[CANCER RESEARCH 44, 915-922, March 1984]

Identification and Partial Characterization of a Low-Molecular-Weight

Inhibitor of Leukotaxis from Fibrosarcoma Cells

H. Warabi, K. Venkat, V. Geetha, L. A. Liotta, M. Brownstein, and E. Schiffmann '

Department of Internal Medicine, Division of Collagen Diseases. Juntendo Medical University, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113, Japan [H. W.¡;Laboratory of

Developmental Biology and Anomalies, National Institute of Dental Research [K. V., V. G., E. S.], Laboratory of Pathology, National Cancer Institute [L. A. L.J, andLaboratory of Clinical Science, National Institute of Mental Health [M. B.], NIH, Bethesda, Maryland 20205

ABSTRACT

Lysate from T-241 murine fibrosarcoma cells contains a low-molecular-weight (M, < 1000), heat-stable peptide factor whichhas antichemotactic activity for both macrophages and polymor-

phonuclear leukocytes in vitro. The tumor factor was partiallypurified from an alcohol extract of the fibrosarcomas by gelfiltration, anión exchange chromatography, and paper chroma-

tography successively. This factor inhibits both the hydrolyticcleavage of the peptide attractant AMormylmethionylleucylphenyl-

alanine by polymorphonuclear leukocytes and the methylation ofboth protein carboxyl groups and membrane phospholipids. Furthermore, the factor does not appear to compete with N-formylmethionylleucylphenylalanine for its receptor. The tumor-

derived material, therefore, affects biochemical reactions believed to have roles in the expression of an adequate leukotacticresponse. These data suggest that depressed inflammatoryresponses at sites of neoplasms may result in part from releaseof small, potent inhibitors of leukotaxis from tumors themselves.

INTRODUCTION

Reduced activities of inflammatory reactions of the immunesystem have been described for neoplastic conditions in humansand animals (18, 37, 39, 42). These conditions may contributeto the increased susceptibility to infections seen in neoplasticstates. Indeed, both the number of macrophages and theirtumoricidal activity at sites of neoplasms are markedly diminishedwhen compared to those which are found at sites of bacterialinfection (41). Macrophages, lymphocytes, and PMNs2 also have

other suppressed functions in tumor-bearing individuals (13). The

presence of tumors is associated with a decreased responsiveness in monocytes and macrophages to complement-derivedchemoattractants which may, in part, be attributed to tumor-

produced antichemotactic agents (6, 21, 23, 33). In support ofthis, it has been found that factors isolated from tumor lysatesobtained from several animal model systems and serum frompatients with neoplasms were able to inhibit the chemotacticresponses by macrophages and PMNs for several attractantsboth in vitro and in vivo (7,11, 14, 17, 32). These effects in cellmotility may contribute in large part to the evasion of immunesurveillance by tumors.

In this report, we demonstrate that the T-241 fibrosarcoma, ahighly malignant murine tumor, produces a low-molecular-weight

factor with peptide properties which is able to inhibit chemotactic1To whom requests for reprints should be addressed.2The abbreviations used are: PMN, polymorphonuclear cell; fMet, AMormylme-

thionyl; fMet-Leu-Phe, W-formylmethionylleucylphenylalanine; EAMS, endotoxin-activated mouse serum; TF-CSM, tumor factor-crude starting material; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IDM, concentration giving half-maximal inhibition of chemotaxis.

Received December 1,1982; accepted November 21,1983.

activities of both macrophages and PMNs in response to complement-derived attractant and fMet peptides, themselves re

lated to a bacterial attractant (30). We have also studied possiblemechanisms of action of this factor upon PMN chemotaxis. Wefind that the tumor factor does not compete with formylatedpeptides for the receptor of the latter, but that it does inhibithydrolysis of the peptide by the cell. Also, the tumor factorinhibits both phospholipid and carboxymethylation in PMNs.These reactions may be involved in the chemotactic response(4, 26, 29, 34).

MATERIALS AND METHODS

Chemoattractants

fMet-Leu-Phe was obtained from Peninsula Laboratories (San Carlos,CA), and the tritiated fMet-Leu-[3H]Phe was from New England Nuclear

(Boston, MA). Crude complement-derived attractant was prepared from

guinea pig serum by activating it with endotoxin (31). EAMS, whichcontained complement-derived chemotactic factor, was prepared by

incubation of syngeneic mouse serum with Salmonella typhosa lipopoly-saccharide W (1.5 to 3.0 mg/ml; Difco Laboratories, Detroit, Ml) at 37°for 90 min. This mixture was then heated at 56° for 30 min andcentrifugea at 40,000 x g for 30 min at 4°.Aliquots of EAMS werestored at -20° until use. Known dilutions of EAMS were used as a

chemotactic agent for macrophages from mice.

Purification of Tumor Factor

Tumor

The transplantable T-241 fibrosarcoma (National Cancer Institute,

Bethesda, MD) was maintained by serial passage from femoral muscleof the 9- to 10-week-okJ male C57BL/6J mouse. This inbred strain (The

Jackson Laboratory, Bar Harbor, ME) minimizes the biological variabilitybetween animals, specifically in variations in the immunological andvascular response to a transplanted tumor. Tumors were initiated byinjection of 106 mechanically disaggregated tumor cells from nonnecrotic

tumor fragments obtained 10 to 15 days after passage (19). The tumorcell viability was determined by the trypan blue dye exclusion test. Acomplete virus screen, including tests for mouse pneumonia virus, mouseminute virus, Polyoma, murine leukemia virus, murine encephalomyelitisvirus (Theiler) GDVII, ectromelia, Reovirus-SM.AD., lymphocytic choreo-

meningitis, and Sendai, was done by the Frederick Cancer ResearchCenter (Frederick, MD) and was found to be negative. The viral screeningprocedure was performed in accordance with protocols developed byMeloy Laboratories, McLean, VA, as a United States Government contractor. The tumor cells also were found to be free of Mycoplasma orbacterial contamination even after prolonged aerobic and anaerobicincubation.

Preparation of Tumor Tissue Extracts

Tumors were dissected under sterile conditions, cut into small pieces,and minced until a fine suspension was obtained. Single-cell suspensions

MARCH 1984 915

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

H. Warabi et al.

were obtained by serial aspirations through 19-, 23-, and 25-gauge

needles. This cell suspension was then cultured in RPMI 1640 withoutserum for 24 hr on tissue culture plastic dishes. The incubated cellsuspension was centrifuged at 800 x g for 20 min. The resultingsupernatant fraction was saved for assay of its antichemotactic activity.The cell pellet was homogenized in RPMI 1640 at 4°using a Teflon

homogenizer, and the homogenate was centrifuged at 10,000 rpm for 1hr at 4°.An aliquot of the clear supernatant fraction was tested for

biological activity. The remaining supernatant fraction was lyophilized,and the residue was extracted with 85% ethanol. The alcohol extractwas evaporated to dryness, and the resulting residue, termed TF-CSM,

was dissolved in distilled water. In the work to be described, we haveprincipally used 85% ethanol extracts of minced tumor, since theseextracts were found to yield more activity than conditioned media.

Gel Filtration

TF-CSM was subjected to Sephadex G-50 (60 x 2-cm column) gel

filtration using 1 mw ammonium carbonate as eluent. Each fraction wastested for biological activity (antichemotactic effect), and the activefractions were pooled and lyophilized. The lyophilized material was againextracted with 85% ethanol. After evaporation, the residue was dissolvedin distilled water and chromatographed on a Sephadex G-15 filtration

column (1 .6 x 70 cm) using 1 mw ammonium carbonate as eluent. Eachfraction was tested for biological activity, and the active fractions werepooled and lyophilized. The residue was dissolved in distilled water andthen filtered on a P-2 (Bio-Gel) column (1 .6 x 70 cm) using 0.01% aceticacid or distilled water. Active fractions from the P-2 filtration were pooledand lyophilized. After extraction of this P-2 material with 85% ethanol

and subsequent evaporation of the solvent, the residue was stored at-60°.

Ion-Exchange Chromatography

Anión exchange resin AG 1-X4 and cation-exchange resin AG 50-X2(Bio-Rad Laboratories, Richmond, CA) were used for further analysis ofmaterial from P-2. The material was applied to each column of resin (1 .5

x 10 cm). The column was eluted with water and then with 2 N aceticacid in the case of AG 1-X4, or 4 N NH4OH in the case of AG 50-X2. The

resulting eluates were each lyophilized. The residues were tested forantichemotactic activity and used for further Chromatographie purificationand amino acid analysis.

Paper Chromatography

Whatman No. 3 Chromatographie paper strips were sequentiallywashed and distilled water, 0.01 N HCI, distilled water, and alcohol, andthen dried. TF-CSM and residues from the active fractions of the Sephadex G-50, G-15, and P-2 Bio-Gel columns were chromatographed.

Butanol, acetic acid, and water (4:1 :1) were used as the solvent system.Chromatography was performed vertically for 15 hr at room temperature.Both ninhydrin and chlorine-toluidine reactions were used to visualize

materials. The visualized zones were cut from paper and eluted withwater or 1% acetic acid and tested for biological activity.

Treatment of Tumor Factor with Enzymes

Tumor factor filtered through P-2 Bio-Gel was concentrated to 2.5

mg/ml and then incubated with 1 mg/ml of each of several enzymes for10 hr at 37°.The enzymes included protease from Streptomyces griseus

type VI (Sigma Chemical Co., St. Louis, MO), pepsin (Boehringer, Mannheim Corp., New York, NY), trypsin (Millipore Corp., Bedford, MA),cathepsin C (Boehringer-Mannheim), carboxypeptidase A (MilliporeCorp.), and carboxypeptidase B from porcine pancreas (Boehringer-

Mannheim). Each reaction was stopped by adding absolute ethanol to afinal concentration of 85%. After centrifugation (1000 rpm for 30 min),the supernatant fraction was lyophilized. The lyophilized material wasdissolved into aliquots of Gey's buffer and tested for biological activity.

As a control, the incubated enzyme without tumor factor was testedunder the same conditions.

Hydrolysis of Tumor Factor from P-2 Filtration

Material from the P-2 preparation (2 mg) was hydrolyzed for 72 hr at110°with 6 N HCI, the hydrolysate was lyophilized, and the residue wasdissolved in Gey's buffer and tested for biological activity.

Peritoneal PMNs and Macrophages

Activated rabbit PMNs were obtained by peritoneal lavage 14 to 16hr after the i.p. injection of 0.2% oyster glycogen (Sigma) in PBS. Thecells were suspended in Gey's solution (0.015 M 4-{2-hydroxyethyl)-1 -

piperazineethanesulfonic acid, pH 7.2) with 2% BSA for chemotaxisassay and in Gey's solution without BSA for binding assay. Inflammatory

macrophages were obtained from the peritoneal cavities of Hartleyguinea pigs 4 days after i.p. injection of 25 ml of 0.2% glycogen in PBS.Macrophages were removed from the peritoneal cavities by lavage withcold PBS containing heparin (10 units/ml). After centrifugation, the cellswere washed once in heparinized PBS and resuspended in Gey's bal

anced salt solution with 2% BSA. Resident macrophages were obtainedin an identical manner from T-241 fibrosarcoma-bearing mouse.

Chemotaxis Assay

The chemotaxis assay was performed with the Boyden chamber (44).For macrophages chemotaxis, different dilutions of EAMS in RPM11640with 25 mw 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer

(Grand Island Biological Co.) were added to lower wells of the chamber,and polycarbonate membrane filters (5-^m pore size) were placed over

the lower wells. The upper wells were filled with 0.2 ml RPM11640 (plus2% BSA) containing 4 x 10s cells. The chambers were incubated for 4hr at 37°in humidified 5% CO2 in air. Viability of the cells was 95% as

determined by trypan blue exclusion. After incubation, the cells from theupper well were removed, and the filter was air-dried and stained withWright's solution and air-dried. Cells that had migrated completely

through the filter and were attached to the lower surface were countedin 20 oil immersion fields. The chemotactic response was expressed asmean number of total macrophages per 20 oil fields for triplicate filters.For rabbit neutrophil chemotaxis, either fMet-Leu-Phe (10~9 M) or crude

complement-derived attractant preparation was used as a chemoattrac-tant. Neutrophils (2.2 x 106 cells/ml) suspended in Gey's buffer contain

ing 2% BSA were used for each assay using a nitrocellulose Milliporefilter (5-MfTipore size; Millipore Corp.). After a 2-hr incubation period, the

filter was stained with hematoxylin (25), and the chemotactic responseof PMNs was measured as described in the macrophage chemotaxisassay. Antichemotactic activity of the tumor factor (% of inhibition ofchemotaxis) was defined as:

No. of cells migrating in the presence of tumor factor

No. of migrating control cells

3H-Formylated Peptide Binding Assay

Rabbit PMNs (4.4 x 106 cells/2 ml) were suspended in Gey's incuba

tion buffer without BSA, to which had been added 0.1 mw L-1-tosylamido-2-phenylethylchloromethyl ketone (Calbiochem, MD), and then the cellswere incubated with 1.5 nM fMet-Leu-[3H]Phe for 1 hr at 4° in the

presence of various concentrations of tumor factor. Some assays wereperformed at 37°in the absence of L-1-tosylamido-2-phenylethyl-chlo-

romethyl-ketone. The reaction was terminated by rapid filtration usingWhatman glass fiber filters attached to a low-pressure filtration unit.

Cells were washed twice with 7 ml of cold PBS (pH 7.4, 0.15 M NaCIand 0.02 M KH2PO4), and the filters were transferred to 10 ml of Aguasolscintillation fluid (New England Nuclear) and counted in a liquid scintillation counter (3, 31 ).

916 CANCER RESEARCH VOL. 44

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Leukotaxis Inhibitor from T-241 Fibrosarcoma Cells

Studies on the Site of Action of Tumor Factor

Inhibition of Peptidase Activity by PMNs

Cells obtained from the peritoneal cavity were freed of erythrocytesby exposure to lysing buffer (0.15 M NH4CI, 0.01 M KHCO3, and 0.11rnw disodium EDTA) for 1 min at 4°, and by subsequent washing inGey's solution with 2% BSA. Cells (2.2 x 107) in Gey's solution were

incubated with gentle shaking for 30 min at 37°together with fMet-Leu-[3H]Phe and "P-2" material (final concentrations, 2.5, 1.25, and 0.625

mg/ml). Similar experiments were performed without P-2 material. The

suspension was centrifugea, and the supernatant fraction was dilutedwith alcohol to a 90% final concentration. After centrifugation, the resulting supernatant fraction was subsequently concentrated to a smallvolume and applied to a thin-layer plate. The reaction products werethen separated by thin-layer chromatography on the silica gel plate withn-butyl alcohol:acetic acid:water (4:1:1, v/v/v) as the mobile phase. After

8 hr, the chromatograms were scanned for radioactivity using a Packardradiochromatogram scanner (3).

Assay of Methylation

Protein carboxymethylation was assayed by measuring the release of[3H]methanol from protein methyl esters formed by the transfer of the3H-methyl group from [mefhy/-3H]methionine (New England Nuclear) to

the carboxyl groups of proteins as described previously (26). Totalphospholipid methylation was assayed by measuring the incorporationof the methyl group from [meffty/-3H]methionine into membrane phos-

pholipids as described previously (29). Briefly, the procedures are asfollows.

Protein Carboxymethylation Determination. After a 15-min preincu-bation with "P-2" tumor factor, 2 x 107 cells in 2 ml of Gey's buffer (0.1 %

BSA) were incubated for 30 min at 37° together with 10 ?Ci of [3H-meffty/Jmethionine (6 x 10~7 M). The reaction was terminated with cold

10% trichloroacetic acid, and the precipitate was washed twice withtrichloroacetic acid containing 10 mw nonisotopic methionine. The pelletwas treated with 0.3 ml of 1 M borate (pH 11.0; 2.6% methanol), and themixture was extracted with toluene:isopropyl alcohol (3:2) (26). Theextract, containing released [3H]methanol, was assayed for radioactivity

in a liquid scintillation counter. Control samples were processed in thesame manner.

Determination of Lipid Methylation. Another aliquot of cells wasprocessed as in the former procedure up to the stage in which the cellpellet was washed with TCA. The pellet was then extracted with 3 ml ofCHC^CHaOH (2:1, v/v), the solvent was evaporated, and the residuewas assayed for radioactivity (29).

Table 1

Chemotactic response of mouse macrophages

Source of peritoneal cells

% of controlchemotactic re

sponse to EAMS"

Normal mice 100

Mice given liver or spleen cell injections6 100 ±10C

Tumor-bearing mice 17 ±15

Cells isolated from normal0 mice and then incubated with 7 ± 8

P-2 fraction of tumor factor

Cells isolated from normal0 mice, incubated with tumor 91 ±12factor for 60 min, and then washed 3 times with Gey's

buffer to remove tumor factor3 Results are given as percentage of positive control cells in 5 fields after

subtracting negative controls (magnification, x900). Values are the average oftriplicate determinations. Positive controls were 270 ±20 cells/5 fields. Negativecontrols were 65 ±10 cells/5 fields.

6 Normal C57BL/6J mice were given injections of liver or spleen cells isolatedfrom tumor-bearing mice; 1 x 106 cells in 1 ml PBS were injected in the femoral

muscle.c Mean ±S.E.a The concentration (0.25 mg/ml) of tumor factor used was that which caused

maximal inhibition of chemotaxis.

Table2Inhibition of rabbit PMN chemotaxis by conditioned medium from tumor cells and

tumor homogenstes% of inhibition of chemotaxis"

Dilution ofsamplesUndiluted

sample1:41:81:20Undiluted sample" dialyzed versus PBSConditioned

medium" of cul

tured tumorcells65

301000Supernatant0

from tumor hc-

mogenates100

5030

00

a Standard chemotactic response to 1 nM fMet-Leu-Phe was 42 ±7 cells/field

(magnification, x900). Negative control gave 7 ±5 cells/field (x900); S.E. did notexceed 10%.

0 Conditioned medium, after desalting, contained 1.7 mg of solids per ml.c Supernatant from homogenates, after desalting, contained 5.2 mg of solids

per ml.d Medium and tissue homogenates were dialyzed against cold PBS and then

tested for biological activity. Medium alone, before and after incubation for thesame period of time as conditioned medium obtained from cultured tumor cells, didnot inhibit chemotaxis.

RESULTS

Antichemotactic Activity of Crude Tumor Extracts

Resident peritoneal macrophages from tumor-bearing mice

were found to be less responsive to chemotactic stimuli (Table1) than those from normal animals. The reduced chemotacticresponse of cells was associated with the presence of tumors,since cells from either liver or spleen when injected into normalmice did not depress the chemotactic responsiveness of residentperitoneal macrophages (Table 1). Furthermore, when activatedmacrophages from normal mice were preincubated with a ho-mogenate of the T-241 fibrosarcoma, their chemotactic response

to EAMS could be completely inhibited. This antichemotacticactivity was also present in the conditioned medium of culturedtumor cells as well as in an 85% ethanol extract of the tumor(Table 2). The antichemotactic material appeared to have amolecular weight of less than 2000 since, after dialysis, the

activity was absent from the dialysate. This conclusion wassupported from the results of gel filtration discussed below. Wehave found that an 85% ethanol extract of normal femoral softtissue from tumor-bearing mice did not inhibit chemotaxis (notshown). The chemotactic response by neutrophils to 1 nM fMet-Leu-Phe in the presence of this extract from normal tissue was

53 ±10 cells/field (S.E.; magnification, x900) equivalent to thatof a positive control. The extract tested was from an amount ofnormal tissue equivalent in weight to 5 tumors, the extract ofwhich completely inhibits leukocyte motility in our assay (4 ±6cells/field). These results suggested that the tumor cells weresynthesizing and secreting inhibitors of phagocyte chemotaxis.However, we cannot eliminate the possibility that, in the tumoritself, some activity might result from infiltrating or activatednormal cells. While both macrophage and neutrophil chemotacticresponses were inhibited by the tumor factor, our results referto the use of neutrophils unless otherwise indicated.

MARCH 1984 917

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

H. Warabi et al.

Isolation of Tumor Factor by Gel Filtration

Gel filtration of TF-CSM on Sephadex G-50 yielded 2 distinct

large peaks that absorbed at 202 nm. One peak representedmaterial with molecular weight larger than 10,000, and thesecond represented low-molecular-weight substances of about

400 to 1000 (Chart 1). Assay of each fraction revealed that mostof the inhibitory activity for PMN chemotaxis was associatedwith the low-molecular-weight fraction. The lyophilized fractions

from the active peak were extracted with 85% ethanol. Thebiological activity of this fraction was:

1050= 0.53 mg = 0.5 A«»™

IDso is that concentration giving half-maximal inhibition of PMNchemotaxis to 1 nw fMet-Leu-Phe as attractant in the Boyden

chamber. Recovery of activity was 72%.The active fraction from Sephadex G-50 was then subjected

to gel filtration on a Sephadex G-15 column and monitored at

202 nm. Two major peaks were resolved, and the biologicallyactive material was associated with the second major peak(Chart 2). This peak was pooled, lyophilized, and extracted with85% ethanol. Following the removal of the ethanol, the specificactivity of this fraction was increased over 4-fold. The biological

activity of this fraction was:

ID»= 0.125 mg = 0.1 A^™

Recovery of activity was 46% based upon the original extraction.This material was then desalted on a P-2 gel filtration column,

and the eluent was assayed for antichemotactic activity (Chart3). Material from the active zone was ninhydrin positive and wasestimated to have a molecular weight of approximately 500 to1000. Recovery of active material was 40% at this stage ofisolation.

Anion-Exchange Chromatography of Tumor Factor

Antichemotactic material from the P-2 gel filtration step wasapplied in 0.5 ml H2O to a cation-exchange column (AG 50-X2).

Biologically active material did not bind appreciably to the column(Table 3). Another sample of the material was applied to ananion-exchange column (AG 1-X4) in water at pH 7.5. Active

material bound to the column and was eluted with 2 N aceticacid. Both active and inactive fractions from each column wereninhydrin positive. These results indicated that the tumor factorhad a net negative charge at neutral pH. Recovery of activematerial from aniónexchange Chromatography was 34% basedupon starting material. Recovery from the cation exchange column was 36%. The steps in purification and respective recoveries and specific activities (ID50s) are given in Table 4. About100-fold purification has been achieved.

Properties of the Purified Tumor Factor

Chemical Properties. The active material was a dialyzablesubstance (Table 2). The activity was destroyed upon hydrolysiswith 6 N HCI but was retained after heating in water at 56°for30 min. The "P-2" material appeared to contain no carbohydrate

(negative anthrone test) and was essentially free of salt (AgNO3test showed minimal reaction). The material did not appear to bea prostaglandin, because a radioimmunoassay for this class oflipids was negative. We sought to determine whether the tumorfactor was a peptide. Paper Chromatographie analysis of the P-

1.5

uzS 1-0

0.5-

100

50

zi

ox

>X

r'ti 25 35

FRACTIONNUMBER(3 ml)

45

Chart 1. Isolation of chemotaxis inhibitor from TF-CSM using Sephadex G-50and the eluent 1 rrtM ammonium carbonate. Arrows, positions of marker usingsubstances (blue dextran, RNase, and fMet-Leu-Phe). , distribution of inhibitory material; , absorbance at 202 nm. Recovery of activity was 72%. Determination of biological activity was performed as described in "Materials andMethods;" 2.2 x 106 cells/ml were incubated with lyophilized fractions in Gey's

solution containing 2% BSA for 15 min at 37° and were used for ordinarychemotaxis assay against complement-derived attractant or fMet-Leu-Phe with themodified Boyden chamber. Percentage of inhibition was calculated as:

100 x [1 - (Migrated cells with tumor factor)/Control]

IDM, activity amount of material that inhibits 50% of control chemotaxis FMLPfMet-Leu-Phe.

2.0

1.5

1.0-

0.5-

100

60

o>x

20 30 40

FRACTIONNUMBER(3ml)

50

Chart 2. Elution of chemotaxis inhibitor from Sephadex G-15 after applicationof active materials for Sephadex G-50. Absorbance, ; 1 ITIMammonium carbonate was used as eluent. Two biologically active zones were demonstrated( ). The most active fraction (secondpeak) was pooled. The biological activityof this fraction is given as:

ID»= 0.125 mg = 0.1

FMLP, fMet-Leu-Phe.

2 material was performed (Chart 4). Assay of the eluted zonesrevealed 2 active materials, one of which was ninhydrin positive(Rf 0.1) and the other of which was ninhydrin negative but waspositive to the chlorine-toluidine reaction (Rf 0.21), a test for

peptides. Results of enzymatic digestion of tumor factor withpeptidases are shown in Table 5. The percentages of inhibitionof a chemotactic response of PMNs to fMet-Leu-Phe after treat

ment of the cells with tumor factor previously exposed to variousenzymes are given. The antichemotactic activity was completelyabolished after tretament of the material with carboxypeptidaseA, pepsin, trypsin, and cathepsin C, and was slightly decreasedafter treatment with carboxypeptidase B and protease fromStreptomyces griseus type VI. These results suggest, but do not

918 CANCER RESEARCH VOL. 44

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Leukotaxis Inhibitor from 7-247 Fibrosarcoma Cells

establish, that the tumor factor has a peptide component that isrequired for its activity. Amino acid analysis of the most purifiedpreparations have not yielded definitive data on the compositionof the material, presumably because of the inadequate size ofthe sample. This suggests that the material is highly potent.

Biological Properties. The tumor factor itself did not stimulatechemotaxis in phagocytes over the range of concentrations from0 to 5 ID,ooS.It was not cytotoxic to either PMNs or macrophagesunder conditions of the chemotactic assay. At least 90% of cellstreated with trypan blue under conditions of the assay for chemotaxis did not show inclusion of the dye. PMNs exposed to thisfactor and then washed to remove it displayed normal chemotaxis, indicating that the inhibition was reversible (Table 1). Inefforts to determine the effect of the tumor factor (purified withBio-Gel P-2) upon other cells, we found that the material was

NINHYDRIN REACTION - --+«f«H»»±

zK»i

5

50

10 15 20 25

FRACTION NUMBER I3 ml)

Charts. Desalting of active fraction from Sephadex G-15 with Bio-Gel P-2.Absorbance, . Antichemotactic activity, . The ninhydrin reaction waspositive in each biologically active fraction.

Table 3Binding of antichemotactic activity to ion-exchange resins

Five IDsoSof tumor factor from P-2 filtration were each applied to the columns.

% of inhibition of chemotaxis3 in:

Resin Nonbound fractions Bound fractionsAG 50-X26' cAG1-X4C'"

720

1469

B Positive control for chemotaxis was 44 ±9 cells/field (x900). Negative control

(buffer in lower chamber) was 6 ±4 cells/field (x900).6 After washing with water, 4 ml of 4 N NH4OH were used to elute the column

to recover bound material.c Blank runs (no antichemotactic factor) from each of the resins did not yield

inhibitory activity." After washing with water, 4 ml of 2 N acetic acid were used to elute the column

to recover bound material.

Table 4

Purification of tumor factor via Chromatographie procedures

Step"85%

ethanol extractSephadex G-50 filtrationSephadex G-15 filtrationBio-Gel P-2 filtrationAG 1-X 4 aniónexchangeSpecific

activity:IDso(mg/ml)0.85

0.530.1250.0940.008%

of recovery610072

464034

a Crude extract of tumors was extracted with 85% ethanol, the solvent was

evaporated, and the residue was reextracted with the same solvent. The residuefrom this was used as starting material.

Based upon the 85% ethanol extract as the starting material.

Rf = 0.1

Rf = 0.21

CRININH.!D00JDE

TOLID.ciZSEPH

G1NINH.00Û^DEX50TOLID.1SEPI-GNINH.VADEX15TOLID.|BICFNINH.0GEL1TOLID.t

Chart 4. Paper Chromatographie analysis of TF-CSM, Sephadex G-50, G-15,and P-2 fractions. Whatman 3-mm paper (40 x 20 cm) was used in descendingchromatograpny. Solvent front was 20 cm from the origin. Fractions were developed for 15 hr at room temperature using butanol:acetic acid:water (4:1:1). Onanalysis of P-2 material, there were 2 biologically active spots, one ninhydrinpositive (R, 0.1) and the other ninhydrin negative, but positive to the chtor-ine:toluidine test (R, 0.21 ). NINH., ninhydrin; TOLID., toluidine; W.O., not determined.

Tables

Effect of enzymatic digestion of tumor factor with peptidase

Enzyme8Carboxypeptidase

APepsinCathepsin CTrypsinProteaseCarboxypeptidase B%

of inhibition ofchemotaxis00

0106869

"Tumor factor was digested with enzymes as described in 'Materials andMethods."

6 Chemotactic assay was performed as described in "Materials and Methods."

Percentage of inhibition was calculated by comparing the migration of cells exposedto enzyme-treated tumor factor with the migration of cells exposed to untreatedtumor factor (a concentration of 0.5 mg/ml of untreated material completely inhibitsmigration of cells). Control experiments, in which enzymes in buffer alone wereallowed to incubate and then inactivated with ethanol (see "Materials and Methods"), did not yield inhibitory activity.

neither a stimulant nor an inhibitor of fibroblast chemotaxis. The"P-2" material provoked an angiogenic response when injected

with the rabbit eye (data not shown).

Studies on the Mechanism of Action of the Tumor Factor

Effect of Increasing Concentrations of Attractant upon Antichemotactic Activity of Tumor Factor. We determinedwhether increasing the concentration of attractant in the lowerwell of the Boyden chamber had any effect upon the inhibitionof chemotaxis by the tumor factor placed in the upper well withthe cells (Table 6). The tumor factor (0.125 mg/ml) completelyinhibited the chemotactic response to 1 nw fMet-Leu-Phe asattractant. A 5-fold increase in the level of fMet-Leu-Phe resultedin only a 14% decrease in this inhibitory effect. Control experi-

MARCH1984 919

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

H. Warabi et al.

ments indicated that concentrations of attractant over the 2 to 5nw range still produced at least 85% of the response given bythe maximally stimulating concentration of 1 nw fmet-Leu-Phe.Concentrations of attractant higher than 5 nM (6 to 10 nw)themselves inhibited chemotaxis (20 to 60% of the responseinduced by 1 nM peptide). The findings suggested both that thetumor factor did not exert its inhibitory effect by competing forthe fMet peptide receptor and that its effect was upon the cell,not upon the attractant.

Receptor-binding Studies. We next tested whether the tumorfactor competed with fMet-Leu-[3H]Phe for binding to the neutro-

phil. This factor, at a concentration that completely inhibitedchemotaxis, did not displace the chemotactic ligand from itsreceptor at either 4°or 37°(Chart 5).

Peptidase Reaction. Since cleavage of fMet peptides bypeptides may be necessary for detection of the chemical gradientby migrating PMNs or macrophages, we determined the effectof the tumor factor upon the hydrolysis of formylated peptide byPMNs. [3H]fMet-Leu-Phe was degraded to an extent of about

17% after incubation with PMNs, but the degradation was inhibited in the presence of tumor factor (Table 7). The degradationproducts [more slowly migrating (R, 0.65) zone of radioactivity]

Table6Effect of increasing concentrations of attractant upon antichemotacticactivity of

tumor factorfMet-Leu-Phea(nM) % of inhibition by tumor factor*

98.29.1485.583.784.5

"One and 2 DM fMet-Leu-Phe gave optimal responses (45 ±5 cells/field;magnification, x900) in rabbit PMNs. Negative controls (Gey's buffer in lowerchamber) were 8 ±4 cells/field. The higher concentrations, 3 to 5 nw, gaveresponses that were not less than 84% of the response induced by 1 nu fMet-Leu-Phe.Quantities are averages of triplicate values, S.E. did not exceed 10%.

A concentration of tumor factor that completely inhibited chemotaxis to 1 nMfMet-Leu-Phe was used for all concentrations of fMet-Leu-Phe. This correspondsto 0.125 mg or 0.1 unit of absórbanosat 210 nm of materialfrom P-2gel filtrations.

30 soTime (mini

Chart 5. Effect of tumor factor upon binding of attractant to cells. • •.binding of labeled peptide to cells at 25°;• •,binding of labeled peptide tocells at 25°in presence of 0.25 mg of tumor factor; O O, binding of labeledpeptides to cells at 4°;O O, bindingof labeledpeptide to cells at 4°in presenceof 0.25 mg of tumor factor. Specific binding was calculated as the differencebetween the total binding and nonspecific binding which occurred in the presenceof excess (10~* M) unlabeled fMet-Leu-Phe. Binding assay was performed as in"Materialsand Methods." Tumor factor (0.25 mg/ml completely inhibitschemotaxis)has no effect upon binding at 37°and 4" compared to control. Data represent theresult of triplicate determinations for each point with an S.E. not greater than 10%.

Table7Effect of tumor factor upon degradation of fMet-Leu-fHJPhe by neutrophils: thin-

layer chromatographyof products

A.B.C.Eluted

zone8R,

0.85CR,0.65"R,

0.85CR,0.65*"R,

0.85°R, 0.65dcpmfH)679,80020,10066,300

31,20080,800

18,700%

of degradation ofpHJfMet-Leu-Phe6016.80

[3H]fMet-Leu-Phe(1 x 105cpm) was incubated: A, in absence of cells; B, inthe presence of cells; and C, in the presenceof cells and tumor factor (0.125 mg/ml).As describedin "Materialsand Methods," radiolabeledproducts were subjectedto thin-layerchromatography, and the radioactivezones were eluted and counted

S.E. did not exceed 10%.c Material eluting at R, 0.85 corresponds to authentic fMet-Leu-Phe.

Material eluting at R, 0.65 is not defined but may contain leucyl[3H]phenylala-nine, which élûtesat this rate.

eCells previously exposed to heat (100°for 5 min) did not degrade fMet-Leu-[3H]Phe(data not shown).

lÃ*̈§1002 9<

so

Mio WOI

0.5 1.0

CONCENTRATION OF TUMORFACTOR mg

Chart 6. Percentageof inhibitionof both protein carboxymethylation and methylation of phospholipids in neutrophils in presence of tumor-derived material fromP-2 gel filtration. Determination of transfer of labeled methyl groups from[ H-CH3]methionineto lipids and proteins was performedas described in "Materialsand Methods." Control cells incorporated 2560 ±150 cpm/107 cells in the lipidfraction and 970 ±45 cpm/10r cells in the protein methyl ester fraction (recoveredas [3H]methanol). Data represent the results of triplicate determinations in 3separate experiments.

were not identified but may contain leucyl-[3H]phenylalaninewhich has this mobility in the thin-layer system. We cannot ruleout some oxidative degradation of fMet-Leu-Phe.

Methylation. Because the chemotactic responses of PMNsand macrophages may involve methylation (28, 33), we determined the effect of the tumor factor upon this reaction in PMNs.Both protein carboxymethylation and methylation of phospholipidwere inhibited by the tumor factor (Chart 6). This effect did notappear to be the result of dilution by peptide-bound or freemethionine in the tumor factor. Highly sensitive determinationsof the amino acid content in hydrolyzed material (o-phthalalde-hyde reagent) did not reveal the presence of methionine. It shouldbe noted that inhibition of methylation is not well-correlated withinhibition of chemotaxis; the ID50 of the "P-2" tumor factor is

0.125 mg/ml for inhibition of chemotaxis, whereas the ID50wouldbe 80 mg/ml for the same amount of tumor factor inhibition ofboth kinds of methylation.

DISCUSSION

The studies reported here demonstrate that material isolatedfrom a fibrosarcoma grown in syngeneic C57BL/6J mice contains

920 CANCER RESEARCH VOL. 44

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Leukotaxis Inhibitor from 7-247 Fibrosarcoma Cells

a low-molecular-weight (500 to 1000) inhibitor of chemotaxis for

PMNs and macrophages. The partially purified material is stableto heat (56°) and is destroyed by acid. A peptide nature is

suggested by its susceptibility to proteolytic enzymes. Thisproperty makes it unlikely that the antichemotactic activity iscaused by a prostaglandin. Such compounds have been shownto produce angiogenic responses in the rabbit eye (5), a characteristic of our partially purified material. Additionally, our antichemotactic activity, unlike prostaglandins, cannot be extractedwith ether from acidified (pH 3) aqueous solution, nor is it solublein absolute ethanol. Finally, the material did not contain detectable prostaglandins according to a radioimmunoassay (notshown). However, this assay procedure (kit from New EnglandNuclear) is only specific for prostanglandins of the E series. Wehave considered these compounds because they have beenreported both to induce angiogenesis (5) and to inhibit chemotaxis (28). Therefore, we cannot exclude from the tumor factorthe presence of other prostaglandins (e.g., prostaglandin F2a) orother metabolites of arachidonate (e.g., leukotrienes).

Our material appears to have properties that are unique incomparison to the tumor-derived materials having immunosup-

pressive activities reported by others. Our tumor factor appearsto be directed against the leukocyte in contrast to the materialof Cohen eíal. (11), which inactivates the attractant, is of muchgreater molecular weight (M, ~ 50,000), and is labile to heating(100°). On the other hand, the inhibition of leukocyte motility

from ascites fluid reported by Friedmann ef a/. (15) does appearto act upon the cell but has a molecular weight of at least twice(1000 to 5000) that of our material. A low molecular activity(1000 to 2000) in media of cultured teratocarcinoma and malignant melanoma cells (Fauve eíal. Ref. 14) has been found toinhibit macrophage motility in vitro, but is not as well-characterized as our tumor factor. A protein containing low-molecular-weight (~10,000) material from oncogenic viruses (Cianciolo ef

a/., Ref. 10) very potently inhibits macrophage migration. Ourmaterial differs from this factor again in possessing a lowermolecular weight and, more importantly, in inhibiting both macrophage and neutrophil migration. In fact, this last property isamong the most significant differences between our tumor factorand the majority of tumor-derived immunosuppressive activities.Another low-molecular-weight activity (M, ~ 500) that inhibits

macrophage migration and that is derived from murine lungcarcinoma (Cheung eíal., Ref. 9) has characteristics of a prostaglandin, whereas our material tested negatively for such acompound in a radioimmunoassay.

While these and other factors reported elsewhere have reportedly varying degrees of purity, our tumor-derived activity hasbeen purified about 100-fold (based upon specific activity deter

minations) from the ethanol extract starting material. The tumorfactor is not homogeneous, since analysis with a high-performance liquid chromatography column (results to be reported) hasindicated the presence of 3 components with antileukotacticactivity. In addition, nonactive materials were found to be presentin material applied to the column. Therefore, the results reportedhere on the effects of the tumor factor upon proteolysis andmethylation must be considered tentative until the more purifiedsubstances can be tested.

Our most purified material has an estimated ID50 of 10 UM,based upon the specific activity of the activity obtained fromanion-exchange chromatography. The ethanol ext act from 5

tumors has the capacity to completely inhibit cell mctility includ

ing random motility in a population of 2 x 106 phagocytes. This

is approximately the concentration both used in the assay forchemotaxis and present in the blood. The same dose of tumorfactor also completely inhibits the chemotactic response in PMNsat twice the aforementioned concentration. On the assumptionthat these are not maximal values, both for potency and yield, itseems reasonable to assume that these fibrosarcomas producehighly potent, physiologically relevant amounts of antileukotacticfactors that may well aid the tumor in its evasion of the host's

immune defense system.The mechanism by which the tumor factor inhibits leukocyte

chemotaxis is not clear. We have found that both the hydrolysisof the chemotactic peptide by the cell and the methylation ofprotein carboxyl groups and phospholipids in PMNs are inhibitedin the presence of the factor. These processes may be necessaryfor chemotaxis. Hydrolysis of the attractant by a cell membrane-

bound peptidase may faciliate detection of the chemical signal(4), and methylation may be involved in amplification of this signal(29, 34). However, the extent of the inhibition of both lipid andcarboxymethylation by the tumor factor is not well correlatedwith its effect upon chemotaxis. Complete inhibition of chemotaxis is achieved with a concentration of tumor factor that inhibitsthe methylation reactions by only 15%. While it is possible thata small fraction of cellular methylation may be necessary forchemotaxis, the observations on the effects of the tumor factoron the inhibition of both methylation of lipids and protein carboxylgroups and the hydrolysis of a peptide attractant could indicatethat the inhibition might act at a site that is antecedent to thoseevents. For example, the tumor factor might interfere with a stepin processing the receptor for chemoattractants that may benecessary for the motile response. In this regard, the tumorfactor does not compete directly with the fMet attractant for abinding site on the cell. It may, however, bind to its own receptor.In some respects, the action of the tumor factor resembles thatof methyl transfer inhibitors, such as 3-deazaadenosine, which

inhibits chemotaxis and methylation in the neutrophil but doesnot compete with fMet peptides for their receptor (29).

A problem often encountered in studies with oncogenic virusesarises from contamination by lactic dehydrogenase viral infection(10,38) which can itself depress macrophage function. However,our antichemotactic factor would appear to be of a much smallersize than products from viral contamination (e.g., proteins). Inaddition, since we have taken precautions to eliminate such viralcontamination from the tumors used here, it is likely that it is thetumor itself which produces a low-mplecular-weight antichemotactic material. Additionally, the T-241 cells in culture were foundto be lactic dehydrogenase virus-negative in multiple tests withmultiple passages (see "Materials and Methods").

There is ample evidence that links a defective inflammatoryresponse to the presence of tumors in the host (20, 22, 24, 25,27). A majority of cancer patients have depressed monocytechemotaxis which improves after surgical removal of the tumor(35, 36). Mice given implants of syngeneic neoplasms show areduced ability to mobilize macrophages to inflammatory areas(33).

Along with lymphocytes (43), phagocytes have a major role intumor killing. Macrophages appear to destroy tumor cells via anantibody-dependent cytotoxicity mechanism and by binding di

rectly to the target cell (1, 2, 8). The first mode is at timesassociated with phagocytic events in the leukocyte, and thesecond, by secretion of a lytic protease that kills the cell. It is

MARCH 1984 921

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

H. Warabi et al.

likely that PMNs use similar mechanisms to kill tumor cells (12,16). In any case, the phagocyte must be in contact with thetarget cell to cause its destruction. Obviously, the secretion ofsubstances which depress phagocyte motility can be a majoradvantage to the tumor in preventing its rejection by the host.Of relevance here are our preliminary indications that the tumorfactor inhibits the formation of active oxygen, a tumoricidal agent,in phagocytes (40). We do not yet have any information on theeffect of the tumor factor upon leukocyte secretion.

Whatever the mechanism is whereby the tumor factor inhibitscell motility, whether by interfering in detection of the chemotacticsignal (which may involve hydrolysis of the attractants) or bypreventing the transmission of that signal to the cytoskeleton(which may involve methylation), the elucidation of the action ofthe tumor factor should contribute to understanding how thetumor becomes established and metastasizes in the host.

REFERENCES

1. Adams, D. O., Johnson, W. J., and Marino, P. A. Mechanisms of targetrecognition and destruction in macrophage-mediated tumor cylotoxicity. Fed.Proc., 41: 2212-2221,1982.

2. Adams, D. O., and Snyderman, R. Do macrophages destroy nascent tumors?J. Nati. Cancer Inst., 62: 1341-1343, 1979.

3. Aswanikumar, S., Corcoran, B., Schiffmann, E., Day, A. R., Freer, R. J.,Showell, H. J., Becker, E. L, and Pert, C. B. Demonstration of a receptor onrabbit neutrophils for chemotactic peptides. Biochim. Biophys. Acta, 74: 810-817, 1977.

4. Aswanikumar, S., Schiffmann, E., Corcoran, B. A., and Wahl, S. M. Role ofpeptidase in phagocyte chemotaxis. Proc. Nati. Acad. Sei. U. S. A. 73: 2439-2442,1976.

5. Ben-Ezra. D. Neovasculogenic ability of prostaglandins, growth factors, andsynthetic chemoattractants. Am. J. Ophthalmol., 86: 455-461, 1978.

6. Boetcher, D. A., and Leonard, E. D. Abnormal monocyte chemotactic responsein cancer patients. J. Nati. Cancer Inst., 52: 1091-1099,1974.

7. Bronza, J. P.. and Ward, P. A. Antileukotactic properties of tumor cells. J. Clin.Invest, 56. 616-623,1975.

8. Cameron, D. J., and Churchill, W. H. Cytotoxicity of human macrophages fortumor cells: enhancement by bacterial lipopolysaccharides (LPS). J. Immunol.,724: 708-712, 1980

9. Cheung, H. T., Cantarow. W. D., and Sundharadas, G. Characteristics of alow-molecular-weight factor from mouse tumors that affects in vitro propertiesof macrophages. Int. J. Cancer, 23. 344-352, 1979.

10. Cianciolo, G. J., Mathews, T. J., Bolognesi, D. P., and Snyderman, R. Macrophage accumulation in mice is inhibited by low molecular weight products frommurine leukemia viruses. J. Immunol., 724: 2900-2905, 1980.

11. Cohen, M. C., Bronza, J. P., and Ward, P. A. In vitro and in vivo production ofchemotactic inhibitors by tumor cells. Am. J. PathoL, 94. 603-614,1979.

12. Dvorak, A. M., Connell, A. B., Proppe, K., and Dvorak, H. J. Immunologierejection of mammary adenocarcinoma (TA3-SI) ¡nC57BL/6 mice: participationof neutrophils and activated macrophages with fibrin formation. J. Immunol.,720: 1240-1248, 1978.

13. Dvorak, H. F., Orenstein, N. S.. and Dvorak, A. M. Tumor-secreted mediatorsand tumor microenvironment. In: E. Pick (ed.), Lymphokines. Vol. 2, pp. 203-233. New York: Academic Press, Inc., 1981.

14. Fauve, R. M.. Hevin, B., Jacob, H., Gaillard, J. A., and Jacob, F. Anti-inflammatory effects of murine malignant cells. Proc. Nati. Acad. Sei. U. S. A.,777:4052-4056, 1974.

15. Friedman, H., Spector, S., Kamao, I., and Katley, J. Tumor-associated immu-nosuppressive factors. Ann. N. Y. Acad. Sci., 276: 417-430,1976.

16. Gale, R. P., and Zighelboim, J. Modulation of polymorphonuclear leukocyte-mediated antibody-dependent cellular cytotoxicity. J. Immunol., 773: 1793-1800, 1974.

17. Hamby, V., and Barrett, J. T. Inhibition of leukocyte chemotaxis by homoge-nates for tumor tissues. Oncology (Basel), 34:13-15, 1977.

18. Huber, S. C., and Lucas, Z. J. Immune response to a mammary adenocarcinoma. V. Sera from tumor-bearing rats contain multiple factors blocking cell-mediated cytotoxicity. J. Immunol., 727: 2485-2489 1978.

19. Liotta, L. A., Kleinerman, J., and Saidel, G. M. Quantitative relationships ofintravascular tumor cells, tumor vessels, and pulmonary métastasesfollowingtumor implantation. Cancer Res., 34: 997-1004,1974.

20. Maderazo, E. G., Anton, T. F., and Ward, P. A. Serum-associated inhibition ofleukotaxis in humans with cancer. Clin. Immunol. Immunopathol., 9:166-176,1978.

21. Meltzer, M. S., and Stevenson, M. M. Macrophage function in tumor-bearingmice: tumoricidal and chemotatic responses of macrophages activated byinfection with Mycobacterium bovis. strain BCG. J. Immunol., Õ78:2176-2181,1977.

22. Nelson, M., and Nelson, D. C. Macrophages and resistance to tumors. I.Inhibition of delayed-type hypersensitivity reactions by tumor cells and bysoluble products affecting macrophages. Immunology, 34: 277-290,1978.

23. Normann, S. J., and Sorkin, E. Cell-specific defect in monocyte function duringtumor-growth. J. Nati. Cancer Inst., 57: 135-140,1976.

24. North, R. J., Kirstein, D. P., and Tuttle, R. L. Subversion of host defensemechanisms by murine tumors. I. A circulating factor that suppresses macrophage-mediated resistance to infection. J. Exp. Med., 743: 559-572, 1976.

25. North, R. J., Kirstein, D. P., and Tuttle, R. L. Subversion of host defensemechanisms by murine tumors. II. Counter-influence of concomitant antitumorimmunity. J. Exp. Med., 743: 574-584,1976.

26. O'Dea, R. F., Viveros, 0. H., Axelrod, J., Aswanikumar, S., Schiffmann, E.,

and Corcoran, B. A. Rapid Stimulation of protein carboxymethylation in leukocytes by a chemotatic peptide. Nature (Lond.), 272: 462-464, 1978.

27. Rhodes, J., Bishop, M., and Benifield, J. Tumor surveillance: how tumors mayresist macrophage-mediated host defense. Science (Wash. D. C.), 203: 178-182,1979.

28. Rivkin, I., Rosenblatt, J., and Becker, E. L. The role of cyclic AMP in thechemotactic responsiveness and spontaneous motility of rabbit peritonealneutrophils. J. Immunol., 775: 1126-1134,1975.

29. Schiffmann, E., O'Dea, R. F., Chiang, P. K., Venkat, K., Corcoran, B., Mirata,

F., and Axelrod, J. Role for Methylation in Leukocyte Chemotaxis in Modulationof Protein Function, pp. 299-313, New York: Academic Press, Inc., 1979.

30. Schiffmann, E., Showell, H. J., Corcoran, B. A., Ward, P. A., Smith, E., andBecker, E. L. The isolation and partial characterization of neutrophil chemotactic factors ¡romEscherichia coli. J. Immunol., 774: 1831-1837,1975.

31. Snyderman, R., and Fudman, E. J. Demonstration of a chemotactic factorreceptor on macrophages. J. Immunol., 724: 2754-2757,1980.

32. Snyderman, R., and Pike, M. C. An inhibitor of macrophage chemotaxisproduced by neoplasms. Science (Wash. D. C.), 792: 370-372,1976.

33. Snyderman, R., Pike, M. C., Blaylock, B. L., and Weinstein, P. Effects ofneoplasms on inflammation: depression of macrophage accumulation aftertumor implanation. J. Immunol., 776: 585-589,1976.

34. Snyderman, R., Pike, M. A., and Kredich, N. M. Role of transmethylationreactions in cellular motility and phagocytosis. Mol. Immunol., 77: 209-218,1980.

35. Snyderman, R., Pike, M. C., McCariey, D., and Lang, L. Quantification ofmouse macrophage chemotaxis in vitro—role of C5 for production of chemotactic activity. Infect. Immun., 77: 488-492, 1975.

36. Snyderman, R.. Pike, M. C., Meadows, L., Hemstreet, G., and Wells, S.Depression of monocyte chemotaxis by neoplasms. Clin. Res., 23: 297,1975.

37. Snyderman, R., and Stahl, C. Defective immune effector function in patientswith neoplastic and immune deficiency diseases. In: J. A. Sellanti and D. H.Dayton (eds.). The Phagocytic Cell in Host Resistance, pp. 267-281. New

York: Raven Press, 1975.38. Stevenson, M. M., Ress, J. C., and Meltzer, M. S. Macrophages function in

tumor-bearing mice: evidence for lactic dehydrogenase-elevating virus-associated changes. J. Immunol., 724: 2892-2899, 1980.

39. Stutman, O. Immunodeficiency and cancer. In: I. Green, S. Cohen, R. T.McCluskey (eds.), Mechanisms of Tumor Immunity, pp. 27-53. New York:

John Wiley & Sons, Inc., 1977.40. Szuro-Sudol, A., and Nathan, C. F. Suppression of macrophage oxidative

metabolism by products of malignant and non-malignant cells. J. Exp. Med.,756:945-961, 1982.

41. Ward, P. A., Dvorak, H. F., Cohen, S., Yoshida, T., Dater, R., and Selvaggio,S. S. Chemotaxis of basophils by lymphocyte-dependent and lymphocyte-independent mechanisms. J. Immunol., 774: 1523-1531, 1975.

42. Winstein, I. B. Cell regulation and cancer. Differentiation, 73: 65-66,1979.43. Yoshida, T., and Cohen, S. Lymphokines in tumor immunity. In: I. Green, S.

Cohen, and R. T. McCluskey (eds.), Mechanisms of Tumor Immunity, p. 87,New York: John Wiley & Sons, Inc., 1977.

44. Zigmond, S. Z., and Hirsch, J. G. Leukocyte locomotion and chemotaxis. Newmethods for evaluation, and demonstration of a cell-derived chemotactic factor.J. Exp. Med., 737: 387-410, 1973.

922 CANCER RESEARCH VOL. 44

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1984;44:915-922. Cancer Res   H. Warabi, K. Venkat, V. Geetha, et al.   Fibrosarcoma CellsLow-Molecular-Weight Inhibitor of Leukotaxis from Identification and Partial Characterization of a

  Updated version

  http://cancerres.aacrjournals.org/content/44/3/915

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

  .pubs@aacr.orgDepartment 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/44/3/915To request permission to re-use all or part of this article, use this link

on April 10, 2021. © 1984 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from