Feb 17, 1986 - glycoproteins (Sachs, 1970; Metcalf, 1977; Burgess and Met- calf, 1980). ... 1985; Lee et al., 1985). More recently, recombinant human GM-.
The EMBO Journal vol.5 no. 5 pp. 871 - 876, 1986
Purification and characterization of human granulocyte colonystimulating factor (G-CSF)
H.Nomura, I.Imazeki, M.Oheda, N.Kubota, M.Tamura, M.Ono, Y.Ueyamal and S.Asano2 New Drug Research Laboratories, Chugai Pharmaceutical Co., 41-8, 3 Takada, Toshima-ku, Tokyo 171, 1Department of Pathology, School of Medicine, Tokai University, Boseidai, Isehara City, Kanagawa 259-11, and 2Department of Pathological Pharmacology, Institute of Medical Science, University of Tokyo, 6-1, 4 Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by L.Sachs
A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19 000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single N112-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological- activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 x 108 colonies/105 non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis. Key words: human granulocyte colony stimulating factor/NH2terminal sequence/purification
Introduction By clonal assay of bone marrow cells using the semi-solid culture system, it has been shown that neutrophilic granulocytes originate from multipotent stem cells and that the proliferation and differentiation in vitro of the progenitor cells of the granulocyte macrophage lineage are regulated by a family of glycoproteins (Sachs, 1970; Metcalf, 1977; Burgess and Metcalf, 1980). These molecules are generally called colonystimulating factors (CSFs) or macrophage and granulocyte inducers (MGIs) (Landau and Sachs, 1971; Sachs, 1978, 1980); on the basis of morphology of the colonies stimulated, these factors are classified into at least four biochemically distinct subtypes, that is, multi-, granulocyte-macrophage (GM)-, macrophage (M)- and granulocyte (G)-CSFs (Metcalf, 1985; Sachs, 1985). Multi-CSF may support the proliferation of all the hemopoietic cells including multipotent stem cells, GM-CSF may stimulate the production of granulocytes and/or macrophages mainly from their committed progenitor cells, M-CSF may be a unipotent factor for monocyte and macrophage lineage and GCSF may be almost specific for granulocytic lineage. In addition, some of the CSFs have recently been shown to be capable of stimulating some of the functions of the mature granulocytes (Gasson et al., 1984; Weisbart et al., 1985) and G-CSF can indirectly (Sachs and Lotem, 1984) induce differentiation of one line (WEHI-3B) of myeloid leukemia cells (Nicola et al., 1983). In the murine system, all the CSFs have been purified to -
IRL Press Limited, Oxford, England
homogeneity (Landau and Sachs, 1971; Nicola et al., 1983; Stanley and Heard, 1977; Burgess et al., 1977; Clark-Lewis et al., 1984). With use of the purified materials, the above actions have been confirmed and analysis of interactions among the factors is now possible. Furthermore, successful isolation of fulllength cDNA coding the multi- (Ihe et al., 1983) or GM-CSFs (Gough et al., 1984) has been reported. This makes it possible to produce large amounts of the corresponding CSF for further study. In contrast, the studies in the human system have not progressed so far. Purification of the CSFs of human origin has been reported only recently, for two of the four subclasses, multi(Welte et al., 1985) and GM-CSFs (Gasson et al., 1984), and only the gene coding GM-CSF has been cloned (Wong et al., 1985; Lee et al., 1985). More recently, recombinant human GMCSF was reported to be a multi-lineage hemopoietin (Sieff et al., 1985). The human counterpart of mouse G-CSF has not been purified to homogeneity although it was shown to be present in the CSF-,B fraction partially purified from the conditioned medium of human placenta (Nicola et al., 1985). For investigations of the regulatory mechanisms of in vivo granulopoiesis in humans, it is necessary to purify completely the human analogue of mouse G-CSF as well as multi- and GM-CSFs. Here we describe the establishment of a human squamous cell line, which we term CHU-2, that produces large amounts of CSF, and the successful purification of a granulocyte-lineage-specific CSF effective on both human and mouse bone marrow cells from serum-free conditioned medium of these cells. The biochemical properties including a unique NH2-terminal amino acid sequence of this human G-CSF is reported.
Results Production of CSFs by CHU-2 cells The CHU-2 cell line constitutively produced CSFs which were active on both human and mouse bone marrow cells. The activities were detectable even 24 h after change to the serum-free medium and became highest after 4 days incubation. Figure la shows the representative dose - response curve of the pooled conditioned medium (CM) assayed on both systems. In both systems, maximum colony formation was observed at ranges of 0.5-1.0% (v/v) CM concentration, and the activity was calculated to be -24 000 colonies/ 105 non-adherent human bone marrow cells/ml, - 10 times as high as those obtained from other human CSF-producing tumors (Okabe et al., 1982a, 1982b; DiPersio et al., 1978; Golde et al., 1978; Wu et al., 1979). In the human system, granulocyte colonies were predominantly observed in the period of 7-10 days culture, but on day 14 various types of colonies including granulocytes, macrophages, eosinophils, and poorly-differentiated cells were observed. This suggests that the CHU-2 CM may contain a wide spectrum of biologically active molecules. In contrast to the human system, granulocyte colonies were predominantly observed in the period of 4-7 days with mouse bone marrow cell culture, and beyond 7 days only macrophage and a small number of granulocyte - macrophage colonies were observed. 871
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Fig. 1. Dose -response curves for the colony stimulating activities (CSAs) of (a) crude CHU-2 conditioned medium and (b) the purified G-CSF. Serially diluted samples were added to the cultures. CSAs were expressed as % of maximum numbers of colonies formed in each experiment. Open circles represent CSAs in the human bone marrow assay, and closed circles represent CSAs in the murine bone marrow assay. In human cell assay, the maximum numbers of colonies were 109 and 243, in (a) and (b), respectively and, in the absence of samples, no colony was formed in both experiments. In the murine assay, the maximum numbers of colonies were 122 and 47, in (a) and (b), respectively and the numbers of colonies formed in the absence of samples, 45 in (a) and 1 in (b), were subtracted from each value.
Purification of G-CSF A representative elution profile when the concentrated CM was applied to Ultrogel AcA-54 is shown in Figure 2. Two distinct activities were separated according to the difference in mol. wt The larger mol. wt (6 x 104) stimulated macrophage colony formation by mouse marrow cells but not by human marrow cells. In contrast, the small one (mol. wt 2 x 104) stimulated mainly granulocyte colony formation by mouse bone marrow cells, and day 7 granulocyte and day 14 various type colony formation by human bone marrow cells. Therefore the low mol. wt fraction (B in Figure 2) was considered to contain the factor specific to the granulocyte lineage. It was then applied to the next purification step, reversed-phase h.p.l.c. Most of the proteins were not retained on the column in the presence of 30% n-propanol, and the bound proteins were separated successfully into five or six peaks by a linear gradient of 30-60% n-propanol as shown in Figure 3a. Fortunately, the activity was demonstrated only in the last peak eluted at a concentration of 40% n-propanol. The result of re-chromatography shown in Figure 3b confirmed this. Further purification was carried out by applying the active fractions onto a TSK-G3000 SW column. As shown in Figure 3c, the proteins were separated into six or more peaks and the activity was found at the major peak corresponding to a mol. wt of -2 x 104. A representative dose - response curve of this purified material assayed on both human and mouse bone marrow cells is shown in Figure lb. In the human system the specific activity was calculated to be -2.7 x 108 colonies/105 nonadherent human bone marrow cells/mg protein. This value was comparable with those reported with the purified form of human GM- and multi-CSFs (Welte et al., 1985; Wong et al., 1985). Maximum colony formation was observed at days 5-7 in the mouse system and at days 7-10 in the human system and the 872
Frc. No. Fig. 2. Gel filtration chromatography. 5 ml of concentrated serum-free CHU-2 conditioned medium (corresponding to 5 1 in original concentration, containing 373 mg protein) was applied onto an Ultrogel AcA-54 column (5 x 90 cm). The elution of proteins was monitored by the A280 (dotted line) and each fraction was assayed for murine CSA in 10-fold dilution (solid line with open circles). Arrows denote the elution positions of bovine serum albumin (BSA, mol. wt 66 200), ovalbumin (OVA, mol. wt 45 000) and cytochrome C (Cyt.C, mol. wt 12 400). The two active fractions (A and B) were pooled separately and concentrated as described in Materials and methods.
morphology of the colonies was exclusively granulocytic in both. The pooled active fractions of the final gel-permeation h.p.l.c. was then analyzed by SDS-PAGE and isoelectric focusing (IEF). Only one silver-stained band was visualized within the range of 0.375-3 yg protein on SDS-PAGE as shown in Figure 4, while on IEF the presence of three different bands (pls 5.5, 5.8 and 6.1) was demonstrated (Figure 5). However, the heterogeneity on IEF was reduced markedly by prior treatment of the sample with neuraminidase or mild acid as described in Materials and methods. Thus, as shown in Figure 5, the majority of the proteins migrated into one band of pI 6.1. Furthermore, co-migration of biological activity with stainable protein bands was clearly demonstrated with the native G-CSF as well as with the asialo form (Figure 6). Analysis ofamino acid composition and amino-terminal sequence The amino acid composition of the purified G-CSF is shown in Table I. Leu residues were the most abundant and 50% of the total amino acid residues were hydrophobic. Basic amino acids were relatively few. From the data, A280 of the 0.1 % (w/v) solution was calculated to 0.85 which is in good agrrement with the observed value of 0.86. There is one GalNH2 but no GlcNH2, indicating that the material was a glycoprotein with 0-linked glycosides. Analysis of the phenylthiohydantoin (PTH) derivatives produced sequentially by Edman degradation using a gas-phase sequencer has revealed that the NH2-terminal 21 amino acid sequence of the purified G-CSF is TPLGPASSLPQSFLLKCLEXV. 'X' represents an unidentified residue and Cys in position 17 was determined only when the performic acid-treated sample was used. As there are only two charged amino acids, the sequence seems to be very hydrophobic.
Purification and characterization of G-CSF
600~~~~~~~~~
(6
0
5
0.1
@1~~~~~~~
0.
..............
0
60
00
120 0
60
30
120 0
60
time min
Fig. 3. H.p.l.c. (a) Half of the concentrated fraction (B) (containing 28 mg protein) in Figure 2 was subjected to reversed-phase h.p.l.c. on YMC-C8 column (8 x 300 mm). The dotted line represents the A280 of the effluent, and the broken line represents % of n-propanol concentration in the effluent. Murine CSA was assayed with several pooled fractions, and the active fraction is indicated with a horizontal bar. (b) The other half of the concentrated fraction (B) in Figure 2 was purified in the same manner as in (a). The active fractions were combined and re-chromatographed under the same conditions as in (a). The solid line with closed circles represents murine CSA assayed in 10 000-fold dilution. (c) The active fraction in b (containing 264 ug protein) was further purified by gel-permeation h.p.l.c. on a TSK-G 3000 SW column (7.8 x 600 mm). Murine CSA was fractionated at the peak corresponding to a mol. wt of 20 000 (yield 140 jig of protein).
Discussion The present work yielded a human active CSF with the specific activity of 2.7 x 108 colonies/105 non-adherent human bone marrow cells/mg protein. The purity of the CSF was confirmed by: (i) coincidence of biological activity and protein peak separated by gel-permeation h.p.l.c., and it was demonstrated by SDSPAGE that this preparation was homogeneous with respect to mol. wt (19 000); (ii) co-migration of biological activity and stainable protein bands was obtained in analytical IEF, and this co-migration was also observed with a neuraminidase-treated preparation; (iii) the specific activity of the G-CSF (2.7 x 108 colonies/105 non-adherent human bone marrow cells/mg protein) is high enough to consider that there was no major contaminant, and the concentration required to obtain a half maximum colony formation (0.45 ng/ml = 2.37 x 10-11 M) is equivalent to human multi-CSF (Welte et al., 1985) and considerably lower than human GM-CSF (-50-fold) (Sieff et al., 1985); (iv) the result of NH2-terminal amino acid sequencing indicated that the purity of the CSF might be >90%, when a large amount (1 mg) of the sample was analyzed (data not shown). In addition to these points Nagata et al. (1986) have recently cloned the cDNA encoding human G-CSF by using oligonucleotide probes derived from the partial amino acid sequences of the G-CSF purified by us. They also have demonstrated that this cDNA was transcribed to synthesize biologically active human G-CSF in a COS cell culture. The purification of this factor resulted from the establishment of the human CSF-producing cell line (CHU-2), which produces large amounts of the CSF activity in the serum-free conditioned medium and the use of high efficiency h.p.l.c.s linked with the conventional purification procedures. The molecule is considered to be a hydrophobic glycoprotein with possible 0linked glycosides. It is apparent that its biochemical properties are different from those of the multi- and GM-CSFs already reported by others (Welte et al., 1985; Gasson et al., 1984). This was also confirmed by the fact that its NH2-terminal amino acid sequence had no homology to the corresponding sequences of these other factors (Ihle et al., 1983; Gough et al., 1984; Wong et al., 1985; Ben-Avram et al., 1985; Gasson et al., 1985; Jacobs et al., 1985).
1
2 3
4 5 -
~|-
KD 92.5 66.2
-451 0
- 31.0
- 21.5 - 14.14 Fig. 4. SDS-PAGE. 3, 1.5, 0.75 and 0.375 lsg of the purified samples were reduced and denatured as described in Materials and methods, then electrophoresed in a 15% Laemmli gel (lanes 1, 2, 3 and 4, respectively). Mol. wt marker proteins, phosphorylase B (92 500), BSA (66 200), ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500) and lysozyme (14 400), were treated as above, and 2 sg of respective markers were co-electrophoresed (lane 5). After electrophoresis, the protein bands were visualized by the silver staining technique.
The factor stimulated mainly granulocyte colony formation by both human and mouse bone marrow cells. Similar results were also obtained when we used a serum-free agar culture system or a methylcellulose culture system for mixed-type colony formation instead of the conventional agar culture system (data not shown). On the basis of these biochemical and biological data, we conclude that the purified CSF is a human-active granulocyte lineage-specific CSF, namely a G-CSF of human origin. Nicola et al. (1983) have purified the G-CSF from the medium conditioned by lung tissues of endotoxin-treated mice. They have also shown that the human analogue exists in a species of CSF (CSF-fl) partially purified from the conditioned medium of human
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3
12
4 5 6
7 8 9
PI 4.15 -4, 55
-5. 20 ;8. .5 -0r 5
-6 , 5
Fig. 5. Analytical IEF. 5 Ag of the purified sample was analyzed by flat bed non-denatured polyacrylamide gel IEF in the pH range of 4-6.5 (lane 2). The pl of the purified sample was determined from the mobilities of standard pI marker proteins, glucose oxidase (pl 4.15), soybean trypsin inhibitor (pl 4.55), fl-lactoglobulin A (pI 5.20), bovine carbonic anhydrase B (pI 5.85) and human carbonic anhydrase B (pl 6.55) (anes 1, 5 and 8). To exclude the effect of terminal sialic acids on pI, the purified sample was treated with neuraminidase (lane 9) or treated at pH 1.5, 80°C for 30, 60, 90 and 120 min as described in Materials and methods (lanes 3, 4, 6 and 7, respectively). U)
t1) 500 0
r
10 Untreated
(Fraction No.) I
[
I
*
a
*
0
Treated 10 En
(Fraction No.)
0.
a) Z., 0 0
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Fig. 6. Analytical IEF. 5 itg of untreated and neuraminidase-treated samples were electrofocussed as in Figure 5, then the gel was fractionated and extracted
as
described in Materials and methods. In the center of this
Figure, a schematic diagram of the protein bands is shown. No visible band corresponding to pl 6.1 was detected with this preparation of untreated GCSF. Cross-hatched bars represent number of mouse granulocytic colonies stimulated by the addition of 10% (v/v) of 100-fold diluted fractions. Dots represent the position of pl markers described in the legend to Figure 5.
placenta (Nicola et al., 1985). It has been reported that purified G-CSF is capable of inducing differentiation of a line of myeloid leukemia cells (WEHI-3B). Therefore, it was interesting to examine whether our CSF had a similar activity. In preliminary experiments, however, we could not detect differentiation of 874
Table I. Amino acid and amino sugar composition of the purified G-CSF Asx (Asp + Asn) Thr Ser Glx (Glu + Gln) Pro Gly Ala 1/2 Cys Val Met
4a
6b.c
13b.c 27 10 13 18 3b.c 7b.d 3b.c
Ile Leu Tyr Phe Lys His
Trp Arg GaiNH2 GicNH2
4b.d 33 3 6 4 5
2b 5 I
b.c
0
aAverage number of residues (mol/mol protein). bValues obtained from the sample hydrolysed with 4 N methanesulfonic acid (see Materials and methods). cCorrected for 0 h value by extrapolation (see Materials and methods). d72 h value (see Materials and methods). KG-1 (Koeffler and Golde, 1978) and HL-60 (Collins et al., 1977) cell lines (data not shown). We could not reach any conclusion from these results because of a possibility that the sensitivity of these cell lines for differentiation-inducing activities might vary among the clones (Sachs, 1980, 1982a, 1982b; Koeffler et al., 1980). In addition to the above, Sachs et al. suggested the possibility that the purified G-CSF might indirectly induce the differentiation of a leukemic cell line such as WEHI-3B via the production of endogeneous differentiation-inducing proteins by these leukemic cells themselves (Symonds and Sachs, 1982; Weisinger and Sachs, 1983; Sachs and Lotem, 1984). Although we cannot determine in the present work whether our CSF is the counterpart of mouse G-CSF or not, undoubtedly it will become a very usefil material for further investigations of human granulopoiesis. Growth of CHU-2 cells from which our G-CSF was originally derived could induce a marked granulopoiesis in the patient and in nude mice transplanted with the cells. Therefore it is expected that the tumor-derived CSF may be effective in vivo as well as in vitro. If so, the CSF merits further study as regards its possible therapeutic usefulness. As mentioned above, using oligonucleotide probes derived from the partial amino acid sequence of the CSF, Nagata et al. have recently isolated cDNA clones that encode human G-CSF from CHU-2 cDNA libraries and have demonstrated the presence of the specific transcripts only in the G-CSF producing cells, but not in other unrelated tumor cells (Nagata et al., 1986). Furthermore they also have obtained several genomic clones which encode exactly the same amino acid sequence as the cDNA mentioned above, from the chromosomal DNA library of human fetal liver, suggesting that the G-CSF is not a pathological but a physiological granulopoietic factor (Nagata et al., in preparation). When a large amount of the recombinant G-CSF becomes available, its in vivo roles in relation to the other CSFs and its clinical usefulness in human diseases will be more clearly defined. Materials and methods Materials
RPMI-1640, McCoy's 5A and 0.25% trypsin solution were purchased from Gibco. Fetal bovine serum (FBS) was purchased from General Scientific Laboratories (GSL) and horse serum was purchased from Flow Lab. These sera were pretested to obtain suitable lots for respective experiments. n-Propanol (sequencing grade) was purchased from Wako Pure Chemicals and trifluoroacetic acid (TFA) was purchased from Aldrich. Reagents for SDS-PAGE and neuraminidase were obtained from Nakarai Chem. Co. Silver stain kit and standard mol. wt marker proteins were purchased from Bio-Rad. Carrier ampholyte (Pharmalyte 4-6.5)
Purification and characterization of G-CSF and standard pl marker kit were purchased from Pharmacia Fine Chemicals. Methanesulfonic acid and 3-(2-aminoethyl)indole were obtained from Pierce Chem. Co. Culture of human CSF-producing tumor cells One of the CSF-producing human tumor cell lines, originally derived from a squamous cell carcinoma developed in the oral cavity of a Japanese female patient, was used in the present experiments. This line has been referred to as CHU-2. The ability of these cells to induce an in vivo neutrophilic granulocytosis had been suggested clinically and has been demonstrated by the successful serial heterotransplantation into nude mice. The cell line was established as follows. At the ninth in vivo passage in nude mice, a part of the transplantable tumor was cut into small pieces (I - 2 mm) followed by treatment with trypsin and EDTA. The resultant single cells were cultured at a concentration of 2 x 106 cells/ml RPMI-1640 medium containing 10% FBS. Four days later when the culture became confluent, the cells were treated with anti-mouse fibroblast antibody plus guinea pig complement (Kyokuto Chem. Co.) to eliminate contaminated mouse fibroblasts according to the method reported by Okabe et al. (1979). The continuous in vitro cell line grown in monolayer has been readily developed by detaching the cells from the surface followed by subeulturing 1/10 in fresh culture medium every 4 days. After the cell line was established, karyotypic analysis was performed, then the human origin of the cell line was confirmed by the observation of metacentric chromosomes, with a mode of 83 chromosomes per cell. The cells have an 'epithelial-like' morphology and a mean population doubling time of - 20 h. Preparation of the conditioned medium (CM) The CM to be used as a starting material for purification of CSF was obtained from large-scale serum-free cultures using the roller bottles as described (Okabe et al., 1982a). For this, 3 days after the cells were re-passaged in the medium containing 10% FBS, the medium was removed and the serum-free RPMI-1640 medium was added. After 4 days incubation the medium was harvested. The serumfree CM were pooled, concentrated 1000-fold by Hollow Fiber DC-10 (HP-05), DC-4(HP-05) and Diaflo PM-10 (Amicon Corp.) ultrafiltrations and then stored at -80°C until use. Gel filtration The concentrated medium was loaded on a column of Ultrogel AcA54 (LKB) (5 x 80 cm) equilibrated with 0.01 M Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and 0.01 % Tween 20, and eluted with the same buffer at a flow-rate of 50 ml/h. Active fractions were pooled and concentrated in a Diaflo cell (PM-10 membrane) for the next fractionation step.
Reversed-phase h.p.1.c. The above sample was mixed with n-propanol and TFA at final concentrations of 30% and 0.1%, respectively, and kept on ice for 30 min. After removal of the precipitate by centrifugation at 25 000 g for 10 min, the supernatant was injected onto a YMC-C8 column (Yamamura Chem. Lab.) equilibrated with 30% n-propanol containing 0.1% TFA and chromatographed at a flow-rate of 1.0 mil/min using Hitachi h.p.l.c. apparatus, Type 838-50. The adsorbed materials were eluted with a linear gadient of 30-60% n-propanol. Active fractions were pooled, re-chromatographed by the same procedure, and then concentrated 10to 20-fold by Centricon-10 membrane (Amicon Corp.). Gel-permeation h.p. . c. The sample from the reversed-phase h.p.l.c. was injected onto a TSK-G300 SW (Toyo Soda) column (7.8 x 600 mm), which was equilibrated with 40% npropanol containing 0.1 % TFA. Separations were performed at a flow-rate of 0.4 mil/min. Pooled active fractions were lyophilized. SDS-PAGE The lyophilized samples were dissolved in an appropriate volume of 62.5 mM Tris-HCl buffer (pH 6.8) containing 2% SDS and 5% 2-mercaptoethanol, and heated for 3 min at 100°C before loading. Electrophoresis was carried out by the method of Laemmli (1970). The gel slab was 1.5 mm thick and contained 15% (w/v) acrylamide. After electrophoresis, the gel was silver-stained by the method of Merril et al. (1981).
Isoelectric focussing (IEF) The lyophilized sample (100 jsg) was dissolved in 100 ul of 6 M urea (Ultra pure;
BRL) solution then 5 isg of samples were analyzed in non-denatured 5% polyacrylamide gel containing 13% (v/v) glycerol and 6.3% (v/v) Pharmalyte 4-6.5. IEF was performed by using FBE-3000 flat bed IEF apparatus (Pharmacia Fine Chem.) for 2 h at a constant power of 30 W. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250 (Sigma). In some cases, 100 ytg of sample was treated with 10 mU of neuraminidase (from Arthrobacter ureafaciens) in 10 mM sodium acetate buffer (pH 5.0) at 37°C for 3 h, alternatively heated at 80°C for 30-120 min in the presence of 6 M guanidineHCI (pH 1.5), as described by Nicola et al. (1979), then analyzed in the same manner as above. To examine the coincidence of biological activity and protein
bands, 5 itg of untreated and neuraminidase-treated samples were separated as above, then the gel was cut into small pieces (5 mm width) followed by extraction with 3 mi of 0.01 M Tris-HCI buffer (pH 7.4) containing 4 M guanidineHCI and 0.1 % (v/v) Tween-20. The resulting fractions were diluted 100-fold with RPMI-1640 medium containing 10% (v/v) FBS, then the biological activity was assayed by murine bone marrow colony formation. Amino acid analysis The CSF protein was hydrolysed under the following conditions: (i) for 24 h at 110°C in 6 N HCI, or (ii) for 24-72 h at 110°C in 4 N methanesulfonic acid containing 0.2 % 3-(2-aminoethyl)indole according to Simpson et al. (1976). Thereafter, the aliquots were analyzed using a Hitachi Automatic Amino Acid Analyzer Type 835. Amino-terminal sequence analysis The purified CSF was subjected to Edman degradation using a gase-phase sequencer (Applied Biosystem Inc.) (Hunkapiller et al., 1981). The resultant PTH derivatives were analyzed by reversed-phase h.p.l.c. using an Ultrosphere ODS column (Beckman Inc.). Each cycle of PTH derivative was injected onto the column, pre-equilibrated with 15 mM Na-acetate buffer (pH 4.5) containing 40% (v/v) acetonitrile and eluted isocratically. The retention time of the MTH derivative was compared with those of standard PTH amino acids separated under the same conditions. Protein estimation The protein content of samples was measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Biological assay for CSF activity As the main CSF derived from CHU-2 was supposed to be effective not only on human but also mouse bone marrow cells as indicated by the in vivo findings, the activities were usually monitored using mouse bone marrow cells which could be prepared more readily. But for the initial assessment and final determination of the CSF, non-adherent human bone marrow cells (Messner et al., 1973) were also used. The mouse assay system was basically similar to the methods originally described by Pluznik and Sachs (1965, 1966; Ichikawa et al., 1966) or Bradley and Metcalf (1966) but with slight modifications. Briefly, 5 x 104 marrow cells from female C3H/HeN mice were cultured in 1 mi of modified McCoy's SA medium containing 0.3% (w/v) purified agar (Difco) and 40% (v/v) horse serum with 0.1 ml of various dilutions of the test samples. The use of horse serum instead of FBS seemed to raise the sensitivity of the assay. The human assay system was described previously (Asano et al., 1977). Numbers of colonies were enumerated by the criteria that the aggregates containing > 50 cells were colonies using an inverted microscope (Olympus CK). The CSF activities were calculated in the linear portion of a dose - response curve, and specific activities were expressed as number of colonies formed/I x 105 non-adherent human bone marrow cells/mg protein. Morphological classification was performed according to the method described by Kubota et al. (1980) by either May -Giemsa staining or double esterase staining and Bieblich Scarlet (for eosinophils) staining (Konwalinka et al., 1980).
Acknowledgements The authors are indebtd to Dr Tatsuji Nomura, Dr Ken-ichi Tamaoki, Dr Nakaaki Ohsawa and Dr Hajime Sano for their continuous encouragements during the course of this work.
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