THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1994 hy The American Society for Biochemistry and Molecular Biology, Inc

1994 Vol. 269,No. 40, Issue of October 7. pp. 2510625119, Printed in U.S.A.

Characterization of a Novel Tumor-derived Cytokine ENDOTHELIAL-MONOCYTE ACTIVATING POLYPEPTIDE II* (Received for publication, May 10, 1994, and in revised form, June 24, 1994)

Janet KaoS, Keith Houck§, Yan Fan, Iris Haehnel, Steven K. Libutti, Mark L. Kaytonn, Tracy Grikscheit, John Chabot, Roman Nowygrod, Steven Greenberg, Wun-Jing KuangQ, David W.LeungO, Joanne R. Hayward§, Walter Kisielll, Mark Heath, Jerold Brett, and David M. Stern$ From the Departments of Physiology, Surgery, Medicine, and Anesthesia, Columbia Uniuersity, College of Physicians and Surgeons, New York, New York 10032, the #Departments of Molecular Biology and Recovery Sciences, Genentech, South San Francisco, California 94080, and //TheBlood Systems Research Foundation Laboratory, Department ofPathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131

Endothelial-monocyte activating polypeptide I1 (EMAP 11) was initially identified in the supernatant of murine methylcholanthrene A-inducedfibrosarcomas (Meth A) by its capacity to activate host effector cells (Kao, J., Ryan, J., Brett, J., Chen, J., Shen, H., Fan, Y-G., Godman, G., Familletti, P., Wang, F., Pan, Y-C., Stern, D., and Clauss, M. (1992) J. BioZ. Chern. 267, 20239-20247). Basedon the NH,-terminal protein sequence, a fulllength cDNA has been cloned which indicates that the precursor ofEMAP I1 is a unique, leaderless, single polypeptide chain with predicted molecular mass 4 4 kDa and that themature form released by Meth A cells corresponds to -20 kDa. Purified recombinant mature EMAP I1 (EMAP 1 1 , 4 0 kDa form) activated endothelial cells with resulting elevation of cytosolic free calcium concentration, release of von Willebrand factor, induction of tissue factor, and expression of the adhesion molecules E-selectin and P-selectin.Neutrophils exposed to EMAP I1 demonstrated elevated cytosolic free calcium concentration, peroxidase generation, and chemotaxis. EMAP I1 also activated mononuclear phagocyteselevating cytosolic free calcium concentration, inducing tumor necrosis factor-a (TNF)and tissue factor, and stimulating chemotaxis. Systemic infusion ofEMAP I1 into C3HMeJ or Balb/c mice was associated with systemic toxicity, pulmonary congestion, and the appearance of TNF,interleukin-1 and -6 in the plasma. A single intratumor injection ofEMAP I1 into Meth A sarcomas induced acute thrombohemorrhage and partial tumor regression.Local injection of EMAP I1 into a tumor resistant to the effects of TNF,murine mammary carcinoma, rendered it sensitive to subsequently administered TNF,which resulted in acute thrombohemorrhage

* This work was supported by United States Public Health Service Grants HL02641, HL42833, HL42507, HL21006, and HL50629 and by grants from the Cancer Research Institute, Surgical Research Fund, Council for Tobacco Research, and Juvenile Diabetes Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement”in accordance with 18 U.S.C.Section1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s1 U10117 (human EMAP II) and U10118 (murine EMAP II). $ To whom correspondenceshould be addressed: Drs. Janet Kao/ David Stern, Dept. of Physiology, P&S 11-518, Columbia UniversityCollege of P & S, 630 W. 168th St., New York, NY 10032. Tel.: 212-3051615; Fax: 212-305-5337. 1 Recipient of American Cancer Society Grant IRG-l77B, support from the Columbia University Comprehensive Cancer Center, and of a medical student fellowship from the American Heart Association New York affiliate.

and partial regression. These data suggest that recombinant EMAP 11, a tumor-derived cytokine, has properties of a proinflammatory mediator with the capacity to prime the tumor vasculature for a locally destructive process.

Tumor vasculature represents a critical link between the host and neoplasm. It serves as a conduit for the delivery of nutrientsnecessarytosustain the tumorandpromoteits growth and spread, but it alsoserves as a portal for the entry of host effector cells and cytotoxic agents (1,2).The vasculature of certain tumors has properties which distinguish it from the normal vasculature; these include an exaggerated response to the systemic infusionof flavone acetic acid or cytokines, such as interleukin 1 (IL-1)’ and tumor necrosis factor-a (TNF), increased permeability, and infiltration by inflammatory/ immune effector cells (3-10). To understand mechanisms responsible for vascular dysfunction in the tumor bed, we have characterized mediators released bythe immunogenic murine methylcholanthrene A-induced fibrosarcoma (Meth A) based on their capacity to modulate properties of endothelial cells(ECs), mononuclear phagocytes (MPs), and polymorphonuclear leukocytes (PMNs), cells which have important roles in tumor vasculature (11-13). Meth A tumors provide a suitable model for assessing the effects of tumor-derived factors which modulate the host response (4,14). The vasculatureof these tumors is distinguished from that of normal tissues by the properties described above. The response of Meth A tumors to systemically administered beginning with cytokines has been well characterized, hemorrhage/thrombosis and increased permeability of tumor vessels, and associated with diminished blood flow as well as subsequent influx of host effector cells (4).Although Meth A cells produce multiple mediators capable of interacting with inflammatoryhmmune effector cells, three polypeptides which are likely to contribute to host-tumor interactions have been characterized in more detail (11-13). One of these proved to be

’The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor-alpha; EMAP, endothelial-monocyte activating polypeptide; r, recombinant; VPFNEGF, vascular permeability factor/vascular endothelial growth factor; Meth A, murine methylcholanthrene A-induced fibrosarcoma; PCR, polymerase chain reaction; LPS, lipopolysaccharide; EC, endothelial cell; MP, mononuclear phagocyte; PMN, polymorphonuclear leukocyte; TF, tissue factor; GAF’DH, glyceraldehyde phosphate dehydrogenase; vWF, von Willebrand factor; FPLC, fast protein liquid chromatography;bp, base paids); MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gelelectrophoresis;ELISA, enzyme-linked immunosorbant assay.

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tides at the 5'-end of the murine EMAP I1 clone.This ClaI (in thevector polylinker) to ScaI (500 bp) fragment was nick-translated and hybridized in formamide (20%),sodium phosphate (50 mM, pH 6.5), SSC (5 x), Denhardt's solution (5 x), SDS (O.l%), and denatured salmon sperm DNA (40 pg/ml) at 42 "C. About 20 positives were obtained, and 10 of these were purified. The three which contained the longest inserts appeared to have identical 1100-bp EcoRI inserts. These inserts were subcloned in pUC219 and sequenced. Northern Analysis of Meth A Cell mRNA for EMAPII ExpressionPoly(A)+ RNAfrom Meth A sarcoma cells was denatured and subjected to electrophoresis on an agarose gel (1.2%)in MOPS-formaldehyde(18). The RNAwas transferred to nitrocellulose (Schleicherand Schuell) and prehybridized in formamide (50%), sodium phosphate (50 mM, pH 6.51, SSC (5 x), Denhardt's solution (5 x), SDS (0.1%),and denatured salmon sperm DNA (40 pg/ml) at 42 "C. A 279-bp DNA fragment was isolated from the murine EMAP I1 clonefollowing XbaI and Sac1 digestion corresponding to nucleotides 652-930. This was nick-translated with 32P-dCTPand hybridized to the blot overnight. Washing was performed at a final stringency of SSC (0.2 x)/SDS (0.1%)at 55 "C. The blot was then exposed overnight for autoradiography. E. coli Expression of Murine EMAP II-In order to confirm the biological activity of the protein encoded by the cloned DNA sequence, the region corresponding to the predicted mature protein, based on the MATERIALSANDMETHODS NH,-terminal sequence obtained from purified EMAP 11, wasexIsolation of Meth A cell RNA-Meth A cells (generously provided by pressed. This was accomplished using a fragment of the murine clone Dr. L.Old, Cancer Research Institute, New York,N y , 14)were grownin extending from the BstBI site (nucleotide 529) to the 3"untranslated RPMI 1640containing fetal bovine serum (10%;Gemini, Calabasas CA) region and synthetic DNA encoding the NHzterminal end, KPIDAand to -90% confluence. Cells (-lo8) were harvested with trypsin, resus- SRLEL (5'-TATGAAACCAATCGATGCATCTCGTCTGGATCTT-3' pended in fetalbovine serum (lo%),and poly(A)' RNA isolated directly 5'-CGAAGATCCAGACGAGATGCATCGATTGGT"CA-3'). This seas described (16).Briefly, cells were lysed in SDS-containingbuffer, and quence, which differs from the NH,-terminal region obtained by microproteins were digested with proteinase K (Boehringer Mannheim) for 3 sequencing because the NH,-terminal residue, serine, was inadverth at 55 "C. The poly(A)+RNA was further purified using oligo(dT)- ently omitted when designing the synthetic DNA, was cloned into the NdeI site, containing the ATG initiation codon, and the BamHI site of cellulose bound tomagnetic beads (Promega, Madison, WI). Isolation of Murine cDNA Clones-Meth A mRNA (1 pg) was dena- the vector PET-3a in which cDNA expression is driven by the T7 protured with MeHgOH and reverse transcribed with avian myeloblastosis moter. The protein was then expressed in the host HMS174(DE3) which virus reverse transcriptase (Invitrogen, San Diego, CAI using oli- contains the T7RNA polymerase gene under control of the lacUV5 go(dT),, as a primer. The first strandcDNA obtained was used as tem- promoter. Following growth to log phase, the T7 polymerase was in(0.4 mM), and the duced with isopropyl-1-thio-0-D-galactopyranoside plate for the polymerase chain reaction (PCR)using degenerate primers based on the NH,-terminal protein sequence obtained for EMAF' I1(13). cells were harvested by centrifugation 3 h later. The pellet was dissolved in Tris (20 mM, pH 7.4)EDTA (2 mM)/ and The sense primer was 5'-GGCGAATTCAARCCNATHGAYGC-3', the antisense primer was 5'-GGCGAATTCYTTNGCNGTNACDAT-3'benzamidine (1mM)/sodium azide (0.02%)/octyl-p-glucoside(0.1%),agiwith EcoRI sites near their 5'-ends to facilitate cloning of the PCR tated a t 4 "C, and sonicated. The supernatant was centrifuged at 20,000 products. The thermocycling parameters consisted of three cycles of revolutions/min for 40 min (4 "C), and dialyzed versus Tris (20 mM,pH (0.1%).After dialysis, the sample (30-40 mg) was 7.4)/octyl-~-glucoside 95 "C for 30 s, 30 s at 37 "C, and 1min at 72 "C. This was followed by 30 cyclesof 30 s at 95 'C, 30 s at 55 OC, and 1min a t 72 "C. Analysisof applied to FPLC Mono Q (HR 5/5),and the column was eluted with an the amplified products on an acrylamide gel showed a DNAfragment of ascending salt gradient (0-1.0 M).Fractions were assayed for their ability to induce EC tissue factor (see below), and active material was the expected size of 77 bp. The PCR products were then digested with EcoRI, run on an acrylamide gel, the appropriate band excised and pooled, concentrated (Centricon 3, Beverly, MA), and subjected to preeluted, and the DNA fragment cloned into the plasmid vector pUC219. parative nonreduced SDS-PAGE (12.%; 19). Gels were then sliced into Plasmids containing EcoRI inserts weresequenced by the Sanger 1-mm portions and eluted during incubation in sodium acetate (0.5 M, pH 8.3,0.5 ml)/octyl-p-glucoside(0.1%)overnight at 4 "C. Samples were dideoxynucleotide method using Sequenase (U. S. Biochemical Corp., Cleveland, OH). The deduced amino acid sequencewas found to match tested for their ability to induce EC tissue factor (see below), subjected exactly that obtained by protein sequencing (13). A 57-mer nucleotide to analytical SDS-PAGE (12.5%)and silver staining, and tested for their probe was designed based on the consensus nucleotide sequence ob- lipopolysaccharide (LPS) content (Endospecy,Seikagaku Corp., Tokyo, tained from sequencing several clones (5'AAGCCCATTGATGCCTC- Japan). According to the latter assay, LPScontamination of the purified CCGGCTGGACCTGCGGATTGGCTGCATTGTGACAGCCAAG-3'). material was