WNT-1 INDUCED SECRETED POLYPEPTIDES: WISP-1, -2 AND -3

Abstract: Wnt-1-Induced Secreted Proteins (WISPs) are provided, whose genes are induced at least by Wnt-1. Also provided are nucleic acid molecules encoding those polypeptides, as well as vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides, and methods for producing the polypeptides.

Description: WNT-1 INDUCED SECRETED POLYPEPTIDES : WISP-1 , "2 AND -3 This invention was made with government suppoπ under grant no. 5POI CA41086. awarded by the National Institutes of Health. National Cancer Institute. The government has certain rights in the invention. FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of novel DN A and to the recombmant production of novel polypeptides having homoiogy to connective tissue growth factor, designated herein as Wnt-1 -Induced Secreted Proteins (WISPs). BACKGROUND OF THE INVENTION Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease. Boring et al.. CA Cancer J Gin.. 43.. 7 ( 1993). Cancer is characterized by the increase in the number of abnormal, or neoplastic. cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation ot malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites ( metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness. Alteration of gene expression is intimately related to the uncontrolled cell growth and de- differentiation which are a common feature of all cancers. The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes. which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes. that act to promote malignant growth. Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype. Hunter. CeJi, 64: 1 129 ( 1991 ); Bishop, CeM, 64: 235-248 (1991 ) A well-known mechanism of gene (c g . oncogene i overexpression in cancer cells is gene amplification. This is a process where in the chromosomeof the ancestral cell multiple copies of a particular gene are produced. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome. Alitalo et al.. Adv. Cancer Res.. 47: 235-281 ( 1986). It is beiieved that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made. Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer. For example, it has been found that the human ErbB2 gene (erbB2. also known as her2. or c-erbB-2). which encodes a 185- kd transmembraneglycoproteinreceptor l 85 HER ~ HER2) related to the epidermal growth factor receptor (EGFR). is overexpressedm about 25% to 30% of human breast cancer. Slamon et al.. Science. 23_5_: 177- 182 ( 1987); Slamon et al. , Science. 244-707-712 ( 1989) It has been reported that gene amplification of a protooncogen is an event typically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome. Schwab et al . Genes Chromosomes Cancer I 181 - 193 ( 1990) A talo et at supra Thus. eibBl overexoression is commonK regarded as a predictorot a poor prognosis, especially in patients w ith pnmarv disease that involves axillarv Iv mph nodes ! Slamon et al Λ 1987) and ( 1989). supra. Ravdin and Chamness. Gene, \___\ 19-27 ( 1995). and Hynes and Stern. 165-184 ( 1994)) and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cvclophosphamide. methotrexate. and Ωuoruracil) and anthracyc lines Baselga et al . Oncology. 1 1 (3 Suppl 1 ) 43-48 ( 1997) However, despite the association ot erbB2 overexpression with poor prognosis, the odds ot HER2-posιtιve patients responding clinical Iv to treatment with taxanes were greater than three times those ot HER2-negatιve patients Baselgaer al . supra A recombinant humanized antι-ErbB2 (antι-HER2) monoclonal antibody (a humanized version ot the muπne antι-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or HERCEPTIN®) has been clinically active in patients with ErbB2-overexpressιng metastatic breast cancers that had received extensive prior anticancer therapy Baselga et a! . J Clin Oncol . 14 737-744 ( 1996) Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dvsfunction has been attributed to a change in amino acid neurotransmitter metabolism. In particular, members ot the transtorminggrowth tactor-β (TGF-β) family have been implicated TGF peptides are small polypeptides that were first identified bv their ability to induce proliferation and transformation in noncancerouscells in culture Although mitiallv defined as a growth factor. TGF-β also inhibits proliferation ot epithelial, endothehal. lymphoid. and hematopoietic cells This cytokme is thought to play an important role in regulating the duration of the inflammatory response, allowing the healing process to proceed. It is also a potent immunomodulator. which has many pleiotrophic effects, including regulating many other cytokines The TGF-β superfamily includes bone morphogenetic proteins (BMP-2. B P-4. BMP-5. BMP-6, BMP-7). activms A &. B. decapentaplegιc(dpp), 60A. OP-2. dorsalin. growth differentiation factors (GDFs). nodal. MIS. Inhibin-α. TGF-β I, TGF-β2, TGF-B3. TGF-β5. and glial-deπved neurotrophic factor (GDNF) Atπsano. et al J Biochemicaet Biophvsica Acta. 1 22 71 -80 ( 1 94) Ot particular interest are the growth differentiation factors, for as their name implies, these factors are implicated in the differentiation ot cells Connective tissue growth factor (CTGF) is a growth factor induced in fibroblasts by many factors. including TGF-β. and is essential for the ability of TGF-β to induce anchorage-independent growth (AIG). a property of transformed cells CTGF was discovered in an attempt to identify the type of platelet-derived growth factor (PDGF) dimers present in the growth media of cultured endothehal cells, and is related immunologicaliy and biologicallv to PDGF See U S Pat No 5,408.040 CTGF also is mitogenic and chemotactic for cells, and hence growth factors in this family are believed to play a role in the normal development, growth, and repair of human tissue Seven proteins related to CTGF, including the chicken ortholog for Cyrό l . CEF10. human, mouse. and Xenopus laevts CTGF, and human, chicken, and Xenopus laevis Nov have been isolated, cloned, sequenced. and characterized as belonging to the CTGF gene family Oemar and Luescher, Arteπoscler Thromb Vase Biol . 17 1483- 1489 (1997) The gene encoding Cyrό l has been found to promote angiogenesis. tumor growth, and vasculaπzation Babic et al . Proc Natl Acad Sci USA. 95- 6355-6360 ( 1998) The nov gene is expressed in the kidney essentially at the embryonic stage, and alterations ot nm e\pressιon relative to the normal kidney have oeen detected in ooth avian nephroblastomas and human ilms tumors Martineπetv α/ Oncogens 2 2 7 29-2732 ( 1994) Wt I downregulateshuman nov expression which downregulation might represent a key element in normal and tumoral nephrogenesis Martineπe et ul . Oncogene 12 1479-1492 ( 1996) It has recently been proposed that the CTGF. and c\r61 genes -> which encode secreted proteins that contain conserved sequences and IGFBP motifs in their N-termini and bind IGFs with low atfinitv represent more members ot the IGFBP supertamilv along with the low-atfinitv mac25TGFBP-7(Yamandkae/ α/ J Biol Chem . - 30729-30734 ( 1997)) and the high-atfinitv lGFBPs 1 -6 CTGF under this proposal would be designated IGFBP-8 eι al Proc Nnti Acad Sci USA 94 12981- 12986 ( 1997) 10 Recentlv a protein was found in the mouse designated ELM 1 that is expressed in low metastatic cells Hashimotoe/ α/ . 187 289-296 ( 1998) The elm 1 gene, a mouse homologue of WISP- I disclosed below is another member of the CTGF. Cyrόl/CeflO. and neuroblastoma overexpressed-gene family and suppresses in vivo tumor growth and metastasis ot K.- 1735 muπne melanoma cells Another recent article on rCop-1, the rat orthologue of WISP-2 described below describes the loss of expression ot i this gene after cell transformation Zhang et al Mol Cell Biol . 18 6131 -6141 ( 1998) CTGF family members (with the exception ot nov) are immediate earlv growth-responsive genes that are thought to regulate cell proliferation, differentiation, embrvogenesis. and wound healing Sequence homology among members of the CTGF gene family is high, however, functions ot these proteins in vitro range from growth stιmulatorv(; e human CTGF) to growth inhibitory (; e . chicken Nov and also possiblv 0 hCTGF) Further, some molecules homologous to CTGF are indicated to be useful in the prevention ot desmopiasia. the formation of highly cellular, excessive connective tissue stroma associated with some cancers, and fibrotic lesions associated with various skin disorders such as scleroderma. keloid. eosinophilic fascutis. nodular fasctitis. and Dupuytren s contracture Moreover. CTGF expression has recentlv been demonstrated in the fibrous stoma ot mammarv tumors, suggesting cancer stroma formation involves the 5 induction ot similar fibroproliferativegrowth factors as wound repair Human CTGF is also expressed at verv high levels in advanced atherosclerotic lesions, but not in normal arteries, suggesting it mav plav a role in atherosclerosis Oemarand Luescher. supra Therefore, molecules homologous to CTGF are ot importance Extracellular and membrane-bound proteins play important roles in the formation, differentiation. and maintenance of multicellularorganisms The fate ofmanv individualcells, e g , proliferation, migration. 0 differentiation, or interaction with other cells, is typically governed by information received from other cells and or the immediate environment This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropepttdes. and hormones), which are. in turn, received and interpreted by diverse cell receptors or membrane-boundproteins These secreted polvpeptidesor signaling molecules normallv pass through the cellular secretory pathwav to 5 reach their site of action in the extracellular environment, usually at a membrane-bound receptor protein Secreted proteins have various industrial applications, including use as pharmaceuticals, diagnostics. biosensors, and bioreactors In fact, most protein drugs available at present, such as thromboivtic agents interferons. interleukins. ervthropoietins.coionv stimulating factors, and various other cvtokines, are secreted proteins Their receptors, which are membrane-bound proteins, also have potential as therapeutic or diagnostic asents Receptor immunoaαhesins. tor instance, can oe employed as tneraoeutic agents to block receptor-ligand interaction. Membrane-bound proteins can also be employed tor screening ot potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. Such membrane-bound proteins and cell receptors include, but are not limited to. cvtokine receptors, receptor kinases. receptor phosphatases. receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and inteεπns. Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphory lation of various cellular proteins. Protein tyrosine kinases. enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor. Efforts are being undertaken by both industry and academia to identify new. native secreted and membrane-boundreceptor proteins, particularly those having homology to CTGF. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature. See, for example, Klein et al.. Proc. Natl. Acad. Sci.. 93. 7108-71 1 ( 1996); and U S. Patent No. 5.536,637. nts are encoded by a large gene family whose members have been found in round worms, insects, cartilaginous fish, and vertebrates. Hollander al . Dev. Suppl.. 125- 133 ( 1994) Wnts are thought to function in a variety of developmentaland physiological processes since many diverse species have multiple conserved Wnt genes. McMahon, Trends Genet.. 8: 236-242 ( 1992); Nusse and Varmus. CeH, 69: 1073- 1087 ( 1992). Wnt genes encode secreted glycoproteinsthat are thought to function as paracrine or autocπne signals active in several primitive cell types. McMahon. supra { 1992); Nusse and Varmus, supra { 1992). The Wnt growth factor family includes more than ten genes identified in the mouse (iVnt-\. -2, -3 A. -3B. -4, -5 A, -5B, -6, -7A, -7B, -8A. -8B. -10B. - l l . - 12. and - 13) (see, ' g , Gavin e. at. Genes Dev . 4. 2319-2332 ( 1990). Lee et al.. Proc. Natl. Acad. Sci USA. 9J . 2268-2272 ( 1995): Christiansen et al.. Mech. Dev.. 5±' 341-350 ( 1995)) and at least nine genes identified in the human ( Wnt- \ . -2. -3. -5 A. -7A. -7B. -8B. - 10B. and - 1 1 ) by cDNA cloning. See. e.g , Vant Veer et al . Mol.Cell Biol . 4. 2532-2534 ( 1984). The Wnt-1 proto-oncogene (ιnt-1) was originally identified from mammary tumors induced by mouse mammary tumor virus (MMTV) due to an insertion of viral D A sequence. Nusse and Varmus, Cell, 31 : 99-109 (1982). In adult mice, the expression level of WntΛ mRNA is detected only in the testis during later stages of sperm development. Wnt- 1 protein is about 42 KDa and contains an ammo- terminal hydrophobic region, which may function as a signal sequence for secretion (Nusse and Varmus. supra. 1992). The expression of Wnt-2/iηp is detected in mouse fetal and adult tissues and its distribution does not overlap with the expression pattern for Wnt- 1. Wnt-3 is associated with mouse mammary tumoπgenesis. The expression of Wnt-3 in mouse embryos is detected in the neural tubes and in the limb buds. Wnt-5a transcripts are detected in the developing fore- and hind limbs at 9.5 through 14.5 days and highest levels are concentrated in apical ectoderm at the distal tip of limbs. Nusse and Varmus, supra ( 1992). Recently, a Wnt growth factor, termed Wnt-x, was described (W095/17416) along with the detection of Wnt-x expression in bone tissues and in bone-derived cells. Also described was the role of Wnt-x in the maintenance of mature osteoblasts and the use ot the W nt-x growth factor as a therapeutic agent or in the development ot other therapeutic agents to treat bone-related diseases Wnts mav plav a role in local cell signaling Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freelv diffusible in the medium Papkott and Schrvver. Mol Cell Biol . 10 2723-2730( 1990) Bradlev and Brown. EMBO J . 9 1569- 1575 ( 1990) Studies ot mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning In Drosophila. windless (v tj) encodes a H- -related gene (Rijsewik et al , Cell. 50 649-657 ( 1987)) and wς- mutations alter the pattern ot embryonic ectoderm, neurogenesis. and imaginai disc outgrowth Morata and Lawerence. Dev B ol . 56 227-240 ( 1977). Baker. Dev Biol , 125 96- 108 (1988). Klingensmtth and Nusse, Dev Biol . 166 396-414 ( 1994) In Caenorhabdttis ele ans, lιn-44 encodes a Wnt homolog which is required for asymmetric cell divisions Herman and Horvitz. Development, 120 1035- 1047 (1994) Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradlev, Cell, 62 1073- 1085 (1990), Thomas and Cappechi. Nature. 346- 847-850 (1990)). and the outgrowth ot embrvonic pπmordia for kidnev (Stark et al . Nature. 372 679-683 ( 1994)), tail bud (Takada et at . Genes Dev . 8 174- 189 ( 1994)). and limb bud Parr and McMahon. Nature. 374 350-353 ( 1995) Overexpressionof Wnts in the mammary gland can result in mammary hyperplasιa(McMahon,.ϊ upra ( 1992). Nusse and Varmus. supra ( 1992)), and precocious alveolar development Bradbury et al . Dev Biol . 170 553-563 ( 1995) Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesoderm and the extraembryonic mesoderm of the day 7-8 muπne embryo Gavin et αl , supra ( 1990) These embryonic domains contribute to the AGM region and yolk sac tissues from which multipotent hematopoietic precursors and HSCs are derived Dzierzak and Medvinsky, Trends Genet , 1 1 359-366 ( 1995); Zon et al , in Gluckman and Coulombel. ed . Colloque, 1NSERM, 235 17-22 ( 1995), presented at the Joint International Workshop on Foetal and Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure. Pans France. April 3-6. 1995, Kanatsu and Nishikawa. Development. 122 823-830 ( 1996) Wnt-Sa. Wnt- \ 0b. and other Wnts have been detected in limb buds, indicating possible roles in the development and patterning of the early bone icroenvironment as shown for Wnt-lb Gavin et αl , supra ( 1990), Christiansen et al , Mech Devel . 51 341-350 ( 1995); Parr and McMahon. supra ( 1995) The Wnt/Wg signal transduction pathway plays an important role in the biological development of the organism and has been implicated in several human cancers This pathway also includes the tumor suppressor gene, APC Mutations in the APC gene are associated with the development of sporadic and inherited forms of human colorectal cancer The Wnt Wg signal leads to the accumulation of beta- catenin/Armadillo in the cell, resulting in the formation of a bipartite transcription complex consisting of beta- catenin and a member of the lymphoid enhancer binding tactor/T cell factor (LEF/TCF)HMG box transcription factor family This complex translocates to the nucleus where it can activate expression of genes downstream of the Wnt Wg signal, such as the engrailed and Ultrabithorax genes in Drosophila The downstream target genes of Wnt- 1 signaling in vertebrates that presumably function in tumoπgenesis however, are currently unknown Tor a most recent review on Wnt see Cadigan and Nusse. enes & Dev . J . 3286-3305 ( 1997) There is a need to elucidate the fuπher members ot the above families including cell-surface molecules that mav be tumor-specific antigens or proteins that serve a regulator y function in initiating the Wnt pathway ot tumoπgenesis These would also include downstream components ot the Wnt signaling pathway that are important to the transformed phenotvpe and the development ot cancer SUMMARY OF THE INVENTION Several putative Wnt- 1 -induced enes have been identified at the mRNA level in a high-throughput cD A substraction experiment Thus, applicants have identified novel cDNA clones ( WISP! H 1SP2 and WISP 3) that encode novel polypeptides ot the WISP family designated as WISP- 1. WISP-2. and WISP-3. respectively This class ot polypeptides was tormerlv referred to as Wnt- 1-lnduced Gene (WIG) polypeptides. with WISP- 1 and WISP-2 formerly designated as WIG- 1 and WIG-2. respectively One of the cDNA clones encodes a novel polypeptide. human WISP-2. having homology to CTGF. wherein the polypeptide is designated in the present application as "human WISP-2" or "PR0261 " The WISP- 1 and WISP-3 molecules also have homology to CTGF In one embodiment, this invention provides isolated nucleic acid comprising D A having at least about 600 nucleotidesand at least about a 75% sequence identity to (a) a DNA molecule encoding a human WISP- 1 polypeptide comprising the sequence ot ammo acids 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3), or (b) the complement of the DNA molecule ot (a) Preferably, this nucleic acid has at least one WISP biological activi'v In a more preferred embodiment, this nucleic acid has at least about a 95% sequence identity to (a) a DNA molecule encoding a human WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3), or (b) the complement of the DNA molecule of (a) More preferred is the nucleic acid comprising DNA encoding a human WISP-1 polypeptide having ammo acid residues 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3), or DNA encoding a human WISP- 1 polypeptide having amino acid residues I to 367 of Figures 3 A and 3B (SEQ ID NO 4), or the complement of either ot the encoding DNAs Further preferred is this nucleic acid comprising DNA encoding a human WISP- 1 polypeptide having amino acid residues 23 to 367 or I to 367 of Figures 3 A and 3B except for an isoieucme residue at position 184 rather than a valine residue or a seπne residue at position 202 rather than an alanine residue (SEQ ID NOS 5-8, respectively) Further preferred also is this nucleic acid comprising DNA encoding a human WISP-1 polypeptide having am o acid residues 23 to 367 or 1 to 367 of Figures 3 A and 3B except for an isoleucine residue at position 184 rather than a valine residue and a seπne residue at position 202 rather than an alanine residue (SEQ ID NOS 21-22, respectively) Also preferred is this nucleic acid comprising DNA encoding a mouse WISP- 1 polypeptide having ammo acid residues 23 to 367 of Figure 1 (SEQ ID NO 1 1 ), or DNA encoding a mouse WISP- 1 polypeptide having amino acid residues 1 to 367 of Figure 1 (SEQ ID NO 12), or the complement of either of the encoding DNAs Also provided by this invention is isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 85% sequence identity to (a) a DNA molecule encoding a mouse WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of Figure 1 (SEQ ID NO 1 1 ), or (b) the complement of the DNA molecule of (a) Preferably this nucleic acid has at least one WISP biological activity More Dreteraolv this nucleic acid comDπses DNA having at least about a 95% seαuence to ( a) a DNA molecule encoding a mouse WISP- 1 polvpeptiαe comprising the sequence ot amino acids 23 to 367 of Figure 1 (SEQ ID NO 1 1 ) or (b) the complement ot the DNA molecule ot (a) In another preferred embodiment, the invention provides an isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding the same tull-length polypeptide encoded bv the human WISP- 1 polypeptide cDNA in ATCC Deposit No 209533 . (pRK5E h WISP- 1 568 38) or (b) the complement ot the DNA molecule ot (a) This nucleic acid preferably comprises DNA having at least about 600 nucleotides and at least about a 95% sequence identity to (a) a DNA molecule encoding the same full-length polvpeptide encoded bv the human WISP-1 polypeptidecDNA in ATCC DepositNo 209533 (pRK5E h WISP- 1 568 38).or (b) the complement of the DNA molecule of (a) In another aspect, the invention provides a process for producing a WISP- 1 polypeptide comprising cultuπng a host cell comprising the above nucleic acid under conditions suitable for expression ot the WISP- 1 polypeptide and recovering the WISP- 1 polypeptide from the cell culture Additionally provided is an isolated WISP- 1 polvpeptide encoded bv the above nucleic acid, including where the polvpeptide is human WISP- 1 or mouse WISP- 1 In another embodiment, the invention provides isolated nucleic acid comprising SEQ ID NO 23.24 25, 26. 27. 28. or 29. and an isolated WISP- 1 polypeptide encoded by such a nucleic acid Also provided by this invention is an isolated nucleic acid having at least about 600 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-1 polypeptidecompπsing the sequence of ammo acids 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3), or (b) the complementof the DNA moleculeof (a), and. if the test DNA molecule has at least about a 75% sequence identity to (a) or (b) isolating the test DNA molecule Furthe. provided is a polypeptide produced by (i) hybridizing a test DNA molecule under conditions with (a) a DNA molecule encoding a human WISP- 1 polypeptide comprising the sequence of ammo acids 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3). or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), (n) cultuπng a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide. and (in) recovering the polypeptide from the cell culture In another aspect, the invention provides isolated nucleic acid comprising D A having at least about an 80% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of Figure 4 (SEQ ID NO 15), or (b) the complementof the DNA molecule of (a) Preferably, this nucleic acid has at ieast one WISP biological activity Also, preferably this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of Figure 4 (SEQ ID NO 15), or (b) the complementof the DNA moleculeof (a) In another preferred embodiment, this nucleic acid comprises DNA encoding a human WISP-2 polypeptide having amino acid residues 24 to 250 of Figure 4 (SEQ ID NO 15). or DNA encoding a human WISP-2 polypeptide having amino acid residues 1 to 250 of Figure 4 (SEQ ID NO 16). or a complement of either ot the encoding DNAs In anotner aspect, tne inv ention provides isolated nucleic acid comprising DNA having at least about an 80% sequence identity to ιa) a DNA molecule encoding a numan WISP-2 polvpeptide comprising the sequence ot amino acids 1 to 250 of Figure 4 (SEQ ID NO 16) or ( b) the complement ot the DNA molecule of(a) In another aspect, the invention provides isolated nucleic acid comprising DNA having at least about 500 nucleotides and at least about an 80% sequence identity to (a) a DNA molecule encoding a mouse WISP- 2 polypeptide comprising the sequence ot amino acids 24 to 251 of Figure 2 (SEQ ID NO 19). or (b) the complement ot the DNA molecule ot (a) In a preferred embodiment, this nucleic acid comprises DNA having at least about a 95% sequence identitv to ( a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 24 to 251 of Figure 2 (SEQ ID NO 19), or (b) the complement of the DNA molecule of (a) More preferably the nucleic acid comprises DNA encoding a mouse WISP-2 polypeptide having amino acid residues 24 to 251 of Figure 2 (SEQ ID NO 19), or DNA encoding a mouse WISP-2 polypeptide having amino acid residues I to 251 of Figure 2 (SEQ ID NO 20), or the complement of either ot these encoding DNAs In a further aspect, the invention provides isolated nucleic acid comprising DNA having at least about 500 nucleotides and at least about an 80% sequence identitv to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence ot ammo acids 1 to 251 of Figure 2 (SEQ ID NO 20), or (b) the complement ot the DNA molecule ot (a) In yet another aspect, the invention provides an isolated nucleic acid comprising DNA having at least abouι400 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-2 polypeptide cDNA in ATCC Deposit No 209391 (DNA33473), or (b) the complement of the DNA molecule of (a) Preferably this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding the same full-length polypep: eencoded by the human WISP-2 polypeptide cDN A in ATCC Deposit No 209391 (DN \33473). or (b) the complement ot the DNA molecule ot (a) In another embodiment, this invention provides an isolated nucleic acid comprising the nucleotide sequence of the full-length coding sequence of clone UNQ228 (DNA33473) deposited under accession number ATCC 209391 In another aspect, the invention provides a process for producing a WISP-2 polypeptide comprising cultunng a host cell comprising the above nucleic acid under conditions suitable for expression of the WISP-2 polypeptide and recovering the WISP-2 polypeptide from the ceil culture Additionallyprovided is a WISP-2 polypeptide encoded by the isolated nucleic acid, including where the polypeptide is human WISP-2 or mouse WISP-2 In a specific embodiment of this, the invention provides isolated native-sequence human WISP-2 polypeptidecomprismgamino acid residues 1 to 250 of Figure 4 (SEQ ID NO 16) or comprising amino acid residues 24 to 250 of Figure 4 (SEQ ID NO 15) In a further embodiment, the invention provides an isolated nucleic acid having at least about 400 nucleotides and produced by hvbπdizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-2 polypeptidecompnsingthe sequence of amino acids 24 to 250 of Figure 4 (SEQ ID NO 15) or (b) the complement ot the DNA molecule ot (a), and. if the test DNA molecule has at least about a 75% sequence identity to (a) or ( b ) isolating the test DNA molecule In a still further embodiment, the invention provides a polvpeptide produced bv ( i) hvbπdizin a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence ot amino acids 24 to 250 of Figure 4 (SEQ ID NO 15) or (b) the complement ot the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence identitv to (a) or (b), (n) cultunng a host cell comprising the test DNA molecule under conditions suitable for expression of the polvpeptide. and (in) recovering the polypeptide from the cell culture In yet another embodiment, the invention provides isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 500 nucleotides to (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of ammo acids 34 to 372 of Figures 6A and 6B (SEQ ID NO.32), or (b) the complement of the DNA molecule of (a) Preferably, this nucleic acid has at least one WISP biological activity Preferably, this nucleic acid comprises DNA encoding a human WISP-3 polypeptide having ammo acid residues 34 to 372 of Figures 6A and 6B (SEQ ID NO 32) or amino acids 1 to 372 of Figures 6A and 6B (SEQ ID NO 33). or the complement thereof In a still further embodiment, the invention provides an isolated nucleic aαd comprising DNA having a 100% sequence identity in more than about 500 nucleotides to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-3 polypeptide cDNA in ATCC Deposit No 209706 (DNA56350-1 176-2), or (b) the complement of the DNA molecule of (a) Asπllfurtheraspect of the invention involves a process for producing a WISP-3 polypeptide comprising cultunng a host cell comprising WISP-3-encodιng nucleic acid under conditions suitable for expression of the WISP-3 polypeptide and recovering the WISP-3 polypeptide from the cell culture Further provided is an isolated WISP-3 polypeptide encoded by the WISP-3-encodmg nucleic acid Prε lerably, this polypeptide is human WISP-3 In another embodiment, the invention provides an isolated nucleic acid produced bv hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 34 to 372 of Figures 6A and 6B (SEQ ID NO 32), or (b) the complementof the DNA moleculeof (a), and, if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 500 nucleotides. isolating the test DNA molecule Also provided is a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of ammo acids 34 to 372 of Figures 6A and 6B (SEQ ID NO 32), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 500 nucleotides, (n) cultunng a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (in) recovering the polypeptide from the cell culture in yet another embodiment, the invention provides isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 400 nucleotides to (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of Figures 7A and 7B (SEQ ID NO 36), or (b) the complement of the DNA molecule of (a) Preferablv this nucleic acid has at least one WISP biological activity Preteraolv this nucleic acid comprises DNA encoding a human W ISP-3 polypeptide having amino acid residues 16 to 355 ot Figures 7A and 7B (SEQ ID NO 36) or amino acid residues 1 to 355 of Figures 7A and 7B (SEQ ID NO 37) or the complement thereof In a still further embodiment, the invention provides an isolated nucleic acid comprising DNA having a 100% sequence identitv in more than about 400 nucleotides to (a) a DNA molecule encoding the same full-length polvpeptide encoded bv the human WISP-3 polypeptide cDNA in ATCC Deposit No 209707 (DNA58800- 1 176-2), or (b) the complement ot the DNA molecule of (a) A still further aspect ot the invention involves a process tor producing a WISP-3 polypeptide of Fig 7A and 7B comprising cultunng a host cell comprising WISP-3-encodιng nucleic acid under conditions suitable for expression ot the WISP-3 polypeptide and recovering the WISP-3 polypeptide from the cell culture Further provided is an isolated WISP-3 polypeptide of Fig 7A and 7B encoded by the WISP-3- encoding nucleic acid Preferably, this polypeptide is human WISP-3 In another embodiment, the invention provides an isolated nucleic acid produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence ot amino acids 16 to 355 of Figures 7A and 7B (SEQ ID NO 36), or (b) the complementof the DNA moleculeof (a), and. it the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 400 nucleotides. isolating the test DNA molecule Also provided is a polypeptide produced by (I) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of Figures 7 A and 7B (SEQ ID NO 36), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 400 nucleotides, (n) cultunng a host ceil comprising the test DNA molecule under conditions suitable for expression of the polypeptide. and (in) recovering the polypeptide from the cell culture Preferably the complements of the DNA molecules herein remain stablv bound to the pπmarv sequence under at least moderate, and optionally, under high stringency renditions Also provided are vectors comprising the above nucleic acids, host cells comprising the vector, preferably wherein the cell is a Chinese hamster ovary (CHO) cell, an E colt cell, a baculovirus-infected cell. or a yeast cell. Additionally provided are a chimeπc molecule comprising one of the above polypeptides or an inactivated variant thereof, fused to a heterologousamino acid sequence, wherein the heterologous amino acid sequence may be. for example, an epitope tag sequence, a polyamino acid such as poly-histidine. or an immunoglobuiin constant region (Fc) Also provided is an antibody which specifically binds to one of the above polypeptides, wherein the antibody can be a monoclonal antibody Further provided are a composition comprising one of the above polypeptides and a carrier therefor, and a composition comprising an antagonist to one of the polypeptides and a carrier theretor In one such embodiment. the invention provides a composition comprising a WISP-1. WISP-2. or WISP-3 polypeptide and a pharmaceutically acceptable carrier Preferably, the polypeptide is a human polypeptide Also, preferably, these compositions mav also comprise a chemotherapeutic agent or growth-inhibitory agent In anotner aspect the invention provides a pharmaceutical product comprising ( a) the composition comprising WISP- 1 W ISP-2. or WISP-3 polypeptide and a pharmaceutically acceptable carrier. (b) a container containing said composition, and (c ) a label affixed to said container or a package insert included in said pharmaceuticaiproduct referring to the use ot said WISP- 1 W ISP-2. or WISP-3 polypeptide in the treatment ot a WISP-related disorder In yet another embodiment, the invention provides a method tor treating a WISP-related disorder in a mammal comprising administering to the mammal an effective amount of anv ot the above compositions. including the composition ot a WISP- 1 WISP-2. or WISP-3 polypeptide in a pharmaceutically acceptable carrier, and including the composition ot an antagonist to a WISP- 1. WISP-2. or WISP-3 polypeptide in a pharmaceutically acceptable carrier Preferablv.the disorder is a maiignantdisorderor arteriosclerosis. More preferably, the malignant disorder is breast cancer, ovarian cancer, colon cancer, or meianoma. A l s o , preferably the mammal is human In another preferred embodiment, the WISP-1 , WISP-2, or WISP-3 polypeptide is administered in comoination with a chemotherapeutic agent, a growth inhibitory agent, or a cytotoxic agent In another embodiment, the invention supplies a process tor diagnosing a disease or a susceptibility to a disease related to a mutation in a nucleic acid sequence encoding a WISP- 1. WISP-2, or WISP-3 polypeptide comprising (a) isolating a nucleic acid sequence encoding a WISP- 1 , WISP-2. or WISP-3 polypeptide from a sample derived from a host: and (b) determining a mutation in the nucleic acid sequence encoding a WISP- 1 , WISP-2. or WISP-3 polypeptide In another embodiment, the invention provides a method of diagr. .^ng a WISP-related disorder in a mammal comprising detecting the level of expression of a gene encoding a WISP- 1 , WISP-2. or WISP-3 polypeptide (a) in a test sample ot tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells ot the same cell type, wherein a higher or lower expression level in the test sample indicates the presence of a WISP-related dysfunction in the mammal from which the test tissue cells were obtained Preferably, such a disorder is a type of cancer and a higher expression level in the test sample indicates the presence of a tumor in the mammal In a still further embodiment, the invention provides an isolated antibody binding a WISP- 1. WISP- 2, or WISP-3 polypeptide Preferablv.the antibody induces death of a cell overexpressing a WISP-1 , WISP- 2. or WISP-3 polypeptide. more preferably a cancer cell Also preferred is an antibody that binds to a human WISP- 1. WISP-2, or WISP-3 polypeptide, and is a human or humanized antibody More preferred is a monoclonal antibody, still more preferred, a monoclonal antibody that has complementary-determining regions and constant immunoglobulin regions, and in other embodiments is an antibody fragment, a single- chain antibody, or an anti-idiotvpic antibody In addition, the antibody is suitably labeled with a detectable label or immobilized on a solid support \Iso provided is a composition comprising an antiDodv to a WISP-1 WISP-2. or WISP-3 polypeptide in admixture with a pharmaceutically accetable carrier Preferably the antibody is to a human WISP- 1 WISP-2. or WISP-3 polypeptide. and is a human or humanized antibody, most preferably a monoclonal antibody against human WISP- 1 Further, the composition mav comprise a growth-infnbitorv amount ot said antibodv In another embodiment, the invention provides a method for treating cancer in a mammal comprising administeπngto the mammal an effective amount ot the above antibodv composition In a preferred aspect ot this method, the cancer is colon cancer, the antibodv is against human WISP- 1 and is a humanized or human monoclonal antibodv, and the mammal is human In another aspect, the invention provides a method for treating a WISP-relateddisorder in a mammal comprising administeπngto the mammal an effective amount of a composition comprising an antagonist to a WISP- 1. WISP-2. or WISP-3 polypeptide in a pharmaceutically acceptable carrier In a further aspect, the invention provides a method for inhibiting the growth of tumor cells compπsingexposing a cell that overexpressesa Wnt- 1 -induced gene to an effective amount of an antagonist that inhibits the expression or activity ot a WISP- 1 WISP-2. or WISP-3 polypeptide A further aspect entails a method tor inhibiting the growth ot tumor cells comprising exposing said cells to an effective amount of the composition with the growth-inhibiting amount of an antι-WISP-1. anti- WISP-2. or antι-WISP-3 antibodv in admixture with the carrier In a preferred aspect of this method, the tumor cells are colon cancer ceils, the antibody is against human WISP- 1 and is a humanized or human monoclonal antibody, and the mammal is human Also provided herein is a kit compπsingone of the above WISP polypeptides or WISP antagonists, such as anti-WISP antibodies, and instructions tor using the polypeptide or antagonist to detect or treat a WISP-related disorder, such as cancer induced by Wnt One such preferred kit is a cancer diagnostic kit comprising an anti-WISP- 1. antι-WISP-2, or antι-WISP-3 and a carrier in suitable packaging Preferablv. this kit further comprises instructions tor using said antibodv to detect the WISP- 1 WISP-2. or WISP-3 polypeptide Also provided is a method for inducing cell death comprising exposing a cell which is induced by Wnt to an effective amount of one of the above WISP polypeptides or WISP antagonists, such as anti-WISP antibodies. Preferably, such cell is a cancer cell. More preferably, the cell is in a mammal, more preferably a human. In addition, an effective amount of another chemotherapeutic antibody is used in the exposure ot the cell, such as an antι-ErbB2 antibody Further, optionally the method comprises exposing the cell to a chemotherapeutic agent, a growth-inhibitoryagent. or radiation Optionally, the cell is exposed to the growth- inhibitory agent prior to exposure to the antibody In a further aspect, the invention provides an article of manufacture, comprising a container, a label on the container: and a composition comprising an active agent contained within the container, wherein the composition is effective for inducingcell death or inhibitingthe growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by oveπnduction of Wnt or a WISP- related disorder or bv overexpressionot a W ISP- 1 W ISP-2 or W ISP-3 polypeptide. and the active agent in the composition is an antagonistto one ot the poivpeptides. that is an agent that inhibits the expression and/or activity ot the WISP- 1 WISP-2 or WISP-3 polypeptide Preferably the active agent in such article ot manufacture is an antι-WlSP- 1 antι-WISP-2 or antι-WISP-3 antibodv and the label on the container indicates that the composition can be used tor treating a WISP-related disorder In another emoodiment. the invention provides a process tor identifying agonists to a WISP- 1 WISP-2. or WISP-3 polypeptide comprising (a) contacting cells and a compound to be screened under conditions suitable for the stimulation ot cell proliferation bv the polypeptide. and (b) measuring the proliferation of the cells to determine if the compound is an effective agonist Additionally, the invention provides an agonist to a WISP- 1. WISP-2. or WISP-3 polypeptide identified by the above process Further, the invention provides a method for identifying a compound that inhibits the expression or activitv ot a WISP- 1 WISP-2. or WISP-3 polypeptide. comprising contacting a candidate compound with a WISP- 1 WISP-2. or WISP-3 polvpeptide under conditions and for a time sufficient to allow the compound and polypeptide to interact In a preferred embodiment, this method comprises the steps ot (a) contacting cells and a compound to be screened in the presence ot the WISP- 1 WISP-2, or WISP-3 polypeptide under conditions suitable for the stimulation ot cell proliferation by polypeptide. and (b) measuπngthe proliferationot the cells to determine if the compound is an effective antagonist Further, a compound identified by this method is provided. In another aspect, this invention provides a compound that inhibits the expression or activity ot a WISP- 1 , WISP-2. or WISP-3 polypeptide in another embodiment, the invention provides a method for determiningthe presence of a WISP-1 , WISP-2, or WISP-3 polypeptide comprising exposing a Cv.il suspected of containing the WISP- 1 , WISP-2. or WISP-3 polypeptide to an anti-WISP- 1 antι-WISP-2. or anti- WISP-3 antibodv and determining binding ot said antibodv to said cell In another preferred embodiment, the invention provides a method of diagnosing a WISP-related disorder in a mammal comprising (a) contacting an anti-WISP- 1 , anti- WISP-2, or anti- WISP-3 antibodv with a test sample of tissue cells obtained from the mammai. and (b) detecting the formation of a complex between the anti-WISP- 1 , anti- WISP-2. or anti- WISP-3 antibody and the WISP- 1. WISP-2, or WISP-3 polypeptide in the test sample Preferably, said test sample is obtained from an individual suspected to have neoplastic cell growth or proliferation Also, preferably the antibody is labeled with a detectable label and/or is immobilized on a solid support BRIEF DESCRIPTION OF THE DRAWINGS Figure I shows the derived amino acid sequence of a native-sequence mouse WISP- 1 protein from ammo acids 1 to 367 (SEQ ID NO 12) and the nucleotide sequence (and complementary sequence) encoding the protein (SEQ ID NOS 9 and 10. respectively) There is a 1 104-bp coding region and 584 bp of 3' untranslated region In the Figure, amino acids 1 through 22 form a putative signal sequence, amino acids 23 through 367 are the putative mature protein (SEQ ID NO 1 1 ) with amino acids 86 to 88. 143 to 145, 284 to 286. and 343 to 345 being potential glycosylation sites. Potential protein kinase C phosphoryiation sites are at amino acios 43-45. 159- 161 , 235-237. 292-294. 295-297. and 345-347 Potential casein kinase II phosphoryiation sites are at amino acids 44-47. 1 1 - 134. 145- 148. and 358-361 Potential N-inyπstovlation sites are at am o acids 18-23.72-77. 127-132. 149-154. 231 -236. and 289-294 A potential amidation site is at amino acids 269-272. A potential prokaryotic membrane poprotein lipid attachment site is at amino acids 1 13- 123. A potential von Willebrand C l domain is at amino acids 130-146 Λ potential thrombospondin 1 domain is at amino acids 223-237. A potential CT module is at ammo acids 301-312. A potential IGF binding protein consensus site is at amino acids 72-80. Figure 2 shows the derived amino acid sequence of a native- sequence mouse WISP-2 protein from amino acids 1 to 251 (SEQ ID NO 20) and the nucleotide sequence (and complementary sequence) encoding the protein (SEQ ID NOS: 17 and 18. respectively) from a clone 1367.3. There are 756 bp of coding nucleotides and 722 bp of 3' untranslated region. In the Figure, amino acids 1 through 23 form a putative signal sequence: amino acids 24 through 251 are the putative mature protein (SEQ ID NO: 19). A potential N-glycosylation site is at ammo acids 197-200. A potential glycosaminoglycan attachment site is at ammo acids 85-88. Potential protein kinase C phosphorylationsites are at am o acids 85-87 and 1 12- 1 14 Potential N-myπstoylation sites are at amino acids 49-54. 81 -86. 126- 131. 210-215. and 245-250 A potential amidation site is at amino acids 103- 106. A potential phospholipase A2 aspartic acid active site is at ammo acids 120- 130 A potential IGF binding protein consensus signature is at amino acids 49-64 A potential von Willebrand C l domain is at amino acids 107- ' 23. A potential thrombospondin 1 domain is at amino acids 202-216. A potential IGF binding protein consensus site is at am o acids 49-57. Figures 3 A and 3B show the derived amino acid sequence of a native-sequence human WISP- 1 protein from amino acids 1 to 367 (SEQ ID NO:4) and the nucieotide sequence (and complementary sequence) encoding the protein (SEQ ID NOS: l and 2, respectively). There are 1 104 bp of coding region in this human clone 568.38. and 1638 bp of 3' untranslated region. In the Figure, ammo acids 1 through 22 form a putative signal sequence, amino acids 23 through 367 are the putative mature protein (SEQ ID NO:3). with amino acids 85 to 87. 143 to 145. 284 to 286. and 343 to 345 being potential glycosylation sites. A potential cAMP- and cGMP-dependent protein kinase phosphoryiation site is from amino acids 171 to 174: potential protein kinase C phosphoryiation sites are at ammo acids 43-45, 235-237, 292-294, and 345-347. Potential casein kinase II phosphoryiation sites are at amino acids 30-33, 145-148. and 358-361. Potential N-myπstoylation sites are at ammo acids 72-77. 127- 132, 149- 154, 201 -206, 231 -236.289-294. and 327-332. A potential amidation site is at amino acids 269-272. A potential prokaryotic membrane lipoprotein lipid attachment site is at ammo acids 1 13- 123. A potentialvon WillebrandCl domain is at amino acids 130-146. A potential thrombospondin 1 domain is at amino acids 223-237 A potential CT (C-Terminal) module is at amino acids 301 -312. A potential IGF binding protein consensus site is at amino acids 72-80. Figure 4 showsthe derived ammo acid sequence of a native-sequence human WISP-2 protein from amino acids 1 to 250 (SEQ ID NO.16) and the nucieotide sequence (and complementary sequence) encoding the protein (SEQ ID NOS: 13 and 14. respectively). The coding region is 753 bp and the 3' untranslated region is 519 bp. The putative signal sequence is from ammo acid residues 1 through 23 and the putative mature region is from 24 through 250 (SEQ ID NO: 15). The clone designated herein as "'LTNQ228" and/or "DNA33473-;>eαmιn ' ( SEQ ID NO 38) begins at nucleotide 13 ot SEQ ID NO 13 Potential protein kinase C phosphoryiation sites are at am o acids 4-6 1 18- 120. and 227-229 A potential casein kinase II phosphoryiation site is at amino acids 98-101. A potential N-myπstoylationsite is at amino acids 3-8. 49-54. 81-86. 85-90. 126-131. 164- 169. 166- 171. 167- 172. 183- 188. and 209-214 A potential IGF binding protein consensus signature is at amino acids 49-64 A potential von W illebrand C l domain is at am o acids 107- 123. A potential thrombospondin 1 domain is at amino acids 201 -215. A potential IGF binding protein consensus site is at ammo acids 49-57. Figure s shows a 841 -bp consensusnucleotιdesequence desιgnated"DNA30843" (SEQ ID NO.39) derived from the nucleotide sequences of twenty different expressed sequence tags from Incyte. When aligned with the other sequences. DNA30843 has 3 gaps. It has 441 bp orf (+ 1 ). DNA30843 was used to design probes for isolation of human WISP-2. Figures 6A and 6B show the derived ammo acid sequence of a native-sequence human WISP-3 protein from amino acids I to 372 (SEQ ID NO:33) and the nucieotide sequence (and complementary sequence) encoding the protein (SEQ ID NOS:30 and 31. respectively). In the Figure, amino acids 1 through 33 form a putative signal sequence, ammo acids 34 through 372 are the putative mature protein (SEQ ID NO:32). with amino acids 196 to 1 8 and 326 to 328 being potential glycosylation sites. Potential protein kinase C phosphoryiation sites are at amino acids 209-21 1. 246-248. 277-279. 308-310. and 342-344. Potential casein kinase II phosphoryiation sites are at ammo acids 47-50, 254-257, and 293-296. Potential N-myπstoylationsites are at am o acids 21 -26. 89-94. 139-144. 166-171 , 180-185. 185-190, 188-193. 242- 247. and 302-307. A potential amidation site is at amino acids 188- 191. Potential prokaryotic membrane lipoprotein lipid attachment sites are at amino acids 130-140 and 160-170. A potential IGF binding protein signature site is at ammo acids 89- 104. A potential IGF binding protein site (less stringent than prosite's) is at am o acids 89-97 Figures 7A and 7B show the der .^d amino acid sequence of a native-sequence human WISP-3 protein from am o acids I to 355 (SEQ ID NO:37) and the nucleotide sequence (and complementary sequence (encoding the protein (SEO ID NOS:34 and 35. respectively). This protein is believed to be a splice variant of the nucleotide sequence shown in Figure 6 with a shorter 5' end. In the Figure, ammo acids 1 through 15 form a putative signal sequence, amino acids 16 through 355 are the putative mature protein (SEQ ID NO:36), with ammo acids 178 to 180 and 308 to 310 being potential glycosylationsιtes. Potential protein kinase C phosphoryiation sites are at amino acids 191-193. 228-230. 259-261, 290-292. and 324-326. Potential casein kinase II phosphoryiation sites are at amino acids 29-32, 236-239. and 275-278. Potential N-myπstoylation sites are at amino acids 3-8, 71 -76. 121 - 126, 148- 153. 162- 167. 167- 172, 170- 175, 224-229, and 284-289. A potential amidation site is at amino acids 170- 173. Potential prokaryotic membrane lipoprotein lipid attachment sites are at ammo acids 1 12-122 and 142-152. A potential IGF binding protein signature site is at amino acids 71 -87. A potential IGF binding protein site (less stringent than prosite's) is at am o acids 71 -79. Figure 8 shows an alignment of the full-length am mo acid sequences of the human and mouse WISP- 1 (SEQ ID NOS:4 and 12. respectively). Figure shows an alignmentot the tull-lengtn amino acid sequencesot the human ano mouse WISP- 2 (SEQ ID NOS 16 and 20 respectively) Figure 10 shows an alignment ot the amino acid sequences of the two clones ot human WISP-3 Figures 1 1 A- 1 1 C show an alignment ot the nucleotide sequences ot human WISP- 1 and the human WISP-3 shown in Fig 6 Figure 12 shows an alignmentot the amino acid sequencesot human WISP- 1 and the human WISP- 3 shown in Fig 6 Figure 13 shows a map of the vector pBabe puro ( 5 1 kb) used to transform cells for purposes ot differential expression The vector includes both unique restriction sites and multiple restriction sites It is shown here in modified form tor Wnt- 1 cloning wherein the Hind l site after the SV40 promoter in the original pBabe puro vector has been removed and a HindUl site added to the multiple cloning site of the original pBabe puro vector Wnt- 1 is cloned from £coRI-//;«dIII in the multiple cloning site Constructs derived from this vector are selected in ampicillin ( 100 μg ml) and the cells infected in culture are selected in 1 0-2 5 μa ml puromvcin Figure 14 shows the sequences ot the PCR-Selecrø cDNA synthesis primer (SEQ ID NO 40) adaptors 1 and 2 (SEQ ID NOS 41 and 42 respectιvelv)and complementarvsequences tor the adaptors(SEQ ID NOS 43 and 44 respectively) PCR primer 1 (SEQ ID NO 45), PCR primer 2 (SEQ ID NO 46), nested PCR primer I (SEQ ID NO 47) nested PCR primer 2 (SEQ ID NO 48), control primer G3PDH 5' primer (SEQ ID NO 49) and control pr mer G3PDH 3' primer (SEQ ID NO 50) used for suppression subtractive hybridization for identifying WISP clones When the adaptors are ligated to Λrøl-digested cDNA. the Rsal site is restored Figure 15 shows the cloning site region of the plasmid pGEM-T used to clone all of the WISP sequences herein (SEQ ID NOS 51 and 52 for 5' and 3' sequences, respectively) Figures 16A- 16D show the sequence (SEQ ID NO 53) of a plasmid that is used to prepare an expression plasmid for expression ot mouse WISP- 1 in mammalian ceils, the latter being designated pRK5 CMV puro-dhfK m WISP- 1 6Hιs Figures 17A-17D snow the sequence (SEQ ID NO 54) of plasmid pb PH IgG, which is used to prepare an expression plasmid for expression of mouse WISP-1 DNA in baculovirus-infected insect cells Figures 18A-18D show the sequence (SEQ ID NO 55) of plasmid pbPH His c. which is used to prepare an expression plasmid for expressionof mouse WISP-1 DNA in baculovirus-infectedinsect cells, the latter being designated pbPH mu 568 8hιs baculo Figures 19A-19D show graphs of the delta CT in nine colon cancer cell lines and DNA from the blood often normal human donors (Nor Hu) as control, for human TNF, human WISP- 1 Lvra and human Apo2 ligand. respectively using the ABI Prism 7700™ Sequence Detection Svstem procedure for testing genomic amplification Figures 20 A-20D show graphs of the delta CT in nine colon cancer celi lines and Nor Hu as control for human DCR1. huFAS, human WISP-2. and Apo3. respectively, using the ABI Prism 7700™ Sequence Detection Svstem procedure tor testing genomic amplification Figures 21 A- 1 D show graphs ot the delta CT in nine coion cancer cell lines and Nor Hu as control, ior three different runs ot human WISP-1 (designated in the figure as huWISP-lc. - l b. and - l a) and the average ot these three runs ot human WISP- 1. respectively, using the ABI Prism 7700 1" ' ' Sequence Detection System procedure tor testing genomic amplification. Figures 22A-22D show graphs ot the delta CT in nine colon cancer cell lines and Nor Hu as control, for three different runs ot human WISP-2 (designated in the figure as huWISP-2c. -2b. and -2a: Figs. 22Λ. C. and D. respectively) and the average of these three runs of human WISP-2 (Fig. 22B). using the ABI Prism 7700 ™ Sequence Detection System procedure for testing genomic amplification Figures 23 A-23C show graphs of the delta CT in nine colon cancer cell lines and Nor Hu as control. for nvo different runs of human DR5 (DR5a and DR5b) and the average of these two runs of DR5. respectively, using the ABI Prism 7700™ Sequence Detection System procedure for testing genomic amplification. Figures 24 A-24D show graphs of the delta CT in nine colon cancer cell lines and Nor Hu as control, for four different runs of c-mvc (c-myc(a 1 ),c-myc(b 1 ), c-myc(b). and c-myc(a)), respectively, using the ABI Prism 7700™ Sequence Detection System procedure for testing genomic amplification Figures 25 A-25D show graphs ot the delta CT in nine colon cancer cell lines and Nor Hu as control. for two different runs of human WISP- 1 (designated in the figure as hu WISP- 1 (a) and huWISP- l (b)) and for two different runs of human WISP-2 (designated in the figure as huWISP-2(a) and huWISP-2(b)), respectively, using the API Prism 7700 Sequence Detection System procedure for testing genomic amplification. Figure 26 shows the sequence (SEQ ID NO:23) of clone 568.13. a potential splice variant of human WISP- 1 obtained by screening with a probe derived from clone 568.15 A, which is the initial clone isolated from a human lung library in the process to obtain full-length human WISP-1 DNA. Figure 27 shows the sequence (SEQ ID NO.24) of clone 568.1 A. a potential human WISP- 1 splice variant, 5' end only, obtained by screening with a probe derived from clone 568.15A Figure 28 shows the sequence (SEQ ID NO:25) of clone 568.39. a potential human WISP- 1 splice variant, 5' end only, obtained by screening with a probe derived from clone 568.15 A. Figure 29 shows the sequence (SEQ ID NO:26) of clone 568.4A, a potential human WISP- 1 splice variant obtained by screening with a probe derived from clone 568.15 A. Figure 30 shows the sequence (SEQ ID N0.27) of clone 568.5 A, a potential human WISP- 1 splice variant, 5' end only, obtained by screening with a probe derived from clone 568.15A. Figure 31 shows the sequence (SEQ ID NO:28) of clone 568.6B, a potential human WISP- 1 splice variant, 5' end only, obtained by screening with a probe derived from clone 568.15A Figure 32 shows the sequence (SEQ ID NO:29) of clone 568.7. a potential human WISP- 1 splice variant. 5' end only, obtained by screening with a probe derived from clone 568.15 A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I Definitions The term 'WISP polvpeptide ' refers to the family ot native- sequence human and mouse WISP proteins and variants described herein whose genes are induced at least bv Wnt- 1 This term includes WISP- I WISP-2. and WISP-3 The terms "WISP-1 polypeptide " . "WISP-1 homologue and grammatical variants thereof, as used herein, encompass native- sequence WISP- 1 protein and variants (which are further defined herein) The WISP- 1 polypeptide may be isolated from a vaπetv of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods, or by anv combination of these and similar techniques The terms "WISP-2 polypeptide". "WISP-2 homologue . "PR0261 ", and "PR0261 polypeptide" and grammatical variants thereof, as used herein, encompass native-sequence WISP-2 protein and variants (which are funher defined herein) The WISP-2 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods, or by anv combination ot these and similar techniques The terms "WISP-3 polypeptide '."WISP-3 homologue ,and grammatical variants thereof, as used herein, encompass native-sequence WISP-3 protein and variants (which are further defined herein) The WISP-3 polypeptide may be isolated from a variety ot sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods, or by any combination of these and similar techniques A "native-sequence WISP- 1 polypeptide" comprises a polypeptide having the same amino acid sequence as a WISP- 1 polypeptide derived from nature Such native-sequence WISP- 1 polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native-sequence WISP- 1 polypeptιde"speuficallyencompassesnaturaily occurring truncated or secreted forms of a WISP- 1 polypeptide disclosed herein, naturally occurring variant forms (e e: , alternatively spliced forms or splice variants), and naturally occurring allelic variants of a WISP- 1 polypeptide In one embodiment ot the invention, the native-sequence WISP-1 polypeptide is a mature or full-length native-sequencehuman WISP-1 polypeptide comprising amino acids 23 to 267 of Figures 3 A and 3B (SEQ ID NO.3) or am o acids 1 to 267 of Figures 3 A and 3B (SEQ ID NO 4), respectively, with or without the N-terminal methioniπe In another embodiment of the invention, the native-sequence WISP- 1 polypeptide is the full-length or mature native-sequence human WISP- 1 polypeptide comprising amino acids 23 to 267 or 1 to 267 of Figures 3 A and 3 B wherein the valine residue at position 184 or the alanine residue at position 202 has/have been changed to an isoleucine or seπne residue, respectively. (SEQ ID NOS 5-8) with or without the N- termmal methionine. In another embodiment of the invention, the native-sequence WISP- 1 polypeptide is the full-length or mature native-sequence human WISP-1 polypeptide comprising amino acids 23 to 267 or 1 to 267 of Figures 3 A and 3B wherein the valine residue at position 184 and the alanine residue at position 202 has/have been changed to an isoleucine or seπne residue, respectively, (SEQ ID NOS.21 and 22. respectιvely)wιth or without the N-terminal methionine In another embodimentof the invention, the native- sequence WISP-1 polypeptide is a mature or full-length native-sequence mouse WISP- 1 polypeptide omprising ammo acids 23 to 367 ot Figure 1 ( SEQ ID NO 1 1 ) or amino acids 1 to 367 of Figure I (SEQ ID NO 12) respectively with or witnout the N-terminal methionine In anotner embodiment ot the invention, the native-sequence WISP- 1 polypeptide is one which is encoded by a nucieotide sequence comprising one ot the human WISP-1 splice or other native-sequence variants, including SEQ ID NOS 23 24. 25. 26. 27. 28. or 29. w ith or without an N-terminal methionine A "native-sequence WISP-2 polypeptide ' or a 'native-sequence PR0261 polypeptide" comprises a polypeptide having the same amino acid sequence as a WISP-2 polypeptide derived from nature Such native-sequence WISP-2 polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native-sequence WISP-2 polypeptide" specifically encompasses naturally occurring truncated or secreted forms of a WISP-2 polypeptide disclosed herein, naturally occurring variant forms (e g . altemativelyspliced forms or splice variants), and naturally occurring allelic variants of a WISP-2 polypeptide. In one embodiment of the invention, the native-sequence WISP-2 polypeptide is a mature or full-length native-sequencehuman WISP-2 polypeptide comprising amino acids 1-24 up to 250 of Figure 4 (SEQ ID NOS 15. 16, and 56-77). including amino acids 24 to 250 and amino acids I to 250 of Figure 4 (SEQ ID NOS 15 and 16. respectively ). with or vv ithoutthe N-terminal methionine In another embodiment ot the invention, the native-sequence WISP-2 polypeptide is a mature or full-length native-sequence mouse WISP-2 polypeptide comprising amino acids 1 -24 up to 251 of Figure 2 (SEQ ID NOS.19. 20. and 78-99). including amino acids 24 to 251 and ammo acids 1 to 2 1 of Figure 2 (SEQ ID NOS 19 and 20. respectively), with or vthout the N-terminal methionine A "native-sequence WISP-3 polypeptide" comprises a polypeptide having the same ammo acid sequence as a WISP-3 polypeptide derived from nature Such native-sequence WISP-3 polypeptides can be isolated from nature or can be produced bv recombinant or synthetic means The term "native-sequence WISP-3 polypeptide" specifically encompasses naturally occurring truncated or other forms ot a WISP-3 polypeptide disclosed herein, naturally occurring variant forms ( g , alternatively spliced forms or : . 'ice variants), and naturaliv occurring allelic variants ot a WISP-3 polypeptide In one embodiment of the invention, the natιve-sequenceWISP-3 polypeptide is a mature or tull-length.native-sequencehuman WISP-3 polypeptide comprising amino acids 34 to 372 of Figures 6A and 6B (SEQ ID NO 32) or ammo acids 1 to 372 of Figures 6A and 6B (SEQ ID NO 33). respectively, with or without the N-terminal methionine In another embodiment of the invention, the native-sequence WISP-3 polypeptide is a mature or full-length. native-sequencehuman WISP-3 polypeptidecompπsing amino acids 16 to 355 of Figures 7A and 7B (SEQ ID NO 36) or ammo acids 1 to 355 of Figures 7A and 7B (SEQ ID NO 37), respectively, with or without the N-terminal methionine The term 'WISP- 1 variant means an active WISP- 1 polypeptide as defined below having at least about 80%, preferably at least about 85%. more preferably at least about 90%. most preferably at least about 95% amino acid sequence identity with human mature WISP- 1 having the deduced amino acid sequence shown in Figs 3 A and 3B (SEQ ID NO 3). and/or with human full-length WISP- 1 having the deduced ammo acid sequence shown in Figs 3 A and 3B (SEQ ID NO 4), and/or with mouse mature WISP- 1 having the deduced ammo acid sequence shown in Fig 1 (SEQ ID NO 1 1 ), and/or with mouse full-length WISP-2 having the deduced amino acid sequence shown in Fig 1 (SEQ ID NO 12) Such variants include, tor instance WISP- 1 oolvpeptioes wnerein one or more am o acid residues are added to or deleted from, the V or L-terminus ot the tull-lengtnor mature sequencesot Figures JA-3B and l (SEQ ID NOS 4 3 12. and 1 1 respectively) lncludingvaπants from other species, but excludes a native-sequence WISP- 1 polypeptide The term WISP-2 variant or "PR0261 variant ' means an active WISP-2 polvpeptide as defined below having at least about 80% preferablv at least about 85% more preferably at least about 90% most preferably at least about 95% amino acid sequence identity with human mature WISP-2 having the putative deduced ammo acid sequence shown in Fig 4 (SEQ ID NO 15 ) and/or with human full-length WISP-2 having the deduced amino acid sequence shown in Fig 4 (SEQ ID NO 16). and/or with mouse mature WISP- 2 having the putative deduced ammo acid sequence shown in Fig 2 (SEQ ID NO 19). and/or with mouse full- length WISP-2 having the deduced amino acid sequence shown in Fig 2 (SEQ ID NO 20) Such variants include, for instance. WISP-2 polypeptides wherein one or more ammo acid residues are added to or deleted from, the N- or C-terminus of the full-length and putative mature sequences of Figures 4 and 2 (SEQ ID NOS 16. 15.20. and 19 respectively), including variants from other species, but excludes a native-sequence WISP-2 polypeptide The term WISP-3 variant means an active WISP-3 polvpeptide as defined below having at least about 80% preferably at least about 85% more preferably at least about 90% most preferably at least about 95% amino acid sequence identitv with human mature WISP-3 having the deduced amino acid sequence shown in Figs όA and 6B (SEQ ID NO 32), and/or with human full-length WISP-3 having the deduced αmino acid sequence shown in Figs 6 A and όB (SEQ ID NO 33). and/or with human tnafre WISP-3 having the deduced am o acid sequence shown in Figs 7A and 7B (SEQ ID NO 36), or with human full-length WISP-3 having the deduced amino acid sequence shown in Figs 7A and 7B (SEQ ID NO 37) Such variants include, for instance. WISP-3 polypeptides wherein one or more amino acid residues are added to. or deleted from, the N- or C-teπrunus ot the full-length or mature sequences of Figures 6A-6B and 7A-7B (SEQ ID NOS 32.33, 36. and 37 respectively), including variants from other species, but excludes a - -tive-sequence WISP-3 polypeptide "Percent (%) ammo acid sequence identitv ' ith respect to the WISP sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are dentical with the amino acid residues in a WISP polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutionsas part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in vanousways that are within the skill in the art, for instance, using publiclv available computer software such as BLAST, ALIGN, or Megahgn (DNASTAR™) software Those skilled in the art can determine appropriate parameters for measuring alignment, including anv algorithms needed to achieve maximal alignment over the full length of the sequences being compared "Percent (%) nucleic acid sequence identity" with respect to the coding region of the WISP sequences identified herein, including UNQ228 (DNA34387-seq min) sequence, and the coding region therein, is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the coding region of the WISP sequence of interest e g . in the UNQ228 (DNA34387-seqmm) sequence (SEQ ID NO 38) or coding region therein (SEQ ID NO 16). after aligning the sequences and introducing gaps it necessarv to achieve the maximum percent sequence identity Alignment tor purposes ot determinin percent nucleic acid sequence identity can be achieved in various wavs that are within the skill in the art. tor instance, using pubiiclv available computer software such as BLAST. ALIGN, or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including anv algorithms needed to achieve maximal alignment over the full length of the sequences being compared 'Stringent conditions are those that ( 1 ) employ low ionic strength and high temperature for washing, for example, 0 015 M sodium chloride 0 0015 M sodium cιtrate/0 1% sodium dodecvl sulfate at 50°C. (2) employ during hybridization a denaturing agent, such as formamide. for example. 50% (vol/voi) formamide with 0 1% bovine serum albumιn/0 1% Fιcoll/0 1% polyvιnylpyrrolιdone/50 mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride. 75 mM sodium citrate at 42°C. (3) employ 50% formamide, 5 x SSC (0 75 M NaCl. 0 075 M sodium citrate). 50 mM sodium phosphate (pH 6 8). 0 1% sodium pyrophosphate. 5 \ Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0 1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0 2 x SSC and 0 1% SDS, or (4) employ a buffer of 10% dextran sulfate.2 x SSC (sodium chloride/sodium citrate Land 50% formamide at 55°C followed bv a high- stringency wash consisting ot 0 1 x SSC containing EDTA at 55°C "Moderatelv stringent conditions ' are described in Sambrook et al . Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989), and include the use of a washing solution and hybridization conditions (e g , temperature, ionic strength, and percent SDS) less stringent than described above An example of moderately stringent conditions is a condition such as overnight incubation at 37°C in a solution comprising 20% formamide. 5 x SSC ( 150 mM NaCl, 15 mM tπsodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA. followed by washing the filters in 1 x SSC at about 37-50°C The skilled artisan will recognize how to adjust the temperature, ionic strength, <c . as necessary to accommodate factors such as probe length and the like "Isolated " when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide. and may include enzymes, hormones, and other proteinaceousor non-proteinaceoussolutes In preferred embodiments, the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal ammo acid sequence by use of a spinning cup sequenator. or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the WISP natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step An "isolated" nucleic acid encoding a WISP polypeptide or "isolated" DNA33473 or "isolated" PR0261 polypeptide-encoding nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the respective nucleic acid Isolated DNA33473 or an isolated WISP-encoding nucleic acid molecule is other than in the form or setting in which it is touno in nature <\n isolated WISP-encoding or DNA33473 nucleic acid molecule therefore is distinguisnedtrom the WlSP-encodmg or DNA33473 nucleic acid molecule respectively. as it exists in natural cells However, an isolated WISP-encoding or DNA33473 nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express WISP- encoding DNA or DNA33473 respectively where, tor example, the nucleic acid molecule is in a chromosomal location different trom that ot natural cells The term control sequences ' refers to DNA sequences necessary tor the expression ot an operablv I inked coding sequence in a particular host organism The control sequences that are suitable for prokarvotes. for example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukaryotic cells are known to utilize promoters, polvadenvlation signals, and enhancers Nucleic acid is "operablv linked" when it is placed into a functional relationshipwith anothernucleic acid sequence For example. DNA for a presequence or secretory leader is operablv linked to DNA for a polypeptide if it is expressed as a preprotem that participates in the secretion of the polypeptide. a promoter or enhancer is operablv linked to a coding sequence if it affects the transcription of the sequence, or a πbosome binding site is operablv linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operablv linked" means that the DN sequences being linked are contiguous, and. in the case ot a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic o gonucleotide adaptors or linkers are used in accordance with conventioml practice The term 'antibodv" is used in the broadest sense and specifically covers single anti-WISP polypeptide. such as antι-PR0261 , monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), and anti-WISP polypeptide. such as antι-PR0261. and antibody compositions with polyepitopic specificity The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. ; e , the individual antibodies cor . ' rising the population are identical except for possible naturallv occurring mutations that mav be present in minor amounts "Active ' or "activity" or "WISP biological activitv", for purposes herein, describes torm(s) of a WISP polypeptide. such as PR0261. including its variants, or its antagonists, which retain the biologic and/or lmmunologic activities of a native or naturally occurring (native-sequence) WISP polypeptide. such as PR0261 , or its antagonist Preferred "activities" for a WISP polypeptide or its antagonist include the ability to inhibit proliferationof tumor cells or to stimulate proliferation of normal cells and to treat arteriosclerosis, including atherosclerosis, as well as to induce wound repair and hematopoiesis, prevent desmoplasia, prevent fibrotic lesions associated with skin disorders such as scleroderma, keloid, eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture, to treat bone-related diseases such as osteoporosis, to regulate anabolism including promotion of growth, to treat immune disorders, to treat Wilms' tumor and kidney- related disorders, to treat testis-relateddisorders. to treat lung-relateddisorders and to treat cardiac disorders An "antagonist" of a WISP polypeptide is a moieculethat inhibits an activity of a WISP polypeptide Preferred antagonists are those which interfere with or block an undesirable biological activity of a WISP polypeptide. such as where a WlSP polypeptidemightact to stimulate cancer cells and the antagonist would serve to inhibit the growth of those cells In some cases, such as with WISP-1 WISP-2. and WISP-3 the antagonist av 0e useiul to inhibit the binding of a WISP pol peptide to an IGF Such molecules include antibodies and small molecules that have such inhibitory capability , as well as WISP polypeptide variants ot. and receptors tor. WISP polypeptide (it available) or portions thereof that bind to WISP For example. antagonists can be derived from receptors of WISP- 1. WISP-2. and WISP-3 using the predicted familv ot receptors tor WISPs- 1. -2, and -3 (the CTGF receptors) Thus, the receptor can be expression cloned from the family, then a soluble form ot the receptor is made by identifying the extracellular domain and excising the transmembrane domain therefrom. The soluble form ot the receptor can then be used as an antagonist. or the receptor can be used to screen tor small molecules that would antagonize WISP polypeptide activity Alternatively, using the muπne sequences shown in Figures 1 and 2 (SEQ ID NOS.1 1. 12. 19. and 20. respectively) or the human sequences shown in Figures 3A-3B. 4, (SEQ ID NOS. 3, 4. 15. and 16. respectively), 6A-6B. and 7A-7B, variants of native WISP-1 WISP-2, or WISP-3, are made that act as antagonists. Using knowledge from the CTGF receptor family, the receptor binding sites of WISP- 1. WISP-2, and WISP-3 polypeptides can be determined by binding studies and one of them eliminated by standard techniques (deletion or radical substitution) so that the molecule acts as an antagonist. Antagonistactivitvcan be determined bv several means, including standard assavs tor induction ot cell death such as that described herein, e g . J H-thymιdιne proliferation assavs. or other mitogenic assays. such as an assay measuring the capability ot the candidate antagonist of inducing EGF-potentiated anchorage independent growth of target eel I lines (Volckaerte/ α/ . Gene. 15 15-223 ( 1981 )) and/or growth inhibition of neoplastic cell lines Roberts et al , Proc Natl Acad Sci uSA, 82- 1 19-123 (1985). Anchorage- independent growth refers to the ability of WISP polypeptide-treated. or TGF-β-treated and EGF-treatednon- neoplastic target cells to form colonies in soft agar. a characteristic ascribed to transformation of the cells. In this assay, the candidate is incubated together with an equimolar amount of a WISP polypeptide otherwise detectable in the EGF-potentiatedanchorage-independent target cell growth assay, and the culture observed for failure to induce anchorage-independentgrowth In addition, an anta^ vnist may be an IGF such as IGF-I or a peptide mimic ot IGF-I or a receptor to IGF or a receptor to an IGFBP "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorderor cor.ditionas well as those in which the disorder or condition is to be prevented. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans. domestic, and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc Preferably, the mammal is human. A "disorder" or " WISP-relateddisorder" is any condition that would benefit from treatment with the WISP polypeptides or WISP antagonists herein. This includes chronic and acute disorders, as well as those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant tumors: ieukemiasand lymphoid malignancies, neuronal, glial. astrocytal. hypothalamic and other glandular, macrophagal. epithelial, stromal. and blastocoelic disorders: hematopoiesis-relateddisorders: tissue-growth disorders: skin disorders: desmoplasia. fibrotic lesions: kidney disorders: bone-related disorders; trauma such as burns, incisions, and other wounds, cataboiic states: testicuiar-related disorders: and inflammatory, angiogenic. and lmmunologic disorders. A3- including aπeπoscierosis A 'Wnt-reιateddιsoroer is one caused at least bv the upreguiationot the Wnt gene pathway including Wnt- 1 and Wnt-4 but preterablv Wnt- 1 and mav include cancer The terms cancer "cancerous '. and 'malignant refer to or describe the physiological condition in mammals that is typically characterized bv unregulated cell growth Examples of cancer include but are not limited to carcinoma including adenocarcinoma. Ivmphoma. blastoma. melanoma, sarcoma, and leukemia More particular examples ot such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer. Hodgkin s and non-Hodgkin s Ivmphoma. pancreatic cancer, glioblastoma. cervical cancer ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma. bladder cancer breast cancer, colon cancer, colorectal cancer, endometπal carcinoma, salivary gland carcinoma, kidnev cancer such as renal cell carcinoma and Wilms tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thvroid cancer, testicular cancer esophageai cancer, and various types of head and neck cancer The preferred cancers for treatment herein are breast, colon, lung, and melanoma The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and or causes destruction of cells The term is intended to include radioactive isotopes (e g , 13 , I '~ 5 I 90 Y, and l ii6 Re). chemotherapeuticagents. and toxins such as enzvmaticallv active toxins ot bacterial, fungal, plant, or animal origin, or fragments thereof A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include Adπamvcin. Doxorubicin. 5-Fluorouracιl. Cytosine arabinoside ("Ara- C"), Cyclophosphamide.Thiotepa. Busulfan. Cv toxin. Ta.xol. Foxotere. Methotrexate. Cisplatin. Melphalan. Vinblastine. Bleomvcin, Etoposide. Ifosfamide. Mitomycin C, Mitoxantrone. Vincreistine. Vinorelbine, Carboplatin. Teniposide. Daunomycin, Carminomycin. Aminopteπn, Dactinomycin. Mitomycins. Esperamιcιns(see U S Pat No 4.675.187), Melphalan. and other related nitrogen mustards Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onapπstone A "growth-inhibitoryagent ' when used herein refers to a compound or composition which inhibits growth of a cell, such as an Wnt-overexpressmg cancer cell, either in vitro or in vivo Thus, the growth- inhibitory agent is one which significantly reduces the percentage of malignant cells in S phase Examples of growth- inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G l arrest and M-phase arrest Classical M-phase blockers include the vincas (vincπstmeand vinblastine), taxol. and topo II inhibitors such as doxorubicin. daunorubicin. etoposide. and bleomvcin Those agents that arrest G l also spill over into S-phase arrest, for example, DNA alkylatmg agents such as tamoxifen, prednisone.dacarbazine.mechlorethamine, cisplatin. methotrexate. 5-fluorouracιl. and ara-C Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds . Chapter 1. entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al (WB Saunders Philadelphia, 1995), especially p 13 The 4D5 antibody (and functional equivalents thereof) can also be employed for this purpose if the cancer involves ErbB2-overexpressιng cancer cells See. e , WO 92/22653 "Northern analysis" or "Northern blot" is a method used to identify RNA sequences that hybridize to a known probe such as an ohgonucleotide.DNA fragment. cDNA or fragment thereof, or RNA fragment The probe is labeled itn a radioisotope such as --P or bv biotinvlation. or with an enzvme The RNA to be analyzed is usually electrophoreticallv separated on an agarose or polvacrviamide gel, transferred to nitrocellulose nv Ion or other suitable memorane. and hvbπdized with the probe using standard techniques well known in the an such as those described in sections 7 39-7 52 of Sambrook et al . supra The technique ot "polymerase chain reaction. ' or PCR." as used herein generally refers to a procedure wherein minute amounts of a specific piece ot nucleic acid. RNA and or DNA. are amplified as described in U S Pat No 4.683.195 issued 28 Julv 1987 Generally, sequence information from the ends ot the region ot interest or bevond needs to be available, such that oligonucleotide primers can be designed, these primers will be identical or similar in sequence to opposite strands of the template to be amplified The 5' terminal nucleotides ot the two primers mav coincide with the ends of the amplified material PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA. and cDNA transcribed from total cellular RNA. bacteπophage, or plasmid sequences, etc See generally Mullis et al , Cold Spring Harbor Svmp Quant Biol . 5 263 ( 1987), Erlich. ed , PCR Technology. (Stockton Press, NY, 1989) As used herein. PCR is considered to be one. but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific piece ot nucleic acid II Compositions and Methods of the Invention A Full-length WISP Polvpeptide The present invention provides newly-identified and isolated nucleotide sequences encoding a polypeptide referred to in the present application as a WISP polypeptide. including a WISP- 1. WISP-2. or WISP-3 polypeptide In particular. cDNAs have been identified and isolated encoding novel muπne and human WISP- 1 and WISP-2. and human WISP-3 splice variants as disclosed in further detail in the Examples below Using BLAST and FastA sequence alignment ^mputer programs, it was found that the coding sequences of mouse and human WISP- 1 and -2. as well as the two coding sequences of human WISP-3 disclosed herein, show significanthomology to DNA sequences disclosed in the GenBank database, including those published by Adams et al , Nature, 377 3-174 (1995) Further, using BLAST and FastA sequence alignment computer programs, it was found that various portions of the coding sequences of mouse and human WISP-1 and WISP-2 show significant homology to CTGF, cef- IO, Cyrόl, and or Nov protein In this regard, mouse WISP-1 is 47% homologous to mouse CTGF and 46% homologous to human CTGF, mouse WISP-2 is 46% homologous to chick cef- 10 protein precursor and 42% homologousto human Cyrόl protein.human WISP- 1 is 47% homologousto mouse CTGF and 48% homologous to human CTGF, and human WISP-2 is 48% homologous to mouse CTGF, 49% homologous to human CTGF precursor, 46% homologous to mouse Nov protein homolog precursor, 49% homologous to human CTGF, and 48% homologous to mouse CTGF precursor Further, apparently the amino acid sequences of mouse WISP-1 and mouse ELM 1 (Hashimoto et al . supra) are identical, and the ammo acid sequences of human WISP-1 and mouse ELM 1 are 84% identical Since these factors have also been correlated with IGFBPs, it is presentlv believed that the WISP- 1 and WISP-2 polypeptidesdisclosed in the present application are newlv identified members of the CTGF or IGFBP family and possess activity relating to development ot normal, iniureo. and cancerous cells and tissue More specifically . WISP- 1 and WISP-2 may be involved in breast cancer, lung cancer, melanoma, and colon cancer, as well as in wound repair. Further, they may be involved in atherosclerosis. Further, using BLAST and FastA sequence alignment computer programs, it was found that various portions of the coding sequences of the two splice variants ot human WISP-3 show significant homology to mouse ELM 1 and CTGF proteins. In this regard, both splice variants of WISP-3 are 45% homologous to mouse ELM 1 and 42% homologousto mouse and human CTGF and its precursor, with the longer variant of Fig. 6 being 43% homologous to Xenopus CTGF and the shorter variant of Fig. 7 being 42% homologous to Xenopus CTGF. B WISP Polvpeptide Variants In addition to the full-lengthnative-sequenceWISP polypeptidesdescnbedherein. it is contemplated that variants of these sequences can be prepared. WISP variants can be prepared by introducing appropriate nucleotide changes into the WISP-encoding DNA. or by synthesis of the desired variant WISP polypeptides. Those skilled in the art will appreciate that amino acid changes may alter post-transiational processes of the WISP polypeptide. such as changing the number or position ot glycosylation sites or altering the membrane- anchoring characteristics, if the native WISP polypeptide is membrane bound. Variations in the native full-length WISP sequences, or in various domains of the WISP polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for insnnce. in U.S. Patent No. 5,364.934. Variations may be a substitution, deletion, or insertion of one or more codons encoding the WISP polypeptide that results in a change in the amino acid sequence as compared with the native-sequence WISP polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in any portion of the WISP polypeptide. Guidance in determining which amino acid residue may be inserted, substituted, or deleted without adversely affecting the desired activity ir . y be found by comparing the sequence of the WISP polypeptide with that of homologous known CTGF protein molecules, in the case of WISP- 1. WISP-2. and WISP-3. and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutionscan be the result of replacing one amino acid with another ammo acid having similar structural and or chemical properties, such as the replacement of a leucine with a seπne, i.e . conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to about 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity in in vitro assays for gene upreguiation or downregulation and in transgenic or knockout animals. The variations can be made on the cloned DNA to produce the WISP DNA or WISP polypeptide variant DNA using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis (Carter et al., Nucl. Acids Res.. 11:4331 (1986); Zoller et al.. Nucl. Acids Res.. 10:6487 ( 1987)), cassette mutagenesis (Wells et al.. Gene, 34-315 (1985)). alanine scanning, PCR mutagenesis, restriction selection mutagenesis(Wells e/ αt. Philos. Trans. R Soc. London SerA. 317:4 5 (1986)), or other known techniques. Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such ammo aciαs include alanine iv cine seπne. and cvsteme Alanine is tv picallv a preferred scanning amino acid among this group because it eliminates the side-chain bevond the beta-carbon and is less likelv to alter the main-chain conformation ot the variant Alanine is also tvpicallv preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed positions T E Creighton Proteins Structure and Molecular Properties ( W H Freeman &. Co San Francisco. 1983). Chothia. J Mol Biol . 150 1 ( 1976) If alanine substitution does not vield adequate amounts ot vaπant. an lsoteπc am o acid can be used Further deletional variants ot the full-length WISP polypeptide include variants from which the N- terminal signal peptide. if anv (such as. for example, those putativelv identified as ammo acids 1 to 22 for WISP- 1. 1 to 23 for WISP-2. 1 -33 for the WISP-3 of Fig 6 and 1 - 15 for the WISP-3 of Fig 7). and/or the initiating methionine has been deleted C Modifications of the ISP Polvpeptide Covalent modifications ot the WISP polypeptides are included within the scope of this invention One tvpe ot covalent modification includes reacting targeted amino acid residues of a WISP polypeptide with an organic deπvatizing agent that is capable ot reacting with selected side chains or the N- or C- terminal residues Deπvatizationwith bifunctional agents is useful, for instance, tor crosslinkmg a WISP polypeptide to a water-insolublesupport matrix or surface for use in the method for purifying anti-WISP antibodies, and vice-versa Commonly used crosslinkmg agents include. c i| , 1 l-bιs(dιazoacetyl)-2-phenylethane, glutaraldehyde. N-hydroxysuccinimide esters, for example, esters with 4-azιdosalιcylιc acid, homobifunctional ιmιdoesters.ιncludιngdιsuccιnιmιdvlesters such as 3.3'-dιthιobιs(succιnιmιdylpropιonate), bifunctional maleimides such as bιs-N-maleιmιdo- 1 8-octane.and agents such as methyl-3-((p-azιdophenvl)- dιthιo)propιoιmιdate Other modificationsincludedeamidationofglutaminvland asparaginylresiduesto the corresponding glutamvl and aspartyl residues, respectively I. Iroxvlationot prolineand lysine. phosphorylationofhvdroxyl groups of servl or threonv I residues, methvlation of the α-ammo groups of ivsine. argmine. and histidine side chains (Creighton. supra, pp 79-86). acetvlation ot the N-terminai amine. and amidation of anv C-terminal carboxyl group Another type ot covalent modification ot the WISP polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosvlation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in the native sequence (either by deleting the underlying glycosylation site or by removing the glycosylationmoietiesby chemical and/or enzymatic means) and or adding one or more glycosylation sites that are not present in the native sequence In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportion of the various sugar residues present Addition ot glycosylation sites to the WISP polypeptide herein may be accomplished by altering the amino acid sequence The alteration may be made, for example, by the addition ot or substitution by, one or more serine or threonine residues to the native sequence (for O-linked glycosylation sites) The ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the ISP polvpeptide at preselected bases such that codons are generated that will translate into the desired amino acids The DNA mutatιon( s) mav be made using methods described above Another means ot increasing the number ot carbohydrate moieties on the WISP polvpeptide is bv chemical or enzvmatic coupling of glvcosides to the polvpeptide Such methods are described in the art. e g in WO 87/05330 published 1 1 September 1987. and in Aplin and Wπston.CRC Cπt Rev Biochem . pp 259- 306 ( 1981 ) Removal of carbohydrate moieties present on the WISP polypeptide mav be accomplished chemically or enzvmaticallvor bv mutational substitution of codons encoding amino acid residues that serve as targets tor glvcosvlation Chemical deglycosvlation techniques are known in the art and described, for instance, bv Hakimuddin.er a/ . Arch Biochem Biophvs . 259 52 ( 1987) and bv Edge et al . Anal Biochem . 1 18 131 ( 1981 ) Enzymatic cleavage of carbohvdrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidasesas described by Thotakura et al , Meth Enzvmol . 138 350 (1987) Another type ot covalent modification comprises linking the WISP polypeptide to one of a variety of nonproteinaceouspolvmers.e g . polvethvlene glycol. polypropylene glycoi. or polyoxyalkvlenes, in the manner set forth, e g in U S Patent Nos 4 640 835 4 496 689 4 301.144 4 670.417 4 791 , 192 or 4 179.337 The WISP polypeptide of the present invention mav also be modified in a wav to form a chimeπc molecule comprising a WISP polypeptide. or a fragment thereof, fused to a heterologous polypeptide or am o acid sequence In one em b odiment, such a chimeπc molecule comprises a fusion of the WISP polypeptidewith a tag polvpeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino- or carboxyl-terminus of a native or variant WISP molecule The presence of such epitope-tagged forms can be detected using an antibodv against the tag polypeptide Also, provision of the epitope tag enables the WISP polypeptides to be readily purified by affinity purification using an anti-tag am. body or another type of affinity matrix that binds to the epitope tag In an alternative embodiment, the chimeπc molecule mav comprise a fusion of the WISP polypeptides. or fragments thereof, with an lmmunoglobulinor a particularregion ot an immunoglobulin For a bivalent form of the chimeπc molecule, such a fusion could be to the Fc region ot an Ig, such as an IgG molecule Various tag polypeptidesand their respectiveantibodies are well known in the art Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-His-Gly) tags, the flu HA tag polypeptide and its antibody 12CA5 (Field et al . Mol Cell Biol . 8 2159-2165 ( 1988)). the c-mvc tag and the 8F9, 3C7, 6E10. G4. B7. and 9E 10 antibodies thereto (Evan e/ α/ . Molecularand Cellular Biology. 5 3610-3616 ( 1985)). and the Herpes Simplex virus glycoprotem D (gD) tag and its antibody Paborsky et al , Protein Engineering. 3(6) 547-553 ( 1990) Other tag polypeptides include the Flag-peptιde(Hopp et al . BioTechnology. 6 1204- 1210 (1988)), the KT3 epitope peptide (Martin et al . Science, 255 192- 194 ( 1992)), an α-tubulin epitope peptide (Skinner et al , J Biol Chem . 266 15163- 15166 ( 1991 )), and the T7 gene 10 protein peptide tag Lutz-Frevermuth et al , Proc Natl Acad Sci USA. 87 6393-6397 (1990) D Preparation of WISP Polvpeptide The description below relates primarily to production of WISP polypeptides bv cultunng cells transformed ortransfectedwith a vector containingat least DNA encodmgthe mature or full-length sequences of human or mouse WISP- 1 (SEQ ID NOS.3.4. 1 l . or 12, respectively ι. or containin at least DNA encoding the mature or full-length sequences of human or mouse WISP-2 (SEQ ID NOS: 15, 16. 19. or 20. respectively), or containingat least DNA encoding the mature or full-length sequences of human WISP-3 of Fig. 6 (SEQ ID NOS:32 or 33. respectively), or containing at least DNA encoding the mature or full-length sequences of human WISP-3 of Fig. 7 (SEQ ID NOS:36 or 37. respectively). It is. of course, contemplated that alternative methods, which are well known in the art. may be employed to prepare WISP polypeptides. For instance, the WISP polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques. See, e.g., Stewart et al.. Solid- Phase Peptide Synthesis (W H. Freeman Co.. San Francisco. CA, 1969): Mernfield. J. Am Chem. Soc. 85_:2149-2154 ( 1963). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems peptide synthesizer (Foster City, CA) in accordance with manufacturer's instructions. Various portions of WISP polypeptides may be chemically synthesized separately and combined using chemical or enzymatic methods to produce a full-length WISP polypeptide. I . Isolation of DNA Encoding WISP Polvpeptide DNA encoding a WISP polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA for WISP polypeptide and to express it at a detectable level. Accordingly, DNA encoding human WISP polypeptide can be conveniently obtained from a cDNA library prepared from human tissue, such as d human fetal liver library or as otherwise described in the Examples. The gene encoding WISP polypeptide may also be obtained from a genomic library or by oligonucleotide synthesis. A still alternative method of cloning WISP polypeptide is suppressive subtractive hybridization. which is a method forgeneratingdifferentiallyregulated or tissue-specific cDNA probes and libraries. This is described, for example, in Diatchenko et al., Proc. Natl. Acad. Sci USA. 93: 6025-6030 ( 1996). The procedure is based primarily on a technique called suppression PCR and combines normalization and subtraction in a single procedure. The norma zationstep equalizes the abundance ofcDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. Libraries can be screened with probes (such as antibodies to a WISP polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al.. supra. An alternative means to isolate the gene encoding WISP polypeptide is to use PCR methodology. Sambrooke/ al., supra: Dieffenbach et al., PCR Primer: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1995). The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that faise positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation. or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al.. supra. Sequences identified in such library screening methods can oe compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases Sequence identity (at either the amino acid or nucleotide level) within defined regions ot the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as ALIGN. DN Astar. and INHERIT which employ various algorithms to measure homology Nucieic acid having polypeptide-codingsequence mav be obtained by screening selected cDN A or genomic libraries using the deduced amino acid sequences disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates ot mRNA that may not have been reverse-transcribed into cDNA 2 Selection and Transformation of Host Cells Host cells are transtected or transformed with expression or cloning vectors described herein for WISP polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants. or amplifying the genes encoding the desired sequences The culture conditions, such as media, temperature. pH, and the like, can be selected by the skilled artisan without undue experimentation In general, principles, protocols, and practical techniques tor maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach. M Butler, ed (IRL Press. 1991) and Sambrook et al . supra Methods of transtection are known to the ordinarily skilled artisan, for example, CaP0 and electroporation Depending on the host cell used, transformation is performed using standard techniques appropπateto such cells The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al , Gene.21 315 ( 1983 ) and WO 89/05859 published 29 June 1989 For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der E Virology, 52456-457 ( 1978) can be employed General aspects of mammalian cell host system transformationshave been described in U S Patent No 4 399.216 Transformations into veast are tvpicallv carried out according to the method of Van Solingen et al , J Bact . 130 946 ( 1977) and Hsiao et ul , Proc Natl Acad Sci (USA). 76 3829 ( 1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or poiycations, e g , polybreneor polyomιthιne,may also be used. For various techniques fortransformingmammalian cells, see Keown et al , Methods m Enzvmology. 85 527-537 (1990) and Mansoure/ al , Nature. 336 348-352 (1988) Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include but are not limited to eubacteπa, such as Gram- negative or Gram-positive organisms, for example, Enterobactertaceae such as Escherichia. e g , E colt. Enterobacter. Erwinta, Klebsiella. Proteus. Salmonella, e g , Salmonella tvphtmurium. Serratia. e g , Serratia marcescans, and Shigella. as well as Bacilli such as B subtilis and B licheniformis (e g , B licheniformis 41 P disclosed in DD 266.710 published 12 April 1989), Pseudomonas such as P aeruginosa. and Streptomvces Various E coli strains are publiclv available, such as E colt K12 strain MM294 (ATCC 31 446). E coli XI 776 (ATCC 31.537), E coli strain W31 10 (ATCC 27,325). and K5 772 (ATCC 53,635) These examples are illustrative rather than limiting Strain W 3 1 10 is one particularly preferred host or parent host because it is a common host strain tor recombinant DN λ product fermentations Preferably the host cell secretes minimal amounts ot proteolvtic enzymes For example, strain W31 10 mav be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host with examples of such hosts including £ t oll W3 1 10 strain I A2. which has the complete genotvpe tonA £ coli W31 10 strain 9E4 w hich has the complete genotvpe tonA ptrS. £ coli W31 10 strain 27C7 (ATCC 55.244) which has the complete genotvpe tonA piri phoA El 5 (urgF-lac) 169 deςP omnT karf. E toll W31 10 strain 37D6 which has the complete genotype tonA ptri phoA El 5 (argF-lac) I69 degP ompT rhs7 dvG kari . E coli W31 10 strain 40B4. which is strain 37D6 with a non-kanamvcin resistant de%P deletion mutation, and an £ coli strain having mutant peπplasmic protease disclosed in U S Patent No 4.946.783 issued 7 August 1990 Alternative I v. in vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable In addition to prokaryotes. eukaryotic microbes such as filamentous fungi or veast are suitable cloning or expression hosts for vectors containing nucleic acid encoding WISP polypeptide Saccharomvces terevisiae is a commonlv used lower eukarvotic host microorganism However, a number ot other genera. species, and strains are commonlv available and useful herein, such as Sthizosaccharomvces pom be (Beach and Nurse. Nature. 290 140 ( 1981 ). EP 139,383 published 2 Mav 1985), kluweromvces hosts (U S Patent No 4.943.529. Fleer et αl . Bio/Technology. Q 968-975 ( 19 1 )) such as. e g . K lαctis (MW98-8C. CBS683. CBS4574 Louvencourt et αl . J Bacteπol . 737 ( 1983)). A, frαgilis (ATCC 12.424) K bulgαncus (ATCC 16.04A k 7S). k nαltu (ATCC 56.500), k drosophilαrum (ATCC 36.906. Van den Berg et αl . Bio/Technology. 8 135 ( 1990)), A. thermotolerαns. and K mαrxiαnus \αrrowια (EP 402,226), Pichiα pαstons (EP 183,070, Sreekπshna et αl . J Basic Microbiol . 2a 265-278 ( 1988)), Candida Trichoderma reesia (EP 244.234). Neurosporacrassa (Case et al , Proc Natl Acad Sci USA. 76 5259-5263 ( 1979)), Schwanniomvces such as Schwanmomxces occidentals (EP 394.538 published 3 1 October 1990). and filamc- .ous fungi such as. e g Neurospora Penicilltum. Tolvpocladium (WO 91 /00357 pubiu .ed 10 Januarv 1991 ), and Aspergillus hosts such as A dulans ( Ballancee/ a/ . Biochem Biophvs Res Commun 1 12 284-289 ( 1983), Tιlbum e/ α/ . Gene, 26 205-221 ( 1983). Yelton c a/ . Proc Natl Acad Sci USA. 81 1470-1474 ( 1984)) and A meer Kelly and Hvnes. EMBO J . 4 475-479 ( 1985) Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula. Candida Kloeckera P chia Saccharomvces. Torulopsis, and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Biochemistry of Methylotrophs. 269 ( 1982) Suitable host cells for the expressionof giycosylated WISP are derived from multicellularorganisms Examples of mvertebratecells include insect cells such as Drosophila S2 and Spodoptera Sf9 as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidnev CV l line transformedby SV40 (COS-7. ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture (Graham et al . J Gen Virol . 36 59 ( 1977)), Chinese hamster ovary cellsΛDHFR (CHO, Urlaub and Chasm. Proc Natl Acad Sci USA. 77 4216 ( 1980)). mouse sertoli cells (TM4. Mather. Biol Reprod . 23 243-251 ( 1980)), human lung cells (Wl 38. ATCC CCL 75) human liver cells (Hep G2. HB 8065) and mouse mammarv tumor ( MMT 060562 ATCC CCL51 ) The selection ot the appropriate host cell is deemed to be within the skill 3 Selection and Use of a Rep cable Vector The nucleic acid (e g oDNA or genomic DNA) encoding the desired WISP polypeptide mav be inserted into a rephcable vector tor cloning (amplification ot the DNA) or tor expression Various vectors are publiclv available The vector mav. for example, be in the form ot a plasmid. cosmid. viral particle, or phage The appropriate nucleic acid sequence mav be inserted into the vector bv a vaπetv ot procedures In general. DNA is inserted into an appropriate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to. one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more ot these components employs standard gation techniques which are known to the skilled artisan The desired WISP polypeptidemav be produced recombinantly not onlv directlv, but also as a fusion polypeptide with a heterologous polvpeptide which mav be a signal sequence, if the WISP polvpeptide is conducive to being secreted or other polypeptide having a specific cleavage site at the N-terminus ot the mature or full-length protein or polypeptide In general, the signal sequence mav be a component ot the vector, or it mav be a part of the DNA encoding the WISP polypeptide that is inserted into the vector The signal sequence mav be a prokaryotic signal sequence such as. for example, the alkaline phosphatase. penicillinase. lpp. or heat-stableenterotoxin II leaders For yeast secretion the signal quence mav be, e g . the yeast lnvertase leader, alpha factor leader ncludingSαcc/raromvcesand Kluweromvces α-factor leaders. the latter described in U S Patent No 5 010, 182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362.179 pubhshed4 April 1990), or the signal described in WO 90/13646 published 15 November 1990 In mammalian cell expression, mammalian signal sequences mav be used to direct secretion ot the rotein, such as signal sequences from secreted polypeptides ot the same or related spec ■., as well as viral secretory leaders, and including signals from WISP polypeptides Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a variety of bacteria, yeast. and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria. the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polvoma. adenovirus. VSV, or BPV) are useful for cloning vectors in mammalian cells Expression and cloning vectors will tvpicallv contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicillin.neomvcin. methotrexate. or tetracvcline. (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e g , the gene encoding D-alanine racemase for Bacilli An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding WISP polypeptide. such as DHFR or thymidine kinase An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity prepared and propagated as described bv Urlaub et al , Proc Natl Acad Sci USA. 77 4216 ( 1980) A suitable selection gene tor use in veast is the trp \ gene present in the veast plasmid YRp7 Stinchcomb et al . Nature. 282.39 ( 1 79). Kingsman et al . Gene. 7 141 ( 1 79): Tschemper et al.. Gene, 10: 157 (1980). The trpi gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan. for example. ATCC No. 44076 or PEP4- 1 Jones. Genetics. 85: 12 ( 1977). Expression and cloning vectors usually contain a promoter operablv linked to the nucleic acid sequence encoding WISP polypeptide to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-iactamase and lactose promoter systems (Chang et al.. Nature. 275.615 ( 1978): Goeddel et al . Nature. 281 544 ( 1979)). alkaline phosphatase. a tryptophan (trp) promoter system (Goeddel. Nucleic Acids Res.. 8.4057 ( 1980): EP 36.776). and hybrid promoters such as the tac promoter. deBoer et al.. Proc. Natl. Acad. Sci. USA. 80:21-25 ( 1983). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operablv linked to the DNA encoding the WISP polypeptide. Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase (Hitzeman et al., J. Biol Chem.. 255:2073 ( 1980)) or other giycoiytic enzymes ( Hess et al . J. Adv Enzyme Reg.. 7 149 ( 1968): Holland. Biochemistry. J_7'4900 ( 1978)). such as enolase. glyceraldehyde-3-phosphate dehydrogenase. hexokinase. pyruvate decarboxylase. phosphofructokinase. glucose-6-phosphate isomerase. 3-phosphoglycerate mutase. pyruvate kinase. tπosephosphate isomerase. phosphoglucose isomerase. and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter region;- for alcohol dehydrogenase 2. isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase. and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. WISP transcription from vectors in mammalian host cells is controile. ' for example, by promoters obtained from the genomes of viruses such as polvoma virus, fowlpox virus ( UK 2.21 1 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus. a retrovιrus,hepatιtis-B virus, and Simian Virus 40 (SV40): from heterologous mammalian promoters, e.g.. the actin promoter or an immunoglobulinpromoter; and from heat-shock promoters, provided such promoters are compatible with the host cell systems. Transcription of a DNA encoding a WISP polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase. albumin, α-fetoprotein. and insulin). Typically, however. one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270). the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the sequence coding for a WISP polypeptide. but is preferably located at a site 5' from the promoter. Expression vectors used in eukarvotic host cells (veast. fungi, insect, plant, animal human, or nucleated cells from other muiucellularorganisms) will also contain sequences necessary tor the termination ot transcription and for stabilizing the mRNA Such sequences are commonlv available from the 5' and. occasionally 3' untranslated regions of eukarvotic or viral DNAs or cDNAs These regions contain nucieotide segments transcribed as polvadenylated fragments in the untranslated portion ot the mRNA encoding WISP polvpeptide Still other methods, vectors, and host cells suitable tor adaptation to the synthesis of WISP polypeptides in recombinant vertebrate cell culture are described in Gething et al . Nature. 293 620-625 ( 1981 ). Mantei et al . Nature, 281 40-46 ( 1979) EP 1 17 060. and EP 1 17.058 4 Detecting Gene Amplification/Expression Gene amplification and or expression may be measured in a sample directly, for example, bv conventional Southern blotting. Northern blotting to quantitate the transcription ot mRNA (Thomas, Proc Natl Acad Sci USA. 77 5201 -5205 ( 1980)), dot blotting (DNA analysis), or m situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies mav be employed that can recognize specific duplexes including DNA duplexes. RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protem duplexes The antibodies in 'urn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibodv bound to the duplex can be detected Gene expression, alternatively, mav be measured by immunological methods, such as immunohistochemicalstaining ot cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal. and may be prepared in any mammal Conveniently, the antibodies mav be prepared against a native-sequence WISP polypeptide or against a synthetic peptide based on the DNA sequences provided herein or agu.i st exogenous sequence fused to DNA encoding WISP polypeptide and encoding a specific antibodv epitope 5 Purification ot Polvpeptide Forms of WISP polypeptide may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e , Tπton-X 100) or by enzymatic cleavage Cells employed in expression of WISP polypeptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication. mechanical disruption, or cell lysing agents It may be desired to purify WISP polypeptide from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanoi precipitation. reverse phase HPLC. chromatographv on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE. ammonium sulfate precipitation, gel filtration using, for example, SEPHADEX™ G-75, protein A SEPHAROSE™ columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of the WISP polypeptide Various methods of protein purification may be employed, and such methods are known in the art and described, for example. in Deutscher. Methods in Enzvmoiogv. 182 ( 1990) and Scopes Protein Purification Principles and Practice ( Springer- Verlag New York. 1982) In one specific example ot purification, either a poiv-His tag or the Fc portion ot human lgG is added to the C-terminal coding region ot the cDNA tor WiSP- 1 WISP-2 or WISP-3 before expression The conditioned media from the transtected cells are harvested bv centπtugation to remove the cells and filtered tagged constructs the protein mav be purified using a Ni-NTA column After loading, the column mav be washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein mav then be desalted into a storage buffer if desired Im unoadhesin (Fc-containing) constructs of the WISP-1 WISP-2, and WISP-3 proteins may be purified from the conditioned media bv pumping them onto a 5-ml Protein A column which had been equilibrated in a phosphate buffer After loading, the column mav be washed extensively with equilibration buffer before elution with citric acid The eluted protein mav be immediately neutralized bv collecting 1-ml tractions into tubes containing TRIS buffer The highly purified protein mav be subsequentlv desalted into storage buffer as described above tor the polv-His-tagged proteins The homogeneity ot the protein mav be assessed bv SDS polyacrviamide gels and bv N-terminal amino acid sequencing by Edman degradation The purification step(s) selected will depend for example, on the nature of the production process used and the particular WISP polvpeptide produced E Uses for WISP Polvpeptide and Its Nucie' Acid Nucleotide sequences ( or their complement ) encoding WISP polypeptides have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping, and in the generation of anti-sense RNA and DNA Nucleic acid encoding WISP polypeptide will also be useful for the preparation of WISP polypeptides bv the recombinant techniques described herein The full-length nucleotide sequences for mouse human WISP- 1 or WISP-2 (SEQ ID NOS 9 1. 17, and 13, respectively), or portions thereot. or the full-length nucleotide sequences tor human WISP-3 of Fig 6 (SEQ ID NO 30) or tor WISP-3 of Fig 7 (SEQ ID NO 34) mav be used as hybridization probes for a cDNA library to isolate or detect the full-length gene encodmgthe WISP polypeptide of interest or to isolate or detect still other genes (for instance, those encoding naturally occurring variants of WISP polypeptide. other WISP polypeptide family members, or WISP polypeptides from other species) which have a desired sequence identity to the WISP polypeptidesequences disclosed in Figures 1 , 2, 3A and 3B, 4, 6A and6B. and 7A and 7B (SEQ ID NOS 3, 4 1 1, 12. 15, 16. 19, 20, 32. 33, 36, or 37) For example, such procedures as in situ hybridization. Northern and Southern blotting, and PCR analysis may be used to determine whether DNA and/or RNA encoding a different WISP is present in the cell type(s) being evaluated Optionally, the length of the probes will be about 20 to about 50 bases For example, the hybridization probes mav be derived from the UNQ228 (DNA33473-seq mm) nucleotide sequence (SEQ ID NO 38) or the full-length human WISP-2 nucleotide sequence (SEQ ID NO 13) as shown in Figure 4 or from genomic sequences including promoters, enhancerelements. and introns of DNA encoding native-sequence WISP polypeptide 55- Bv wav ot example, a screening method will comprise isolating the coding region ot the WISP gene using the known DNA sequence to svnthesize a selected probe ot about 40 bases Hv bπdization probes mav be labeled bv a vaπetv ot labels, including radionucleotides such as J -P or J - S. or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems Labeled probes having a sequence complementary to that ot anv ot the genes encoding WISP polypeptides ot the present invention can be used to screen libraries ot human cDNA genomic DNA or mRNA to determine to which members ot such libraries the probe hv bπdizes Hv bπdization techniques are described in further detail in the Examples below The probes may also be employed in PCR techniques to generate a pool of sequences for identification ot closelv related WISP sequences Nucleotide sequences encoding a WISP polypeptide can also be used to construct hybridization probes tor mapping the gene which encodes that WISP polypeptide and for the genetic analysis of individuals with genetic disorders The nucleotide sequences provided herein may be mapped to a chromosome and specific regions ot a chromosome using known techniques, such as in situ hybridization, linkage analvsis against known chromosomal markers, and hy bridization screening with libraries If the amplification ot a given gene is functionally relevant, then that gene should be amplified more than neighboring genomic regions which are not important for tumor survival To test this, the gene can be mapped to a particular chromosome, e g by radiation-hvbπd analysis The amplification level is then determined at the location identified. and at neighboπnggenomic region Selective or preferential amplification at the genomic region to which to gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival Chromosome mapping includes both framework and epicentermapping For further details see e g . Stewart et al , Genome Research 7. 422-433 ( 1997) Nucleic acid encoding a WISP polypeptidemav be used as a diagnostic to determine the extent and rate of the expression ot the DNA encoding the W I :; P polypeptide in the cells of a patient To accomplish such an assay, a sample of a patient's cells is treated, via in suit hybridization. or bv other suitable means, and analyzed to determine whether the sample contains mRNA molecules capable of hybridizing with the nucleic acid molecule. Nucleic acids which encode WISP polypeptides or any of their modified forms can also be used to generate either transgenic animals or "knock-out" animals which, in turn, are useful in the development and screening of therapeutical ly useful reagents A transgenic animal (e g . a mouse or rat) is an animal having cells that contain a transgene. which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e , an embryonic stage A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops In one embodiment, cDNA encoding a WISP polypeptide can be used to clone genomic DNA encodmgthe WISP polypeptide in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding the WISP polypeptide. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4.736.866 and 4 870 009 and WO 97/38086 Tvpicallv. particular cells would be targeted for WISP transgene incorporation with tissue-specific enhancers Transgenic animals that include a copy ot a transgene encoding the WISP polvpeptide introduced into the germ line ot the animal at an embrvonic stage can oe used to examine the effect ot increased expressionof DNA encodmgthe WISP polvpeptide Such animals can be used as tester animals for reagents thought to confer protection from tor example pathological conditions associated with its overexpression In accordance with this facet ot the invention, an animal is treated with the reagent and a reduced incidence ot the pathological condition compared to untreated animals bearing the transgene w ould indicate a potential therapeutic intervention tor the pathological condition Alternatively non-human homologues of WISP polypeptides can be used to construct a WISP polypeptide "knock-out ' animal which has a defective or altered gene encoding a WISP polvpeptide as a result of homologous recombination between the endogenous gene encoding the WISP polypeptide and altered genomic DNA encoding the WISP polypeptide introduced into an embryonic cell of the animal For example cDNA encoding the WISP polypeptide can be used to clone genomic DNA encoding the WISP polypeptide in accordance with established techniques A portion of the genomic DNA encoding the WISP polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration Typically several kiiobases ot unaltered flanking DNA (both at the 5 and 3' ends) are included in the vector See e g , Thomas and Capecchi Cell. 51 503 ( 1987) tor a description ot homologous recombination vectors The vector is introduced into an embrvonic stem cell line (e g , by electroporation )and cells in which the introducedDNA has homologouslv recombined with the endogenous DNA are selected See e g Li ei cl . Cell. 69 915 ( 1992) The selected cells are then injected into a blastocvst of an animal (e g , a mouse or rat) to form aggregation chimeras See e g , Bradley, in Teratocarcinomas and Embryonic Stem Cells A Practical Approach. E J Robertson, ed (IRL, Oxford, 1987), pp 1 13-152 A chimeπc embrvo can then be implanted into a suitable pseudopregnaπt female foster animal and the embryo brought to term to create a "knock-out ' animal Progeny harboring the homologouslv recombined DNA in their germ cells ca." .ιe identified bv standard techniques and used to breed animals in which all cells of the animal contain the homologouslv recombined DNA Knockout animals can be characterized tor instance, bv their ability to defend against certain pathological conditions and bv their development of pathological conditions due to absence of the WISP polypeptide In particular, assays in which CTGF, IGFBPs. and other members of the CTGF superfamily and other growth factors are usually used are preferably performed with the WISP- 1 and WISP-2 polypeptides For example, an assay to determine whether TGF-β induces the WISP polypeptide. indicating a role in cancer, may be performed as known in the art as well as assavs involving induction of cell death and • 'H-thymidine proliferation assays Mitogenic and tissue growth assays are also performed with the WISP polypeptide as set forth above The results are applied accordingly The WISP polypeptides of the present invention mav also be used to induce the formation ot anti- WISP polypeptide antibodies, which are identified bv routine screening as detailed below In addition to their uses above, the WISP-1, WISP-2, and WISP-3 polypeptides of the present invention are useful as the basis for assays of IGF activitv Importantly, since such an assav measures a physiologically significant binding event. ; a that ot an IGF to its IGFBP triggering a detectable change {such as phosphoryiation cleavage, chemical modification etc ) it is hkelv to be both more sensitive and more accurate than immunoassav s w hich detect the pnvsioiogicailvnon-sigmficantbinding ot an IGF to anti- WISP polvpeptide antibodv Although more sensitive and accurate than antibodies the WISP- 1 WISP-2 and WISP-3 molecules ot the invention can be used to assav IGF (such as IGF-I or IGF-I I) levels in a sample in the same ways in which antibodies are used For diagnostic purposes the WISP- 1 W ISP-2 or W ISP-3 polypeptide can be used in accordance w ith immunoassav technology Examples of immunoassavs are provided bv Wide at pages 199-206 of Radioimmune Assav Method Kirkham and Huner ed E &. S Livingstone. Edinburgh 1970 Thus, in one embodiment WISP- 1 WISP-2. and WISP-3 polypeptides can be detectablv labeled and incubated with a test sample containing IGF molecules (such as biological fluids, e ? , serum, sputum. urine, etc ). and the amount of WISP- 1 WISP-2 or WISP-3 molecule bound to the sample ascertained Immobilizationof reagents is required for certain assav methods Immobilization entails separating the WISP- 1 WISP-2. or WISP-3 polvpeptide from any analvte that remains free in solution This conventionally is accomplished bv either msolubilizingthe WISP-1 WISP-2, or WISP-3 polypeptide before the assav procedure as bv adsorption to a water-insolublematπ or surface (Bennich e/ α/ . U S 3.720.760) bv covalent coupling (tor example using glutaraldehvde cross-linking), or bv msolubilizing the molecule afterward, e g , by immunoprecipitation The foregoing are merely exemplary diagnostic assavs for IGF Other methods now or hereafter developed for the determination of these analvtes are included within the scope hereof WISP- 1 WISP-2 - ' id WISP-3 polypeptides are also useful in radioimmunoassavs to measure IGFs such as IGF-I or IGF-II Such a radioimmunoassav would be conducted as described in the literature using the naturally purified or recombinant WISP- 1 WISP-2, or WISP-3 as the WISP element In addition WISP polypeptidesare useful for screening for compounds that bind to them as defined above Preferably these compounds are small molecules such as organic or peptide molecules that exhibit one or more of the desired activ es Screening assavs of this kind are conventional in the art. and any such screening procedure mav be employed whereby the test sample is contacted with the WISP polypeptide herein and the extent of binding and biological activity of the bound molecule are determined More specifically, this invention encompasses methods of screening compounds to identify those that mimic the WISP polypeptide (agonists) or prevent the effect of the WISP polypeptide (antagonists) Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the WISP polypeptidesencoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assavs will include assavs amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates The assays can be performed in a variety of formats, including protein-protein binding assays biochemical screening assavs immunoassavs. and cell- based assavs which are well characterized in the art All assays for antagonists are common in that thev call for contacting the drug candidate with a WISP polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact In binding assavs. the interaction is oinding and the complex formed can be isolated or detected in the reaction mixture In a particular embodiment, the W ISP polvpeptide encoded bv the gene identified herein or the drug candidate is immobilized on a solid phase, e g . on a microtiter plate, bv covalent or non- covalent attachments Non-covalentattachment generally is accomplished bv coating the solid surtace with a solution ot the W ISP polypeptide and drving Alternatively an immobilized antibody, e g a monoclonal antibodv, specific for the WISP polypeptide to be immobilized can be used to anchor it to a solid surtace The assav is performed bv adding the non-immobilized component, which mav be labeled bv a detectable label, to the immobilized component e g the coated surtace containing the anchored component When the reaction is complete, the non-reacted components are removed, e g by washing, and complexes anchored on the solid surface are detected When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilized on the surtace indicates that complexing occurred Where the originally non-immobt zed component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex If the candidate compound interacts with but does not bind to a particular WISP polypeptideencoded by a gene identified herein, its interaction with that polvpeptide can be assaved bv methods well known tor detecting protein-protein interactions Such assavs include traditional approaches, such as. e g , cross-linking, co-immunoprecipitation. and co-purification through gradients or chromatographic columns In addition, protein-protein interactions can be monitored by using a yeast-based genetic svstem described by Fields and co-workers (Fields and Song, Nature ( London), 340 245-246 ( 1989). Chien ei al . Proc Natl Acad Sc, USA. 88 9578-9582 ( 1991 » as disclosed bv Chevrav and Nathans. Proc Natl Acad Sc USA. 89 5789- 5793 ( 1991 ) Many transcπptionalactivators. such as veast GAL4. consist of two physically discrete modular domains, one acting as the DNA-bindmg domain, the other one functioning as the transcription-activation domain The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid svstem") tikes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-bindmg domain of GAL4. and another, in which candidate activating proteins are tu^ed to the activation domain The expression of a GA \-lacZ reporter gene under control ot a GAL4-actιvated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. Compounds that interfere with the interaction ot a gene encoding a WISP polypeptide identified herein and other intra- or extracellular components can be tested as follows usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound I n addition, a placebo may be added to a third reaction mixture, to serve as positive control The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as descriDeo hereinaoove The formation ot a complex in the control reaction! s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction ot the test compound and its reaction partner If the W ISP polypeptide has the ability to stimulate the proliferation ot endothehal cells in the presence ot the co-mitogen ConA. then one example ot a screening method takes advantage ot this ability Specifically, in the proliferation assav. human umbilical vein endothehal cells are obtained and cultured in 96-well fiat-bottomed culture plates (Costar. Lambπdge. MA) and supplemented with a reaction mixture appropriate tor facilitating proliferation ot the cells, the mixture containing Con-A (Calbiochem. La Jolla. CA) Con-A and the compound to be screened are added and after incubation at 37°C. cultures are pulsed with J" H-thymιdιne and harvested onto glass fiber filters (phD. Cambridge Technology. Watertown. MA) Mean J "(H)thymιdιne incorporation (cpm) ot triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments. Irvine. CA) Significant ■5" (H)thymιdιne incorporation indicates stimulation of endothehal cell proliferation To assav tor antagonists, the assav described above is performed, however, in this assay the WISP polypeptide is added along with the compound to be screened and the ability ot the compound to inhibit J" (H)thymιdιne incorporation in the presence ot the WISP polypeptide indicates that the compound is an antagonist to the WISP polypeptide Alternatively, antagonists mav be detected bv combining the WISP polypeptide and a potential antagonist with membrane-bound WISP polypeptide receptors or recombinant recepιc r s under appropriate conditions tor a competitive inhibition assay The WISP polypep"de can be labeled, such as by radioactivity such that the number of WISP polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist The gene encoding the receptor can be identified by numerous methods known to those of skill in the art. for example. Iigand panning and FACS sorting Cohgan / / . Current Protocols n Immun . 1(21 Chapter s ( 1991 ) Preferably, expression cloning is employe . wherein polyadenvlated RNA is prepared from a cell responsive to the WISP polypep: e and a cDNA library created from this RNA is divided into pools and used to transtectCOS cells or other cells that are not responsive to the WISP polvpeptide Transtected cells that are grown on glass slides are exposed to labeled WISP polypeptide The WISP polypeptide can be labeled by a variety of means including lodination or inclusion of a recognition site tor a site-specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographicanalysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screen g process, eventually yielding a single clone that encodes the putative receptor As an alternative approach for receptor identification, labeled WISP polypeptide can be photoaffinity- nked with cell membrane or extract preparations that express the receptor molecule Cross- linked material is resolved by PAGE and exposed to X-ray film The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing The amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor In anotner assav tor antagonists mammalian cells or a membrane preparation expressing tne receptor would be incuoated with labeled WISP polvpeptide in the presence ot the candidate compound The abihtv ot the compound to enhance or block this interaction could then be measured The compositions useful in the treatment ot WISP-related disorders include without limitation antibodies, small organic and inorganic molecules peptides phosphopeptides antisense and πbozvme molecules, triple-helix molecules etc , that inhibit the expression and or activity of the target gene product More specific examples ot potential antagonists include an oligonucleotide that binds to the WISP polypeptide. (poly)peptιde-ιmmunoglobuhn fusions and. in particular, antibodies including without limitation poly- and monoclonal antibodies and antibodv fragments, single-chain antibodies, anti-idiotvpic antibodies and chimeπc or humanized versions ot such antibodies or fragments, as well as human antibodies and antibodv fragments Alternatively, a potential antagonist mav be a closely related protein, for example. a mutated form of the WISP polvpeptide that recognizes the receptor but imparts no effect, thereby competitivelv inhibiting the action of the WISP polypeptide Another potential WISP polvpeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology where, e g , an antisense RNA or DNA molecule acts to block directly the translation ot mRNA bv hybridizing to targeted mRNA and preventing protein translation Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA both of which methods are based on binding ot a polvnucleotideto DNA or RNA For example, the 5' coding pirtion of the polynucleotide sequence, which encodes the mature WISP polypeptides r rein. is used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcπptιon(tπple helix - see Lee et al Nucl Acids Res . 6 3073 (1979) Cooney et al Science. 241 456 (1988), Dervan et al , Science. 251 1360 ( 1991 )), thereby preventing transcription and the production of the WISP polypeptide The antisense RNA oligc.ucleotidehvbπdizesto the mRNA in vivo and blocks translation of the mRNA molecule < f o the WISP polypeptide (antisense - Okano Neurochem . 56 560 ( 1991 ) Ohgodeoxynucleotidesas Antisense Inhibitors ot Gene Expression (CRC Press Boca Raton. FL 1988) The ohgonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production ot the WISP polypeptide When antisense DNA is used, oligodeoxvπbonucleotides derived from the translation- initiation site, e g , between about - 10 and + 10 positions of the target gene nucieotide sequence, are preferred Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the WISP polypeptide. thereby blocking the normal biological activity of the WISP polypeptide Examples of small molecules include, but are not limited to small peptidesor peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidvl organic or inorganic compounds Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA followed bv endonucleolyticcleavage Specific πbozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biology. 4 469-471 ( 1 94) and PCT publication No WO 97/33551 (published September 18 1997) Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single- stranded and composed ot deoxvnucleotides The base composition ot these ohgonucieotides is designed such that it promotes tπple-heiix formation via Hoogsteen base-paiπngrules, which generally require sizeable stretches ot puπnes or pyπmidines on one strand of a duplex For further details see. e g , PCT publication \o WO 97/3355 \ . supra These small molecules can be identified bv any one or more of the screening assavs discussed hereinabove and or bv any other screening techniques well known tor those skilled in the art WISP- 1 WISP-2. and WISP-3 polypeptides are additionally useful in affinity purification ot an IGF that binds to WISP- 1. WISP-2. or WISP-3 (such as. for example. IGF-I) and in purifying antibodies thereto The WISP- 1. WISP-2. or WISP-3 is tvpicallv coupled to an immobilized resin such as Affi-Gel 10™ (Bio- Rad, Richmond. CA) or other such resins (support matrices) by means well known in the art. The resin is equilibrated in a buffer (such as one containing 150 mM NaCl. 20 mM HEPES. pH 74 supplemented to contain 20% glycerol and 0 5% NP-40) and the preparation to be purified is piaced in contact with the resin. wherebv the molecules are selectively adsorbed to the WISP- 1 WISP-2. or WISP-3 on the resin The resin is then sequentially washed with suitable buffers to remove non-adsorbed material. including unwanted contaminants, from the mixture to be purified, using, e g , 150 mM NaCl, 20 mM HEPES. pH 7 4. containingO 5% NP-40 150 mM NaCl, 20 mM HEPES, pH 7 4 contai ngO 5 M NaCl and 0 l% NP-40. 150 mM NaC1.20 mM HEPES. pH 7 4 containin 0 1% deoxychoiate. 150 mM NaCl. 20 mM HEPES. pH 7 4 containing 0 1% NP-40. and a solution ot 0 1% NP-40. 20% glycerol and 50 mM glycine. pH 3 The resin is then treated so as to elute the IGF using a buffer that will break the bond between the IGF and WISP- 1. WISP-2. or WISP-3 (using, e g . 50 mM glycine. pH 3. 0 1% NP-40. 20% glycerol. and 100 mM NaCl) It is contemplated that the WISP polypeptidesof the present invention may be used to treat various • onditions. including those characterized bv overexpression and/or activation of at lea- f he Wnt pathway Further, since the WISP- 1. WISP-2. and WISP-3 molecules respond to hormone-expressed breast cancer in mice and are abnormally expressed in human cancer, and are over-amplified in var'ous colon cancer cell lines. they are useful in diagnosing cancer, for example, as a marker for increased susceptibi tv to cancer or for having cancer Exemplary conditions or disorders to be treated with the WISP polypeptides include benign or malignant tumors (e g , renal, liver, kidney, bladder, testicular. breast, gastric, ovarian, colorectal. prostate. pancreatic, lung, esophageal, vulval, thyroid, hepatic carcinomas, sarcomas, glioblastomas. and various head and neck tumors), leukemias and iymphoid malignancies, other disorders such as neuronal. glial. astrocytal. hypothalamic, and other glandular, macrophagal, epithelial, stromal. and blastocoe c disorders, cardiac disorders; renal disorders: catabolic disorders, bone-related disorders such as osteoporosis, and inflammatory angiogenic. and lmmunologic disorders, such as arteriosclerosis, as well as connective tissue disorders. including wound healing The WISP polypeptides of the invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal. intracerebrospinal. subcutaneous, lntra-articular. lntrasynovial. trathecal. oral, topical, or innalation routes. Intravenous or subcutaneous administration ot the polypeptide is preferred. Therapeutic formulations of the WISP polypeptide are prepared for storage by mixing the polypeptide having the desired degree ot purity with optional pharmaceuticallyacceptable carriers, excipients. or stabilizers ( Remington's Pharmaceutical Sciences 16th edition. Osol. A. Ed. (1980)). in the form of lyophihzed formulationsor aqueous solutions. Acceptable earners, excipients. or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids: antioxidants including ascorbic acid and methionine: preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride: benzalkonium chloride. benzethonium chloride phenol, butyl or benzyl alcohol: alky I parabens such as methyl or propyl paraben: catechol: resorcinol; cyclohexanol: 3-pentanol: and m-cresol): low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins: hydrophilic polymers such as polyvinylpyrroiidone: amino acids such as glycine. glutamme. asparagine. histidine. arginine. or lysine: monosacchaπdes. disacchaπdes. and other carbohydrates including glucose, mannose. or dextπns: chelating agents such as EDTA; sugars such as sucrose, mannitol. trehalose. or sorbitol: salt-tomningcounter- IOΠS such as sodium; metal complexes (e . Zn-protein complexes): and/or non-ionic surtactants such as TWEEN™. PLURONICS™. or polyethylene glycol (PEG). Other therapeutic regimens may be combined with the administration of the WISP polypeptides of the instant invention. For example, the patient to be treated with the poiypepti ' s disclosed herein may aiso receive radiation therapy if the disoideπs cancer. Alternatively, or in addition, a chemotherapeuticagent may be administered to the patient with cancer. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service. Ed.. M.C. Perry (Williams & Wilkins: Baltimore, MD, 1992). The cher..otherapeutιc agent may precede or follow administration of the polypeptide or may be given simultaneously therewith. The polypeptide may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapπstone (see, EP 616812) in dosages known for such molecules. It may be desirable also to co-administer with the WISP polypeptide (or anti-WISP polypeptide) antibodies against other tumor-associatedantigens. such as antibodies which bind to HER-2. EGFR. ErbB2. ErbB3, ErbB4, or vascular endothehal factor (VEGF). Alternatively, or in addition, two or more different anti-cancerantibodies.such as antι-ErbB2 antibodies, may be co-administered to the patient with the WISP polypeptιde(or anti-WISPpolypeptιde antibody). Sometimes. it may be beneficial also to administer one or more cytokines to the patient. In a preferred embodiment, the WISP polypeptide is co-administeredwith a growth-inhibitoryagent to the cancer patient. For example, the growth-inhibitory agent may be administered first, followed by the WISP polypeptide. However, simultaneous administration or administration of the WISP polypeptide first is also contemplated. Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and polypeptide. The antibodies, cv totoxic agents, cytokines. or growth-inhibitorv agents are suitably present in combination in amounts that are effective tor the purpose intended. The active ingredients mav also be entrapped in microcapsules prepared, tor example, bv coacervation techniques or by intertacial polymerization, tor example, hvdroxymethvlceliulose or gelatin microcapsulesand poly-(methvlmethacvlate)mιcrocapsules. respectively, in colloidal drug delivery systems (tor example, hposomes. albumin microspheres. microemulsions. nano-particles. and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington s Pharmaceutical Sciences. 16th edition. Osol. A Ed. ( 1980). supra The formulations to be used tor in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes Sustained-release preparationsmay be prepared. Suitableexamplesof sustained-releasepreparations include semipermeablematπces ot solid hydrophobic polymers containing the polypeptide. which matrices are in the form ot shaped articles, e g , films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example. polv(2-hvdroxvethyl-methacrylate). or poly(vιnylalcohol)). polylactides (U S Pat No 3.773,919), copolymers ot L-glutamic acid and γ ethyl-L-glutamate. non- degradable ethylene-vinyl acetate, degradable lactic acid-glyco c acid copolymers such as the LUPRON DEPOT™(ιnjectab!emιcrospherescomposed of lactic acid-glycohc acid copolymer and leuprolide acetate). an poly-D-(-)-3-hydroxybutyπcacιd While polymers such as ethylene-vinylacetateand lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C. resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be lntermolecular S-S bond formation through thιo-dιsulfιdeιnterchange,stabιlιzatιonmay be achieved by modifyin. u I fhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions For the prevention or treatment of disease or disorder, the appropriate dosage of WISP polypeptide will depend on the type of disorder to be treated, as defined above, the severity and course of the disorder. whether the polypeptide is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the polypeptide, the route of administration, the condition of the patient, and the discretion of the attending physician. The polypeptide is suitably administered to the patient at one time or over a series of treatments Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e g , 0 1-20 mg kg) of WISP polypeptide is an initial candidate dosage for administration to the patient, whether, for example. by one or more separate administrations.or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms of the disorder occurs. However, other dosage regimens may be useful The progress of this therapy is easilv monitored by conventional techniques and assays In another embodiment ot the invention an article ot manutacture containing materials useful for the treatment ot the disorders described above is provided The article of manutacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a variety ot materials such as glass or plastic. The container holds a composition which is effective tor treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent in the composition is the WISP polypeptide The label on. or associated with, the container indicates that the composition is used for treating the condition or disorder of choice. The article of manutacture may turther comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline. Ringer's solution, and dextrose solution. It mav turther include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. F Anti-WISP Polvpeptide Antibodies The present invention turther provides anti-WISP polypeptide antibodies. Exemplary antibodies include polvclonal, monoclonal, humanized, bispecific. and heteroconjugate antibodies. I Polvclonal Antibodies The anti-WISP polypeptide antibodies ot the present invention may comprise polvclonal antibodies Methods ot preparing polvclonal antibodies are known to the skilled artisan. Polvclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and. if desired, an ad j uvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrapeπtoneal injections The immunizing agent may include the WISP polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocvanin. serum albumin, bo . <e thyroglobu n. and soybean trypsin inhibitor Examples of ad j uvants which mav be employed include Freund's complete ad|uvant and MPL-TDM adjuvant (monophosphoryl Lipid A. synthetic trehalose di orynomvcolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. 2. Monoclonal Antibodies The anti-WISP polypeptide antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybπdoma methods, such as those described by Kohler and Milstein. Nature. 256-495 ( 1975) In a hybπdoma method, a mouse, hamster, or other appropriate host animal is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include the WISP polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as PEG. to form a hybπdoma cell. Coding. Monoclonal Antibodies Principles and Practice (Academic Press- New York. 1986) pp 59-103 Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually . rat or mouse myeloma cell lines are employed. The hybπdomacells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused. immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanιnephosphoπbosyltransferase( HGPRT or HPRT). the culture medium for the hybπdomas tvpicallv will include hypoxanthine. aminopteπn. and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. Preferred immortaiizedcell lines are those that fuse efficiently. support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortaiizedcell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center. San Diego, California, and the American Type Culture Collection. Manassas. Virginia. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of human monoclonal antibodies. Kozbor. J. Immunol.. 133:3001 (1984); Brodeur et al.. Monoclonal Antibodv Production Techniques and Applications (Marcel Dekker. Inc.: New York. 1987) pp. 51-63. The culture medium in which the hybπdomacells are cultured can then be assayed for the presence of monoclonal antibodies directed against a WISP polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybπdoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassa; (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard. Anal. Biochem.. 107:220 ( 1980). After the desired hybπdoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Coding, supra. Suitable culture media for this purpose include. for example. Dulbecco's Modified Eagle's Medium md RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as. for example, protein A-Sepharose, hydroxylapatite chromatography. gel electrophoresis, dialysis, or affinity chromatography. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4.816.567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e ., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and ght chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, CHO cells. or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4.816.567: Morrison et al , Proc Natl. Acad. Sci. USA. 81 6851 -6855 ( 1984)) or bv covalentlv loinin to tne immunoglobulin coding sequence all or part ot the coding sequence tor a non-immunoglobulinpolvpeptide Such a non-immunoglobulinpolvpeptidecan be substituted for the constant domains ot an antibodv ot the invention, or can be substituted for the variable domains ot one antigen-combining site of an antibodv ot the invention to create a chimeπc bivalent antibodv The antibodies mav be monovaient antibodies Methods tor preparing monovalent antibodies are well known in the art For example one method involves recombinant expression ot immunoglobulin light chain and modified heavv chain The heavv chain is truncated generailv at anv point in the Fc region so as to prevent heavv-chain crosslinkmg Alternatively the relevant cvste e residues are substituted with another am o acid residue or are deleted so as to prevent crosslinkmg In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof particularlv Fab fragments, can be accomplishedusing routine techniques known 3 Humanized Antibodies The anti-WISP antibodies ot the invention mav turther comprise humanized antibodies or human antibodies Humanized forms ot non-human . g , murine ) antibodies are chimeric immunoglobulins immunoglobulin chains or fragments thereof (such as Fv Fab. Fab' F(ab') 2 , or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibodv) in which residues from a complementary-determining region (cDR) ot the recipient are replaced bv residues from a CDR of a non- human species (donor antibodv) such as mouse, rat. or rabbit having the desired specificity, affinity, and capacity In some instances. Fv framework residues ot the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies mav also comprise residues which are found neither in the recipientantibodv nor in the importedCDR or framework sequences In general the humanized antibodvwill comprise substantially ail of a. least one and tvpicallv two variable domains in which all or substantially all of the CDR regions correspond to those ot a non-human immunoglobulin and all or substantially all of the FR regions are those ot a human lmmunoglobuhnconsensus sequence The humanized antibody preferably also will comprise at least a portion of an Fc, typicallvthat of a human immunoglobulin Jones et al , Nature. 321 522-525 ( 1986), Riechmann et al , Nature, 312323-329 ( 1988), Presta. Curr Op Struct Biol . 2 593-596 ( 1992) Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers (Jones et al . Nature. 321 522-525 ( 1986), Riechmann et al , Nature. 312 323-327 ( 1988) Verhoeyen et al , Science. 239 1534- 1536 ( 1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibodv Accordingly, such "humanized" antibodies are chimeric antιbodιes(U S Patent No 4,816.567), wherein substantially less than an intact human variable domain has been substituted bv the correspondingsequence from a non-human species In practice, humanized antibodies are tvpicallv human antibodies in which some CDR residues and possiblv some l-R residues are substituted bv residues from analogous sites in rodent antibodies Human antibodies can also be produced using various techniques known in the art, including phage- display libraries Hoogenboom and Winter. J Mol Biol .222 381 ( 1991 ) Marks et al . J Mol Biol . 222 581 ( 1 1 ) The techniques of Cole ei al and Boerner et al are also available tor the preparation ot human monocionalantibodies Cole et al . Monoclonal Antibodies and Cancer Therapy. Alan R Liss. p 77 (1985). Boemer et al . J Immunol . 14^( 1 ) 86-95 ( 1991 ) 4 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities tor at least two different antigens In the present case, one of the binding specificities is for a WISP polypeptide. the other one is for anv other antigen, and preferably for a cell-surface protein or receptor or receptor subunit Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production ot bispecific antibodies is based on the co-expression ot two immunoglobulin heavv-chain/ ght- chain pairs, where the two heavv chains have different specificities Milstein and Cuello Nature. 305 537- 539 ( 1983) Because of the random assortment of immunoglobulin heavy and light chains, these hybπdomas (quadromas ) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification ot the correct molecule is usually accomplished by affinity chromatography steps Similar irocedures are disclosed in WO 93/08829, published 13 May 1993. and in Traunecker et al . EMBO J . JO 3655-3659 ( 1991 ) Antibody variable domains with the desired binding specιficιtιes(antιbody-antιgencombιnιng sites) can be fused to immunoglobulin constant-domain sequences The fusion preferably is with an immunoglobulin heavy-chain constant domain, compπsingat least part of the hinge, CH2. and CH3 regions It is preferred to have the first heavv . ham constant region (CH I ) containing the site necessary for light-chain binding present in at least one ot the fusions DNAs encoding the immunoglobulin heavv-chain fusions and. if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism For further details of generating bispecific antibodies see, for example, Suresh et al , Methods in Enzvmology. 121 210 ( 1986) 5 Heteroconiugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalentlv joined antibodies Such antibodies have, tor example, been proposed to target immune system cells to unwanted cells (U S Patent No 4,676.980), and for treatment of HIV infection WO 91/00360. WO 92/200373, EP 03089 It is contemplated that the antibodies mav be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross- linking agents For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioetherbond Examples of suitable reagents for this purpose include ιmιnothιolateand methvl-4- mercaptobutyπmidate and those disclosed, for example, in U S Patent No 4.676.980 G. Uses tor anti-WISP Polv peptide Antibodies The antibodies of the invention may be used as affinity purification agents. In this process, the antibodies are immobilized on a solid phase such a SEPHADEX ' M resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the WISP polypeptide (or fragment thereof) to be purified, and thereafterthe support is washed with a suitable solvent that will remove substantially all the material in the sample except the WISP protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer. pH 5.0. that will release the WISP polypeptide from the antibody. Anti-WISP polypeptide antibodies may also be useful in diagnostic assays for WISP polypeptide. g., detecting its expression in specific cells, tissues, or serum. Thus, the antibodies may be used in the diagnosis of human malignancies ( see, for example, U.S. Pat. No. 5.183.884). For diagnostic applications, the antibody typically will be labeled with a detectable moiety. Numerous labels are available which can be preferably grouped into the following categories: (a) Radioisotopes. such as •3:> S. 1 C. '- 5 I, 3 H. and 13 1 I The antibody can be labeled with the radioisotopeusing the techniques described in Current Protocols in Immunology. Volumes I and 2. Coligen et al.. Ed.. (Wiley-interscience: New York. 1991 ), for example, and radioactivity can be measured using scintillation counting. (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescem and its derivatives, rhodamine and its derivatives, dansyl. Lissamine. phycoerythπn, and Texas Red are available. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra. Coligen, ed.. for example. Fluorescence can be quantified using a fluoπmeter. (c) Various enzyme-substrate labels are available, and U.S. Patent No. 4,275.149 provides a review of some of these. The enzyme preferably catalyzesa chemical alteration of the chromogenic substrate which can be measured using vaπcu.-. techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometπcally. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiiuminescentsubstrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase. urease, peroxidase such as horseradish peroxidase (HRPO). alkaline phosphatase, β- galactosidase. giucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, gaiactose oxidase, and glucose-6-phosphate dehydrogenase), heterocychc oxidases (such as uncase and xanthine oxidase), lactoperoxidase, microperoxidase. and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et a!.. Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassav, in Methods in Enzvm.. Vol. 73. Langone and Van Vunakis. eds. (New York: Academic Press, 1981 ), pp. 147-166. Examples of enzyme-substrate combinations include: (I) Horseradish peroxidase (HRPO) with hvdrogen peroxidase as a substrate wherein the hvdrogen peroxidase oxidizes a dve precursor .e , orthophenvlene diamine (OPD) or 3 3', 5 5'-tetramethvl benzidine hvdrochloπde (TMB)), (n) alkaline phosphatase (AP) with para-nitrophenvl phosphate as chromogemc substrate, and (in) β-D-galactosidase (β-D-Gal) with a chromogemc substrate (e g , p-mtrophenvl-β-D- galactosidase) or fluorogenic substrate (4-methvlumbellιfervl-β-D-galactosιdase) Numerous other enzvme-substrate combinations are available to those skilled in the art For a general review of these, see. for example. U S Patent Nos 4 275,149 and 4,318,980 Sometimes, the label is indirectly conjugated with the antibody The skilled artisan will be aware of various techniques for achieving this For example, the antibody can be conjugated with biotin and anv of the three broad categories of labels mentioned above can be conjugated with avidm, or vice versa Biotin binds selectively to avidm, and thus, the label can be conjugated with the antibody in this indirect manner Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e g dιgoxιn)and one of the differenttvpes of labels mentioned above is conjugated with an anti-hapten antibodv (e g anti-digoxin antibodv) Thus, indirect conjugation of the label with the antibodv can be achieved In another embodiment of the invention the anti-WISP polypeptide antibodv need not be labeled and the presence thereof can be detected using a labeled antibody which binds to the anti-WISP polypeptide antibody The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques (New York CRC Press, lnc , 1987), pp 147-158 Competitive binding assavs rely on the ability of a labeled standard to compete with the test sample analyte for binding . ith a limited amount of antibody The amount of WISP protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound Sandwich assavs involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected In a sandwich assay, the test sample analvte is bound by a first antibody which is immobilizedon a solid support, and thereafter a second antibody binds to the analyte. thus formingan insolublethree-partcomplex See, e g , U S Pat No 4,376, 1 10 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moietv is an enzvme For lmmunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example The antibodies mav also be used for in vivo diagnostic assavs Preferablv, the antibodv is labeled wιth a radιonuclιde(such as ' "in 99 Tc 1 C l j 'l '-- 3 H 3 -P or -^S) so that the tumor can be localized using immunoscintiography Addιtιonally,antι-WISP polypeptide antibodies mav be useful as antagonists to WISP polypeptide functions where WISP polypeptide is upregulated in cancer cells or stimulates their proliferation or is upregulated in atherosclerotic tissue Hence, for example, the anti-WISP polypeptide antibodies may by themselves or with a chemotherapeuticagent or other cancer treatment or drug such as antι-HER-2 antibodies be effective in treating certain forms ot cancer such as breast cancer, colon cancer, lung cancer, and melanoma Further uses for the antibodies include inhibiting the binding of a WISP polypeptide to its receptor, if applicable, or to an IGF, if applicable For therapeutic use. the antibodies can be used in the formulations, schedules, routes, and doses indicated above under uses for the WISP polypeptides In addition. anti-WISP polypeptide antibody may be administered into the lymph as well as the blood stream As a matter of convenience, the anti-WISP antibody of the present invention can be provided as an article of manufacture such as a kit An article of manufacture containing a WISP polypeptide or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers mav be formed from a variety of materials such as glass or plastic The container holds a composition that is effective for diagnosing or treating the condition and may have a sterile access port (for example, the container i"ay be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermic irjection needle) The active agent in the composition is the WISP polypeptide or an agonist or antagonistthereto The label on. or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice The article of manufacture may further comprise a second container comprising a pharmaceuticAy-acceptable buffer, such as phosphate-buffered saline. Ringer's solution, and dext' . se solution It mav further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use The article ot manufacture may also comprise a second or third container with another active agent as desc-.bed above A kit format generally is a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic or treatment assay If the active agent is an antibody that is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e g , a substrate precursor which provides the detectable chromophore or fluorophore) In addition, other additives may be included such as stabilizers, buffers (e g , a block buffer or lysis buffer), and the like The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially maximize the sensitivity of the assay Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way All patent and literature references cited in the present specification are hereby incorporated bv reference in their entirety EXAMPLES Commerciallvavailable reagents referred to in the examples were used according to manufacturer s instructionsunless otherwise indicated The source of those cells identified in the following examples, and throughoutthe specification bv ATCC accession numbers is the American Type Culture Collection, 10801 University Blvd Manassas. Virginia EXAMPLE 1 Isolation of cDNA Clones Encoding Mouse WISP- 1 Several putative WISP genes have been identified at the mRNA level in a high-throughput PCR- select cDN A substraction experiment carried out using a mouse mammary cell line (C57MG), which has been transformed by a Wnt- 1 retroviral vector and compared with the parental cell line The WISP family disclosed herein, including the mouse WISP-1 gene, was induced only m the transformed cell line C57MGWnt- l 1 Suppression Subtractive Hybridization Mouse WISP-1 was isolated independently by Wnt- 1 differential screening using suppression subtractive hvbπdization (SSH), as described bv Diatchenko et al , Proc Natl Acad Sci USA. 93 6025- 6030 (1996) SSH was carried out using the PCR-SELECT®cDNA Subtraction Kit (Clontech Laboratories, Inc ) according to the manufacturer s protocol Driver double-stranded (ds) cDNA was synthesized from 2 microgramsof polyA+ RNA isolated from a mouse mammary cell line (C57MG), obtainable t r om a mouse breast cancermyoepithelialcell line This cell line is described in Brown et al , CelL 46 1001 1009 (1986), Olson and Papkoff, Cell Growth and Differentiation. 5 197-206 ( 1994), Wong et al , Mol Cell Biol , 14 6278-6286 ( 1994), and Jue et al , Mol Cell Biol . 12 321-328 (1992), and is responsive to Wnt- 1 but not to Wnt-4 Tester ds cDNA was synthesized from 2 microgramsof polyA+ RNA isolated from a transformed version . f C57MG, called C57MG/wnt- l The C57MG/wnt- l mouse mammarv derivative cell line was prepared by first transforming the parent line with a Wnt- 1 retroviral vector. pBabe Puro (5 1 kb) This vector has a 5' LTR. packaging elements, a multiple cloning site, the puromycin-resistancegene driven off the SV40 promoter, a 3' LTR, and the bacterial elements for replication and ampicillin selection The vector was modified slightly for Wnt- 1 cloning by removing the Hmdlll site after the SV40 promoter and adding a HindUl site to the multiple clonmg site Wnt- 1 is cloned from £coRI-///«dIII in the multiple cloning site Figure 13 shows a map of the vector The transformed derivative cells were grown up in a conventional fashion, and the final cell population was selected in DMEM + 10% FCS with 2 5 μg ml puromycin to stabilize the expression vector PCR was performed using the Clontech kit, including the cDNA synthesis primer (SEQ ID NO 40), adaptors 1 and 2 (SEQ ID NOS 41 and 42, respectιvely)and complementary sequences for the adaptors (SEQ ID NOS 43 and 44. respectively) PCR primer 1 (SEQ ID NO 45), PCR primer 2 (SEQ ID NO 46), nested PCR primer I (SEQ ID NO 47) nested PCR primer 2 (SEQ ID NO 48), control primer G3PDH5' primer (SEQ ID NO 49), and control primer G3PDH3' primer (SEQ ID NO 50), shown in Figure 14 Products generated from the secondary PCR reaction were inserted into the cloning site region of pGEM-T vector (Promega), shown in Figure 15 (SEQ ID NOS.51 and 52 for 5' and 3' sequences, respectively) Plasmid DNAs were prepared using the WIZARD MINIPREP™ Kit (Promega) DNA sequencing of the subcloned PCR fragments was performed manually by the chain termination reaction (SEQUENASE2.0™ Kit, Pharmacia). Nucleic acid homology searches were performed using the BLAST program noted above. A total of 1384 clones were sequenced out of greater than 5000 found. A total of 1996 DNA templates were prepared. A program was used to trim the vector off. and a different program used to cluster the clones into two or more identical clones or with an overlap of 50 bases the same. Then a BLAST was performed of a representative clone from the cluster. Primers were designed for RT-PCR to see if the clones were differentially expressed. 2 Semi-quantitative RT-PCR One of the clones was clone 568 having 71 bp, which was identified as encoding mouse WISP-1 There were six clones in this cluster The nucleotide sequence and putative amino acid sequence of full- length mouse WISP- 1 are shown in Figure I (SEQ ID NOS 9 and 12, respectively) RT-PCR primers were designed for confirmingdifferential expression, screeningfor full-length mouse clone, and screening for the human clone. These primers were 568. PCR.top l (nucleotides 909-932 of the full-length nucleotide sequence encoding mouse WISP- 1 (SEQ ID NO.9) of Figure 1 ) and 568 PCR.botl (nucleotides 955-978 of the full- length complementary nucleotide sequence encoding mouse WISP- 1 (SEQ ID NO lϋ i of Figure I ), which are as follows- 568.PCR.top l : 5'-CCAGCCAGAGGAGGCCACGAAC (SEQ ID NO: 100) 568.PCR.bot I 3'-TGTGCGTGGATGGCTGGGTTCATG (SEQ ID NO: 101 ) For the RT-PCR procedure, cell lines were grown to subconfluence before extracting the RNA. T; .al RNA was extracted using Stat-60™ (TEL-TEST™ B) per manufacturer's instruct . s. First-strand cDN A was prepared from 0.1 μg - 3 μg ot total R A with the SUPERSCRIPT™ RT kit (Gibco. BRL). PCR amplificationof 5 μl of first-strand c DNA was performed in a 50-μl PCR reaction. The above primers were used to amplify first-strand cDNA. As controls, primers corresponding to nucleotide positions 707-729 (sense, 5'-GTGGCCCATGCTCTGGCAGAGGG (SEQ ID NO: 102)) or 836-859 (sense; 5'- GACTGGAGCAAGGTCGTCCTCGCC (SEQ ID NO: 103)) and 1048-1071 (anti-sense; 5'- GCACCACCCACAAGGAAGCCATCC (SEQ ID NO: 104)) of human tnosephosphate isomerase (huTPI) (Maquate/ α/ . J. BioI Chem.. 260: 3748-3753 ( 1985): Brown et al . Mol Cell Biol . 5 1694-1706 ( 1985)) were used to amplify first-strand cDNA. For mouse tnosephosphate isomerase, primers corresponding to nucleotide posιtιons433-456 (sense, 5'-G ACG AAAGGG AAGCCGGCATC ACQSEQ ID NO 105)) or 457- 480 bp (sense, 5'-GAGA AGGTCGTGTTCGAGCAAACC (SEQ ID NO- 106)) and 577-600 bp (antisense: 5'-CTTCTCGTGTACTTCCTGTGCCTG (SEQ ID NO: 107)) or 694-717 bp (antisense, 5'- CACGTCAGCTGGCGTTGCCAGCTC (SEQ ID NO.108)) were used for amplification. Briefly, 4 μCi of ( 32 P-)CTP (3000 Ci/mmol) was added to each reaction with 2.5 U of TAKARA EX TAQ™ (Panvera. Madison, WI) and 0.2 μM of each dNTP The reactions were amplified in a 480 PCR THERMOCYCLER™ (Perkm Elmer) using the following conditions 94°C for 1 mm . 62°C for 30 sec. 72 °C for 1 mm. for 18-25 cvcles 5 ul of PCR products were electrophoresed on a 6% polvacrvlamide gel The gel was exposed to film Densitometrv measurements were obtained using ALPHA EASE VERSION 3 3a™ software (Alpha Innotech Corporation) to quantitate the WISP- or TPI-specific gene products 3 Northern Blot Analysis Adult multiple-tissue Northern blots (Clontech) and the Northern blot of the C57MG parent and C57MG/Wnt- 1 derivative polyA+RNA (2 μg/lane) were hybridized with a 70-bp mouse WISP-1 probe (amino acids 278 through 300 of Fig 1 , QPEEATNFTLAGCVSTRTYRPKY. SEQ ID NO 109) generated using the primers 568 PCR top 1 and 568 per bot 1 noted above The membranes were washed in 0 1 X SSC at 55-65 °C and exposed for autoradiography Blots were rehybπdizedwith a 75-bp synthetic probe from the human actin gene See Godowski et al , Proc Natl Acad Sci USA. 86 8083-8087 ( 1989) for a method for making a probe with overlapping oligos, which is how the actm probe was prepared 4 cDNA Library Screening Clones encodmgthe full-length mouse WISP-1 were isolated by screening a λgtlO oligodT primed mouse embryo library (Clontech) with the primers 568 PCR top l and 568 PCR bot 1 noted above The inserts of 13 of these clones were subcloned into pBLUESCRIPT™ IISK+ and their DNA sequences determined by dideoxy DNA sequencing on both strands 5 Results The recently described technique of SSH combines a high subtraction efficiency with an equalized representation of differentially expressed sequences This method is baser' on specific PCR reactions that permit exponential amplification of cDNAs which differ in abundance, whereas amplification of sequences of identical abundance in two populations is suppressed. The SSH technique was used herein to isolate genes expressed in a mouse mammary myoepithelial cell transformed with Wnt- l whose expression is reduced or absent in the parental myoepithelial cell The polyA + RNA extracted from both types of cells was used to synthesize tester and driver cDNAs The degree of subtraction efficiency wa- monitored by Southern blot analysis ofunsubtractedand subtracted PCR products using a β-actm probe No β-actin mRNA was apparent in the subtracted PCR products, confirming the efficiency of the subtraction The subtracted cDN A library was subcloned into a pGEM-T vector for further analysis A random sample of 1996 clones was sequenced from the transformed colonies obtained To determine if the clones obtained were differentially expressed, PCR primers were designed for selected clones and sem l-quantitative RT-PCR and Northern analyses were performed using mRNA from the mouse mammary cell line and its derivative It was found that expression of Wnt-1 in C57MG cells leads to elongated cell morphology and loss of contact inhibition One clone (m568 19A) of those that fulfilled the criteria for differential expression was found to encode full-length mouse WISP- 1 By both RT-PCR analysis and Northern analysis, it was found that this clone provided an about three-fold induction in the Wnt- 1 cell line over the parent cell line The cDNA sequence of this clone and deduced amino acid sequence of full-length mouse WISP-1 are shown in Figure 1 (SEQ ID NOS 9 and 12. respectively) The sequence alignment of human and mouse WISP-1 (SEQ ID NOS 4 and 12. respectively) is shown in Figure 8 In-situ analysis of the clone is presented below, along with thymidine incorporation assay and angiostatic assay results This clone was placed in pRK5E an £ co//-deπved cloning vector having a human cvtomegalovirus intermediate early gene promoter, an SV40 origin and polvA site an sp6 transcription initiation site, a human immunoglobulin splice acceptor and XhollNotl cD A cloning sites It is a progeny of pRK5D that has an added S el site Holmes et αl . Science. 253 1278-1280 (1991 ) Upon transformation into JM 109 cells, the plasmid rendered the cells ampicillin resistant Upon digestion with λbαl and Bαm l. a 1 140-bp fragment was obtained, and the mouse insert size was 1 122 base pairs, from the ATG to the stop codon, including a 3' tag of six histidines EXAMPLE 2 Isolation of a cDNA Clone Encoding Mouse WISP-2 The cDNA for mouse WISP-2 was isolated independently by Wnt-1 differential screening using the procedure described in Example 1 The initial clone isolated was 3 18 bp in length and was designated clone 1367 There were four clones in this cluster The clone was sequenced as described above and RT-PCR primers were designed as follows 1367 per top 1 nucleotides 1604- 1627 of Figure 2 3'-GGTGTGAAGACCGTCCGGTCCCGG (SEQ ID NO 1 10) and 1367 per bot 1 nucleotides 1438- 1461 of Figure 2 5'-GTGTGCCTTTCCTGATCTGAGAAC (SEQ ID NO 1 1 1 ) After RT-PCR and Northern blot procedures were carried out as described in Example 1 to confirm differential expression, a five-fold induction in the Wnt- 1 cell line "/as observed Clones encoding full-length mouse WISP-2 were isolated from RNA library 21 1 C57MG/Wnt- 1 mRNA for construction of this library was isolated from the C57MG/Wnt- 1 cell line described in Example 1 The RNA was used to generate an oligo-dT-pπmed cDNA library in the cloning vector pRK5E using reagents and protocols from Life Technologies. Gaithersburg, MD (SUPERSCRIPT PLASMID SYSTEM™) In this procedure, the double-strandedcDN A was primed with o go dT containing a Noll site, linked with blunt-to-4?a/I hemikinased adaptors cleaved with Noll, sized to greater than 1000 bp appropriately by gel electrophoresιs,and cloned in a defined orientation into the v oI//Vo/I-cleavedpRK5E vector The library was screened by colony hybridization with a probe 1367 50mer 1 of bases 1463- 1512 of Figure 2 3'-GGGACGGGCCGACCCTTCTTAAAAGACCCTTGTACTTCTCTACCTTAGTG (SEQ ID NO 1 12) The full-length mouse WISP-2 clone was obtained, designated clone 1367 3 The cDNA for mouse WISP-2, like the mouse WISP-1 molecule, encodes a novel secreted protein that belongs to the CTGF family and is the mouse homologue of SST DNA33473 of Example 4 (The alignmentofhuman and mouse WISP-2 (SEQ ID NOS 16 and 20, respectively) is shown in Figure 9 ) The mouse WISP-2 gene is 38% identical in sequence to mouse WISP-1 , disclosed in Example 1 but lacks the C-termιnal95 amino acids thought to be involved in dimeπzation and receptor binding Mouse WISP-2 was highly expressed in the lung In-situ analysis of the clone is noted below The nucleotide sequence and putative amino acid sequence of full-lengthmouse WISP-2 are shown in Figure 2 (SEQ ID NOS 17 and 20, respectively) The putative signal sequence is from am o acid positions 1 to 23 of SEQ ID 20 _>- The clone was inserted into pRK5E described above Upon transformation ot J 109 cells, the plasmid rendered the cells ampicillin resistant Upon digestion with BamHl and Notl a 1770-bp fragment was obtained, having a mouse insert of 756 bp from ATG to the stop codon EXAMPLE 3 Isolation of a cDNA Clone Encoding Human WISP-1 To isolate the full-length human clone correspondingto m568 19A (mouse WISP- 1 ) a human lung cDNA library (Clontech). treated with the SUPERSCRIPT™ kit using the pRK5E vector as described above, was screened with a 70-bp probe at low stringency (20% formamide. 1 X SSC, 55 °C wash) The probe had the sequence from nucleotides 909-978 of the full-length mouse WISP-1 nucleotide sequence of Figure 1 ( e . the sequence 5'-CCAGCCAGAGGAGGCCACGAACTTCACTCTCGCAGGCTGTGTCAGCACACGCACCTACC GACCCAAGTAC (SEQ ID NO 1 13) Only one clone was identified, hL 568 15 A The insert to this clone was subcloned into pBLUESCRIPT™ IISK+ and its DNA sequence determined by dideoxy DNA sequencmgon both strands This clone was found to be missing about 280 amino acids Hence, a new probe (hu 568 50mer 1 ) was designed from clone 15 A having the nucleotides 750-799 ot the full-length human WISP-1 nucleotide sequence shown in Figures 3 A and 3B. / e 5'-GCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACTCGG (SEQ ID NO 1 14) This probe was used to screen a human fetal kidney cDNA library (Clontech), treated with the SUPERSCRIPT' M kit using the pRK5E vector as described bove, by colony hybridization A number of clones were obtained by screening this human fetal kidney cDNA library (clones without the A or B desιgnatιon)or by screening a human fetal kidney λgtlO library (clones with the A or B designation) using the same probes described above The inserts of these clones were subcloned into pBLUESCRIPT ' IISK+ and their DNA sequences determined by dideoxy DNA sequencing on both strands Two of these clones, designated as 568 1 A and 568 4 A have their respective sequences (SEQ ID NOS 24 and 26) shown in Figures 27 and 29 These clones are missing the von Willebrand C l domain, the variable domain, and the thrombospondin 1 domain, and have a frameshift Other clones designated as 5b8 13, 568 39, 568 5 A, 568 6B. and 568 7 (SEQ ID NOS 23, 25, 27, 28, and 29, respectively, Figs 26, 28, and 30-32, respectively), were obtained that lack one or more domains and or short amino-acid stretches (e , an 8- ammo-acid deletion) or contain additional short ammo-acid stretches and may contain introns or alternative splice variants Two clones sharing a significant amount of sequence with the full-length clone 568 38 were identified 568 23 and 568 35 Human clone 568 38 encoded the full-length human WISP- 1 The nucleotide sequence and putative amino acid sequence for clone 568 38 are shown in Figures 3A and 3B (SEQ ID NOS 1 and 4, respectively) The aligning sequences of clones 568 38 and 568 35 differ from the correspondingalignmg sequences of clones 568 15A and 568 23 in that the respective sequences of the latter two clones have an isoleucine residue at amino acid position 184 of Figs 3 A and 3B. whereas the respective corresponding sequences of clones 568 38 and 568 35 have a valine residue at this position Further, the aligning sequences of clones 568 35 and 568 38 differ from each other in that the sequence of done 568 35 has a serine residue at am o acid position 202 of Figs 3 A and 3B whereas the corresponding sequence ot clone 568 38 has an alanine residue at this position The human WISP- 1 polypeptide. by homology searching, is also found to be a member ot the CTGF family The clone was placed in a pRK5E plasmid as described above and deposited with the ATCC Upon transformation into JM 109 cells, the plasmid rendered the cells ampicillin resistant Digestion with Clal and £coRV yielded a 1435-bp fragment with an insert size of 1 104 basepairs from ATG to the stop codon In situ hybridization of human WISP- 1 was performed, with the results given below Northern analysis ot human WISP- 1 showed high expression in adult heart tissue and ovary tissue, and in fetal kidnev tissue Also presented below are thymidine incorporation assay, gene amplification assav. and angiostatic assay results The chromosomal location of the human WISP genes was determined by radiation hybrid mapping using the Stanford G3™ and the MIT Genebπdge 4 Radiation HybrW panels WISP- 1 resides at approximately 3 48 cR from the meiotic marker AFM259xc5 (LOD score 16 31) on the Genebπdge map This places WISP-1 ιn band 8q24 1 to 8q24 3. roughly four megabases distal to c-/wvc located at chromosome band 8q24 12-8q24 13 Takahashie/ al . Cvtogenet Cell Genet . 57 109- 1 1 1 (1991 ) c-m\c is a region that is a recurrent site ot amplification in non-small cell lung carcinoma EXAMPLE 4 Isolation of a cDNA Clone Encoding Human PRQ261 (designated herein as human WISP-2) The extracellulardomaιn (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the SWISS-PROT™ pi'blic protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ™ Incyte Pharmaceuticals, Palo Alto, CA) The search was performed using the computerprogram BLAST or BLAST2 (Altshule/ α/ . Methods in Enzvmology 266 460- 480 ( 1996)) as a comparison of the ECD protein sequences to a 6-frame translation of the EST sequence Those comparisons resulting in a BLAST score of 70 (oi ,ι some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program 'phrap ' (Phil Green. University of Washington. Seattle. Washington http.//bozeman mbt Washington edu/phrap docs/phrap html) A consensus DNA sequence was assembled relative to other EST sequences using phrap The EST sequences used (from Incyte) were Nos 2633736.21 18874, 360014.2316216, 1985573,2599326, 1544634, 2659601 , 1319684, 783649, 627240, 1962606. 2369125, 939761. 1666205, 69291 1 , 984510, 1985843 2104709, and 2120142 This consensus sequence is herein designated DN A30843 (see Fig 5) Based on the DNA30843 consensus sequence, oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0261 (human WISP-2) A pair of PCR primers, forward and reverse, were synthesized having the respective sequences 5'-AAAGGTGCGTACCCAGCTGTGCC (SEQ ID NO 1 15) and 3'-TCCAGTCGGCAGAAGCGGTTCTGG (SEQ ID NO 1 16) Additionally, a synthetic oligonucleotidehybπdization probe was constructed from the consensus DNA30843 sequence, which probe has the sequence 5'-CCTGGTGCTGGATGGCTGTGGCTGCTGCCGGGTATGTGCACGGCGGCTGGG (SEQ ID NO 117) For screening several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience. N Y . 1989). ith the PCR primer pair identified above A positive library was then screened by colony hybridization to isolate clones encoding PR0261 (human WISP- 2) using the probe oligonucleotide and one of the PCR primers RNA for construction of the cD A libraries was isolated from human fetal lung tissue The cDNA libraries used to isolate the cDN A clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDN A was primed with oligo dT containing a Notl site, linked with blunt-to-Sα/I-hemιkιnased adaptors, cleaved with Notl. sized appropriately by gel electrophoresis.and cloned in a defined orientation into a suitable cloning vector (such as pRK5B or pRK5D, pRK5B is a precursor of pRK5D that does not contain the Sβl site, see Holmes et al , Science, 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites DNA sequencing of the clones isolated as described above gave the DNA sequence for PR0261 (herein designatedas UNQ228(DNA33473-seqmιn), SEQ ID NO 38). which begins at nucleotide 13 of SEQ ID NO 13 (Fig 4) and the derived amino acid sequence tor PR0261 (SEQ ID NO 16) The entire nucleotide sequence encoding human WISP-2 is shown in Figure 4 (SEQ ID NO 13) This sequence contains a single open reading frame with an apparent translational initiation site at nucleotide positions 22-24 of SEQ ID NO 13 and ending at the stop codon after nucleotide 770 of SEQ ID NO 13 (Figure 4) The predicted polypeptide precursor is 250 amino acids long (Figure 4) The putative signal sequence spans from amino acid positions 1 to 23 of SEQ ID NO 16 Clone UNQ228 (DNA33473-seq mm) has been deposited with ATCC and is assigned ATCC deposit no 209391 Analysis of the ammo acid sequence of the full-length PR0261 polypeptide suggests that portions of it possess significant homology to CTGF. tr' >eby indicating that PR0261 is a novel growth factor In situ hybridization of human WISP-2 is given below The chromosomal location of the human WISP-2 gene was determined as described above for human WISP- 1 Specifically. WISP-2 is linked to the marker SHGC-33922, with a LOD scsre of 1000 This places WISP-2 in band 20q l2-20q l 3 1 Human chromosome 20ql2 is a frequent site of DNA amplification in human breast cancer In a Xenopus assay, injection of human WISP-2 RNA partially induced axis duplication (see Example 1 1 ) Also presented below are thymidine incorporation assay, gene amplification assay, and angiostatic assay results for human WISP-2 EXAMPLE 5 Isolation of cDNA Clones Encoding Human WISP-3 In this example, the gene encoding WISP-3 was cloned twice essentially in parallel First, it was determined whether the databases described above contained any new members of the WISP family Two EST homologies to the WISPs were found and both were cloned Full-length clones were isolated corresponding to each of these EST homologies The efforts resulted in two full-length clones of the same gene (the original EST homologies had been from distinct regions of the same gene) The first clone obtained was designated as DNA56350 and the second as DNA58800 DNA56350 Based on the sequence of INCYTE 3208053 a virtual DNA 48917 was obtained and o gonucleotideswere synthesized for use as probes to isolate a clone of the full-length coding sequence for PR0956 (human WISP-3) A pair of PCR primers, forward and reverse, were synthesized having the sequences 5'-GTCTTGTGCAAGCAACAAAATGGACTCC (SEQ ID NO 1 18) 3'-GACACAATGTAAGTCGGAACGCTGTCG (SEQ ID NO 1 19) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the INCYTE sequence, which probe has the sequence 5'-GCTCCAGAACATGTGGGATGGGAATATCTAACAGGGTGACCAATGAAAAQSEQ ID NO 120) A human fetal kidney library primed with oligo dT containing a Xhol-Notl size cut greater than 3700 kb was screened for a source of a full-length clone by PCR amplification with the PCR primer pair identified above The positive library was then used to isolate clones encoding PR0956 (human WISP-3) using the probe oligonucleotide and one of the PCR primers DNA sequencing of the clone isolated as described above gave the DNA sequence tor PR0956 (herein designated as UNQ462 (SEQ ID NO 30), and the derived amino acid sequence for PR0956 (SEQ ID NO 33) The entire nucleotide sequence encoding human WISP-3 from this clone is shown in Figure 6 (SEQ ID NO 30) This sequence contains a single open reading frame with an apparent translational initiation site at nucleotide positions 46-48 of SEQ ID NO 30 and ending at the stop codon after nucleotide 1 161 of SEQ ID NO 30 (Figure 6) The predicted polypeptide precursor is 372 amino acids long (Figure 6) The putative signal sequence is from am o acid positions 1 to 33 of SEQ ID NO 33 Clone UNQ462 (DNA56350- 1 176- 2) has been deposited with ATCC and is assigned ATCC deposit no 209706 Analysis of the amino acid ..quence of the full-length PR0956 polypeptide suggests that portions of it possess significant homology to CTGF. thereby indicating that PR0956 is a novel growth factor This clone has an additional methionine lust 5' of the first methionine in this clone The amino acid sequence of this clone is 42% homologous to that of human WISP- 1, and 32% homologous to that of human WISP-2 In situ hybridization of human WISP-3 is shown below Using the mapping techniques set forth above, it was found that human WISP-3 was localized to chromosome 6q22-6q23 and was linked to the marker A FM21 lze5 with a LOD score of 1000 WISP-3 is approximately 18 megabases proximal to CTGF and 23 megabases promimal to the human cellular oncogene MYB, which are also located at 6q22-6q23 Martineπe et αl , Oncogene. 7 2529-2534 ( 1992), Meese et αl , Genes Chromosomes Cancer, 1 88-94 ( 1989) The clone was inserted into pRK5E. described above Upon transformation of JM 109 cells, the plasmid rendered the cells ampicillin resistant Upon digestion with BαmYU and Notl. a fragment was obtained having a human insert from ATG to the stop codon as indicated in Figure 6 DNA58800 Based on the sequence ot HS 142L7, a virtual DNA 56506 was obtained and oligonucieotides were synthesized for use as probes to isolate a clone ot the full-length coding sequence for PRO790 (human WISP- 3) To this end. a pair of PCR primers, forward and reverse, were synthesized having the sequences 5'-CCTGGAGTGAGCCTGGTGAGAGA (SEQ ID NO 121 ) 3'-ACACTGGGTGTGTTTCCCGACATAACA (SEQ ID NO 122) Additionally. a synthetic oligonucleotidehvbπdization probe was constructed from the HS142L7 sequence, which probe has the sequence S'-TGGTTGCTTGGCACAGATTTTACAGCATCCACAGCCATCTCTCA (SEQ ID NO 123) A human bone marrow library primed with oligo dT containing a Xhol-Notl size cut of 1 -3 kb was screened for a source of a full-length clone by PCR amplification with the PCR primer pair identified above The positive library was then used to isolate clones encoding PRO790 (human WISP-3) using the probe oligonucleotide and one of the PCR primers DNA sequencingof the clone isolated as described above gave the DNA sequence for PRO790 (SEQ ID NO 34). and the derived amino acid sequence tor PRO790 (SEQ ID NO 37) The entire nucleotide sequence encoding human WISP-3 from this clone is shown Figure 7 (SEQ ID NO 34) This sequence contains a single open reading frame with an apparent translational initiation site at nucleotide positions 16- 18 of SEQ ID NO 34 and ending at the stop codon after nucleotide 1077 of SEQ ID NO 34 (Figure / ) The predicted polypeptide precursor is 355 amino acids long (Figure 7) The putative signal sequence spans from amino acid positions 1 to 15 of SEQ ID NO 37 This clone DNA58800-1 176-2 has been deposited with ATCC and is assigned ATCC deposit no 209707 Analysis of the amino acid sequence of the full-length PRO790 polypeptide suggests that portions of it possess significant homology to CTGF. thereby indicating that, like PR0956 which is a splice variant thereof, PRO790 is a novel growth factor In situ hybridization of human WISP-3 is shown below The clone was inserted into pRK5E. described above Upon transformation of JM 109 cells, the plasmid rendered the cells ampicil n resistant Upon digestion with BamHl and Notl, a fragment w as obtained having a human insert from ATG to the stop codon as indicated in Figure 7 EXAMPLE 6 Use of WISP-Encoding DNA as a Hybridization Probe The following method describes use of a nucleotide sequence encoding a WISP polypeptide as a hybridization probe DNA comprising the coding sequence of full-length or mature human WISP-1 (as shown in Figures 3A and 3B, SEQ ID NOS 4 or 3, respectively), or full-length or mature mouse WISP- 1 (as shown in Figure 1 , SEQ ID NOS 12 or 1 1, respectively), or of full-length or putative mature human WISP-2 (as shown in Fig 4, SEQ ID NOS 16 or 15, respectively), or full-length or putative mature mouse WISP-2 (as shown in Figure 2, SEQ ID NOS 20 or 19, respectively) is employed as a probe to screen for homologous DNAs (such as those encoding naturally occurring variants of these particular WISP proteins in human tissue cDNA libraries or human tissue genomic libraries Hybridization and washing of filters containing either library DNAs is performed under the following high-stπngencyconditions Hvbπdizationof radiolabeled WISP-polypeptide-deπved probe (such as UNQ228 (DN A33473-seqmιn)-deπved probe) to the filters is performed in a solution of 50% formamide. Sx SSC. O 1% SDS. 0 1% sodium pyrophosphate.50 mM sodium phosphate. pH 6 8 2x Denhardt's solution. and 10% dextran sulfate at 42°C for 20 hours Washing of the filters is performed in an aqueous solution of 0 Ix SSC and 0 1 % SDS at 42°C DNAs having a desired sequence identity with the DNA encoding a full-length, native-sequence WISP polypeptide can then be identified using standard techniques known in the art EXAMPLE 7 Expression of WISP Polvpeptide in £ coli This example illustrates preparation of an unglycosvlatedform of WISP polypeptide bv recombinant expression in £ coli The DNA sequence encoding WISP polypeptide is initially amplified using selected PCR primers The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector A variety of expression vectors may be employed An example of a suitable vector is pBR322 (derived from £ coli. see Bolivar et al . Gene, 2 95 ( 1977)) which contains genes for ampicillin and tetracyclineresistance The vector is digested with restriction enzyme and dephosphorv'ated The PCR-ampIifiedsequencesare then ligated into the vector The vector will preferably include sequences which encode an antibiotic-resistance gene, a tip promoter, a polyhis leader (including the first six STII codons, polyn-s sequence, and enterokinase cleavage site), the WISP-coding region, lambda transcπpti nal terminator, and an argil gene The ligation mixture is then used to transform a selected £ coli strain using the methods described in Sambrook et al . supra Transformantsare identified by their ability to grow on LB plates, and antibiotic- resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antib'otics The overnight culture mav subsequently be used to inoculate a larger- scale cultu r e The cells are then grown to a desired optical density, during which the expression promoter is turned o.i After the cells are cultured for several more hours, the cells can be harvested by centnfugation The cell pellet obtained by the centnfugation can be solubi zed using various agents known in the art, and the WISP polypeptide can then be purified using a metal-chelating column under conditions that allow tight binding of the protein EXAMPLE 8 Expression of WISP Polvpeptide in Mammalian Cells This example illustrates preparation of a potentially glycosylated form of WISP polypeptide by recombinant expression in mammalian cells The vector, pRK5E. was employed as the expression vector The appropriate DNA encoding WISP polypeptide was ligated into pRK5E with selected restriction enzymes to allow insertion of the DNA for WISP polypeptide using ligation methods as described in Sambrook et al , supra The resulting vectors were pRK5E h WIG- 1 568 38, pRK5E m WIG-1 568 6hιs. pRK5E WIG-2 1367 3, plasmid encoding human WISP-2. DNA56350- 1 176-2 and DNA58800- 1 176-2. all deposited at the ATCC These vectors are conveniently referred to collectively as pRK5E WISP in the general description below In one embodiment, the selected host cells mav be 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplementedwith fetal calf serum and optionally nutrient components and/or antibiotics About 10 μg pRK5E WISP DNA is mixed with about 1 μg DNA encoding the VA RNA gene (Thimmappaya et al , Cell, H 543 (1982)) and dissolved in 500 μl of 1 mM Tπs-HCl. 0 1 mM EDTA. 0 227 M CaCl 2 To this mixture is added, dropwise. 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl 1 5 mM NaPO^, and a precipitate is allowed to form for 10 minutes at 25°C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C The culture medium is aspirated off and 2 ml of 20% glycerol in phosphate-buffered saline (PBS) is added for 30 seconds The 293 cells are then washed with serum-free medium, fresh medium is added, and the cells are incubated for about 5 days Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml and 200 μO/mr 5 S- methionine After a 12-hour incubation the conditioned medium is collected, concentrated on a spin filter and loaded onto a 15% SDS gel The processed gel mav be dried and exposed to film for a selected period of time to reveal the presence of the WISP polypeptide The cultures containing transfected cells may undergo further incubation (in serum-free medium) and the medium is tested in selected bioassays In an alternative technique, the WISP polypeptide may be introduced into 293 cells transiently using the dextran sulfate method described bv Somparvrac e/ α/ . Proc Natl Acad Sci . 12 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5E WISP DNA is added The cells are first concentrated from the spinner flask by centnfugation and washed with PBS The DNA-dextran precipitate is incubated on the cell pellet for four hours The cells are treated with 20% glyceroi for 90 seconds, washed with tissue 4.ulture medium, and re-introduced into the spinner flask containing tissue culture mec .. m, 5 μg/ml bovine insulin, and 0 1 μg/ml bovine transfemn After about tour davs, the conditioned media are centπfugedand filtered to remove cells and debris The sample containing expressed WISP polypeptide can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography In another embodiment, the WISP polypeptide can be expressed in CHO cells The pRK5E WISP can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3 :> S-methιonιne After determining the presence of the WISP polypeptide, the culture medium may be replaced with serum-free medium Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested The medium containing the expressed WISP polypeptide can then be concentrated and purified by any selected method Epitope-tagged WISP polypeptide may also be expressed in host CHO cells The WISP polypeptide may be subcloned out of the pRK5 vector Suva et al , Science. 237 893-896 ( 1987), EP 307,247 published 3/15/89 The subclone insert can undergo PCR to fuse in-frame with a selected epitope tag such as a poly-his tag into a baculovirus expression vector The poly-his-taggedWISP polypeptide insert can then be subcloned into a SV40-dπven vector containing a selection marker such as DHFR for selection of stable clones Finally the CHO cells can be transfected (as described above) with the SV40-dπven vector Labeling mav be performed, as described above, to verify expression The culture medium containing the expressed poly-His- tagged WISP can then be concentratedand purified by any selected method, such as by Nr + -chelate affinity chromatography In particular, mouse WISP- 1 cDNA for insertion into mammalian expression vectors was created via PCR using clone m568 19A (see above) pure phage DNA as template and using as primers m 568 per top4 (5'-TGACTTCCAGGCATGAGGTGGCTCCTG, SEQ ID NO 124) and m 568 per bot3 (5'- ATTGGCAATCTCTTCGAAGTCAGGGTAAGATTCC, SEQ ID NO 125) for the 6-hιs version, or m 568 pcr top4 (SEQ ID NO 124) and 568 pcr bot5, which has a 3'-termιnal Xba site (5'- GGTACGTCTAGACTAATTGGCAATCTCTTCGAAGTCAGGG. SEQIDNO 126)forthenon-hιsversιon The insert integrity was confirmed by sequencing and analyzed The PCR was run using Pfit polymerase and the conditions were temp time denaturation 94°C 1 mm annealing 62°C 30 sec extension 72°C 1 5 mm # of cycles 25 For transient expression in 293 cells analyzed by Western blot, the above inserts were ligated into the pRK5 vector referred to above at the Bam l/Xbal site using the BOEHRINGER iv' ANNHEIM™ rapid ligationkit The resulting plasmids were designated pRK5 mu WISP-1 6hιsand pRK5 mu WISP-1 nohis for the His-taggedand non-His-tagged inserts, respectively Then the 293 cells were plated and allowed to reach approximately 85% confluency overnight (37°C/5% C0 2 ) The plated cells were transfected with plasmid DNA pRK5 mu WISP-1 όhis or pRK5 mu WISP- 1 nohis by using lipofectamine (Gibco BRL) at a 4 5 1 iid DNA ratio Transfection efficiency (>70% usually) was monitored using a GFP expression plasmid (pGREEN LANTERN™ from Gibco BRL) Approximately 5 hours post-transfection. the medium was changed to fresh SF media (50 50 with IX L-Glu and IX P/S) for protein production Tro conditioned media was allowed to accumulate for specified amounts of time (depending on the experiment) before harvesting The medium was harvested and concentrated in the presence of 1 mM PMSF using the CENTRICON- 10™ concentrator The concentrated, conditioned media (usually 1 5 ml) was then bound to Nι - NTA agarose beads (Qiagen) for 2 hours (for the His-tagged version only) Protein was eluted from the beads by boiling for 10 minutes in 2X SDS loading buffer (Novex) with or without beta-mercaptoethanol for reduced vs non-reduced protein, respectively The protein was visualized by SDS-PAGE using 4-20% gradient TRIS-glycinegels, 10-wells. 1 mm thickness (Novex) Gels ran at 125 volts (constant) for 95 minutes Western transfer was achieved using a NOVEX™ transfer apparatus to PVDF membranes (Novex) at 200 mA (constant) for 45 minutes The blots were blocked for a minimum of 1 hour at room temperature in blocking buffer (PBS + TWEEN-20™ (0 5%), 5% dry milk, and 3% goat serum) Blots were incubated in primary antibody (for His-tagged protein INVITROGEN™ antι-hιs(C-termιnai)-HRP-conjugatecantιbodv or for the non-His version polvclonal anti- murine WISP- 1 antibodv prepared as described below) at a 1 2000 dilution in fresh blocking buffer for 1 hour at room temperature The non-His-tagged protein blots were incubated in secondary antibodv (PIERCE rr ' goat anti-rabbit IgG(H+L) HRP conjugated) diluted 1 2000 in fresh blocking buffer The blots were incubated in chemiluminescentsubstrate (ECL™ from Amersham or SUPERSIGNAL™ from Pierce) for 1 minute before exposing to film For transient expression analyzed by antibodv staining. 293 cells were cultured plated, and transfected as described above The cells were fixed to culture dishes for 2 minutes in 1 1 methanol acetone solution Fixed cells were then incubated in pπmarv antibodv (for His-tagged protein INVITROGEN™ antι-hιs(C-term)-HRP-conjugated antibody or for the non-His version polyclonal anti-muπne WISP-1 antibody prepared as described below) diluted 1 1000 in PBS with 10% fetal bovine serum for 2 hours The non-His-tagged protein blots were then incubated in secondary antibody (PIERCE™ goat anti-rabbit IgG(H+L) HRP conjugated) diluted 1 150 in PBS with 10% fetal bovine serum for 1 hour The incubation was in color reagent substrate for HRP for up to 1 hour ( 1 0% O-dianisidine-saturated ETOH, 0 01% hvdrogen peroxide in PBS) For stable expression of mouse WISP- 1 in mammalian cells, the starting vector employed was pRK5 CMV puro-dhfR. the sequence of which is shown in Figures 16A- 16D This vector has two SAR sequences cloned into kpnl. Sap sites of the SVID5 splice-donor vector, and has the pSVI backbone with the pRK5 cloning linker (pSVI5) and the intron made from pSVI WTSD D by adding a linearization linker (LL) into the Hpal site The sequence is edited to include changes in vector pucl 18 backbone derived from the sequence of pRK5 and includes a four-base insertion after MCS characteristic of the SVI vector The above inserts were ligated into pRK5 CMV puro-dhfR at the Bam l/Xbal site using the BOEHRINGER MANNHEIM™ rapid ligation kit. producing pRK5 CMV puro-dhfR mu WISP- 1 6hιs or pRK5 CMV puro-dhfR mu WISP- 1 nohis This constructallows for stable selection ofexpressingcells using either puromycin (2 μg/ml in 293 cells or 10 μg ml in CHO-DP12 cells) or the " ^HFR deletion in the CHO- DP12 line, which allows for subsequent amplification in methotrexate Isolated colonies representative of stably transfected cells were picked, cultured under selective pressure, and analyzed by antibodv staining or Western blot as described above EXAMPLE 9 Expression of WISP Polvpeptide in Yeast The following method describes recombinant expression of a WISP polypeptide in yeast First, yeast expression vectors are constructed for intracellular production or secretion of a WISP polypeptide from the ADH2/GAPDH promoter DNA encoding a WISP polypeptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression For secretion, DNA encoding a WISP polypeptide can be cloned into the selected plasmid, together with DNA encodmgthe ADH2/GAPDH promoter, a native WISP signal peptide or other mammalian signal peptide or yeast alpha-factor or mvertase secretory signal/leader sequence, and linker sequences (if needed) for expression Yeast cells, such as veast strain ABI 10 can then be transformed with the expression plasmids described above and cultured in selected fermentation media The transformed yeast supernatants can be analyzed bv precipitation with 10% tπchloroaceticacid and separation bv SDS-PAGE. followed bv staining of the gels with Coomassie Blue stain Recombinant WISP polypeptide can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centnfugation and then concentrating the medium using selected cartridge filters The concentrate containing the WISP polypeptide may further be purified using selected column chromatography resins EXAMPLE 10 Expression of WISP Polvpeptide in Baculovirus-lnfected nsect Cells and Purification Thereof The following method describes recombinant expression of a WISP polypeptide in baculovirus- infected insect cells, and purification thereof General The sequence coding for WISP polypeptide is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL 1393 (Novagen) Briefly, the sequence encoding WISP polypeptide or the desired portion ot the coding sequence (such as the sequence encoding the mature protein if the protein is extracellular) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vectoi Recombinant baculovirus is generated by co-transfectingthe above plasmid and BACULOGOLD™ virus DNA (Pharmingen) into Spodoptera frugφerda ("Sf9") cells (ATCC CRL 171 1 ) using pofectin (commercially available from Gibco-BRL) After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al , Baculovirus Expression Vectors \ Laboratory Manual (Oxford Oxford University Press. 1994) Expressed poly-His-tagged WISP polypeptide can then be purified, for example, bv Nr + -chelate affinity chromatography as follows Extracts are prepcied from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature. 362 175-179 ( 1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL HEPES, pH 7 9, 12 5 mM MgCl 2 , 0 1 mM EDTA. 10% glycerol, 0 1% NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centnfugation. and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate. 300 mM NaCl, 10% glycerol, pH 7 8), and filtered through a 0 45 μ filter A Ni -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water, and equilibrated with 25 mL of loading buffer The filtered cell extract is loaded onto the column at 0 5 mL per minute The column is washed to baseline A-) 8 Q with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate. 300 mM NaCl, 10% glycerol, pH 6 0), which elutes non- specifically bound protein After reaching A 280 baseline again, the column is developed with a 0 to 500 mM lmidazole gradient in the secondary wash buffer One-mL fractions are collected and analyzed by SDS- PAGE and silver staining or Western blot with Nr + -NTA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted HiS j Q -tagged WISP polypeptide are pooled and dialvzed against loading buffer Alternatively purification of the IgG-tagged (or Fc-tagged) WISP polypeptide can be performed using known chromatographv techniques, including, tor instance Protein A or protein G column chromatography Specific 1 Expression In particular, mouse WISP- 1 polypeptide was expressed in a baculovirus expression svstem similar to that described above using as the baculovirus transfer vector pb PH mu 568 9 IgG baculo or pbPH mu 568 8hιs baculo Figures 17A- 17D show the sequence (SEQ ID NO 54) of plasmid pb PH IgG, which was used to prepare pb PH mu 568 9 IgG baculo Figures I 8A- 18D show the sequence (SEQ ID NO 55) of plasmid pbPH His c, which was used to prepare pbPH mu 568 8hιs baculo Both of these baculovirus transfer vectors are based on pVL1393 (Pharmingen), which has neither the His nor Fc tags The pb PH IgG vector (Fig 17) allows the expression of foreign proteins under control of the AcNPV polyhedπn promoter which is active in the verv late phase of virus infection The foreign protein can be expressed as a C-terminal human IgG fusion protein The Hιs(8)-tag will not be translated as a result of the IgG stop codon just 5' of the Hιs(8)-tag The sequence encoding the foreign protein should be inserted as a 3' blunt-ended fragment into the unique Stul site preceeding the His-tag In that case an additional proline residue will be added The 5' site car be either BamHl £coRI, Notl. Ncol, and Nhel The IgG vector was constructed by Ndel digestion of the pVL 1393 IgG plasmid followed by Klenow treatmentto fill in the sticky end site This is followed by a Ncol digest and insertion into the pbPH His c x /vcoI/Smαl-digested vector The sequence of pbPH H is c shown in Figs 18 A- 18D contains the backbone sequence of the vector pVL1392, which contains approximately the £coRI Λ/nαIII ^gment of AcMNPV C-6, from position 0 0 to 5 7 mu Possee et al . Virology. I 85 229-241 (1991 ) It allows the expression of foreign proteins under control of the Autographacalifornica nuclear polyhedrosisvirus (AcNPV) polyhedπn gene promoter, which is active in the very late phase of virus infection The foreign protein can be expressed as a C-terminallyHis- or a IgG (Fc region on ly)-tagged protein The sequence encoding the foreign protein should be inserted as a 3'-blunt-ended fragment into the unique Smal site preceding the His-tag or the Stul site for IgG In that case an additional glycine residue will be added for His tags and a proline will be added for IgG tags The 5' site can be either βαmHI, Notl, £coRI, oτ Ncol Bam HI was used for both The vectors were constructed by inserting a PCR insert into BamHllSmal for the His vector and Bam l/Stul for the IgG vector The PCR insert was made using 5'-phosphorvlated primers as follows m 568 per top6(5'-TTTCCCTTTGGATCCTAAACCAACATGAGGTGGCTCCTGCCC. SEQ ID NO 127) and m 568 per bot3(SEQ ID NO 125), 5' phosphorvlated A twenty-cycle PCR reaction with Pfu polymerase enzyme was performed using the following conditions 1 mm at 95°C, 30 sec at 60°C, 3 5 mιn at 72°C The PCR product was purified with QIAQUICK™ and digested with BamHl at 37°C for 1 hr The digested PCR insert was ligated into the digested vector using a 1 3 ratio of insert to vector with 1 ul T4 DNA gase (Bio Labs) ULTRA MAX rM DH5a FT competent cells 100 u I, (Gibco BRL Cat 10643-013) were added to the ligation product, and the mixture was incubated on ice for 30 min followed bv a heat shock at 42°C for 45 sec Individual colonies were picked and miniscreen DNA was prepared using QIA PREP™ (Qiagen) Construct sequencing was performed using ABI Prism's dRHODAMINE DYE " V terminator cycle sequencing The plasmid pb PH IgG has a polylιnkerβαwHI- Vo/I-£coRI-ιVcoI-5/-yi-4S/«l-(IgG Fc region only)- Stop-Λ/'αl-.Sp' el- s/I-βg/II The location of particular regions in this plasmid is as follows Insertion ot poly nker/foreign gene 4129-4912 (BamHl-Bgll ). polh coding 4913-5479, ORF 1629 7134-4820, ORF 588 (PK1) 7133-7723, ColEl origin of replication 7973-8858. and ampicillin coding 9779-8230 The plasmid pbPH His c has a polylιnkerβαmHI-Vo/I-£coRI-Λ'coI-5/7l-5/nαI-(Hιs8)-Stop-Λ6αI-5/?eI-/ , 5/I-βg/II The Ncol site of pbPH His c resides within a Kozak sequence The location of particular regions in this plasmid is as follows ORF 504 (PTP) 76-582, ORF 984 (ORF2) 1600-614, ORF 453 (ORF3) 2323-1868, conotoxm 1818- 1657, ORF 327 (ORF4) 2352-2681. ORF 630 (lef-2) 2662-3294, ORF 603 3937-3332. ORF polh 4093 (mutated codon ATG/ATT), insertion of polylinker/foreigngene 4129-4218(βamHI-βg/II), polh coding 4224-4790, ORF 1629 6445-4820. ORF 588 (P 1 ) 6444-7034. ColEl origin of replication 7284-8169, and ampicillin coding 9090-8230 The mouse WISP-1 cDNA disclosed herein was inserted into the vectors pbPH His c and pb PH IgG to produce the respective expression plasmids by creating a 3' blunt-ended fragment for cloning into the unique Smal site precedmgthe His-tag or IgG-tag An additional glycine residue was added to the His protein produced An additional proline was added to the IgG protein The 5' site of the cDNA insert was Bam l 2 Purification For purification purposes, either a polv-His tag or the Fc portion of human IgG was added to the C-terminal coding region of the cDN A before expression The conditioned media from the transfected cells (0 5 to 2 L) was harvested by centnfugation to remov. .he cells and filtered through 022 micron filters For the poly-His-tagged constructs, the protein was purified using a Nι + - -NTA column (Qiagen) Before purification, lmidazole was added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6-ml Nι + --NTA column equilibrated in 20 mM HEPES, pH 7 4, buffer containing 0 3 M NaCl and 5 mM ιmιdazoleat a flow rate of4-5 ml/mιn at 4°C After loading, the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was subsequently desalted into a storage buffer containing 10 mM HEPES.0 14 M NaCl, and 4% manmtol, pH 6 8. with a 25 ml G25 SUPERFINE™ (Pharmacia) column and stored at -80 °C Immunoadhesin (Fc-containing) constructs of WISP- 1 protein were purified from the conditioned media as follows The conditioned media was pumped onto a 5-ml Protein A column (Pharmacia) which had been equilibrated in a 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elution with lOO mM citric acid, pH 3 5 The eluted protein was immediately neutralized by collecting 1-ml fractions into tubes containing 275 uL of 1 M Tπs, pH 9 buffer The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His- tagged proteins The homogeneity ot the protein was assessed by SDS polyacrylamidegels and by N-terminal amino acid sequencing by Edman degradation EXAMPLE 1 1 Axis Duplication Assay Xenopus embryos were injected with human WISP-2 mRNA into either a presumptive ventral or presumptive dorsal vegetal blastomere at the 8- to 16-cell stage to overexpress locally the encoded protein and assay for its developmental effects. The methods used are described in Sokol et al , Cell. 67 741-752 ( 1991 ) More specifically, for synthesis of capped RNA. human WISP-2 and mouse Wnt- 1 cDNAs were cloned into the pGEMHE vector (gift of Dr. Todd Evans. AECOM) to prepare pGEMHE.hu.WISP-2.8H and pGEMHE.mu.Wnt- 1 , respectively. The constructs were linearized at the 3' end using the Sph restriction enzyme. Capped RNAs were synthesized using AMBION's T7 MESSAGEMACHINE™ RNA synthesis kit. For obtaining mature oocytes, an adult female Xenopus laevis was injected with 200 I U pregnant mare serum 3 days before use. The night before the experiment, the female frog was injected with 800 LU ofhuman choπonic gonadotropin. Fresh oocytes were squeezed from temaie frogs the next morning. In vitro fertilization of oocytes was performed by mixing oocytes with minced testes from a sacrificed male frog. Fertilized eggs were dejellied with 2% cysteine (pH 7.8) for 10 minutes. Dejel ed eggs were washed once with distilled water and transferred to 0.1 x Modified Barth's Solution (MBS) (Methods in Cell Biology. Volume 36. Xenopus laevis: Practica 1 uses in Cell and Molecular Biology, Kay and Peng, Eds (New York: Academic Press, 1991)) with 5% Ficoll. Eggs were lined on injection trays which contained 0.1 x MBS with 5% Ficoll for injection. After injection, embryos were kept in 0.1 X MBS in an 18"C incubator. Embryos were staged according to Nieuwkoop and Faber. Normal Table of Xenopus laevis- (Daudin) (Amsterdam: North-Holland. 1967). For animal cap assays, embryos were injected at the 2-cell stage with 1 ng of capped RNA, and animal caps were isolated at stage 8 and cultured in 1 x MMR for another 24 hours for the RT-PCR assay. Total RNA was isolated from harvested animal caps using a RNEASY™ kit (Qiagen) RNA samples (approximately 1 μg) were reverst transcribed using random hexamer and GIBCO BRL SUPERSCRIPTII™ reverse transcπptase. The annealing temperature for the PCR reactions was 55 °C unless noted otherwise. For axis duplication assays, embryos at the 8-cell stage were injected with 1 ng capped RNA at either the dorsal or ventral vegetal blastomere and incubated in 0.1 X MBS for 72 hours. The sequences of PCR primers used in this experiment were: EF-la.U: 5'-CAGATTGGTGCTGGATATGC (SEQ ID NO: 128) EF-l a.D: 5'-ACTGCCTTGATTACTCCTAC (SEQ ID NO: 129) noggin.U: 5'-AGTTGC AG ATGTGGCTCT (SEQ ID NO: 130) noggin.D- 5'-AGTCCAAGAGTCTCAGCA (SEQ ID NO: 131 ) goosecoid.U. 5'-ACAACTGGAAGCACTGGA (SEQ ID NO: 132) goosecoid.D 5'-TCTTATTCCAGAGGAACC (SEQ ID NO: 133) cardiac-actin.U 5'-TCCCTGTACGCTTCTGGTCGTA (SEQ ID NO: 134) cardiac-actin.D. 5'-TCTCAAAGTCCAAAGCCACATA (SEQ ID NO: 135) NCAM U 5 -CACAGTTCCAGCAAATAC (SEQ ID NO 136) NCAM D 5'-GGAATCAGGCGGTACAGT (SEQ ID NO 137) It was found that human WISP-2 can partially induce axis duplication in this assay EXAMPLE 12 Thvmidine Incorporation Assav In a ( J H)-thymιdιne incorporation assay, 19 different cell lines, including RAG (renal adenocarcinoma. mouse) and NRK-49F (normal kidney fibroblasts. rat) cells, identified in Table I below, were plated in 96-well plates at 3x 10 in HGDMEM with 10% serum Twenty four hours after plating, the medium was changed to HGDMEM with 0 2% serum before adding the test proteins WISP proteins were added to a final concentration of approximately 3 6 ng/ul Serial dilutions were made in a total volume of 70 μl/well of fresh media After 18 hr incubation at 37°C. 5u Ci/ml ( 3 H)thymιdιne was added for 5 hrs Medium was aspirated and cells were removed with 1 X trypsin onto a GF/C filter using Packard's™ 1 96-well FILTERMATE 196™ The filters were dried and 40 μl of scintillation fluid was added for counting on a top count, micropiate scintillation counter (Packard) The results are shown in Table I TABLE 1 It is seen that WISP-1 and WISP-2 exhibit both stimulatory and inhibitory effects on proliferation of normal and tumor cells, depending on the cell line employed EXAMPLE 13 Preparation of Antibodies that Bind WISP Polvpeptide 1 Polvclonal Antibodies Polyclonal antisera were generated in female New Zealand White rabbits against murine WISP- 1 and human WISP-2 The antigens used were proteins fused with histidine for murine WISP- 1 and proteins fused with the Fc portion of IgG for human WISP-2 The same protocol was used for both proteins Each protein was homogenized with Freund's complete adjuvant for the primary injection and with Freund's incompleteadjuvant for all subsequent boosts For the primary immunization and the first boost, 3 3 μg per kg body weight was injected directly into the popliteal lymph nodes as described in Bennett et al , J Biol Chem . 266 23060-23067 (1991 ) and "Production of Antibodies by Inoculation into Lymph Nodes" by Morton Sigel et al in Methods in Enzvmology. Vol 93 (New York Academic Press, 1983) For all subsequent boosts, 3 3 μg per kg bodv weight was injected into subcutaneous and intramuscular sites Injections were done every 3 weeks with bleeds taken on the following two weeks 2 Monoclonal Antibodies Techniques for producing monoclonal antibodies that can specifically bind a WISP polvpeptide are known in the art and are described for instance in Goding, supra Immunogens that mav be employed include purified WISP polypeptide fusion proteins containing WISP polypeptide, and cells expressing recombinant WISP polypeptide on the cell surface Selection of the immunogen can be made by the skilled artisan without undue experimentation Mice, such as Balb/c, are immunized with the WISP immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intrapeπtoneally in an amount from 1 to 100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research Hamilton MT) and injected into the animal's hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples mav be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect antibodies to WISP polypeptide After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of a WISP polvpeptide Three to four davs later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% PEG) to a selected murine myeloma cell line such as P3X63AgU 1 available from ATCC. No CRL 1597, or x63 Ag8 653 (Kearney et al , J_ Immunology. 123 1548 ( 1979)) The fusions generate hybπdoma cells which can then be plated in 96-well tis-ue culture plates containing HAT (hypoxanthine, aminopteπn, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids The hybπdoma cells will be screened in an ELISA for reactivity against a WISP polypeptide Determination of "positive" hybπdoma cells secreting the desired monoclonal antibodies against a WISP polypeptide is within the skill in the art The positive hybπdomaceils can be injected mtrapeπtoneally into syngeneic Balb/c mic to produce ascites containing the anti-WISP polypeptide monoclonal antibodies Alternatively, the hvbπdoma cells can be grown in tissue culture flasks or roller bottles Purification of the monoclonal antibod'es produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel-exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed Specifically, for each of the human WISP- 1 antibodies, five female Balb-c mice were pre-bled and then injected via their hind foot pads with purified human WISP-1, tagged with the Fc portion of IgG and emulsified prior to injection in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) in a 1 1 ratio of WISP antigen to adjuvant The dosing schedule for the WISP-1 immunogen was as follows Iniection Date Dose/Site Dose/Animal Concentration Day 16 of 50 μl/site 100 μl/animal 6 μg/animal month 1 Day 12 50 μl/site 100 μl/animal 6 μg/animal of month 2 Dav 21 50 μl/site 100 μl/animal 6 μg/animal ot month 2 Day 28 50 μl/site 100 μl/animal 2 μg/animal of month 2 Day 4 of 50 μl/site 100 μl/animal 2 μg/animal month 3 Day 1 1 of 50 μl/site 100 μl/animal 2 μg animal month 3 Day 18 of 50 μl/site 100 μl/animal 2 μg/animal month 3 Day 25 of 50 μl/site 100 μi/animal 2 μg/animal month 3 For WISP- 1. the mice were bled on Day 10 of month 4 After the mice were bled, the monoclonal antibodies were made by harvesting their spleens and by fusion as indicated above, using as the murine myeloma cell line X63 Ag8 653 The five monoclonal antibodies generated to human WISP-1 are I0F2 2A7 gamma 2b/kappa 10 A9 2B 1 gamma 2a/kappa 8F7 1 B 1 gamma 1 /kappa 1 H 1 1 D5 gamma 1 /kappa 2G7 2H4 gamma 1 /kappa For WISP-2 monoclonal antibodies the same regimen is employed except that purified human WISP- 2 is used as immunogen in the above protocol rather than purified human WISP-1 and the dosing schedule for the WISP-2 immunogen is as follows Injection Date Dose/Site Dose/Animal Concentration Day 16 of 50 μl/site 100 μl/animal 6 μg/animal month 1 Day 21 50 μl/site 100 μl/animal 1 μg/animal of month 2 Day 28 50 μl/site 100 μl/animal 1 μg/animal of month 2 Day 4 of 50 μl/site 100 μl/animal 1 μg/animal month 3 Day 1 1 of 50 μl/site 100 μl/animal 1 μg/animal month 3 Day 18 of 50 μl/site 100 μl/animal 1 μg/animal month 3 Day 25 of 50 μl/site 100 μl/animal 1 μg/animal month 3 FXAMPLE 14 Uses of Antibodies that B nd WISP Polypeptide 1 Cell lines The established human breast tumor cells BT474 and MDA-MB-231 (which are available from ATCC) are grown in minimum essential medium (Gibco, Grand Island. NY) supplemented with 10% heat- inactivated fetal bovine serum (FBS) (HyClone. Logan, UT), sodium pyruvate. L-glutamine (2mM). non- essential amino acids, and 2x vitamin solution and maintained at 37°C in 5% C0 2 Zhang et al Invas & Metas . 1 1 204-215 ( 1991 ). Price et al Cancer Res . 50 717-721 ( 1990) 2 Antibodies Anti-WISP- 1 or antι-WISP-2 monoclonal antibodies that may be prepared as described above are harvested with PBS containing 25mM EDTA and used to immunize BALB/c mice The mice are given injections i p of 10 7 cells in 0 5 ml PBS on weeks 0, 2, 5 and 7 The mice with antisera that ιmmunoprecιpιtated 3 -P-labeled Wnt- 1 are given i p injections of a wheatgerm agglutinin-SEPHAROSE™ (WGA)- purified Wnt membrane extract on weeks 9 and 13 This is followed by an I v injection of 0 1 ml of the Wnt-1 preparation, and the splenocvtesare fused with mouse myeloma line X63-Ag8 653 Hybπdoma supernatants are screened for Wnt- 1 binding by ELISA and radioimmunoprecipitation MOPC-21 (IgG 1 ) (Cappell. Durham. NC) is used as an isotype-matched control Additionally, the antι-ErbB2 IgG j K murine monoclonal antibodies 4D5 (ATCC CRL 10463 deposited May 24, 1990) and 7C2, specific for the extracellulardomain of ErbB2. may be used with the above antibodies They are produced as described in Fendly et al Cancer Research. 50 1550- 1558 ( 1990) and WO89/06692 3 Analysis of cell cycle status and viability Cells are simultaneously examined for viability and cell cycle status by flow cytometry on a FACSTAR PLUS™ (Becton Dickinson Immunocytometry Systems USA, San Jose. CA) Breast tumor cells are harvested by washing the monolayer with PBS. incubating cells in 0 0- o trypsin and 0 53 mM EDTA (Gibco). and resuspendingthem in culture medium The cells are washed twice with PBS containing 1% FBS and the pellet is incubated for 30 minutes on ice with 50 μl of 400 μM 7-amιnoactιnomycιn D (7AAD) (Molecular Probes, Eugene, OR), a vital dye which stains all permeable cells Cells are then fixed with 1 0 ml of 0 5% paraformaldehydein PBS and simultaneouslypermeabiiizedand stained for 16 hours at 4°C with 220 μl of 10 μg ml HOECHST 33342™ dye (also a DNA binding dye) containing 5% TWEEN 20™ The data from 1 x 10 4 cells are collectedand stored using LYSYS II™ software and analyzed using PAINT-A-GATE™ software (Becton Dickinson) Darzynkiewica et al , Cytometry, 12 795-808 ( 1992), Picker et al . J Immunol , 150 1 105-1 121 (1993) The viability and percentage of cells in each stage of the cell cycle are deter ined on gated single cells using 7 AAD and Hoechst staining, respectively (Cell doublets are excluded by pulse analysis of width vs area of the Hoechst signal ) Cell numbers are determined using a hemocytometer 4 DNA synthesis (( HVThvmidine Incorporation Assav) The assay was performed exactly as described in Example 12, except that the WISP polypeptides used as test proteins were replaced by the polyclonal antibodies generated in New Zealand White rabbits against murine WISP- 1 and human W ISP-2 described in Example 13 and not all the cell lines in Example 12 were tested The results are shown in Table II TABLE II J H-Thvmιdιne Incorporation Assay Results It is seen that the polyclonal antibodies to mouse WISP- 1 and to human WISP-2 exhibited both stimulatory and inhibitory effects on proliferation of normal and tumor cells, depending on the cell line employed 5 Affinity of binding to putative receptor Radioiodinated anti-WISP- 1 and antι-WISP-2antιbodιesare prepared by the IODOGEN™ method Fracker et al Biochem Biophvs Res Comm , 80 849-857 ( 1978) Binding assays are performed using appropπatereceptor-expressιngcells (such as, for mouse anti-WISP antibodies. MLG. a mouse lung cell line available from the ATCC) cultured in 96-well tissue culture ;-'ιtes (Falcon, Becton Dickinson Labware, Lincoln Park, N J ) The cells are trypsinized and seeded in wells of 96-well plates at a density of 10 cells/well and allowed to adhere overnight The monolavers are washed with cold culture medium supplemented with 0 1% sodium azide and then incubat p d in triplicate with 100 μl of serial dilutions of '- 5 I- anti-WISP- l or WISP-2 antibodies in cold culture medium containing 0 1% sodium azide for 4 hours on ice Non-specific binding is estimated by the premcubation of each sample with a 100-fold molar excess of nonradioactive antibodies in a total volume of 100 μl Unbound radioactivity is removed by two washes with cold medium containing 0 1% sodium azide. The cell-associatedradioactivity is detected in a gamma counter after solubi zationof the cells with 150 μl of 0 1 M NaOH/well The WISP- 1 and WISP-2 binding constants (K_) and anti-WISP antibody binding affinities are determined by Scatchard analysis It is expected that the antibodies against WISP-1 and WISP-2 will affect the growth of these cells EXAMPLE 15 Further Uses of Antibodies that Bind WISP Polvpeptide 1 WISP-1 and WISP-2 This example shows that the WISP- and WISP-2 genes are amplified in the genome of certain human lung, colon, and/or breast malignant tumors and/or cell lines Amplification is associated with overexpression of the gene product, indicating that the WISP- 1 and WISP-2 proteins are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast, and other cancers A therapeutic agent mav take the form of antagonists of WISP molecules, for example, muπne-human. chimeric. humanized, or human antibodies against WISP-1 and WISP-2. such as the antibodies prepared as described above The starting material for the screen was genomic DNA isolated from a variety of cancers The DNA is quantιtatedprecιsely, g , fluorometπcaily As a negative control, DNA was isolated from the cells of ten normal healthy individuals, pooled, and used as an assay control for the gene copy in healthy individuals The 5' nuclease assay (for example, TAQMAN™) and real-time quantitative PCR (for example, ABI PRIZM 7700™ Sequence Detection System™ (Perkin Elmer, Applied Biosystems Division. Foster City. CA)), were used to find genes potentially amplified in certain cancers The results were used to determine whetherthe DNAs encoding WISP- 1 and WISP-2 are over-represented in any of the primary lung or colon cancers or cancer cell lines or breast cancer cell lines that were screened The primary lung cancers were obtained from mdiviuals with tumors of the type and stage as indicated in Table III An explanation of the abbreviations used for the designation of the primary tumors listed in Table III and the primary tumors and cell lines referred to throughout this example is given below Human lung carcinoma cell lines include A549 (SRCC768), Calu- 1 (SRCC769), Calu-6 (SRCC770), H I 57 (SRCC771 ). H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774) and SW900 (SRCC775), all available from ATCC Primary human lung tumor cells usually derive from adenocarcinomas. squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas and include, for example. SRCC724 (squamous cell carcinoma abbreviated as "SqCCa"), SRCC725 (non-small cell carcinoma, abbreviated as "NSCCa"), SRCC726 (adenocarcinoma.abbreviatedas "AdenoCa"), SRCC727 (adenocarcinoma), SRCC728 (squamous cell carcinoma), SRCC729 (adenocarcinoma). SRCC730 (adeno/squamous cell carcinoma), SRCC731 (adenocarcinoma), SRCC732 (squamous cell carcinoma), SRCC733 (adenocarcinoma), SRCC734 (adenocarcinoma), SRCC735 (broncho alveolar caπ..ιoma. abbreviated as "BAC"), SRCC736 (squamous cell carcinoma), SRCC738 (squamous cell carcinoma), SRCC739 (squamous cell carcinoma), SRCC740 (squamous cell carcinoma), and SRCC740 (lung cell carcinoma, abbreviated as "LCCa") Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcιnoma.SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), COLO320 (adenocarcinoma. SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (carcinoma, SRCC780), CaWiDr (adenocarcinoma. srcc781), HCTl 16 (carcinoma. SRCC782), SKC01 (adenocarcιnoma,SRCC783), SW403 (adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), and HM7 (a high mucin producing variant of ATCC colon adenocarcmomacell line LS 174T, obtained from Dr Robert Warren, UCSF) Primary colon tumors include colon adenoocarcιnomasdesιgnatedCT2 (SRCC742),CT3 (SRCC743),CT8 (SRCC744),CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT17 (SRCC750), CT1 (SRCC751 ), CT4 (SRCC752), CT5 (SRCC753),CT6 (SRCC754),CT7 (SRCC755),CT9 (SRCC756),CT1 1 (SRCC757),CT18 (SRCC758), and DcR3, BACrev, BACfwd. T160. and T159 Human breast carcinomacell lines include, for example, HBL 100 (SRCC759), MB435s (SRCC760), T47D (SRCC761 ), MB468(SRCC762),MB 175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), and SKBR3 (SRCC767) The results are reported in delta (Δ) CT units One unit corresponds to one PCR cycle or approximately a 2-fold amplification relative to normal two units corresponds to 4-fold 3 units to 8-fold amplification and so on Quantitation was obtained using primers derived from the 3'-untranslated regions of the WISP- and WISP-2 cDNAs and a TAQMAN™ fluorescent probe corresponding to the respective intervening sequences Using the 3' region tends to avoid crossing intron-exon boundaries in the genomic DNA. an essential requirement for accurate assessment of gene amplification using this method The sequences for the primers and probes (forward, reverse, and probe) used for the WISP-1 -encodingand WISP- 2-encodιng gene amplification were as follows WISP- 1 probe and primers hu WISP1 TMP (probe) 5'-AGCCTTTCCAAGTCACTAGAAGTCCTGCTGG (SEQ ID NO 138) hu WISP1 TMF (forward primer) 5'-CTGGACTACACCCAAGCCTGA (SEQ ID NO 139) hu WISP1 TMR (reverse primer) 5'-CATTTCTTGGGATTTAGGCAAGA (SEQ ID NO 140) WISP-2 probe and primers DNA33473 3utr-5 (forward primer) 5'-TCTAGCCCACTCCCTGCCT (SEQ ID NO 141 ) DNA33473 3utr-3 (reverse primer) 5'-GAAGTCGGAGAGAAAGCTCGC (SEQ ID NO 142) DNA33473 3utr-probe 5'-CACACACAGCCTATATCAAACATGCACACG (SEQ ID NO 143) The 5' nuclease assav reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor amplification in real time Two oltgonucleotidepπmers are used to generate an amp con typical of a PCR reaction A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dve Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe During the amplification reaction, the Taq DNA polymerase enzvme cleaves the be in a template-dependent manner The resultant probe fragments disassociate in solution and signal from the released reporter dye is free from the quenching effect of the second fluorophore One moleculeof reporter dve is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRIZM 7700™ Sequence Detection System™ The system consists of a thermocyler, laser, charge-coupled device (CCD), camera and computer The system amplifies samples in a 96-well format on a thermocvcler During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD T e system includes software for running the instrument and for analyzing the data 5'-Nuclease assay data are initially expressed as Ct, or the threshold cycle This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence The ΔCt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer DNA results to normal human DNA results The results of the first run performed are shown in Figures 19A-D and 20A-D for WISP-1 and WISP-2, respectively, and controls Note the pattern shown in Fig 19B (marked hu WISP-1 ) The standard deviation tor two samples ot normal human DNA is shown in the column marked Nor Hu This was used as a quality control tool If the standard deviation was unacceptablv large, the entire run was repeated The nine additional columns correspondedto the human colon cancer cell lines noted above The delta CT's for HT29 and WIDrwere >3 correspondingto an about 8-fold over-representation of the WISP- l gene in these samples compared to the normal samples Similarly Fig 19B suggests an about 4-fold amplification of WlSP- l in the HCT116, SKCo- 1 and SW403 cell lines As a comparison, see Fig 20B (marked huFASr) The generally small delta CT values indicate that this gene was not significantlvamplified in anv of the cell lines (the value ot 1 for SW620 corresponding to 2-fold amplification is within the noise level for the assay) The WlSP- l result was confirmed in three replicate reactions See Figures 21 A-D, 22A-D, and 23A-C The pattern and delta CT values obtained were very similar in Figures 21 A-C (marked huWISP-l c. huWISP- lb,and huWISP-la. respectively) The result was essentially identical to that obtained in the first run HT29 and WIDr showed the highest levels of WlSP-l amplification, while HCT1 16, SKCo-1, and SW403 cell lines showed somewhat lower levels of WlSP- l gene amplification Two additional reactions trom a third run were confirmatory See Figs 25A and 25B The WISP- gene is located on chromosome 8. in the general vicinity of the mvc gene, which is known to be amplified in some colon cancer cell lines The pattern obtained using primers and probe for the mvc gene, namely, hu c-myc tm p 5'-CTTGAG - CTGAAAGATTTAGCCATAATGTAAAC rGCCT (SEQ ID NO 144) hu c-mvc tm f 5'-CAAATGCAACCTCACAACCTTG (SEQ ID NO 145), and hu c-myc tm r 5'-TTCTTTTATGCCCAAAGTCCAATT (SEQ ID NO 146), is consistent with a published report (Cancer Research. 57 1769- 1775 ( 1997)). tending to validate the 5' nuclease assay method, but is clearly different from that obtained for WISP- l These data prove that the mvc gene is not the target of the amplif.i jtion detected using the primers and probes for WISP-l The data using primers and probes based on the WISP-2 DNA sequence suggest that this gene may be the target ot low-level gene amplification in most ot the cell lines examined See Figs 20C. 22A-D, and 25C and D Hence, antibodies to both WISP- 1 and WISP-2, particularly humanized antibodies, are expected to be of benefit in combating certain types of cancer such as colon cancer, similar to the humanized antι-HER-2 antibody in clinical use 2 WISP-2 Description of Tumors and Cell Lines Amplification using several different tumor types was performed for human WISP-2 (PR0261), as described below Table III describes the stage, T stage, and N stage of various primary tumors which were used to screen the WISP-2 compound of the invention TABLE III Pπmarv Lung and Colon Tumor Profiles DNA Preparation DNA was prepared from cultured cell lines, primary tumors, and normal human blood (controls and framework and epicenter mapping) The isolation was performed using purification kit # 13362 (which incli' iss 10 purification tips with a capacity of 400 μg genomic DNA each), buffer set # 1960 . nd protease # 19155 and # 19101 , all from Quiagen. according to the manufacturer's instructions and the description below Cell culture Ivsts Cells were washed and trypsinizedat a concentrationof 7 5 x 10° per tip and pelleted by centnfuging at lOOO rpm for 5 minutes at 4°C. followed by washing again with 1/2 volume of PBS recentπfugation The pellets were washed a third time, and the suspended cells collected and washed 2x with PBS The cells were then suspended into lO mL PBS BufferC l was equιhbratedat 4°C Protease # 19155 (Quiagen) was diluted into 6 25 ml cold ddH- > 0 to a final concentration of 20 mg/ml and equilibrated at 4°C 10 mL of G2 Buffer was prepared by diluting RNAse A stock (Quiagen) ( 100 mg ml) to a final concentration of 200 μg/ml Buffer C 1 ( 10 mL, 4 °C) and ddH20 (40 mL, 4 °C ) were then added to the 10 mL of cell suspension, mixed by inverting and incubated on ice for 10 minutes The cell nuclei were pelleted by centnfuging in a BECKMAN™ swinging bucket rotor at 2500 rpm at 4°C for 15 minutes The supernatant was discarded and the nuclei were suspended with a vortex into 2 mL Buffer C l (at 4 °C) and 6 mL ddH^O. followed by a second 4°C centnfugation at 2500 rpm for 15 minutes The nuclei were then resuspended into the residual buffer using 200 μl per tip G2 buffer ( 10 ml) was added to the suspended nuclei while gentle vortexing was applied Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds Quiagen protease (200 μl, prepared as indicated above) was added and incubated at 50 °C for 60 minutes The incubation and centnfugation were repeated until the lysates were clear ( g , incubating an additional 30-60 minutes, pelleting at 3000 x g for 10 mm , 4°C) Solid human tumor sample preparation and Ivsis Tumor samples were weighed and placed into 50-ml conical tubes and held on ice Processing was limited to no more than 250 mg tissue per preparation ( 1 tip/preparation) The protease solution was freshlv prepared by diluting into 6 25 ml cold ddH 2 0 to a final concentration of 20 mg/ml and stored at 4°C G2 buffer (20 ml) was prepared by diluting DNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock) The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds using the large tip of the polytron in a laminar-flow TC hood to order to avoid inhalation of aerosols, and held at room temperature Between samples, the polytron was cleaned by spinning at 2 x 30 seconds each in 2L ddHiO. followed by G2 buffer(50 ml) If tissue was still present on the generator tip. the apparatus was disassembled and cleaned Protease (Quiagen), prepared as indicated above, 1 0 ml, was added, followed by vortexing and incubation at 50°C for 3 hours The incubation and centnfugation were repeated until the lysates were clear (e , incubating additional 30-60 minutes, pelleting at 3000 x g for 10 mm , 4°C) Human blood preparation ana lysis Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated into 10 ml samples per tip Protease (Quiagen) was freshly prepared by dilution into 6 25 ml cold ddH- > 0 to a final concentration of 20 mg/ml and stored at 4°C G2 buffer was prepared by diluting RNAse A to a final concentration of 200 μg/ml from 100 mg/ml stock The blood ( 10 ml) was placed into a 50-ml conical tube, and 10 ml C 1 buffer and 30 ml ddH 2 0 (both previously equilibratedto 4°C) were added, and the components mixed by invertingand held on ice for 10 minutes The nuclei were pelleted with a BECKMAN M swinging bucke' rotor at 2500 φm, 4°C for 15 minutes and the supernatant was discarded With a vortex, the nuclei were suspended into 2 ml C l buffer (4°C) and 6 ml ddH->0 (4°C) Vortexing was repeated until the pellet was white The nuclei were then suspended into the residual buffer using a 200-μl tip G2 buffer ( 10 ml) was added to the suspended nuclei while gently vortexing, followed by vigorous vortexing for 30 seconds Protease (Quiagen) was added (200 μl) and incubated at 50°C for 60 minutes The incubation and centnfugation were repeated until the lysates were clear (e g , incubating an additional 30-60 minutes, pelleting at 3000 x g for 10 mm . 4°C) Purification of cleared Ivsates, Isolation of genomic DNA Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) with 10 ml QBT buffer QF elution buffer was equilibrated at 50°C The samples were vortexed for 30 seconds, then loaded onto equilibrated tips and drained by gravity The tips were washed with 2 x 15 ml QC buffer The DNA was eluted ιnto 30-ml sιlanιzed,autoclaved 30-ml COREX™ tubes with 15-ml QF buffer (50°C) Isopropanol ( 10 5 ml) was added to each sample, and the tubes were covered with paraffin and mixed by repeated inversion until the DNA precipitated Samples were pelleted by centnfugation in the SS-34 rotor at 15,000 rpm for 10 minutes at 4°C The pellet location was marked, the supernatant discarded, and 10 ml 70% ethanol (4°C) was added Samples were pelleted again by centnfugation on the SS-34 rotor at 10,000 rpm for 10 minutes at 4 °C The pellet location was marked and the supernatant discarded The tubes were then placed on their side in a drying rack and dried 10 minutes at 37°C, taking care not to overdry the samples After drying, the pellets were dissolved into 1 0 ml TE (pH 8 5) and placed at 50°C for 1-2 hours Samples were held overnιghtat 4°C as dissolution continued The DNA solution was then transferred to 1 5- ml tubes w ith a 26-gauge needle on a tuberculin svπnge The transfer was repeated 5x in order to shear the DNA Samples were then placed at 50°C for 1 -2 hours Ouantitation of genomic DNA and preparation for gene amplification assav The DNA levels in each tube were quantifiedbv standard A260. A280 spectrophotometry on a 1 20 dilution (5 μl DNA • * • 95 μl ddH 2 0) using the 0 1 -ml quartz cuvettes in the BECKMAN DU640™ spectrophotometer A260/A280 ratios were in the range of 1 8-1 9 Each DNA sample was then diluted further to approximately 200 ng/ml in TE (pH 8 5) If the original material was highly concentrated (about 700 ng/μl), the material was placed at 50°C for several hours until resuspended Fluorometπc DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer s guidelines as modified below This was accomplished by allowing a HOEFFER DYNA QUANT 200™ fluorometer to warm up for about 15 minutes The HOECHST M dve working solution (#H33258, 10 μl, prepared within 12 hours of use) was diluted into 100 ml 1 x TNE buffer A 2-ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed pGEM 3Zf(+) (2 μl. lot #360851026) was added to 2 ml of fluorometersolution and calibrated at 200 units A second 2 μl ofpGEM 3Zf(+) DNA was then tested and the reading confirmed at 400 +/- 10 units Each sample was then read at least in triplicate When 3 samples were found to be within 10% of each other, their average was taken and this value was used as the quantification value The fluorometπcally-determined concentration was then used to dilute each sample to 10 ng/μl in ddH- > 0 This was done simultaneously on all template samples for a single TAQMAN™ plate assay, and with enough material to run 500-1000 assays The samples were tested in triplicate with both B-actin and G APDH on a single plate with normal human DNA and no-template controls The diluted samples were used, provided that the CT value of normal human DNA subtracted from test DNA was +/- 1 CT The diluted, lot- qualified genomic DNA was stored in 1 0-ml alιquotsat -80°C A quots which were subsequently to be used in the gene amplification assav were stored at 4°C Each 1-ml aliquot is enough for 8-9 plates or 64 tests Framework Mapping and Epicenter Marking Human WISP- 1 was reexaminedwith both frameworkand epicentermapping Selected tumors from the above initial screen were reexammed with both framework and epicenter mapping Table IV indicates the chromosomal mapping of the framework markers that were used in the present example The framework markers are located approximately every 20 megabases along Chromosome 8 and were used to control for aneuploidy TABLE IV Framework Markers Table V describes the epicenter markers that were employed in association with WISP- 1 These markers are located in close proximity to the gene for WISP- 1 and are used to assess the amplification status of the region of chromosome 8 in which the gene for WISP- 1 is located The distance between individual markers is measured in centιrays(cR), which is a radiation breakage unit approximately equal to a 1% chance of a breakage between two markers. One cR is very roughly equivalentto 20 kilobases. The marker SHGC- 32958 is the marker found to be the closest to the location on chromosome 8 to which the gene encoding WISP- 1 most closely maps TABLE V Epicenter Markers The framework markers for human WISP-2 are located approximately every 20 megabases along Chromosome 20, and are used to control for aneuploidy The markers are shown in Table VI TABLE VI Framework Markers The marker SHGC-33922 is the marker to which human WISP-2 DNA most closely maps. This marker is between the framework markers Framework analysis showed that all markers were u7p in tumors, thus, chromosome20 was aneuploid in many tumors Since the markers were up due to aneuploidy, epicenter analysis was not done for human WISP-2 gene The ΔCt values of the above described framework markers along Chromosome 8 relative for WISP- 1 are indicated for selected tumors in Tables VII and VIII. TABLE VII Amplification of framework markers relative to Human WISP- 1 DNA Framework Markers (Δct) TABLE VIII Amplification of framework markers relative to Human WISP-1 DNA Framework Markers (Δct) Gene Amplification Assav Results The human WISP-2 (PR0261 ) compound of the invention was screened in the following primary tumors and the resulting ΔCt values are reported in Table IX TABLE IX ΔCt values in lung and colon primary tumor models The ΔCt values for DNA33473 (PR0261 , human WISP-2) in a variety of pπmary lung and colon tumors as well as lung tumor cell lines are reported in Table IX A ΔCt value of > 1 was tvpicallv used as the threshold value for amplification scoring, as this represents a doubling of the gene copy Table IX indicates that significant amplification of DNA33474 occurred in ( 1 ) primary lung tumors LTla, LT10 LT12. LT15 LT17 and LT19 (2) pπmarv colon tumors CT2 CT3 CT14 and CT5 (3) colon tumor cell hnes SW480 SW620. HT29, WiDr HCT1 16 SKCOl SW403 and LSI 74T and (4) breast tumor cell lines HBL 100 MB435s BT20 and SKBR3 The ΔCt and average ΔCt values for the primary lung tumors were the following 1 08. 1 16. 1 17 1 64, 1 50 and 1 47. respectively those for the pπmarv colon tumors were 1 16. 2 14, I 03 and 1 07, respectively, those for the colon tumor cell lines were 1 67, 1 54. 1 73, 1 24, 1 32. 1 35. 1 65, and 1 48. respectively, and those for the breast tumor cell lines were 1 40 1 43 1 66 and 1 73 respectively For the lung tumors, this represents approximately a 2 1-, 2 2-, 2 2-. 3 1-, 2 8-, and 2 8-, respectively, fold increase in gene copy relative to normal tissue For the colon tumors, this represents a 22-, 44-, 2 0-, and 2 1-, respectively fold increase in gene copy relative to normal tissue For the colon tumor cell lines, this represents a 3 2- 2 9-, 3 3-. 2 4-. 2 5-, 2 5-. 3 1-, and 2 8-. respectively, fold increase in gene copy relative to normal tissue For the breast tumor cell lines, this represents a 2 6-, 2 7-, 3 2-, and 3 3-, respectively, fold increase in gene copy relative to normal tissue Because amplificationof DNA33473 (PR0261 ) occurs in various tumors it is likelv associated with tumor formation or growth As a result. antagonists (e g , antibodies) directed against the protein encoded bv DNA33473 (PR0261 ) would be expected to be useful in cancer therapy EXAMPLE 16 In Situ Hybridization In situ hybridization is a powerful and versatile technique for the detectio- and localization of nucleic acid sequences within cell or tissue preparations It mav be useful, for example, in identifying sites of gene expression, analyzing the tissue distribution of transcription, identifying and localizing viral infection, following changes in specific mRNA synthesis, and aiding in chromosome mapping In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett. Cell Vision I 169- 176 ( 1994), using PCR-generated 33 P-labeled πboprobes Br"*fiy. formalin- fixed, paraffin-embeddedhuman tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37°C and further processed for in situ hybridization as described bv Lu and Gillett, supra A ( 33 -P)UTP-labeled antisense nboprobe was generated from a PCR product and hybridized at 55°C overnight The slides were dipped in KODAK NTB2™ nuclear track emulsion and exposed for 4 weeks 33 P-Rιboprobe synthesis 6 0 μl ( 125 mCi) of 33 P-UTP ( Amersham BF 1002. SA<2000 Ci/mmol) were speed- vacuum dried To each tube containing dried 33 P-UTP, the following ingredients were added 2 0 μl 5x transcription buffer 1 O μl DTT ( lOO mM) 2 0 μl NTP mix (2 5 mM 10 μl each of 10 mM GTP. CTP & ATP + 10 ul H-,0) 1 0 μl UTP (50 μM) 1 0 μl RNAsin 1 0 μl DNA template ( 1 ug) 1 0 ul H 2 0 1 0 μl RNA polvmerase (for PCR products T3 = AS. T7 = S usually) The tubes were incubated at 37°C for one hour A total of 1 O μl RQl DNase was added, followed by incubation at 37 °C for 15 minutes A total of 90 μl TE ( 10 mM Tπs pH 7 6/1 mM EDTA. pH 8 0) was added, and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a MICROCON-50 ™ ultrafiltration unit, and spun using program 10 (6 minutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, a total of 100 μl TE was added Then 1 ul of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™ 1 The probe was run on a TBE/ureagel A total of 1 -3 μl of the probe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a -70 C C freezer one hour to overnight 33 P-Hvbndιzatιon A Pretreatment of frozen sections The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes The trays were placed in a 55°C ιncubaιo r for five minutes to reduce condensation The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0 5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml s c H 2 0) After deproteination in 0 5 μg/ml proteinase K for 10 minutes at 37°C ( 12 5 μl of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0 5 x SSC for 10 minutes at room temperature The sections were dehydrated in 70%. 95%. and 100% ethanol, _ ninutes each B Pretreatment ot paraffin-embedded sections The slides were deparaffinized. placed in s c H- > 0, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNAse-free RNAse buffer, 37°C, 15 minutes) for human embryo tissue, or 8 x proteinase K ( 100 μl in 250 ml RNAse buffer.37°C, 30 minutes) for formalin tissues Subsequent rinsing in 0 5 x SSC and dehydration were performed as described above C. Prehvbπdization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) The filter paper was saturated The tissue was covered with 50 ul of hybridization buffer (3 75 g dextran sulfate + 6 mi s c H-iO). vortexed, and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18 75 ml formamide.3 75 ml 20 x SSC, and 9 ml s c H- ι O were added, and the tissue was vortexed well and incubated at 42 °C for 1 -4 hours D Hybridization 1 0 x I 0 6 cpm probe and 1 0 ul tRNA (50 mg/ml stock) per slide were heated at 95°C for 3 minutes The slides were cooled on ice and 48 ul hybridization buffer was added per slide After vortexing, 50 ul j P mix was added to 50 μl prehv bπdization on the slide The slides were incubated overnight at 55°C E Washes Washing was done for 2x 10 minutes with 2xSSC. EDTA at room temperature (400 ml 20 x SSC 16 ml 0 25 M EDTA V =4L), followed bv RNAseA treatment at 37°C for 30 minutes (500 μl of 10 mg/ml in 250 ml RNAse buffer = 20 μg/ml) The slides were washed 2x 10 minutes with 2 x SSC, EDTA at room temperature The stringency wash conditions were as follows 2 hours at 55°C, 0 1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, Vf=4L) F Oligonucleotides In situ analysis was performed on DNA sequences disclosed herein The oligonucleotides employed for these analyses are as follows ( 1 ) Mouse WISP- 1 (Clone 568) Notπm-p 1 5'-GGA TTC TAA TAC G AC TCA CTA TAG GGC GTC CCT GGC CAG TGC TGT G AG-3' (SEQ ID NO 147) Notπm-p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GGG CCA GGC TTT GCT TCC ATT-3' (SEQ ID NO 148) (2) Human WISP-1 hm WISP- 1 p i 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC TGG AGG CAT GGC ACA GGA AC-3' (SEQ ID NO 149) hm WISP-1 p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TCC GGA TCA GGC TTG GGT GTA-3' (SEQ ID NO 150) (3) Mouse WISP-2 (Clone 1367 3) 1367 p1 5'-GGATTC TAATAC GACTCA CTA TAG GGC AGC TTG GGA TGG AGG TCTTTC-3' (SEQ ID NO 151) 1367 p2 5'-CTATGA AATTAA CCCTCA CTA AAG GGA GGG CAC TGG GGT GGT GT-3' (SEQ ID NO 152) (4) Human WISP-2 (DNA33473) DNA33473-p 1 5'-GG A TTC TAA TAC GAC TCA C TA TAG GGC GCG AGG ACG GCG GCT TCA-3' (SEQ ID NO 153) DNA33473-p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA AGA GTC GCG GCC GCC CTT TTT-3' (SEQ ID NO 154) (5) Human WISP-3 WISP3-p 1 5'-GG A TTC TAA TAC GAC TCA CTA TAG GGC GGG GCT CCT CTT CTC CAC TCT-3' (SEQ ID NO 155) WISP3-p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GCT GTC GC A AGG CTG AAT GTA-3' (SEQ ID NO 156) G Results In situ analysis was performed on the above DNA sequences disclosed herein The results from these analyses are as follows ( 1 ) Mouse WISP- 1 Expression in Mouse Tissues Mouse Fetal Tissues In situ hybridization of mouse WISP-1 showed strong expression in embryonic mesenchymal tissues At El 0 5 expression was observed in tissues that would develop into skeletal elements in the adult.this patternwas maintainedat laterstagesofembryonicdevelopment In later stages (El 2 5 and E15 5), expression was highest in osteoblasts at the sites of bone formation Expression was also observed in the embryonic heart, where the signal was particularly strong in the atria at E12 5 (atria were not included in sections at E15 5) Mouse Adult Tissues No expression was observed in anv of the adult tissues examined, including heart, lung, kidney, adrenal, liver, pancreas, cerebrum, and cerebellum These results do not correlate with the Northern data Additional sites of expression in the fetus were the walls of developing blood vessels and in fibroblast- ke cells within the hepatic portal tract mc^enchyme Expression in Normal and Wnt- 1 Transgenic Tumors Expression with the antisense probe was observed over fibroblast-hke cells lying adjacent to the subcutaneous skeletal muscle in P 10 (post-natal day 10 pups) and in pregnant females Expression was not observed over breast epithelial cells at any of the time points examined in the study Expression of mouse WISP-1 was high in all three of the Wnt- 1 transgenic tumors tested and appeared to be confined to the supporting fibroblast-likecells within the delicate connective tissue stroma Some expression was seen over the tumor cells themselves, however, this likely represents overspill from tumor fibroblasts, rather than true expression by tumor cells In summary, mouse WISP-1 was expressed in embryonic skeletal mesenchymeand at sites of bone formation It was additionally expressed in fibroblasts in the sub-cutus of growing pups and pregnant females It is likely to play a role in osteogenesis, and may be involved in repair after injury Expression was also observed in the embryonic heart (2) Human WISP-1 Expression in Human Tissues Human Fetal Tissue The fetal tissues examined (E12-E16 weeks) included placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach, small intestine. spleen, thy us. pancreas, brain, eye, spinal cord, body wall, pelvis, and lower limb Human WISP- 1 exhibited expression at sites of connective tissue interfaces in the fetus, for example, developing portal tracts fascial planes in muscle, and connective tissue surrounding developing skeletal elements and tendons Expression also was seen in the epithelium of the developing renal cortex and in spindle-shapedfibroblast-likecells in the fetal adrenal Human WISP- 1 was strongly expressed by osteoblasts at sites of bone formation in the fetal limb Human Adult Tissue The adult tissues examined were liver, kidney, adrenal, myocardium, aorta, spleen, lung, skin, chondrosarcoma. eve, stomach, gastric carcinoma, colon, colonic carcinoma, renal ceil carcinoma, prostate, bladder mucosa. and gall bladder, as well as tissue with acetominophen-induced liver injury and hepatic cirrhosis No expression was seen in normal or diseased adult tissues in this study In summary, the overall pattern of expression of human WISP- 1 was broadly similar to that observed for the mouse gene as noted above The human WISP- 1 probe did not cross react with the mouse embryo section Expression in Human Breast Carcinoma and Normal Breast Tissue Human WISP- 1 was negative on benign and malignant epithelial cells, but showed specific hybridization in mesenchmal cells, particularly in areas of tissue repair, including dystrophic ossification Most positive cells had the morphology of fibroblasts. smooth muscle cells appeared to be negative In summary, this studv shows expression of human WISP- 1 RNA in mesenchymal cells involved in tissue repair and or collagen deposition The signal was particularly strong in benign fibroblast-hke cells adjacent to either infiltrating breast carcinoma cells or tissue destruction due to benign, inflammatory conditions (duct rupture) Of note is the fact that deposition of benign osteoid seemed to correlate with strong expression of the RNA (3) Mouse WISP-2 Expression in Normal Mouse Tissues Mouse Fetal Tissues Expressionof mouse WISP-2 was observed in osteoblasts in an E l 5 5 mouse embryo, within the developing mandible Mouse Adult Tissues Expression of mouse WISP-2 was observed in stromal cells around the origin, and within the cusps of the mitral and tncuspid valves of the adult heart Expression was also observed in the adventitialcells ofthe renal artery, expression was presumed to be present at this site in ail arteries All other tissues were negative Expression in Wnt- 1 Tumors The results demonstratedspecific expressionof mouse WISP-2 in the stroma of all Wnt- 1 tumors examined There was a signal over mononuclear cells with open vesicular nuclei, possibly macrophages No expression was observed in either the benign or the malignant epithelium (4) Human WISP-2 Expression in Human Tissues Strong expression of the WISP-2-encodιng gene was observed in dermal fibroblasts in normal adult skin Additionally, strong expression was seen in two cirrhotic livers at sites of active hepatic fibrosis Moderate expression was found over tasiculatacells ot adrenal cortex This localization supports a role tor human WISP-2 in extracellular matrix formation or turnover Expression in Human Breast Carcinoma and Normal Breast Tissue, and in Lung Carcinoma Human WISP-2 showed a similar hybridization pattern to human WISP-1 (descπbedabove) ιn the two breast tumors examined It was negative on benign and malignant epithelial cells, but showed specific hybridization in mesenchmal cells, particularly in areas ot tissue repair, including dystrophic ossification The signal appeared to localize to the same cell population for both probes WISP-1 and WISP-2, however, in some areas (breast tumor 02), the signal for WISP-2 was significantly stronger than that for human WISP- 1 Most positive cells had the morphology of fibroblasts. smooth muscle cells appeared to be negative The signal for human WISP-2 was less intense in the lung tumor tissue, however, this section also showed less tissue repair compared with the breast tumor slides Normal lung and kidney tissue were essentially negative for human WISP-2, as for human WISP- 1 In summary, this study shows expression of human WISP-2 RNA in mesenchymal cells involved in tissue repair and/or collagen deposition The signal was particularly strong in benign fibroblast-hke cells adjacent to either infiltrating breast carcinoma cells or tissue destruction due to benign, inflammatory conditions (duct rupture) Of note is the fact that deposition ot benign osteoid seemed to correlate with strong expression of the RNA (5) Human WISP-3 Expression in Normal Adult and Fetal Tissues and in Human Breast Carcinoma and Normal Breast Tissue and in Colon Carcinoma The analysis shows strong expression ot human WISP-3 in dermal fibroblasts in normal adult skin and in cirrhotic livers at sites of active hepatic fibrosis This localization pattern supports a role for this growth factor in extracellular mat: .> formation and turnover The probe for human WISP-3 was negative on most tissues examined It showed a weak, diffuse positivity on sectionsof an r.steosarcoma.some of the positive cells do represent malignant cells WISP-3 was negative on all normal and fetal tissues examined EXAMPLE 17 Ability of WISP Polypeptides to Inhibit VEGF-StimulatedProhferationof Endothe alCell Growth The ability of mouse and human WISP- 1 and human WISP-2 polypeptides to inhibit VEGF- stimulated proliferation of endothehal cells was tested Specifically, bovine adrenal cortical capillary endothehal (ACE) cells (from primary culture, maximum 12-14 passages) were plated on 96-well microtiter plates (Amersham Life Science) at a density of 500 cells/well per 100 μL in low-glucose DMEM, 10% calf serum, 2 mM glutamine. 1 x pen/strept, and fungizone, supplemented with 3 ng/mL VEGF Controls were plated the same way but some did not include VEGF A test sample of either mouse WISP- 1 , human WISP- 1 conjugated to IgG, or human WISP-2 (PR0261 ) conjugated to poly-His was added in a 100-μl volume for a 200-μL final volume Cells were incubated for 5-7 days at 37°C The media were aspirated and the cells washed 1 x with PBS An acid phosphatase reaction mixture ( 100 μL. 0 1 M sodium acetate, pH 5 5 0 1% TRITON-100™ 10 mM p-mtrophenyi phosphate) was added After incubation for 2 hours at 37°C the reaction was stopped bv addition ot 10 μL 1 N NaOH OD was measured on a microtiter plate reader at 405 nm Controls were no cells cells alone cells + FGF (5 ng/mL) cells + VEGF (3 ng/mL). cells + VEGF (3 ng ml) + TGF-β ( I ng/ml). and cells + VEGF (3 ng mL) + LIF (5 ng/mL) (TGF-β at a 1 ng/ml concentration is known to block 70-90% of VEGF-stimulated cell proliferation ) The results were assessed bv calculating the percentage inhibition of VEGF(3ng/ml)-stιmulated cell proliferation, determined bv measuring acid phosphatase activity at OD405 nm ( 1 ) relative to cells without stimulation, and (2) relative to the reference TGF-β inhibition of VEGF-stimulated activity The results, as shown in Table X below, are indicative of the utility ot the WISP polypeptides in cancer therapy and specifically in inhibiting tumor angiogenesis The numerical values (relative inhibition) shown in Table X are determined by calculating the percent inhibition of VEGF-stimulated proliferation by the mouse WISP- 1 , human WISP-1 -IgG, and human WISP-2-poly-Hιs polypeptides relative to cells without stimulation and then dividing that percentage into the percent inhibition obtained by TGF-β at 1 ng/ml, which is known to block 70-90% of VEGF-stimulatedcell proliferation Human WISP-1 and human WISP- 2 appear to be particularly useful as angiostatic agents Table X Polvpeptide Concentration (r iM) Relati ive Inhibition Mouse WISP- 1 0 1 1 13 1 0 108 10 0 109 Human WISP- 1 -IgG I 1 1 I I 0 0 95 1 10 0 0 9 Human WISP-2-poly-Hιs 0 01 % 0 95 0 01% 1 1 0 1 0 62 1 0 1 03 1 0 0 5 1 0 0 6 Deposit of Material The following materials have been deposited with the American Type Culture Collection. 10801 University Blvd , Manassas, VA, USA (ATCC) Material ATCC Pep No Deposit Date pRK5E h WIG-1 568 38 209533 December 10, 1997 pRK5E m WIG-1 568 6hιs 209537 December 10, 1997 Plasmid (encoding human WISP-2) 209391 October 17 1997 pRKE m WIG-2 1367 3 209538 December 10 1997 DNA56350- 1 176-2 209706 March 26. 1998 DNA58800- 1 176-2 209707 March 26. 1998 These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) This assures maintenance of viable cultures of the deposits for 30 years from the date of deposit The deposits will be made availableby ATCC under the terms ot the Budapest Treaty, and subject to an agreement between Genentech. Inc and ATCC, which assures permanent and unrestricted availability of the progeny of the cultures of the deposits to the public upon issuance of the pertinent U S patent or upon laying open to the public ot any U S or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U S Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner'srules pursuant thereto (including 37 CFR § 1 14 with particular reference to 886 OG 638) The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same Availability of the deposited materials is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws The foregoing written specification is considered to be sufficient to enable one skilled n ihe art to practice the invention The present invention is not to be limited in scope by the constructs deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionallv equivalent are within the scope of this invention The deposits of materials herein do not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims

Claims: WHAT IS CLAIMED IS 1 Isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 75% sequence identitv to (a) a D A molecule encoding a human WISP- 1 polypeptide comprising the sequence of amino acids 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3). or (b) a complementof the DNA molecule of (a) 2 The nucleic acid of claim 1 having at least one WISP biological activity 3 The nucleic acid of claim 1 comprising DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a human WISP- 1 polypeptide comprising the sequence of amino acids 23 to 367 of Figures 3A and 3B (SEQ ID NO 3), or (b) a complement of the DNA molecule of (a) 4 The nucleic acid of claim 3 comprising DNA encoding a human WISP- l polypeptide having amino acid residues 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3), or DNA encoding a human WISP- 1 polypeptide having amino acid residues 1 to 367 of Figures 3 A and 3B (SEQ ID NO 4), or a complement of either of the encoding DNAs 5 The nucleic acid of claim 3 comprising DNA encoding a human WISP-1 polypeptide having ammo acid residues 23 to 367 or 1 to 367 of Fιgures 3A and 3B except for an isoleucine residue at position 184 rather than a valine residue (SEQ ID NOS 5 and 6, respectively) 6 The nucleic acid of claim 3 comprising DNA encoding a human WISP-1 polypeptide having amino acid residues 23 to 367 or 1 to 367 of Figures 3 A and 3 B except tor a serine residue at position 202 rather than an alanine residue (SEQ ID NOS 7 and 8, respectively) 7 The nucleic acid of claim 3 compπsing DNA encoding a human WISP- 1 polypeptidehaving amino acid residues 23 to 367 or 1 to 367 of Figures 3 A and 3 B except for an isoleucine residue at position 184 rather than a valine residue and except for a serine residue at position 202 rather than an alanine residue (SEQ ID NOS 21 and 22, respectively) 8 Isolated nucleic acid comprising SEQ ID NO 23, 24, 25, 26. 27. 28, or 29 9 The nucleic acid of claim 1 comprising DNA encoding a mouse WISP- 1 polypeptidehaving amino acid residues 23 to 367 of Figure 1 (SEQ ID NO 1 1 ), or DNA encoding a mouse WISP-1 polypeptide having amino acid residues 1 to 367 ot Figure 1 (SEQ ID NO 12) or a complement of either ot the encoding DNAs 10 Isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 85% sequence identity to (a) a DNA molecule encoding a mouse WISP- 1 polypeptide comprising the sequence of amino acids 23 to 367 of Figure 1 (SEQ ID NO 1 1 ) or (b) a complement of the DNA molecule of (a) 1 1 The nucleic acid of claim 10 having at least one WISP biological activity 12 The nucleic acid of claim 10 comprising DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a mouse WISP- 1 polypeptide comprising the sequence of amino acids 23 to 367 of Figure 1 (SEQ ID NO 1 1), or (b) a complement of the DNA molecule of (a) 13 Isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded bv the human WISP- 1 polypeptidecDNA in ATCC Deposit No 209533 (pRK5E h WISP-1 568 38).or (b) a complement of the DNA molecule ot (a) 14 A vector comprising the nucleic acid of claim 1 15 A host cell comprising the vector of claim 14 16 A process for producing a WISP- 1 polypeptide comprising cultunng the host cell of claim 15 under conditions suitable for expression of the WISP- 1 polypeptide and recoveringthe WISP- 1 polypeptide from the cell culture 17 Isolated WISP- 1 polypeptide encoded bv the nucleic acid of claim 1 18 The polypeptide of claim 17 that is human WISP- 1 or mouse WISP- 1 19 Isolated WISP- 1 polypeptide encoded bv a nucleic acid of claim 8 20 A chimeric molecule compπsinga WISP- 1 polypeptide fused to a heterologous amino acid sequence 21 The chimeric molecule of claim 20 vvherein said heterologous amino acid sequence is an epitope tag sequence, a polv-amino acid sequence or an Fc region 22 An antibodv which specifically binds to a WISP- 1 polypeptide 23 The antibodv ot claim 22 wherein said antibody is a monoclonal antibodv 24 Isolated nucleic acid having at least about 600 nucleotides and produced bv hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP- 1 polypeptide comprising the sequence of amino acids 23 to 367 of Figures 3A and 3B (SEQ ID NO 3), or (b) a complementof the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), isolating the test DNA molecule 25 A polvpeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP- 1 polypeptidecompπsing the sequence of am o acids 23 to 367 of Figures 3 A and 3B (SEQ ID NO 3), or (b) a complement of the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), (iΛ cultunng a host cell comprising the test DNA molecule under conditions suitable for expression of the polvpeptide, and (in) recovering the polypeptide from the cell culture 26 Isolated nucleic acid comprising DNA having at least about an 80% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of ammo acids 24 to 250 of Figure 4 (SEQ ID NO 15), or (b) a complement of the DNA molecule of (a) 27 The nucleic acid of claim 26 having at least one WISP biologic?, activity 28 The nucleic acid of claim 26 comprising DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of Figure 4 (SEQ ID NO 15), or (b) a complement of the DNA molecule of (a) 29 The nucleic acid of claim 26 comprising D A encoding a human WISP-2 polypeptide having ammo acid residues 24 to 250 of Figure 4 (SEQ ID NO 15), or DNA encoding a human WISP-2 polypeptide having ammo acid residues 1 to 250 of Figure 4 (SEQ ID NO 16), or a complement of either of the encoding DNAs 30 Isolated nucleic acid comprising DNA having at least about an 80% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 1 to 250 of Figure 4 (SEQ ID NO: 16), or (b) a complement of the DNA molecule of (a). 31. Isolated nucleic acid comprising DNA having at least about 500 nucleotides and at least about an 80% sequence identity to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 24 to 251 of Figure 2 (SEQ ID NO: 19). or (b) a complement of the DNA molecule of (a). 32. The isolated nucleic acid of claim 31 comprising DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 24 to 251 of Figure 2 (SEQ ID NO: 19), or (b) a complement of the DNA molecule of (a). 33. The nucleic acid of claim 32 comprising DNA encoding a mouse WISP-2 polypeptide having amino acid residues 24 to 251 of Figure 2 (SEQ ID NO: 19). or DNA encoding a mouse WISP-2 polypeptide having ammo acid residues I to 251 of Figure 2 (SEQ ID NO.20). or a complement of either of these encoding DNAs. 34. Isolated nucleic acid comprising DNA having at :sast about 500 nucleotides and at least about an 80% sequence identity to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 1 to 251 of Figure 2 (SEQ ID NO:20), or (b) a complement of the DNA molecule of (a). 35. Isolated nucleic acid comprising DNA having at least about 400 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-2 polypeptide cDN A in ATCC DepositNo. 209391 (DNA33473). or (b) a complement of the DNA molecule of (a). 36. The nucleic acid of claim 35 comprising the nucleotide sequence of the full-length coding sequence of clone UNQ228 (DN A33473) deposited under accession number ATCC 209391. 37. A vector comprising the nucleic acid of claim 26. 38. A host cell comprising the vector of claim 37 39. A process for producing a WISP-2 polypeptide comprising cuituπng the host cell of claim 38 under conditions suitable for expression of the WISP-2 polypeptide and recoveringthe WISP-2 polypeptide from the cell culture. 40 Isolated WISP-2 polypeptide encoded bv the nucleic acid of claim 26 41 The polypeptide of claim 40 that is isolated native-sequence human WISP-2 polypeptide comprising amino acid residues 1 to 250 of Fιgure 4 (SEQ ID NO 16) or comprising ammo acid residues 24 to 250 of Figure 4 (SEQ ID NO 15) 42 A chimeric molecule comprising a WISP-2 polypeptide fused to a heterologous amino acid sequence 43 An antibody which specifically binds to a WISP-2 polypeptide 44 The antibody of claim 43 that is a monoclonal antibody 45 Isolated nucleic acid having at least about 400 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of ammo acids 24 to 250 of Figure 4 (SEQ ID NO 15), or (b) a complement of the DNA molecule of (a), and. if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), isolating the test DNA molecule 46 A polypeptide produced by (l) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-2 polypeptidecompnsing the sequence of amino acids 24 to 250 of Figure 4 (SEQ ID NO 15). or (b) a complement ot the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence ldentty to (a) or (b), (n) cultunng a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide. and (in) recovering the polypeptide from the cell culture 47 Isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 500 nucleotides to (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of ammo acids 34 to 372 of Figures 6 A and 6B (SEQ ID NO 32), or (b) a complement of the DNA molecule of (a) 48 The nucleic acid of claim 47 having at least one WISP biological activity 49 The nucleic acid of claim 47 comprising DNA encoding a human WISP-3 polypeptide having amino acid residues 34 to 372 of Figures 6A and 6B (SEQ ID NO 32) or amino acid residues 1 to 372 of Figures 6A and 6B (SEQ ID NO 33), or a complement thereof 50 A vector comprising the nucleic acid of claim 47 51 A host cell comprising the vector of claim 50 52 A process for producing a WISP-3 polypeptide comprising cultunng the host cell of claim 51 under conditions suitable for expression of the WISP-3 polypeptide and recoveringthe WISP-3 polypeptide from the cell culture 53 Isolated WISP-3 polypeptide encoded by the nucleic acid of claim 47 54 The polypeptide of claim 53 that is human WISP-3 55 A chimeric molecule comprising the WISP-3 polypeptide of claim 53 fused to a heterologous amino acid sequence 56 An antibodv which specifically binds to the WISP-3 polypeptide of claim 53 57 Isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 500 nucleotides to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP- 3 polypeptide cDN A in ATCC Deposit No 209706 (DNA56350- 1 176-2),or (b) a complement of the DNA molecule of (a) 58 Isolated nucleic acid produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptidecompnsingthe sequence of amino acids 34 to 372 of Figures 6A and 6B (SEQ ID NO 32), or (b) a complement of the DNA molecule of (a), and, if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 500 nucleotides, isolating the test DNA molecule 59 A polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encodinga human WISP-3 polypepttdecompπsing the sequence of amino acids 34 to 372 of Figures 6A and 6B (SEQ ID NO 32), or (b) a complement of the DNA molecule of (a), and if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 500 nucleotides (n) cultunng a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide. and (in) recovering the polypeptide from the cell culture 60 Isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 400 nucleotides to (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of Figures 7A and 7B (SEQ ID NO 36), or (b) a complement of the DNA molecule of (a) 61 The nucleic acid of claim 60 having at least one WISP biological activity 62 The nucleic acid of claim 60 comprising DNA encoding a human WISP-3 polypeptide having amino acid residues 16 to 355 of Figures 7A and 7B (SEQ ID NO 36) or amino acid residues 1 to 355 of Figures 7A and 7B (SEQ ID NO 37), or a complement thereof 63 Isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 400 nucleotides to (a) a DNA molecule encoding the same full-length polypeptideencoded bv the human WISP- 3 polypeptide cDN A in ATCC Deposit No 209707 (DN A58800- 1 176-2),or (b) a complement of the DNA molecule of (a) 64 A vector comprising the nucleic acid of claim 60 65 A host cell comprising the vector of claim 64 66 A process for producing a WISP-3 polypeptide comprising cultunng the host cell of claim 65 under conditions suitable for expression of the WISP-3 polypeptide and recoveringthe WISP-3 polypeptide from the cell culture 67 Isolated WISP-3 polypeptide encoded by the nucleic acid of claim 60 68 The polypeptide of claim 67 that is human WISP-3 69 A chimeric molecule comprising the WISP-3 polypeptide of claim 67 fused to a heterologous amino acid sequence 70 An antibody which specifically binds to the WISP-3 polypeptide of claim 67 71 Isolated nucleic acid produced by hvbπdizinga test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptidecompnsingthe sequence of amino acids 16 to 355 of Figures 7A and 7B (SEQ ID NO 36) or (b) a complement of the DNA molecule of (a), and. if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 400 nucleotides isolating the test DNA molecule 72 A polypeptide produced by (i) hvbπdizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of Figures 7A and 7B (SEQ ID NO 36), or (b) a complement of the DNA molecule of (a) and if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 400 nucleotides (n) cultunng a host cell compπsingthe test DNA molecule under conditions suitable for expression of the polypeptide and (in) recovering the polypeptide from the cell culture 73 A composition comprising the polypeptide of claim 17 and a earner therefor 74 A composition comprising an antagonist to the polypeptide of claim 17 and a carrier therefor 75 A composition comprising the polypeptide ot claim 19 and a carrier therefor 76 A composition comprising an antagonist to the polypeptide of claim 19 and a carrier therefor 77 A composition comprising the polypeptide of claim 40 and a carrier therefor 78 A composition comprising an antagonist to the polypeptide of claim 40 and a carrier therefor 79 A composition comprising the polypeptide ot claim 53 and a carrier therefor 80 A composition comprising an antagonist to the polypeptide of claim 53 and a carrier therefor 81 A composition comprising the polypeptide of claim 67 and a carrier therefor 82 A composition comprising an antagonist to the polypeptide of claim 67 and a carrier therefor 83 A composition comprising a WISP- 1 , WISP-2, or WISP-3 polypeptide and a pharmaceutically acceptable carrier 84 The composition of claim 83 that further comprises a chemotherapeutic agent or growth-inhibitorv agent 85 The composition of claim 83 wherein the WISP- 1. WISP-2. or WISP-3 polypeptide is a human polypeptide. 86. A pharmaceutical product comprising: (a) a composition of claim 83, (b) a container containing said composition: and (c) a label affixed to said container, or a package insert included in said pharmaceutical product referring to the use of said WISP- 1 , WISP-2. or WISP-3 polypeptide in the treatment of a WISP- related disorder. 87. A process for diagnosinga disease or a susceptibility to a disease related to a mutation in a nucleic acid sequence encoding a WISP- 1 , WISP-2, or WISP-3 polypeptide comprising: (a) isolating a nucleic acid sequence encoding a WISP- 1. WISP-2. or WISP-3 polypeptide from a sample derived from a host: and (b) determιnιnga mutation in the nucleic acid sequence encoding a WISP- 1. WISP-2. or WISP-3 polypeptide . 88. A method of diagnosing a WISP-related disorder in a mammal comprising detecting the level of expression of a gene encoding a WISP- 1 , WISP-2. or WISP-3 polypeptide(a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample indicates the presence of a WISP-related dysfunction in the mrmmal from which the test tissue cells were obtained. 89. A method for treating a WISP-related disorder in a mammal comprising administering to the mamiral an effective amount of the composition of claim 83. 90. The method of claim 89 wherein the disorder is a malignant disorder or arteriosclerosis and the mammal is human. 91. The method of claim 90 wherein the malignant disorder is breast cancer, ovarian cancer, colon cancer. or melanoma. 92. An isolated antibody binding a WISP- 1 , WISP-2, or WISP-3 polypeptide. 93. The antibody of claim 92 that induces death of a cell overexpressing a WISP- 1. WISP-2. or WISP-3 polypeptide. 94 The antibodv ot claim 93 wherein said cell is a cancer cell 95 The antibody of claim 92 that binds to a human WISP- 1 WISP-2. or WISP-3 polypeptide. and is a human or humanized antibody 96 The antibodv of claim 92 that is a monoclonal antibody 97 The antibody of claim 96 that is an antibody fragment, a single-chain antibody, or an anti-idiotypic antibody 98 A composition comprising an antibody of claim 92 in admixture with a pharmaceutically acceptable carrier 99 The composition of claim 98 comprising a growth-inhibitory amount of said antibody 100 A method for determining the presence of a WISP- 1. WISP-2. or WISP-3 polypeptide comprising exposing α cell suspected of containing the WISP- l . WISP-2, or WISP-3 polypeptide to an antι-WιSP-1 , WISP-2, or WISP-3 antibody and determining binding of said antibody to said cell 101. A method for treating a WISP-related disorder in a mammal comprising administering to the mammal an effective amount of a composition comprising an antagonist to a WISP- 1 , WISP-2. or WISP-3 polypeptide in a pharmaceutically acceptable carrier . 102. A method for inhibiting the growth of tumor cells comprising exposing a cell that overexpresses a Wnt- 1-ιnduced gene to an effective amount of an antagonist that inhibits the expression or activity of a WISP- 1 , WISP-2. or WISP-3 polypeptide. 103. A method for inhibiting the growth of tumor cells comprising exposing said cells to an effective amount of the composition of claim 99 104 The method of claim 103 wherein the tumor cells are colon cancer cells, the antibody is against human WISP- 1 and is a humanized or human monoclonal antibody, and the mammal is human 105 A kit comprising a WISP- 1. WISP-2, or WISP-3 polypeptide or antagonist and instructions for using the polypeptide or antagonist to detect or treat a WISP-related disorder 106 The kit ot claim 105 comprising an anti-WISP- 1 WISP-2 or WISP-3 antibodv and a carrier in suitable packaging 107 A method for inducing cell death comprising exposing a cell that is induced by Wnt to an effective amount of a WISP- 1 , WISP-2, or WISP-3 polypeptide or antagonist 108 An article of manutacture comprising a container, a label on the container, and a composition comprising an active agent contained within the container, wherein the composition is effective for inducing cell death or inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by oveπnduction of Wnt or a WISP-related disorder or by overexpression of a WISP- 1 WISP-2, or WISP-3 polypeptide. and the active agent in the composition is an antagonist that inhibits the expression or activity of the WISP- l WISP-2. or WISP-3 polypeptide 109 The article of manufacture of claim 108 wherein the active agent is an anti-WISP- 1. anf WISP-2, or anti- w'ISP-3 antibody, and wherein the label on the container indicates that the composition can be used for treating a WISP-related disorder 1 10 A process for identifying agonists to a WISP- l , WISP-2. or WISP-3 polypeptide comprising •' ) contacting cells and a compound to be screened under conditions suitable for the stirr .'ation of cell proliferation by the polypeptide. and (b) measuring the proliferation of the cells to determine if the compound is an effective agonist 1 1 1. An agonist to a WISP-1, WISP-2, or WISP-3 polypeptide identified by the process ot claim 1 10 1 12. A method for identifying a compound that inhibits the expression or activity of a WISP-1 , WISP-2, or WISP-3 polypeptide, comprising contacting a candidate compound with a WISP- 1 , WISP-2, or WISP-3 polypeptide under conditions and for a time sufficient to allow the compound and polypeptide to interact 1 13 The method of claim 1 12 comprising the steps of (a) contacting cells and a compound to be screened in the presence of the WISP-1. WISP-2. or WISP-3 polypeptide under conditions suitable for the stimulation of cell proliferation by polypeptide. and (b) measuπngthe proliferationof the cells to determine if the compound is an effective antagonist 1 14 A compound identified bv the method of claim 1 12 1 15 A compound that inhibits the expression or activity of a WISP- 1, WISP-2, or WISP-3 polypeptide 1 16 A method of diagnosing a WISP-related disorder in a mammal comprising (a) contacting an anti- WISP- 1 , anti-WISP-2. or antι-WISP-3 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-WISP- 1 , anti-WISP-2. or anti- WISP-3 antibody and the WISP-1 , WISP-2, or WISP-3 polypeptide in the test sample 1 17 The method of claim 1 16 wherein said test sample is obtained from an individual suspected to have neoplastic cell growth or proliferation

Inventors: BOTSTEIN DAVID A, COHEN ROBERT L, GODDARD AUDREY, GURNEY AUSTIN L, HILLAN KENNETH, LAWRENCE DAVID A, LEVINE ARNOLD J, PENNICA DIANE, ROY MARGARET ANN, WOOD WILLIAM I, BOTSTEIN, DAVID, A., COHEN, ROBERT, L., GODDARD, AUDREY, GURNEY, AUSTIN, L., HILLAN, KENNETH, LAWRENCE, DAVID, A., LEVINE, ARNOLD, J., PENNICA, DIANE, ROY, MARGARET, ANN, WOOD, WILLIAM, I.
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 1998
Main classification: C12N15
IPC codes: A61K38, A61K39, A61K45, A61P35, C07K14, C07K16, C07K19, C12N5, C12N15, C12P21