[CANCER RESEARCH 47, 5025-5030, October 1, 1987]

Production of Human Hematopoietic Survival and Growth Factor by a MyeloidLeukemia Cell Line (KPB-M15) and Placenta as Detected by a MonoclonalAntibody1

Atsunobu Hiraoka,2 Tadashi Ohkubo, and Minoru Fukuda

Department of Intentai Medicine, Osaka Dental University, 1-4 Kyobashi, Higashi-ku, Osaka 540, Japan

ABSTRACT

Fifty-five hematopoietic cell lines, including 19 T-, 16 B-, S pre-B-, 5non-T non-B-, 1 erythroid, and 9 myeloid-monocytoidcells, were screenedfor production of human hematopoietic survival and stem cell growthfactor (SCGF) by enzyme immunoassay using anti-SCGF monoclonalantibody. The KPB-M15 myeloid cell line constitutionally secreted aconsiderable quantity of SCGF, while other T- or myeloid-monocytoidcell lines did not secrete SCGF. Other biomaterials investigated werefetal calf, horse, and human serum; granulocyte-macrophage colony-stimulating factor and erythropoietin preparations;human placental conditioned medium; lectin (phytohemagglutinin, concanavalin A, and poke-weed mitogen); and mixed leukocyte reaction-stimulated leukocyte-conditioned medium.SCGF was detected only in humanplacental conditionedmedium. SCGF produced by the KPB-M15 cells was a protein with amolecular weight of 20,000. The molecule, highly purified by immunoad-sorbent affinity chromatography, retained SCGF activity in vitro, e.g.,erythroid burst-promoting activity and granulocyte-macrophage-colonypotentiation. With the availability of purified SCGF, it is now possibleto study in detail the mechanisms regulating hematopoietic stem cells.

INTRODUCTION

In vitro clonal growth of human hematopoietic stem cells orearly primitive progenitors is mediated by specific regulators.A classical concept has indicated that human GM-CSF3 is

essential for survival, proliferation, and differentiation of CMprogenitors (CFU-GM) in vitro and functional capacity of theirmature progeny (1). As for the erythroid lineage, early erythroidprogenitors (BFU-E) are regulated by BPA distinct from erythropoietin acting on late progenitors; (CFU-E) (2). The factorstimulating both BFU-E and CFU-E growth is also describedas EPA (3), which is produced by human monocyte cell lines,U937 (4) and GCT-C (5), and most HTLV-transformed T-lymphoblast cell lines (6, 7). As recombinant human GM-CSFbecame available by molecular cloning of cDNA for GM-CSFfrom HTLV type H-transformed T-lymphoblast cell line, Mo

Received 2/19/87; revised 6/18/87; accepted 7/1/87.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 Grant-in Aid (58771792) from the

Ministry of Education, Japan, and the Osaka Dental University EndowmentFund.

*To whom requests for reprints should be addressed, at Department of InternalMedicine, Osaka Dental University, 1-4 Kyobashi, Higashi-ku, Osaka 540, Japan.

3The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor, CFU-GM, colony-forming unit-granulocyte-macrophage; CFU-E,colony-formingunit-erythroid; BFU-E, burst-forming unit erythroid: BPA, burst-promoting activity; SCGF, stem cell growth factor, LCM, leukocyte-conditionedmedium; MoAb, monoclonal antibody; HPCM, human placentnl conditionedmedium; FCS, fetal calf serum; EIA, enzyme immunoassay; EPA, erythroid-potentiating activity; HTLV, human T lymphoma virus; Con A, concanavalin A;l'I IA. phytohemagglutinin; PHA-P, phytohemagglutinin-protein fraction; GPA,granulocyte-promoting activity; PWM, pokeweed mitogen; MLR, mixed leukocyte reaction; TBS, Tris-buffered saline; BSA, bovine serum albumin; GAM-HRP, goat anti-mouse IgG antibody conjugated with horseradish peroxidase;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; I 1Its,Tween-20 in TBS; D-PBS(-), Dulbecco's Ca- and Mg-free phosphate-bufferedsaline, pH 7.4; BM, bone marrow; IMDM, Iscove's modified Dulbecco's medium;rh, recombinant human, IL-3, interleukin-3.

(8), Con A-activated T-cells (9), and HUT-102 and PHA-stimulated T-cells (10), pure GM-CSF was demonstrated tocross-stimulate BFU-E and pluripotent stem cells (11,12). Thefactor capable of stimulating pluripotent stem cell growth isalso reportedly produced by the 5637 bladder carcinoma cellline (13) and the SK-HEP-1 human hepatoma cell line (14).

Partially purified human hematopoietic survival and growthfactor (SCGF), originally detected in phorbol myristate acetate-stimulated LCM, has diverse biological activities on hematopoietic stem cells of both granulocyte and erythroid lineages(15); GM colony potentiation (16, 17), BPA, and AGPA (18)and ABPA, but lacks GM-CSF activity. Analysis so far hasshown that the protein has a molecular mass of M, 18,000-25,000, similar to that of most lymphokines. Recently, MoAbsspecific for SCGF have been produced (19), providing a usefultool for biological and biochemical characterization of themolecule. As more is learned of the properties of SCGF, regulating systems operating at the stage of hematopoietic stemcells can be well defined.

Here we report the production of SCGF in vitro by the KPB-M15 myeloid cell line and placenta as evidenced using anti-SCGF MoAb, and its immunoadsorbent affinity purification.

MATERIALS AND METHODS

Culture Media Conditioned by Hematopoietic Cell Lines and OtherBiomaterials. Fifty-five hematopoietic cell lines were screened for SCGFproduction. Most of the cell line supernatants, obtained at each mediumexchange once or twice a week, were a generous gift from Dr. K.Minato, National Cancer Center, Tokyo, consisting of 19 T-, 16 B-, 5pre-B-, 5 non-T non-B-, 1 erythroid, and 9 myeloid-monocytoid cells(refer to Table 1 for each cellular origin and classification). Otherbiomaterials investigated included HPCM (four lots) (20), FCS (fivebatches), horse serum (two batches), normal human serum (threebatches), partially purified GM-CSF, CSF-Chugai (6370 units/mg protein; Chugai, Tokyo, Japan) derived from the T3M-5 thyroid cancercell line (21), and erythropoietin from human urine (49 units/mgprotein; Toyobo, Osaka, Japan).

Lectin-stimulated Leukocyte-conditioned Medium. Human peripheralblood mononuclear cells were separated by density-gradient centrifuga-tion on Ficoll-Paque (Pharmacia, Uppsala, Sweden). The low densitycell suspension (2 x Hf/nil) was cultured in Lux 35-mm l'etri dishesat 37°Cfor 1 to 6 days in a fully humidified atmosphere of 5% CO: in

air. A 2 nil culture medium per dish contained 5% FCS (Commonwealth Serum Lab., Melbourne, Australia) and 0.5-10 nn/m\ PHA-P(Difco, Detroit, MI) for PHA-LCM, 0.5-10 /.g/ml Con A (type IV;Sigma, St. Louis, MO) for Con A-LCM, 0.1-2% PWM (GIBCO,Grand Island, NY) for PWM-LCM in RPMI 1640 medium supplemented with 50 fiM 2-mercaptoethanol, 1 HIMsodium pyruvate, 2 HIML-glutamine and kanamycin 60 mg/liter. Lectin-free LCM was used ascontrol. MLR-LCM (three lots) were obtained by incubating a mixtureof allogene,it- mono nuclei!r cells (1 x U("/nil each) in RPMI 1640

medium with 5% FCS for 2 to 4 days.Enzyme Immunoblotting. An immunoblotting system was used to

detect SCGF; antigens passively bound (dot blotting) or electrophoret-ically transferred (Western blotting) to a nitrocellulose membrane weredetected by EIA (22) using anti-SCGF MoAb. All procedures for

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Table 1 Hematopoietic cell line-conditioned medium

WellA-lA-2A-3A-4A-5A-6A-7A-8A-9A-10A-llA-l

2B-lB-2B-3B-4B-5B-6B-7B-gB-9B-10B-llB-12C-lC-2C-3C-4C-5C-6C-7C-8C-9C-10C-llC-12D-lD-2D-3EMD-5D6D-7D-8D-9D-IOD-llD-I2E-lE-2E-3E-4E-5E-6E-7E-8E-9E-10E-llE-l

2F-lF-2F-3F-4Cell

lineRPMI

8402(Sommer)P12/IchikawaHPB-ALLHPB-MLTPeerMT-1P30/OhkuboHUT78HUT102DND41CCRF-HSB-2CCRF-CEMATL-5SMOLT-3K-562KG

1ML-3HL-60THP-l-OP39/TsuganeU-937KYO-1-A(37)KYO-1-D(37)KPB-M15(38)RajiDaudiP3-HR1TonikaKOBK-101P32/IshidaSK-DHLALL-SOhnoA3/KawalcamiA4/FukudaARH77U-266RPMI-8226BALL-1NALL-1RehKM-3NALM-16KOPN-KKOPN-1NALM-1NALM-6LAZ-221SKW-3P43/AndoKE-37KOPT-K1JMP40/OzawaAS/TakayamaHPCM,

lot 2*(20)HPCM,

lot3HPCM,lot4'HPCM,lot5'PCS

(CSLS023)FCS(HyClone407)PCS

(X-397)PCS(Boehringer678168)FCS(Boehringer679313)OriginALL-ALLALLALLALLATLALLSézary

syndromeMycosisfungoidesALLALLALLATLALLCML

atBCAMLAMLAPLAMoLAMMoLHistiocytic

TCMLatBCCMLatBCCMLatBCBurkitt

lymphomaBurkittlymphomaBurkittlymphomaBurkittlymphomaBurkittlymphomaBurkittlymphomaDHLALL

(Burkitt?)IleocecalmalignantlymphomaGastric

malignantlymphomaIleocecalmalignantlymphomaMultiple

myelomaMultiplemyelomaMultiplemyelomaALLALLALLALLALLALLALLALLALLALLCLLALLLeukemic

NHLALLNormalMalignant

lymphomaOtherbiomaterialsF-5F-6F-7F-8F-9F-10F-llF-l

2ClassificationTTTTTTPre-T

orcommonTTTTTTTErythroidMyeloidMyeloidMyeloidMonocytoidMyelomonocytoidMonocytoidMyelomonocytoidMyelomonocytoidMyeloid*BBBBBBBBBNon-T

non-BBBBBBNon-T

non-BNon-Tnon-BNon-Tnon-BNon-Tnon-BPre-BPre-BPre-BPre-BPre-BTTTTTBBHorse

serum (Irvine1056)Horseserum (Flow 29211087)BlankHuman

serum-1Humanserum-2Humanserum-3CSF-Chugai(21)Erythropoietin

(Toyobo)

* ALL, acute lymphoblastic leukemia; ATL, adult I cell leukemia; CML at BC, chronic myeloid leukemia at blastic crisis; AML, acute myeloblastic leukemia;

APL.Õlymphocytk leukemia; NHL, non-Hodgkin's lymphoma.

* Highly positive.' Faintly positive.

enzyme immunoblotting were done at room temperature unless otherwise indicated.

Dot Blotting (19, 23, 24). A 0.45-ftm nitrocellulose membrane (15 x9.2 cm), equilibrated with 20 mMTris-0.5 M NaCl, pH 7.5 TBS for 10min, was laid over the gasket of the dot-blotting apparatus (Bio-Rad,Richmond, CA). Various samples (400 .iludí) were applied to eachwell of the sample template, and were gravity filtered for 120 min. Theantigen-bound membrane was blocked with 250 dir, BSA (Armour,Chicago, IL, Fr. V) in TBS (blocking solution) for 30 min. Anti-SCGFMoAb 241 (100 M')diluted 1:26 with blocking solution was added to

allow antibody binding for 90 min. Then, 100 n\ of l:2000-dilutedaffinity purified GAM-HRP (Bio-Rad) was loaded. After 100 min ofincubation, the membrane was drained completely, air dried, washed,and stained with ice cold 0.015% I!..().. and 4-chloro-l-naphthol (0.5mg/ml) in methanol and TBS. Blue to purple dots were developedwithin 7 to 15 min in the positive cases.

Western Blotting (24-26). Crude materials, found positive for SCGFby dot blotting, were applied to SDS-PAGE. After electrophoresis, thegel was soaked in 25 mM Tris-192 mM glycine, pH 8.3, with 15%methanol (transfer buffer) for 30 min, and placed between the assembly

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HUMAN HEMATOPOIETIC SURVIVAL AND GROWTH FACTOR

of the transfer-blotting sandwich. Western blotting was performed in atank filled with 2500 ml prechilled transfer buffer, under the followingconditions: 20 V (0.1 A) for 30 min, 37.5 V (0.17 A) overnight, and 70V (0.44-0.6 A) for 2 h at 4°C.The nitrocellulose membrane was cut

into strips along each lane, one of which was stained with 0.2%Coomassie brilliant blue-R250. Other strips were washed twice with0.05% TTBS and blocked with 3% BSA in TBS at 37'C for 60 min.

After five washes with 0.1% TTBS, the strips were assayed by threemethods of EIA. First, the strips were floated in Protein A-purifiedanti-SCGF MoAbs 241 and 311 diluted 1:4 with 1% BSA in TBS for60 min, washed, and reacted with l:1875-diluted GAM-HRP for 60min. Second, the strips were incubated in 1:8- and 1:4 diluted biotiny-

lated MoAbs 241 and 311, respectively, for 60 min, washed, and reactedwith 1:300-diluted HRP-streptavidin (Amersham, UK) for 30 min.

Third, after reaction with MoAbs 241 and 311 for 60 min, the stripswere incubated in 1:250 diluted biotinylated sheep anti-mouse IgGserum (Amersham) for 60 min, washed, and further incubated in 1:300-diluted streptavidin-HRP for 30 min. After each of these three proce

dures, the strips were washed three times with 0.1 % TTBS, and stainedwith 0.015% H2O2 and 4-chloro-l-naphthoI (0.5 mg/ml) for 7-15 minor 0.015% H2O2and 0.02% 3-amino-9-ethylcarbazole in 50 mM acetatebuffer, pH 5.0, for 2-5 min (27).

Biotin Labeling of MoAb. Biotinylation of MoAb was performed asdescribed (28, 29). Briefly, lyophilized preparations of Protein Apurified MoAb IgG were reconstituted with 0.1 M NaHCO3. Thesolutions (1 mg protein/ml) of 1.3 ml MoAb-241 and 1.1 ml MoAb-311 were mixed with 78 and 60 ¿ilof biotinyl-jV-hydroxysuccinimide (1mg/ml) in dimethylformamide, respectively, then incubated at roomtemperature for 4 h, and dialyzed at 4°Cfor 24 h against D-PBS(—).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. SDS-

PAGE was carried out according to the method of Laemmli (30). Testsamples were reduced with 2% SDS and/or 5% 2-mercaptoethanol(and/or 20 mM dithiothreitol in some cases) in 25 mM Tris-HCl, pH6.8, at 37°Cfor 2 h, and applied to 12.5% polyacrylamide gel. Protein

bands were visualized by silver staining (31).Anti-SCGF MoAb Immunoadsorbent Affinity Chromatography.

MoAb was coupled with Affi-Gel 10 (Bio-Rad) (32). Ammonium sul-fate-precipitated MoAb 241 was dissolved in 2.5 ml 0.1 M NaHCO3-0.15 M NaCl, pH 8.0 (coupling buffer), and dialyzed at 4°Cfor 24 h

against 500-ml coupling buffer. It was mixed with packed Affi-Gel 10,4.5 ml, i.e., at the rate of 34.4 mg protein/ml Affi-Gel 10, and incubatedat room temperature for 90 min with gentle end-to-end swirling. Thiscoupling technique achieved 75.3% fixation of total proteins on Affi-

Gel 10. After washing twice with coupling buffer, the gel was incubatedwith 0. l M ethanoIamine-HCl, pH 8.0, for 60 min to block the rest ofactivated residues. SCGF-containing samples, 0.5 ml, were applied at4°Cto a MoAb 241 immunoadsorbent Affi-Gel 10 column (1x5 cm)

that had been equilibrated with D-PBS(-). When absorbance at 280nm reached baseline, bound proteins were eluted with 0.2 M acetate-0.15 M NaCl, pH 2.5. The flow rate was 6 ml/h; 2-ml fractions were

collected.Biological Activity of Purified SCGF. Immunoadsorbent affinity-

purified SCGF was tested for BPA and GM colony potentiating activityas representative of the SCGF activities. For the BPA assay, soft agarculture consisted of normal nonadherent BM cells (5 x 104/ml) inIMDM (GIBCO, OH) supplemented with 30% PCS, affinity-purified

erythropoietin from human urine, (1 unit/ml; Snow Brand Milk Products, Tokyo, Japan, 81,600 units/mg protein) and various concentrations of affinity-purified SCGF. In this assay system without BPA-containing materials, only a few erythroid bursts (4.7 ±1.7/5 x IO4

BM cells) were formed. Maximally stimulated BFU-E culture contained30% PCS (Irvine, selected lot), 1% deionized BSA (Armour, Fr. V),and semipurified erythropoietin from human urine, 1 unit/ml (Toyobo)in IMDM. After 14 days of incubation, hemoglobinized erythroid burstswere counted. GM colony potentiating activity was assayed as describedpreviously (16). Various concentrations of affinity-purified SCGF wereadded to the soft agar culture of BM cells (5 x 104/ml) for CFU-GM,

maximally stimulated with 10% CSF-Chugai in IMDM and 20% FCS.

RESULTS

Screening of Hematopoietic Cell Line-conditioned Mediumand Other Biomaterials for SCGF by Enzyme Immunoblotting.Of 55 hematopoietic cell line culture supernatants investigated,only the KPB-M15 myeloid cell line produced SCGF (Fig. 1;well B-12). Well E-5 (JM) appeared to be faintly positive, butreexamination failed to validate it. The KPB-M15 myeloid cellline was established from the peripheral blood leukocytes of a38-yr-old male patient with chronic myelogenous leukemia atblastic crisis (38); KPB-M15 cells are hyperdiploid with positivePh1 chromosomes, negative for peroxidase, periodic acid-Schiff,a-naphthyl butyrate esterase, a-naphthyl acetate esterase, andchloroacetate esterase activities, positive for acid phosphataseactivity, C3R and FcyR, and reactive to OKM1, MCS2, MY4,and MY7 MoAbs. No differentiation is induced by treatmentof the cells with phorbol myristate acetate, dimethyl sulfoxide,sodium butyrate, or 1-a-hydroxycholecalciferol. Other mediaconditioned by T-, B-, non-T-, non-B-, and myeloid-monocytoidcells were all negative. Other biomaterials, including partiallypurified GM-CSF and erythropoietin, were negative exceptHPCM; one lot (well E-8) was highly and two lots (wells E-10and 11) were faintly positive. Anti-SCGF MoAb did not cross-react with various dilutions of the purified rh GM-CSF preparation (Sumitomo, Osaka, Japan) (data not shown).

Anti-SCGF MoAb Titer Tested Using the KPB-M15 CultureSupernatant. As shown in Fig. 2, MoAbs 241 and 311 preparations at each purifying step developed positive dots, depending upon the dilution; particularly, Protein A-purified MoAbs241 and 311 (19) (Fig. 2, peaks 1 and 2 and legend), wereintensely positive within 1:10 dilution; crude and Protein Aflow-through fractions were moderately positive up to 1:200-1000 dilution; hybridoma culture supernatants were positivebetween 1:20-40 dilution.

Screening of a Variety of LCM for SCGF by Enzyme Immunoblotting. PHA-LCM (0.5,1, 2, 5, and 10 Mg/ml), PWM-LCM(0.1, 0.3, 0.6, 1, and 2%), MLR-LCM and lectin-free LCMwere all negative for SCGF (data are not shown, since no visibledots were developed). Con A-LCM (0.5, 1, 2, 5, and 10 Mg/ml)appeared to be dimly positive but the findings were not reproducible.

Enzyme Immunoblotting of KPB-M 15-derived SCGF. TheSCGF molecule in the crude KPB-M 15 culture supernatantwas identified by three methods of EIA using two types ofMoAb after Western blotting (Fig. 3). MoAbs 241 and 311clearly identified the SCGF molecule with molecular weight

1234 56 789 10 11 12

Fig. 1. Screening of the culture media conditioned by various hematopoieticcell lines and other biomaterials for SCGF using enzyme immunoblotting. Proteins in the conditioned medium, bound to a nitrocellulose membrane, werereacted with 1:26 dilutai anti-SCGF MoAb after blocking with \% BSA, andincubated with l:2000-diluted GAM-HRP. Wells A-l to F-12, each cell line-conditioned medium and biomaterials analyzed (Table 1).

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HUMAN HEMATOPOIETIC SURVIVAL AND GROWTH FACTOR

6 7 8 9 1OA

B

C

D

Fig. 2. KPB-M15-derived SCGF reactive with various dilutions of MoAbs.The KPB-M 15 culture supernatant bound to a nitrocellulose membrane werereacted with various dilutions of MoAbs 241 and 311 at each purifying step, anddetected by enzyme immunoblotting. For each well dot, refer to the followingexplanatory diagram:

Mo Ab 241 ascitic fluids

Culturesupernatants

Protein A

Crude Flow-through Peak 1 Peak 2

ABCDE11:11:101:201:401:10021:101:1001:2001:4001:10003:3:30:60:120:3004:1:5:10:20:305:1:5:10:20:30

MoAb 3 11 asciticfluidsCulturesupernatants

CrudeProtein

AFlow-through

Peak 1Peak 2

ABCDE6:1:10:20:40:1007:IO:100:200:400:1000g:3:30:60:120:3009:1:5:10:20:3010:1:5:10:20:30

Figures indicate dilution. Most of MoAb IgG bound to Protein A-Sepharosewas eluted with 0.l M glycine-HCl, pH 2.8 (peak 1), and partly with SOHIMTris-150 HIMNaCI, pH 8.6, at an early reequilibrating phase (peak 2) (19).

Mr

94,000-

67.000-

43.000-30,000-

20.100-

14,400-

BCDEFGHIJK

Ir

l-MoAb241-J LMoAb241-"-MoAb311J

C N AEC AECFig. 3. Enzyme immunoblotting of KPB-M 15-derived SCGF. The KPB-M 15

myeloid cell line was cultured in RPMI 1640 medium with 10% horse serum,and half of the medium including cells was weekly exchanged with fresh medium.The SCGF molecule in the crude KPB-M 15 culture supernatant was detected bythree methods of EIA using two types of anti-SCGF MoAb. SCGF/anti-SCGFMoAb/GAM-HRP complex EIA, lanes C, F, and /; SCGF/biotinylated anti-SCGF MoAb/streptavidin-HRP complex EIA, lanes D, G, and /; SCGF/anti-SCGF MoAb/biotinylated sheep anti-mouse IgG antibody/streptavidin-HRPcomplex EIA, lanes E, H, and A. Lane A, silver-stained SDS-PAGE not subjectto Western blotting; lane B, Coomassie brilliant blue-R250-stained nitrocellulosemembrane. Anti-SCGF MoAb 241 was used for lanes C to //, and MoAb 311 forlanes I to K; color development substrate for HRP staining was 4-chloro-l-naphthol (lanes C to E) or 3-amino-9-ethyIcarbazole (lanes F to K).

20,000-22,000. As the color-developing substrate for HRPstaining, 4-chloro-l-naphthol was less sensitive than 3-amino-9-ethylcarbazole; bands were hardly detected in Fig. 3, lanes Cand D. No relevant protein bands were observed when theblotted nitrocellulose membrane was stained with Coomassiebrilliant blue-R250. The SCGF/anti-SCGF MoAb/biotinylatedsheep anti-mouse IgG antibody/streptavidin-HRP complexEIA system (Fig. 3, lanes E, H, and K) was the most sensitive,yielding the clearest bands among the three EIA methods. Morenoteworthy were the two distinct MoAb-defined protein bandswith molecular weights 20,000 and 22,000 (Fig. 3, lanes E, H,I, J, and A'). Other relevant protein bands tend to be blurred

within the range of M, 20,000-22,000. Nevertheless, they essentially consisted of two single bands (Fig. 3, lanes F and G).

Purification of the KPB-M 15-derived SCGF by MoAb Im-munoadsorbent Affinity Chromatography. A small amount ofthe KPB-M 15-conditioned medium, concentrated 15-fold byan Amicon TCP-10 apparatus (PM-10 filter membrane), wasapplied to an immunoadsorbent column packed with Affi-Gel10 coupled with MoAb 241 IgG. Proteins bound to the gel weremostly eluted with 0.2 M acetate-0.15 M NaCI, pH 2.5 (peakI), but a small portion was eluted with D-PBS(—)at an early

reequilibrating phase (peak II, Fig. 4). These two SCGF preparations (peaks I and II) were of nearly identical quality, i.e.,an SCGF molecule with a molecular weight of 20,000 wasobserved as a single band by SDS-PAGE (Fig. 5). Affinity-purified SCGF preparations were incubated with or without 5%2-mercaptoethanol and/or 20 mM dithiothreitol for SDS-PAGE. The SCGF preparations had been highly purified; particularly, peak II fractions were almost purified to a homogeneity. In any case, the electrophoretic patterns of fractionatedprotein bands were not altered irrespective of whether they werereduced or not.

Biological Activities of Affinity-purified, KPB-M 15-derivedSCGF. Of biological importance was to test whether the affinity-purified SCGF preparations could retain SCGF activities invitro. BPA and GM-colony potentiating activity were tested asrepresentative of the SCGF activities. As shown in Fig. 6,affinity-purified SCGF (peak II fractions) showed a linear BPAelevation in a dose-dependent manner up to the plateau level

1.0

0.8

- -22,000 I °6- -20.000 8

<M

«0.4

0.2 IVIAcetate-0.15M NaCI,pH 2.5

Peak-1

Peak-H

10 20 30

Fraction Number40 50

Fig. 4. Purification of KPB-M 15-derived SCGF by immunoadsorbent affinitychromatography. A 0.5-ml culture supernatant of 15-fold-concentrated KPB-M15 was applied at 4 ( to a MoAb 241-coupled Affi-Gel 10 column (1x5 cm).Bound proteins, as monitored by absorbance at 280 nm ( ), were eluted with0.2 M acetate-0.15 M NaCI, pH 2.5 (ear). The column was reequilibrated withD-PBS (-).

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Mr 1234567

94,000-

67.000- j ....__

43000-

30000-

20J CO-**,* A A

14,400-

2%SDS + •»-+ + +-»-+ +

5% 2-ME H h - H 1

20mM DTT \- -\ h +Fig. 5. SDS-PAGE of affinity-purified SCGF of KPB-M15 origin. Affinity-

purified SCGF preparations, peaks I and II fractions, were reduced at 37'C for 2h with 2% SDS and/or 5% 2-mercaptoethanol and/or 20 HIMdithiothreitol incombination, and applied to 12.5% polyacrylamide gel. The protein bands weredetected by silver staining. Lanes I to 4 and 9, peak I fractions (1.5 Mgproteinloaded/lane); Itine* 5 to S and 10, peak II fractions (400 ng protein loaded/lane).Standard marker proteins were used for molecular weight determination; phos-phorylase-b (M, 94,000), albumin (M, 67,000), ovalbumin (M, 43,000), carbonicanhydrase (M, 30,000), trypsin inhibitor (M, 20,100) and a-lactalbumin (M,14,400).

200

SCGFFig. 6. Dose-response curve of SCGF for BPA and GM-colony potentiation.

Various concentrations of affinity-purified KPB-M l S SCGF, peak II fractions,were assayed for BPA (•)and GM colony potentiating activity (O). Basal HI-1E growth in the absence of BPA-containing materials was 4.7 ±1.7 erythroidhursts/5 x IO4BM cells (negative control). Maximally stimulated BFU-E cultureformed 92.3 ±3.9 erythroid bursts/5 x IO4BM cells (positive control). Negativecontrol CFU-GM cultures formed 119 ±6.9 GM colonies/5 x IO4BM cells.

of 800% of the negative controls. After formation of the plateau,peculiarly, BPA decreased to the level of the negative controlsat a 50-fold higher range than a half-maximal one. The dose-response curve for GM colony potentiation showed a closesimilarity to the situation with BPA. Affinity-purified SCGFpreparation of placenta! origin exhibited similar biological activities.

DISCUSSION

The KPB-M 15 myeloid cell line and human placenta werefound to produce SCGF. Monocytoid cell line U937 andHTLV-transformed T-lymphoblast cell lines MT-1, ATL-5S,and HUT 102 reportedly elaborating EPA or GM-CSF (4, 6,7,10), were expected to be positive, but were uniformly negativefor SCGF production. SCGF was not detected in the purified

rhGM-CSF preparation. In this respect, anti-SCGF MoAbs241 and 311 did not cross-react with EPA or GM-CSF, eliminating the possibility that SCGF is immunologically analogousto EPA or GM-CSF. Also SCGF activities differ from those ofEPA or GM-CSF, while they all have BPA. As yet, no regulatorspecific for BPA has been discovered, and all GM-CSF or EPAhave BPA cross-reactivity (3, 11, 12). Because a myeloid cellline, KPB-M 15, produces SCGF, does not appear to mean thatall other myeloid cell lines such as KG-1, ML-3, HL-60, orKYO-1 do so. We have no explanation to specify the attributesexclusive for the KPB-M 15 cells to produce SCGF. Recently,human IL-3 cDNA has been cloned using cDNA probe for IL-3 from the UCD-144-MLA cell line (33). Biological activitiesof rhIL-3 are similar to that of rhGM-CSF, while the formerappears to act on the more immature hematopoietic stem cells.It is possible but presently uncertain if SCGF could be analogous or identical to human IL-3. Molecular cloning is warrantedto further define the protein in reference to other hematopoieticregulators, particularly human IL-3 or GM-CSF.

Partially purified GM-CSF and erythropoietin were negativefor SCGF, and so were lectin (PHA, Con A, or PWM)-stimu-lated, lectin-free, and MLR-LCM. Particularly, both erythropoietin from human urine (Toyobo) and PHA-LCM are knownto be good sources of BPA and GM-CSF activity. Here, thesame situation described above should be considered whetherGM-CSF cross-stimulates as BPA or, less likely, two separablemolecules, GM-CSF and BPA, coexist in the preparations. Itcannot be excluded that SCGF present in various materialsmight be overlooked, considering the present sensitivity ofenzyme immunoblotting systems. However, materials containing a trace amount of SCGF, otherwise negligible by the presentEIA method, are unsuitable as a sufficient source of SCGF.

EIA with Western blotting of KPB-M 15-conditioned medium demonstrated two distinct anti-SCGF MoAb-defined protein bands with molecular weights of 20,000 and 22,000. Theprotein bands with different mobility could be explained by theextent of glycosylation or some degradation products of SCGF.Similar findings were reported; rat anti-mouse IL-3 inumino-globulin immunoprecipitated [35S]methionine-labeled WEHI-3-derived IL-3 to form two protein bands (34). Digestion withglycosidase or tunicamycin treatment lower the molecularweight of glycoproteins (35, 36), indicating that two distinctSCGF bands are principally related to glycosylation. In fact,the SCGF molecule has no subunit structures; affinity-purifiedSCGF presented a single protein band with a molecular weightof 20,000 even under various reducing conditions (Fig. 5).

Affinity-purified KPB-M 15 SCGF exhibited biological activities in vitro, i.e., BPA and GM colony potentiation. However,the most important finding was that both BPA and GM colonypotentiating activity were unexpectedly decreased at higherconcentrations of even highly purified SCGF. This so-calledhigh dose inhibition is ascribed to a minute amount of inhibitorspossibly contaminating the preparation. Usually, regulators,e.g., GM-CSF, purified to complete homogeneity, never exhibithigh dose inhibition. Possible explanations for the observationsare: first, incomplete purification or the presence of an unknownartifact (further purification is now underway); second, theinvolvement of some physiologically intriguing mechanism,since a similar high dose inhibition is observed in purified EPA(3). As neither of these explanations are satisfactory, biologicalaspects of the regulator acting on the intermediate stage between early hematopoietic events should be further studied.

The KPB-M 15 myeloid cell line stably provides a considerable amount of SCGF of leukemia cell origin. HPCM is another

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HUMAN HEMATOPOIETIC SURVIVAL AND GROWTH FACTOR

source of SCGF of normal cell origin. Whether these twoSCGFs are different from each other in their molecular structure remains to be clarified. SCGF, obtained by one-step im-munoadsorbent affinity purification, may be useful in elucidating the mechanisms involved in human early hematopoiesis,including self-generation of the stem cells.

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1987;47:5025-5030. Cancer Res   Atsunobu Hiraoka, Tadashi Ohkubo and Minoru Fukuda  as Detected by a Monoclonal Antibody

PlacentaFactor by a Myeloid Leukemia Cell Line (KPB-M15) and Production of Human Hematopoietic Survival and Growth

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