Proc. Nati. Acad. Sci. USAVol. 83, pp. 9739-9743, December 1986Medical Sciences

Oncostatin M: A growth regulator produced by differentiatedhistiocytic lymphoma cells

(cytokine/tumor inhibitor/differentiation)

JOYCE M. ZARLING, MOHAMMED SHOYAB, HANS MARQUARDT, MARCIA B. HANSON, MARIO N. LIOUBIN,AND GEORGE J. TODAROOncogen, 3005 First Avenue, Seattle, WA 98121

Contributed by George J. Todaro, August 29, 1986

ABSTRACT A polypeptide termed oncostatin M, whichinhibits the replication of A375 melanoma and other humantumor cells, but not normal human fibroblasts, has beenisolated from serum-free supernatants of U-937 histiocyticlymphoma cells that have been induced to differentiate intomacrophage-like cells following treatment with the phorbolester phorbol 12-myristate 13-acetate. No such growth inhib-itory activity is detected in the supernatant of untreated U-937cells, indicating that the protein is induced or increased inexpression in the phorbol ester-induced differentiated cells.Oncostatin M is stable between pH 2 and 11 and after heatingfor 1 hr at 560C but is not stable at 90TC. Purification ofoncostatin M has been achieved by gel chromatography andreversed-phase HPLC, using sequentially acetonitrile andn-propanol in the presence of aqueous trifluoroacetic acid. Theapparent molecular weight of oncostatin M is -18,000, asdetermined by gel chromatography, and 28,000, as determinedby polyacrylamide gel electrophoresis. The amino-terminalamino acid sequence of the purified polypeptide has beendetermined.' No substantial sequence homology betweenoncostatin M and other proteins was found, including othertumor cell inhibitory proteins produced by mononuclear cells.Oncostatin M, therefore, appears to represent a distinct cellgrowth regulator.

Mononuclear cells including T cells, natural killer cells, andmacrophages have been shown to inhibit growth of cells ofmany different tumors (1-6) either by direct contact or bysoluble factors they produce. There has been considerableeffort expended to elucidate factors produced by mononu-clear cells that can directly inhibit tumor cell replicationand/or can augment T-cell immune responses or natural killercell and macrophage activity against tumors. Mononuclearcell-derived factors with such activities include the interfer-ons (for review see ref. 7), tumor necrosis factor (8-10),lymphotoxin (11, 12), type ,B transforming growth factor (13),interleukin 1 (14-19), interleukin 2 (for review see ref. 20),leukoregulin (21), and some less well-defined factors (22, 23).Since each ofthese factors have different spectra of activitiesand may interact differently in conjunction with other factors,there remains a strong interest in isolating and characterizingadditional factors that could be used to suppress tumorgrowth. Following treatment of the U-937 histiocytic lym-phoma cell line (24) with the phorbol ester phorbol 12-myristate 13-acetate (PMA), growth is inhibited, and the cellsare induced to differentiate, expressing cell surface markers,enzymes, and activities such as the phagocytic activityassociated with macrophages (25). We have thus undertakenstudies aimed at isolating, purifying, and characterizingtumor inhibitory factors produced by these macrophage-like

cells. We report here that PMA-treated U-937 cells release apolypeptide that inhibits the growth of A375 melanoma andother tumor cells, but augments the growth of normalfibroblasts. This factor, termed oncostatin M, has beenpurified by gel chromatography and reversed-phase HPLCand appears to be a distinct cell growth regulator based on itsamino-terminal amino acid sequence and its biological activ-ities.

MATERIALS AND METHODS

Cell Lines and Propagation. All cell lines used were ofhuman origin. U-937 cells, derived from a histiocytic lym-phoma (24), were grown in RPMI 1640 medium with 10%(vol/vol) fetal bovine serum, L-glutamine, and penicil-lin-streptomycin. All of the following cell lines were grownin Dulbecco's modified Eagle's medium (DMEM) supple-mented with 10% (vol/vol) fetal bovine serum, L-glutamine,and penicillin-streptomycin. The A375 cell line was derivedfrom a human melanoma (26); A549, from a lung carcinoma(26); HTB10, from a neuroblastoma (27); SK-MEL-28, froma melanoma (28); and WI-26 and WI-38 cells were derivedfrom human embryonic lung (29, 30).

Cell Growth Inhibition Assay. The assay was performed inflat 96-well plates (3596; Costar, Cambridge, MA). HumanA375 melanoma cells were used as a sensitive indicator cellline. Cells (3 x 103 cells) in 0.1 ml of DMEM supplementedwith 10% (vol/vol) fetal bovine serum and penicillin-streptomycin were placed in each well. Three hours later, 0.1ml of test samples was added to each well. Plates wereincubated at 37°C for 3 days. Then 0.025 ml (0.5 ,uCi; 1 Ci =37 GBq) of a solution of [3H]thymidine (specific activity,27,Ci/,ug; New England Nuclear) was added to each well forthe final 6 hr of incubation and [3H]thymidine incorporationwas determined by liquid scintillation counting. One unit ofgrowth inhibitory activity (GIA) was defined as the amountof factor required to inhibit [3H]thymidine incorporation intoA375 cells by 50%.

Soft Agar Colony Inhibition Assay. A 0.5-ml base layer of0.5% agar (Agar Noble; Difco) in DMEM containing 10%(vol/vol) fetal bovine serum was added to 24-well Costartissue culture plates. Then 0.5 ml of 0.3% agar containing thesame medium, 1-2.5 x 103 A375 cells, and the factor to betested at various concentrations, were overlaid on the baselayer of agar. The plates were incubated at 37°C in ahumidified atmosphere of 5% C02/95% air and refed after 7days by addition of 0.5 ml of 0.3% agar containing the samemedium and concentrations of the factor. Colonies wereenumerated unfixed and unstained.

Source of Oncostatin M. The U-937 cell line was grown insuspension in RPMI 1640 medium supplemented with 10%

Abbreviations: PMA, phorbol 12-myristate 13-acetate; GIA, growthinhibitory activity.

9739

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

May

25,

202

0

9740 Medical Sciences: Zarling et al.

(vol/vol) fetal bovine serum, L-glutamine, and penicil-lin-streptomycin. When the cells had reached a density of-1.5 x 106 cells per ml, the cells were seeded in fresh mediumcontaining 5% (vol/vol) fetal bovine serum at a density of 7.5x 105 cells per ml in tissue culture flasks containing PMA (10ng/ml). Three days later, after the cells had become adher-ent, the cell monolayers were washed extensively withserum-free RPMI 1640 medium and were then incubated inserum-free medium supplemented with amino acids for 24 hr,at which time the supernatants were collected and replacedwith fresh serum-free medium. A second collection of super-natant was made the following day, and the supernatantswere concentrated by ultrafiltration (PM10 filter, molecularweight cut-off, 10,000; Amicon). The concentrated superna-

tant was the starting material for the purification ofoncostatinM.

Purification of Oncostatin M. The retentate, after ultrafil-tration, was dialyzed against 1 M acetic acid, and thesupernatant, after centrifugation, was concentrated bylyophilization and reconstituted in 0.1% trifluoroacetic acid(CF3COOH) containing 40% (vol/vol) acetonitrile for subse-quent gel chromatography on a column (21.5 x 600 mm) ofBio-Sil TSK-250 (Bio-Rad). The column was equilibratedwith 0.1% CF3COOH containing 40% (vol/vol) acetonitrile ata flow rate of 2.5 ml/min. Fractions comprising the majorgrowth inhibitory activity were pooled.The final purification of oncostatin M was achieved by

reversed-phase HPLC. The separations were performed on a

1LBondapak C18 column (10-,im particle size, 7.8 x 300 mm;Waters) at room temperature. The primary mobile phase was0.1% CF3COOH, and the secondary mobile phase wasacetonitrile containing 0.1% CF3COOH. The concentrationof acetonitrile was increased linearly (0.1% per min) duringiS0 min at a flow rate of 2.0 ml/min at room temperature forelution of proteins. Oncostatin M-containing fractions were

rechromatographed on an analytical gBondapak-C18 column(3.9 x 300 mm), using acetonitrile for elution of proteins, andrechromatographed on the same column, with n-propanolcontaining 0.1% CF3COOH as the mobile-phase modifier.The n-propanol concentration was increased linearly (0.05%per min) during 4 hr at a flow rate of 0.5 ml/min.Amino Acid Sequence Determination of Oncostatin M. For

amino-terminal amino acid sequence analysis, oncostatin M(1.6 ,ug) was reduced with 20 mM dithiothreitol in 100 jLI of0.4 M Tris.HCl/6M guanidine HCl/0.1% Na2EDTA, pH 8.5,for 2 hr at 50°C and subsequently S-carboxamidomethylatedwith 45 mM iodoacetamide for 30 min at 22°C. The S-carboxamidomethylated oncostatin M was desalted on a

RP-300 column (2.1 x 30 mm; Applied Biosystems, FosterCity, CA). The peptide was eluted with a gradient of aqueousacetonitrile containing 0.1% CF3COOH. The concentrationof acetonitrile was increased linearly (1% per min) during 1 hrat a flow rate of 100 gl/min at 35°C.Automated repetitive Edman degradation (31) of S-

carboxamidomethylated oncostatin M was performed in themodel 470A protein sequencer (32) (Applied Biosystems).Sequenator fractions were analyzed by reversed-phaseHPLC with a model 120A phenylthiohydantoin analyzer(Applied Biosystems), using a sodium acetate buffer/tetra-hydrofuran/acetonitrile gradient for elution (33).NaDodSO4/PAGE and Electroelution. Oncostatin M (ap-

proximately 2 jig) was subjected to preparative NaDodSO4/PAGE on a 12-20% acrylamide gradient gel (34). The gel wasstained with Coomassie brilliant blue R-250 (Serva, GardenCity Park, NY) and was destained in a solution of acetic acid[5% (vol/wt)] and methanol [17% (vol/vol)]. The proteinband was excised with a razor blade, electroeluted, andelectrodialyzed as described (35).

RESULTS AND DISCUSSION

Source, Concentration, and Initial Characterization ofOncostatin M. U-937 histiocytic lymphoma cells, after treat-ment with PMA, become adherent and acquire many of thecharacteristics of macrophages (25). Serum-free superna-

tants collected from U-937 cells treated for 3 days with PMAwere found to inhibit the proliferation of A375 melanomacells (Table 1). In several experiments, 50% inhibition of[3H]thymidine incorporation into A375 cells was observedwith concentrations of supernatants ranging from 2 to 6%(vol/vol). In contrast, neither undiluted supernatant nor

supernatant concentrated -50-fold from untreated cells pos-

sessed detectable anti-proliferative activity against A375cells (data not shown). These results indicate that growthinhibitory factors are induced, or increased in expression, inU-937 cells that have differentiated into macrophage-likecells. The anti-proliferative activity was found to be stable totreatment with 1 M acetic acid or 1 M ammonium hydroxide(Table 1) and to treatment at 560C for 1 hr; however, theactivity was abrogated by treatment for 1 hr at 90'C and bytreatment with trypsin (data not shown).

Purification of Oncostatin M. A summary of the stepsleading to the isolation and recovery of oncostatin M ispresented in Table 2. The supernatant of the PMA-treatedU-937 cells was concentrated, dialyzed against 1 M aceticacid, and lyophilized, and the acid-soluble oncostatin Mcontaining crude material was subjected to gel chromatogra-phy (Fig. 1). A major peak of anti-proliferative activityagainst A375 cells, with an apparent molecular weight of18,000, was found.

Oncostatin M was further purified by reversed-phaseHPLC. The biologically active fractions, after gel chroma-tography, were diluted 1:2 with 0.1% CF3COOH, and thenchromatographed on a preparative ,Bondapak C18 column.A typical elution pattern is illustrated in Fig. 2A. GIA ofindividual fractions was determined and the four fractionswith peak activity were taken for rechromatography on an

analytical ,uBondapak C18 column (Fig. 2B). Oncostatin Meluted at approximately 41% (vol/vol) acetonitrile. A 140-fold purification after gel chromatography was obtained.Rechromatography of the biologically active fractions on

1LBondapak support was chosen for the final two purificationsteps using n-propanol as the organic solvent for the elutionof peptides. A typical elution pattern is shown in Fig. 2C.Rechromatography of the active fraction, using more shallowgradient conditions, is shown in Fig. 2D. GIA copurified witha distinct absorbance peak. Oncostatin M was eluted from a

,uBondapak C18 column at =26% (vol/vol) n-propanol. Thepurification of oncostatin M was -3500-fold with a yield of8% of the initial total GIA. The overall recovery of oncostatinM from the gel permeation step through the final reversed-

Table 1. Effect of various treatments of serum-free supernatantsfrom PMA-induced U-937 cells on growth inhibitory activity ofA375 melanoma cells

[3H]Thymidine incorporation into A375

Treatment of cells, cpmsupernatant 25% 12.5% 6.2% 0O

Medium control 39,780Untreated 7206 13,896 16,0001 M acetic acid 6670 17,073 18,7831 M NH3 6956 15,016 13,923The supernatants were treated with 1 M acetic acid or 1 M

ammonium hydroxide (NH3) for 1 hr and then dialyzed againstmedium. Medium, untreated supernatant, and acetic acid- or am-monium hydroxide-treated supernatants were tested for their abilityto inhibit [3H]thymidine incorporation into A375 cells in triplicatewells for the indicated concentrations (vol/vol).

Proc. Natl. Acad. Sci. USA 83 (1986)

Dow

nloa

ded

by g

uest

on

May

25,

202

0

Proc. Natl. Acad. Sci. USA 83 (1986) 9741

Table 2. Summary of purification of oncostatin M

SpecificProtein, GIA,* activity, Yield,

Fraction mg units x 10-3 units x 10-3/mg %

Crude 220 750 3 100TSK-250 27.5 200 7 27HPLC A 0.610 125 205 17HPLC B 0.130 135 1,038 18HPLC C 0.008 64 8,000 9HPLC D 0.005 60 12,000 8

Serum-free supernatant from PMA-treated U-937 cells was col-lected, concentrated, and purified. HPLC A, B, C, and D refer to theHPLC steps depicted in Fig. 2 A, B, C, and D, respectively. GIA wasdetermined by testing the unfractionated, concentrated (crude)U-937 supernatant and aliquots of fractions from the TSK-250 andHLPC C18 columns for inhibition of [3H]thymidine incorporation intoA375 melanoma cells.*One unit of GIA was defined as the amount of factor required toinhibit [3H]thymidine incorporation into A375 cells by 50%.

0-8 A

060=6C~~-40

-- 20~~~-2Q~~~~~~~40 8 1 160 , I040 80 120 160

0.16

CI 0.12

0.08

0.04

phase HPLC step was approximately 30% (Table 2), and thespecific activity of purified oncostatin M was 12 x 106units/mg of protein. This purified protein was used fordetermining its amino-terminal amino acid sequence.Amino-Terminal Amino Acid Sequence of Oncostatin M.

Automated Edman degradation of reduced and S-carboxami-domethylated oncostatin M was performed with 90 pmol(based on the initial yield of the identified phenylthiohydan-toin derivative of isoleucine-3). Unambiguous identificationof phenylthiohydantoin derivatives of amino acids was pos-sible up to residue 26. The partial amino-terminal amino acidsequence of oncostatin M is shown in Fig. 3.The partial amino acid sequence of oncostatin M was

compared with each known sequence stored in the protein

1.6

1.2

0fn)c<J

69K 43K25K 137K 6K

f((

I'

II

'I

I I

I

I

fl

0.41-

20 40 60

Retention Time (min)

16

I12 l

ro)0

x

8 .00D

H4 O:

O0

FIG. 1. Gel chromatography of supernatant from PMA-treatedU-937 cells on a Bio-Sil TSK-250 column. Elution pattern of 20 mgof acid-soluble protein. The elution was performed with 0.1%CF3COOH containing 40% (vol/vol) acetonitrile at 22°C at a flow rateof 2.5 ml/min, and 5-ml fractions were collected. Aliquots of theindicated fractions were evaporated and assayed for GIA on A375cells (dashed lines). The solid line gives the protein absorbance at 230nm. The following proteins were used as markers: bovine serumalbumin (Mr, 69,000), ovalbumin (Mr, 45,000), chymotrypsinogen(Mr, 25,000), bovine pancreatic ribonuclease A (Mr, 13,700), insulin(Mr, 6000). The elution volumes of the standard proteins areindicated.

20 i

0

ro0

60 In

40 D

20 H0D

0

80 160 240Retention Time (min.)

FIG. 2. Purification of oncostatin M by reversed-phase HPLC on,Bondapak C18 support. (A) Elution pattern of 27.5 mg of proteinfrom gel chromatography-purified oncostatin M (protein collectedfrom 11 gel chromatographic preparations, Fig. 1) on a preparative,Bondapak C18 column with 0.1% CF3COOH. Elution was achievedwith a linear 20-min gradient of 0-30% (vol/vol) acetonitrile with0.1% CF3COOH, followed by a linear 150-min gradient of 30-45%(vol/vol), a linear 20-min gradient of 45-55% (vol/vol), and a linear10-min gradient of 55-100% (vol/vol) acetonitrile with 0.1%CF3COOH. Fractions (4-ml) were collected. Aliquots of the indicat-ed fractions were evaporated and assayed for GIA (dashed lines). (B)Rechromatography of the four active fractions shown in A. Elutionof 610 jug of protein on an analytical ,uBondapak C18 column wasachieved with a linear 10-min gradient of0-35% (vol/vol) acetonitrilewith 0.1% CF3COOH, followed by a linear 100-min gradient of35-45% (vol/vol), and a linear 10-min gradient of 45-100% (vol/vol)acetonitrile with 0.1% CF3COOH. Fractions (2 ml) were collected.Aliquots of the indicated fractions were evaporated and assayed forGIA (dashed line). (C) Rechromatography of the four active fractionsshown in B. Elution of 130 ,ug of protein on an analytical ,uBondapakC18 column was achieved with a linear 40-min gradient of 23-35%n-propanol, followed by a linear 240-min gradient of 23-35% n-propanol with 0.1% CF3COOH. Fractions (2 ml) were collected.Aliquots of the indicated fractions were evaporated and assayed forGIA (dashed line). (D) Rechromatography of the active fractionshown in C. Elution of 8 ,ug of protein on an analytical ,uBondapakC18 column was achieved with a linear 40-min gradient of 0-21%(vol/vol) n-propanol, followed by a linear 200-min gradient of21-30% (vol/vol), and by a linear 20-min gradient of 30-35%n-propanol with 0.1% CF3COOH. Fractions (2 ml) were collected,and aliquots of the indicated fractions were evaporated and assayedfor GIA (dashed lines). UV-absorbing material was measured at 214nm (dashed lines); (---) denotes the concentration of the organicmodifier.

data base* (36) containing 3557 protein sequences, as de-scribed (37). The amino-terminal amino acid sequence ofoncostatin M did not reveal any substantial sequence homol-

*National Biomedical Research Foundation (1986) Protein SequenceDatabase, Protein Identification Resource (Natl. Biomed. Res.Found., Washington, DC), Release 9.0.

20

80 60 40

Medical Sciences: Zarling et al.

Dow

nloa

ded

by g

uest

on

May

25,

202

0

9742 Medical Sciences: Zarling et al.

1 5 10

Ala -Ala-Ile-Gly-Ser-Cys-Ser - Lys -Glu -Tyr

15 20Arg-Vol-Leu-Leu-Gly-GIn - Leu-GIn - Lys-GIn

25Thr - Asp-Leu-Met-GIn-Asp

FIG. 3. Amino-terminal amino acid sequence of oncostatin M.

ogies with any other known sequence including those ofotherproteins with anti-tumor activity that are produced by mono-nuclear cells, namely the interferons (38-44), tumor necrosisfactor (12), various proteins with interleukin 1 activity (16,18, 19), lymphotoxin (12), or type ,B transforming growthfactor (45).NaDodSO4/PAGE Analysis of Oncostatin M. Purified

oncostatin M, subjected to NaDodSO4/PAGE performedunder reducing conditions, was found to have an apparentmolecular weight of %28,000 (Fig. 4). The band was excisedfrom the gel, electroeluted, electrodialyzed, and found tohave the same amino-terminal amino acid sequence as thatshown in Fig. 3. Oncostatin M, subjected to PAGE undernonreducing conditions, also has an apparent molecularweight of28,000, and protein electroeluted from the band wasfound to inhibit proliferation of A375 cells (data not shown).

Biological Characterization of Oncostatin M. As shown inTable 3, proliferation of HTB10 neuroblastoma cells, A549lung carcinoma cells, as well as A375 and SK-MEL-28melanoma cells was inhibited following treatment withoncostatin M. In these and other experiments, oncostatin Mdid not inhibit, but rather augmented, [3H]thymidine incor-poration into WI-26 or WI-38 normal embryonic fibroblasts.Furthermore, whereas viable cell recovery from oncostatinM-treated A375 tumor cells was reduced, viable cell recoveryfrom oncostatin M-treated normal fibroblasts was increasedby 20-30%o. Oncostatin M, therefore, appears to inhibit thegrowth of certain human tumor cells but not normal humanfibroblasts. In addition, purified oncostatin M also inhibitedcolony formation of A375 cells in soft agar (Table 4).

Since mononuclear cells produce tumor cell inhibitoryfactors including tumor necrosis factor, interleukin 1, type /3

transforming growth factor, and the interferons, oncostatinM was tested for activities associated with these factors.

A B

43,000

1 2

25,7004

18,400

14,300

6,200-1

FIG. 4. NaDodSO4/PAGE of oncostatin M (lane B). The follow-ing proteins were used as standards (lane A) ovalbumin (Mr, 43,000);chymotrypsinogen a (Me, 25,700); lactoglobulin (Mr, 18,400);lysozyme (Mr, 14,300); bovine trypsin inhibitor (Mr, 6200); insulin Aand B chain (Mr 3200 and 3400, respectively).

Table 3. Inhibition of proliferation of tumor cells andaugmentation of proliferation of normal fibroblasts byoncostatin M

ProliferationGIA, % inhibition % stimulationunits _ _ _ _ _ _

Exp. per well A375 HTB10 A549 SK-MEL-28 WI-38 WI-26

1 16 83 254 62 301 46 46

2 27 NT 28 469 87 22 363 76 11 52

3 75 89 3025 85 228 71 16

4 20 87 445 75 251 59 11

Tumor cells were seeded at 3 x 103 cells per well, and normalfibroblasts were at 1.5 x 103 cells per well in 96-well plates for 3 hr.Various concentrations of purified oncostatin M (GIA units), ob-tained from the HPLC fraction C with peak anti-proliferative activityagainst A375 cells, were added, and 3 days later [3H]thymidineincorporation into cells was measured in triplicate wells at eachconcentration. Results shown are % inhibition or % stimulation of[3H]thymidine incorporated into tumor cells (A375, HTB10, A549,and SK-MEL-28) and normal fibroblasts (WI-26 and WI-38), respec-tively. NT, not tested.

First, highly purified oncostatin M was found not to inhibitproliferation of mouse L-929 cells, the prototype tumornecrosis factor-sensitive cell line (8-10), whereas crudesupernatant from PMA-treated U-937 cells had anti-prolifer-ative activity against L-929 cells. However, at concentrationsof tumor necrosis factor that inhibited replication of L-929cells by 99%, no inhibition ofA375 cell growth was observed.A375 melanoma cells have also been reported by others (17)to be resistant to the anti-proliferative activity of humantumor necrosis factor. The lack of cytotoxic activity againstL-929 cells, together with the amino-terminal amino acidsequence, suggests that oncostatin M is not related to tumornecrosis factor or lymphotoxin (12). Second, although inter-leukin 1 has been reported to be produced by U-937 cells (19)and to inhibit replication of A375 cells (17), oncostatin M, atconcentrations up to 50 GIA units/ml, lacked interleukin 1activity as measured by augmentation of mouse thymocyteproliferation (data not shown). Third, type (3 transforminggrowth factor inhibits A375 melanoma cells and mink lungcells; however, oncostatin M only minimally effected thereplication of mink lung cells compared to its marked inhib-itory activity for A375 cells. Furthermore, whereas type P3transforming growth factor is resistant to heating at 90°C,activity of oncostatin M is lost at 90°C. Fourth, interferons-a,

Table 4. Inhibition of A375 melanoma cell colony formation insoft agar by purified oncostatin M

GIA, % inhibition ofunits per well Colonies, no. colony formation

250 4 9683 6 9427 11 894.5 32 690 106

A375 cells were plated in soft agar, with or without purifiedoncostatin M from the HPLC column C fraction with peak activityfor inhibiting [3H]thymidine incorporation into A375 cells. Elevendays later, the number of colonies was determined. A colony wasdefined as a cluster of at least six cells.

Proc. Natl. Acad. Sci. USA 83 (1986)

Dow

nloa

ded

by g

uest

on

May

25,

202

0

Proc. Natl. Acad. Sci. USA 83 (1986) 9743

-p, and --yaffect the replication of encephalomyocarditis virus(7); however, oncostatin M, at concentrations of up to 50 GIAunits/ml, had only a negligible effect on the replication ofencephalomyocarditis virus. The biological activities de-scribed, together with the amino-terminal amino acid se-quence presented, would, therefore, suggest that oncostatinM is a cell growth regulator not resembling other factors.

Several growth regulatory polypeptides have been foundthat inhibit the replication of certain cells while not inhibitingand sometimes stimulating the growth of other cells (7-13, 17,21-23, 46-48). Those molecules that selectively inhibit can-cer cells and not the surrounding normal tissue cells are ofparticular interest as potential cancer therapeutic agents.Oncostatin M appears to be such a molecule, although thestudies so far have been restricted to human tumor cell linesin culture and to normal human diploid fibroblasts. With theprotein sequence determined, at least in part, it becomespossible to isolate the gene encoding the factor to determinewhat physiological role oncostatin M normally plays andwhat pharmacologic activities the molecule might possess.The biological assay used to purify oncostatin M depended onits ability to inhibit the replication of a human tumor cell line.Other peptides with an analogous property such as theinterferons, interleukins, and tumor necrosis factor havemultiple, diverse biological activities when tested in vivo. Wewould expect the same to be the case for oncostatin M.

We thank Dr. Joseph P. Brown and Dr. Timothy M. Rose for theirassistance in the computer searches, and Ms. Nancy Olfs for herassistance in the preparation of this manuscript.

1. Fidler, I. J., Sone, S., Fogler, W. E. & Barnes, Z. L. (1981)Proc. Natl. Acad. Sci. USA 78, 1680-1684.

2. Fidler, I. J. (1985) Cancer Res. 45, 4714-4726.3. Alexander, P. & Evans, R. (1971) Nature (London) New Biol.

232, 76-78.4. Hammerstrom, J. (1979) Scand. J. Immunol. 10, 575-582.5. Mantovani, A., Jerrells, T. R., Dean, J. H. & Herberman,

R. B. (1979) Int. J. Cancer 23, 18-25.6. Herberman, R. B. (1982) in NK Cells and Other Natural

Effector Cells, ed. Herberman, R. B. (Academic, New York),pp. 683-776.

7. Taylor, J. L., Sabran, J. L. & Grossberg, S. E. (1984) inInterferons and Their Applications, Handbook of Experimen-tal Pharmacology, eds. Came, P. E. & Carter, W. A.(Springer, Berlin), Vol. 71, pp. 169-195.

8. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore,N. & Williamson, B. (1975) Proc. Natl. Acad. Sci. USA 72,3666-3670.

9. Ruff, M. R. & Gifford, G. E. (1981) in Lymphokines, ed. Pick,E. (Academic, New York), Vol. 2, pp. 235-275.

10. Sugarman, B. J., Aggarwal, B. B., Hass, P. E., Figari, I. S.,Palladino, M. A., Jr., & Shepard, H. M. (1985) Science 230,943-945.

11. Gately, M. K., Mayer, M. M. & Henney, C. S. (1976) Cell.Immunol. 27, 82-93.

12. Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H.,Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B.& Goeddel, D. (1984) Nature (London) 312, 724-728.

13. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche,N. S. & Stern, D. F. (1985) Proc. Natl. Acad. Sci. USA 82,119-123.

14. Blyden, G. & Handschumacher, R. E. (1977) J. Immunol. 118,1631-1638.

15. Onozaki, K., Matsushima, K., Aggarwal, B. B. & Oppenheim,J. J. (1985) J. Immunol. 135, 3962-3968.

16. March, C. J., Mosley, B., Larson, A., Cerretti, D. P., Braedt,S. V., Price, S., Gillis, S., Henney, C. S., Kronheim, S. R.,Grabstein, K., Conlon, P. J., Hopp, T. P. & Cosman, D.(1985) Nature (London) 315, 641-647.

17. Lachman, L. B., Dinarello, C. A., Llansa, N. D. & Fidler,I. J. (1986) J. Immunol. 136, 3098-3102.

18. Rimsky, L., Wakasugi, H., Ferrara, R., Robin, P.,Capdevielle, J., Tursz, T., Fradelici, D. & Bertoglio, J. (1986)J. Immunol. 136, 3304-3310.

19. Knudsen, P. J., Dinarello, C. A. & Strom, T. B. (1986) J.Immunol. 136, 3311-3316.

20. Rosenberg, S. (1985) J. Natl. Cancer Inst. 75, 595-603.21. Ransom, J. H., Evans, C. H., McCabe, R. P., Pomato, N.,

Heinbaugh, J. A., Chin, M. & Hanna, M. G. (1985) CancerRes. 45, 851-862.

22. Sone, S., Mutsuura, S., Ishii, K., Shirahama, T. & Tsubura, E.(1984) Gann 75, 920-928.

23. Degliantoni, G., Murphy, M., Kobayashi, M., Francis, M. K.,Perussia, B. & Trinchieri, G. (1975) J. Exp. Med. 162,1512-1530.

24. Sundstrom, C. & Nilsson, K. (1976) Int. J. Cancer 17,565-577.

25. Harris, P. E., Ralph, P., Litcotsky, P. & Moore, M. A. J.(1985) Cancer Res. 45, 9-13.

26. Giard, D. J., Aaronson, S. A., Todaro, G. J., Arnstein, P.,Kersey, J. H., Dosik, H. & Parks, W. P. (1973) J. Natl.Cancer Inst. 51, 1417-1423.

27. Spengler, B. A., Biedler, J. L., Helson, L. & Freedman, L. S.(1973) In Vitro 8, 410-416.

28. Carey, T. E., Toshitada, T., Resnick, L. A., Oettgen, H. F. &Old, L. J. (1976) Proc. Natl. Acad. Sci. USA 73, 3278-3282.

29. Hayflick, L. (1965) Exp. Cell Res. 37, 614-636.30. Hayflick, L. & Moorhead, P. S. (1961) Exp. Cell Res. 25,

585-621.31. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91.32. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer,

W. J. (1981) J. Biol. Chem. 256, 7990-7997.33. Hunkapiller, M. W. & Hood, L. E. (1983) Science 219,

650-659.34. Laemmli, U. K. (1970) Nature (London) 227, 680-685.35. Hunkapiller, M. W., Lujan, E., Ostrander, F. & Hood, L. E.

(1983) Methods Enzymol. 91, 227-236.36. Dayhoff, M. O., Hunt, L. T., Barker, W. C., Orcutt, B. C.,

Yeh, L.-S., Chen, H. R., George, D. G., Blomquist, M. C. &Johnson, G. C. (1983) Protein Sequence Database (Nati.Biomed. Res. Found., Washington, DC).

37. Wilbur, W. J. & Lipman, D. J. (1983) Proc. Natl. Acad. Sci.USA 80, 726-730.

38. Allen, G. & Fantes, K. H. (1980) Nature (London) 287,408-411.

39. Zoon, K. C., Smith, M. E., Bridgen, P. J., Anfinsen, C. B.,Hunkapiller, M. W. & Hood, L. E. (1980) Science 207,527-528.

40. Levy, W. P., Shively, S., Rubinstein, M., DeValle, U. &Pestka, S. (1980) Proc. Natl. Acad. Sci. USA 77, 5102-5104.

41. Fiers, W., Remaut, E., Devos, R., Cheroutre, H., Contreras,R., Gheysen, D., Degrave, W., Stanssens, P., Tavernier, J.,Taya, Y. & Content, J. (1982) Philos. Trans. R. Soc. LondonSer. B. 299, 29-38.

42. Gray, P. W., Leung, D. W., Pennica, D., Yelverton, E.,Najarian, R., Simonsen, C. C., Derynck, R., Sherwood, P. J.,Wallace, D. M., Berger, S. L., Levinson, A. D. & Goeddel,D. V. (1982) Nature (London) 295, 503-508.

43. Taniguchi, T., Mantei, N., Schwartzstein, M., Nagata, S.,Muramatsu, M. & Weissman, C. (1980) Nature (London) 285,547-549.

44. Weissenbach, J., Chernajovsky, Y., Zeen, M., Shulman, L.,Soreg, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P.& Revel, M. (1980) Proc. Natl. Acad. Sci. USA 77, 7152-7156.

45. Derynck, R., Jarrett, J. A., Chen, E. Y. & Goeddel, D. V.(1986) J. Biol. Chem. 261, 4377-4379.

46. Iwata, K. K., Fryling, C. M., Knott, W. B. & Todaro, G. J.(1985) Cancer Res. 45, 2689-2694.

47. Fryling, C. M., Iwata, K. K., Johnson, P. A., Knott, W. B. &Todaro, G. J. (1985) Cancer Res. 45, 2695-2699.

48. Horowitz, J. B., Kaye, J., Conrad, P. J., Katz, M. & Janeway,C. A., Jr. (1986) Proc. NatI. Acad. Sci. USA 83, 1886-1890.

Medical Sciences: Zarling et al.

Dow

nloa

ded

by g

uest

on

May

25,

202

0