human GM-CSF on proliferation of clonogeniccells in acute myeloblastic leukemia. Blood. 67: 1448-1453, 1986.

7. Hoang, T., Nara. N., Wong, G.. Clark. S., Minden, M. D., and McCulloch.E. A. Effects of recombinant GM-CSF on the blast cells of acute myeloblasticleukemia. Blood, 68: 313-316, 1986.

8. Vellenga, E.. Delwel, H. R., Touw, I. P., and Lowenberg, B. Patterns of acutemyeloid leukemia colony growth in response to recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Exp. Hematol.. 75:652-656, 1987.

9. Vellenga, E., Young, D. C., Wagner. K.. Wiper, D., Ostapovicz, D., andGriffin. J. D. The effects of GM-CSF and G-CSF in promoting growth ofclonogenic cells in acute myeloblastic leukemia. Blood, 69:1771 -1776, 1987.

10. Nara, N., Murohashi, L, Suzuki. T., Yamashita, Y.. Maruyama, Y., Aoki,N., Tanikawa. S., and Onozawa, Y. Effects of recombinant human granulo-cyte colony-stimulating factor (G-CSF) on blast progenitors from acutemyeloblastic leukaemia patients. Br. J. Cancer, 56: 49-51, 1987.

11. Miyauchi, J.. Wang, C, Kelleher. C. A., Wong. G. G., Clark, S. C., Minden,M. D., and McCulloch, E. A. The effects of recombinant CSF-1 on the blastcells of acute myeloblastic leukemia in suspension culture. J. Cell. Physiol.,Â»5:55-62, 1988.

12. Begley, C. G.. Metcalf, D., and Nicola. N. A. Primary human myeloidleukemia cells: comparative responsiveness to proliferative stimulation byGM-CSF or G-CSF and membrane expression of CSF receptors. Leukemia(Baltimore), /: 1-8, 1987.

13. Park, L. S., Waldron, P. E., Friend, D., Sassenfeld, H. M., Price, V.,Anderson, D., Cosman, D., Andrews. R. G.. Bernstein. I. D., and Urdal, D.L. lnterleukin-3, GM-CSF, and G-CSF receptor expression on cell lines andprimary leukemia cells: receptor heterogeneity and relationship to growthfactor responsiveness. Blood, 74: 56-65, 1989.

14. Bennett, J. M., Catovsky, D.. Daniel, M. T.. Flandrin. G., Galton, D. A. G.,Gralnick, H. R., and Sultan. C. Proposed revised criteria for the classificationof acute myeloid leukemia. A report of the French-American-British cooperative group. Ann. Intern. Med., 103: 620-625, 1985.

15. Ogura, M.. Morishima, Y., Ohno, R., Kalo, Y., Hirabayashi, N., Nagura,H., and Saito, H. Establishment of a novel human megakaryoblastic leukemia

7708



G-CSF-, M-CSF- RESPONSIVE CELL LINE

cell line, MEG-O1, with positive Philadelphia chromosome. Blood, 66:1384-1392, 1985.

16. Morishima, Y., Kobayashi. M., Yang, S. Y., Collins, N. H., Hoffmann, M.k., and Dupont, B. Functionally different T lymphocyte subpopulationsdetermined by their sensitivity to complement-dependent cell lysis with themonoclonal antibody 4A. J. Immunol., 129: 1091-1098, 1982.

17. LJeda, R., Tanimoto, M.. Takahashi, T.. Ogata, S., Nishida. K., Namikawa,R., Nishizuka, Y.. and Ota, K. SerologieÂ«)analysis of cell surface antigensof null cell acute lymphocytic leukemia by mouse monoclonal antibodies.Proc. Nati. Acad. Sci. USA, 79: 4386-4390, 1982.

18. Hayashi, K., and Furukawa, K. Analysis of cell surface molecules on humanplatelets with monoclonal antibodies: identification of four platelet specificcell surface molecules. Nagoya Igaku, 106:171-179, 1984.

19. Park, B. H., Fikrig, S. M., and Smithwick, E. M. Infection and nitroblue-tetrazolium reduction by neutrophils. Lancet, /: 532-534, 1968.

20. Souza, L. M., Boone, T. C., Gabrilove, J., Lai, P. H., Zsebo, K. M., Murdock,D. C., Chazin, V. R., Bruszewski, J., Lu, H., Chen, K. K., Barendt, J.,Platzer, E., Moore. M. A. S., Mertelsmann, R., and Welle, K. Recombinanthuman granulocyte colony-stimulating factor: effects on normal and leukemicmyeloid cells. Science (Washington DC), 232.-6ÃŒ-65,1986.

21. Wong, G. G., Temple, P. A., Leary, A. C., Witek-Giannotti, J. S., Yang, Y.,riarietta. A. B., Chung, M., Murtha, P., Kriz, R., Kaufman. R. J., Ferenz,C. R., Sibley, B. S., Turner, K. J., Hewick, R. M., Clark, S. C., Yanai, N.,Yokota, H., Yamada, M., Saito, M., Motoyoshi, K., and Takaku, F. Human('SI I : molecular cloning and expression of 4-kb cDNA encoding the humanurinary protein. Science (Washington DC), 235: 1504-1508, 1987.

22. Burgess, A. W., Begley, C. G., Johnson, G. R., Lopez, A. F., Williamson, D.J., Mermod, J. J., Simpson, R. J., Schmilz, A., and DeLamarter, J. F.Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor. Blood, 69:43-51, 1987.

23. Yang, Y. C., Ciarletta, A. B., Temple, P. A., Chung, M. P., Kovacic, S.,Witek-Giannotti, J. S., Leary, A. C., Kriz, R., Donahue, R. E., Wong, G. G.,and Clark, S. C. Human IL3 (multi-CSF): identification by expressioncloning of a novel hematopoietic growth factor related to murine IL3. Cell,47:3-10, 1986.

24. Mosmann, T. Rapid colorimetrie assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immunol. Methods,65:55-63,1983.

25. Nara, N., and McCulloch, E. A. The proliferation in suspension of theprogenitors of the blast cells in acute myeloblastic leukemia. Blood, 65:1484-1493, 1985.

26. Cannistra, S. A., Rambaldi, A., Spriggs, D. R., Herrmann, F., Kufe, D., andGriffin, J. D. Human granulocyte-macrophage colony-stimulating factorinduces expression of the tumor necrosis factor gene by the U937 cell lineand by normal human monocytes. J. Clin. Invest., 79: 1720-1728, 1987.

27. Nicola, N. A., and Metcalf, D. Binding of the differentiation- inducer. gran-ulocyte-colony-stimulating factor, to responsive but not unresponsive leu

kemic cell lines. Proc. Nati. Acad. Sci. USA, SI: 3765-3769, 1984.28. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J.

Isolation of biologically active ribonucleic acid from sources enriched inribonuclease. Biochemistry. 18: 5294-5299. 1979.

29. Scatchard, G. The attraction of proteins for small molecules and irons. Ann.N. Y. Acad. Sci., 51: 660-672. 1949.

30. Sherr, C. J. Colony-stimulating factor-1 receptor. Blood. 75: 1-12. 1990.31. Piao, Y. F., Tojo, A., Urabe, A., Takaku, F., Suzuki, T., and Nara, N. Effects

of recombinant human G- and GM-CSF on the proliferation of leukemicblast progenitors from acute myeloblastic leukemia patients. Acta Haematol.Jpn., 51: 1115-1121, 1988.

32. Begley, C. G., Metcalf, D., and Nicola, N. A. Purified colony stimulatingfactors (G-CSF and GM-CSF) induce differentiation in human HL60 leukemic cells with suppression of clonogenicity. Int. J. Cancer, 39: 99-105,1987.

33. Santoli, D., Yang, Y., Clark, S. C., Kreider, B. L., Caracciolo, D., andRovera, G. Synergistic and antagonistic effects of recombinant human inter-leukin (IL) 3, IL-la, granulocyte and macrophage colony-stimulating factors(G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic celllines. J. Immunol., 139: 3348-3354, 1987.

34. Lange, B., Valtieri, M., Santoli, D., Caracciolo, D., Mavilio, F., Gemperlein,I., Griffin, C., I .manuel. B., Finan, J., Nowell, P., and Rovera, G. Growthfactor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines. Blood, 70: 192-199, 1987.

35. Valtieri, M., Santoli, D., Caracciolo, D., Kreider, B. L., Altmann, S. W.,Tweardy, D. J., Gemperlein, I., Mavilio, F., Lange, B., and Rovera, G.Establishment and characterization of an undifferentiated human T leukemiacell line which requires granulocyte-macrophage colony stimulatory factorfor growth. J. Immunol., 138:4042-4050, 1987.

36. Matsuda, S., Shirafuji, N., and Asano, S. Human granulocyte colony-stimulating factor spirit Â¡rallybinds to murine myeloblastic MS 60 cells andactivates their guanosine triphosphate binding proteins/adenylate cyclasesystem. Blood, 74: 2343-2348, 1989.

37. Dubreuil, P., Torres, H., Courcoul, M., Birg, F., and Marinoni. P. c-fmsexpression is a molecular marker of human acute myeloid leukemias. Blood,72:1081-1085,1988.

38. Parwaresch, M. R., Kreipe, H., Feigner, J., Heidorn. K.. Jaquel, K., BÃ¶de-wadt-Radzun. S., and Radzun, H. J. M-CSF and M-CSF-receptor geneexpression in acute myelomonocytic leukemias. Leuk. Res., 14: 27-37,1990.

39. Ralph, P., Prichard, J., and Cohn. M. Reticulum cell sarcoma: an effectorcell in antibody-dependent cell-mediated immunity. J. Immunol., 114: 898-905, 1975.

40. Guilbert, L. J., and Stanley, E. R. Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells. J. Cell Biol. 85: 153-159, 1980.

41. Budel, L. M., Touw, I. P., Delwel, R., and LÃ¶wenberg, B. Granulocytecolony-stimulating factor receptors in human acute myelocytic leukemia.Blood, 74: 2668-2673, 1989.

7709



1990;50:7703-7709. Cancer Res Takae Kataoka, Yoshihisa Morishita, Michinori Ogura, et al. and Macrophage Colony-stimulating Factor Receptors

ReceptorsCoexpressing Granulocyte Colony-stimulating Factor A Novel Human Myeloid Leukemia Cell Line, NKM-1,

Updated version

http://cancerres.aacrjournals.org/content/50/23/7703

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/50/23/7703To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



a novel human myeloid leukemia cell line, nkm-1 ...growth factors, leukemic cells proliferate in...

Documents