Chondrosarcoma associated genes

The invention features a nucleic acid molecule encoding a chondrosarcoma associated polypeptide and methods for diagnosing patients with chondrosarcoma. the gene.

BACKGROUND OF THE INVENTION
 The invention relates to bone malignancies.
 Chondrosarcoma, which usually occurs in late adulthood and old age, is the
 second most common form of bone malignancy. Conventional chondrosarcoma
 tumors are graded from stage I through stage III, stage III being the most
 advanced. In addition to conventional chondrosarcoma, there are other
 types of chondrosarcoma with distinguishing characteristics: myxoid,
 mesenchymal, clear cell, and dedifferentiated (spindle cell)
 chondrosarcoma.
 Diagnosis and grading of chondrosarcoma has been problematic. For example,
 the criteria used to distinguish benign enchondroma from low grade
 chondrosarcoma include parameters which are difficult to quantify such as
 increased cellularity and more than occasional binucleate cells. The
 histologic criteria are not absolute, and the diagnosis is frequently made
 by taking into account clinical features such as pain, rate of growth,
 location, and radiologic features. Furthermore, the location of the tumor
 may affect clinical assessment. For example, lesions in the hand can
 appear aggressive histologically and yet behave benignly. In contrast,
 lesions occurring in the pelvis are likely to represent a malignancy
 despite a relatively innocuous histologic appearance. Notwithstanding
 attempts to integrate clinicopathologic criteria, it has not been possible
 to predict which tumors will metastasize or recur.
 SUMMARY OF THE INVENTION
 The invention is based on the discovery of a novel gene which is
 differentially expressed in chondrosarcoma cells. Accordingly the
 invention features an isolated nucleic acid (e.g., genomic DNA, cDNA or
 synthetic DNA) encoding a chondrosarcoma associated (CSA) polypeptide such
 as human CSA-1. The term "chondrosarcoma associated" refers to the
 property of differential expression in chondrosarcoma cell compared to
 normal cartilage cells. For example, a CSA gene product is expressed at a
 detectably higher or lower level compared to the level at which it is
 expressed in normal cartilage cells. A CSA gene product may be expressed
 solely in chondrosarcoma cells (and not in normal cartilage cells).
 The nucleic acid molecule contains a nucleotide sequence encoding a
 polypeptide having an amino acid sequence that is at least 80% identical
 to the amino acid sequence of CSA-1 (SEQ ID NO:2). Preferably, the nucleic
 acid molecule contains the nucleotide sequence of SEQ ID NO:1 or a
 degenerate variant thereof. For example, the nucleic acid contain the
 nucleotide sequence of SEQ ID NO:3. The invention also includes a nucleic
 acid molecule which contains a strand which hybridizes at high stringency
 to a DNA having the sequence of SEQ ID NO:1, or the complement thereof. A
 substantially pure DNA having at least 50% sequence identity (preferably
 at least 70%, more preferably at least 80%, and most preferably at least
 90%) to SEQ ID NO:1, and encoding a polypeptide having the differential
 pattern of expression of a CSA-1 polypeptide is also within the invention.
 For expression of a CSA polypeptide, a CSA polypeptide encoding nucleic
 acid molecule is operably linked to regulatory sequences, e.g., a
 promoter.
 The invention also includes a substantially pure CSA polypeptide such as
 human CSA-1 or a fragment thereof. CSA-1 fragments, e.g., a fragment
 containing the amino acid sequence of SEQ ID NO: 8), are useful as
 immunogens for raising anti-CSA antibodies. The CSA polypeptide preferably
 contains an amino acid sequence that is at least 50% identical to the
 amino acid sequence of SEQ ID NO:2. More preferably the amino acid
 sequence of the polypeptide is 75%, 85%, 95%, 98%, and most preferably
 100% identical to the amino acid sequence of SEQ ID NO:2. A cell
 containing a CSA polypeptide-encoding nucleic acid molecule is also within
 the invention, as is a method of making a CSA polypeptide. Such a method
 may involve the following steps: (a) providing cell containing a CSA
 polypeptide-encoding nucleic acid molecule, and (b) culturing it under
 conditions permitting expression of the nucleic acid molecule.
 By "isolated nucleic acid molecule" is meant a nucleic acid molecule that
 is free of the genes which, in the naturally-occurring genome of the
 organism, flank a csa gene. The term therefore includes, for example, a
 recombinant DNA which is incorporated into a vector; into an autonomously
 replicating plasmid or virus; or into the genomic DNA of a prokaryote or
 eukaryote; or which exists as a separate molecule (e.g., a cDNA or a
 genomic or cDNA fragment produced by PCR or restriction endonuclease
 digestion) independent of other sequences. It also includes a recombinant
 DNA which is part of a hybrid gene encoding additional polypeptide
 sequence. The term excludes large segments of genomic DNA, e.g., such as
 those present in cosmid clones, which contain a gene of interest, e.g., a
 csa gene, flanked by one or more other genes which naturally flank it in a
 naturally-occurring genome.
 Nucleic acid molecules include both RNA and DNA, including cDNA, genomic
 DNA, and synthetic (e.g., chemically synthesized) DNA. Where
 single-stranded, the nucleic acid molecule may be a sense strand or an
 antisense strand. The term therefore includes, for example, a recombinant
 DNA which is incorporated into a vector, into an autonomously replicating
 plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote at
 a site other than its natural site; or which exists as a separate molecule
 (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain
 reaction (PCR) or restriction endonuclease digestion) independent of other
 sequences. It also includes a recombinant DNA which is part of a hybrid
 gene encoding additional polypeptide sequence.
 Hybridization is carried out using standard techniques such as those
 described in Ausubel et al., Current Protocols in Molecular Biology, John
 Wiley & Sons, (1989). "High stringency" refers to DNA hybridization and
 wash conditions characterized by high temperature and low salt
 concentration, e.g., wash conditions of 65.degree. C. at a salt
 concentration of approximately 0.1.times.SSC. "Low" to "moderate"
 stringency refers to DNA hybridization and wash conditions characterized
 by low temperature and high salt concentration, e.g. wash conditions of
 less than 60.degree. C. at a salt concentration of at least 1.0.times.SSC.
 For example, high stringency conditions may include hybridization at about
 42.degree. C., and about 50% formamide; a first wash at about 65.degree.
 C., about 2.times. SSC, and 1% SDS; followed by a second wash at about
 65.degree. C. and about 0.1% .times.SSC. Lower stringency conditions
 suitable for detecting DNA sequences having about 50% sequence identity to
 csa-1 gene are detected by, for example, hybridization at about 42.degree.
 C. in the absence of formamide; a first wash at about 42.degree. C., about
 6.times. SSC, and about 1% SDS; and a second wash at about 50.degree. C.,
 about 6.times. SSC, and about 1% SDS.
 Where a particular polypeptide or nucleic acid molecule is said to have a
 specific percent identity to a reference polypeptide or nucleic acid
 molecule of a defined length, the percent identity is relative to the
 reference polypeptide or nucleic acid molecule. Thus, a peptide that is
 50% identical to a reference polypeptide that is 100 amino acids long can
 be a 50 amino acid polypeptide that is completely identical to a 50 amino
 acid long portion of the reference polypeptide. It might also be a 100
 amino acid long polypeptide which is 50% identical to the reference
 polypeptide over its entire length. Of course, many other polypeptides
 will meet the same criteria. The same rule applies for nucleic acid
 molecules.
 For polypeptides, the length of the reference polypeptide sequence will
 generally be at least 16 amino acids, preferably at least 20 amino acids,
 more preferably at least 25 amino acids, and most preferably 35 amino
 acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length
 of the reference nucleic acid sequence will generally be at least 50
 nucleotides, preferably at least 60 nucleotides, more preferably at least
 75 nucleotides, and most preferably 100 nucleotides or 300 nucleotides.
 In the case of polypeptide sequences which are less than 100% identical to
 a reference sequence, the non-identical positions are preferably, but not
 necessarily, conservative substitutions for the reference sequence.
 Conservative substitutions typically include substitutions within the
 following groups: glycine and alanine; valine, isoleucine, and leucine;
 aspartic acid and glutamic acid; asparagine and glutamine; serine and
 threonine; lysine and arginine; and phenylalanine and tyrosine.
 Sequence identity can be measured using sequence analysis software (for
 example, the Sequence Analysis Software Package of the Genetics Computer
 Group, University of Wisconsin Biotechnology Center, 1710 University
 Avenue, Madison, Wis. 53705), with the default parameters as specified
 therein.
 By "promoter" is meant a minimal DNA sequence sufficient to direct
 transcription. Promoters may be constitutive or inducible, and may be
 coupled to other regulatory sequences or "elements" which render
 promoter-dependent gene expression cell-type specific, tissue-specific or
 inducible by external signals or agents; such elements may be located in
 the 5' or 3' region of the native gene, or within an intron. DNA encoding
 a CSA polypeptide may be operably linked to such regulatory sequences for
 expression of the polypeptide in prokaryotic or eukaryotic cells. By
 "operably linked" is meant that a coding sequence and a regulatory
 sequence(s) are connected in such a way as to permit gene expression when
 the appropriate molecules (e.g., transcriptional activator proteins) are
 bound to the regulatory sequence(s).
 A protein is substantially free of naturally associated components when it
 is separated from those contaminants which accompany it in its natural
 state. (proteins and other naturally-occurring organic molecules) which
 naturally accompany it. Typically, the polypeptide is substantially pure
 when it constitutes at least 60%, by weight, of the protein in the
 preparation. Preferably, the protein in the preparation is at least 75%,
 more preferably at least 90%, and most preferably at least 99%, by weight,
 CSA-1 polypeptide. A substantially pure CSA-1 polypeptide may be obtained,
 for example, by extraction from a natural source (e.g., a chondrosarcoma
 cell); by expression of a recombinant nucleic acid encoding an CSA-1
 polypeptide; or by chemically synthesizing the protein. Purity can be
 measured by any appropriate method, e.g., column chromatography,
 polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly,
 substantially pure polypeptides include recombinant polypeptides derived
 from a eukaryote but produced in E. coli or another prokaryote, or in a
 eukaryote other than that from which the polypeptide was originally
 derived.
 The invention also features CSA polypeptide binding species, such as an
 antibody or antibody fragment which specifically binds to a CSA
 polypeptide, e.g., a CSA-1-specific antibody. Antibodies specific for a
 CSA polypeptide are useful to diagnose chondrosarcoma.
 Chondrosarcoma is diagnosed by measuring expression of csa gene expression
 in patient tissue samples. Expression of CSA-1 is detectable in
 chondrosarcoma cells but not in normal cells (or in certain other types of
 tumors which were tested). Thus, the use of CSA polypeptides and CSA
 polypeptide-encoding nucleic acid molecules in diagnosing and grading of
 chondrosarcoma is also within the invention. For example, a method for
 diagnosing the presence of a chondrosarcoma cell in a tissue sample is
 carried out by measuring expression of a csa gene, e.g., a gene encoding
 CSA-1, in the tissue sample and a control sample such as a normal
 nonneoplastic cartilage cell. An increase in expression of the csa gene in
 the tissue sample compared to the control sample indicates that the tissue
 sample contains a chondrosarcoma cell. A method of grading a
 chondrosarcoma tumor may be carried out by determining the level of csa-1
 gene expression in a test sample and comparing it to the level of csa-1
 gene expression in a control sample. The level of expression in the test
 sample compared to the control sample is directly proportional to the
 grade or stage of tumor, i.e., the greater the level of expression of a
 csa gene the more advanced the stage of the tumor.
 In addition to evaluating tissue biopsy samples for csa gene expression,
 csa gene expression may be detected in vivo. For example, a diagnostically
 effective amount of a detectably labeled CSA-1-specific binding species
 may be administered to a patient, followed by a determination of whether
 the species specifically binds to cartilage cells of the patient. Binding
 of the CSA-1 binding species, e.g., a CSA-1-specific antibody, antibody
 fragment, or non-antibody CSA-1 binding compound, to patient cells is an
 indication of the presence of chondrosarcoma in the patient. The level of
 binding correlates with the grade of the chondrosarcoma, i.e., a greater
 amount of binding compared to a normal control of known low grade tumor
 indicates that the patient's tumor is of a high grade. Similarly, a method
 of detecting progressive chondrosarcoma in a patient may be carried out as
 follows: (a) successively administering to a patient suspected of having a
 chondrosarcoma a diagnostically effective amount of a detectably labeled
 CSA-1-specific binding species (e.g., an antibody labelled with a
 radioisotope or a paramagnetic label), and (b) comparing the amount of the
 species that binds to cartilage cells of the patient in each successive
 administration to detect an increase of binding of the binding species
 over time. An increase in binding over time is an indication of
 progressive chondrosarcoma in said patient. Where the detecting step is
 quantitative, the amount of binding would correlate with and allow
 diagnosis of the severity of the disease. A diagnostic method carried out
 multiple times by repeatedly administering at spaced intervals the
 labelled binding species to the patient, with the administrations spaced
 by, e.g., a day, a week, a month, several months, or even years, is a
 useful method for detecting progression of disease in a patient.
 Compounds capable of inhibiting expression of a CSA polypeptide may be
 therapeutically useful to treat chondrosarcoma. Accordingly, the invention
 includes a compound capable of inhibiting expression of a CSA polypeptide,
 e.g., CSA-1, by (a) providing a chondrosarcoma cell expressing a CSA
 polypeptide, (b) contacting the cell with the candidate compound, and (c)
 determining the amount of expression of the CSA polypeptide by the cell. A
 decrease in the amount of CSA polypeptide expression in the presence of
 the candidate compound compared to that in the absence of the candidate
 compound indicates that the compound inhibits expression of the CSA
 polypeptide.
 The invention also includes a method of inhibiting the expression or
 activity of CSA-1. Suitable antagonists include a nucleic acid molecule
 that interfere with transcription or translation of csa-1, for example,
 antisense nucleic acid molecules and ribozymes. Also included are
 antibodies or other suitable antagonistic molecules, that specifically
 binds CSA-1 polypeptide and "neutralize" its activity.
 In addition to diagnostic methods, such as described above, the present
 invention encompasses methods and compositions for evaluating appropriate
 treatment, and treatment effectiveness of malignancies associated with
 expression of csa-1. For example, the csa-1 can be used as a probe to
 classify cells in terms of their level of csa-1 expression, or as primers
 for diagnostic PCR analysis in which mutations and allelic variation of
 csa-1 can be detected.
 The invention also includes non-human transgenic animals that express human
 CSA-1 and non-human transgenic mammal with a null mutation in its
 endogenous CSA-1 gene. These animals can serve as new and useful models of
 chondrosarcoma. The invention also includes a transgenic non-human mammal,
 e.g., a rodent such as a mouse, the germ cells and somatic cells of which
 contain a null mutation, e.g., a deletion, in DNA encoding a csa gene. By
 "null mutation" is meant an alteration in the nucleotide sequence that
 renders the gene incapable of expressing a functional protein product. The
 mutation could be in csa gene regulatory regions or in the coding
 sequence. It can, e.g., introduce a stop codon that results in production
 of a truncated, inactive gene product or it can be a deletion of all or a
 substantial portion of the coding sequence.
 The invention also features an isolated nucleic acid (e.g., genomic DNA,
 cDNA or synthetic DNA) encoding a cartilage associated (CAA) polypeptide
 such as human CAA-1. The term "cartilage associated" refers to the
 property of differential expression in cells of the cartilage lineage
 compared to cells of other tissue specificities. For example, a CAA gene
 product is expressed in normal cartilage cells and chondrosarcoma cells
 but not cells of other tissue specificities or other tumor types.
 The nucleic acid molecule contains a nucleotide sequence encoding a
 polypeptide having an amino acid sequence that is at least 80% identical
 to the amino acid sequence of CAA-1 (SEQ ID NO:7). Preferably, the nucleic
 acid molecule contains the nucleotide sequence of SEQ ID NO:6 or a
 degenerate variant thereof. For example, the nucleic acid may have the
 nucleotide sequence of SEQ ID NO:5. The invention also includes a nucleic
 acid molecule which contains a strand which hybridizes at high stringency
 to a DNA having the sequence of SEQ ID NO:6, or the complement thereof. A
 substantially pure DNA having at least 50% sequence identity (preferably
 at least 70%, more preferably at least 80%, and most preferably at least
 90%) to SEQ ID NO:7, and encoding a polypeptide having the activity of
 CAA-1. By the activity of CAA-1 is meant inhibition of interferon gamma
 induced upregulation of HLA class II antigens. For expression of a CAA
 polypeptide, a CAA polypeptide encoding nucleic acid molecule is operably
 linked to regulatory sequences, e.g., a promoter.
 The invention also includes a substantially pure CAA polypeptide such as
 human CAA-1 or a fragment thereof. The CAA-1 polypeptide preferably
 contains an amino acid sequence that is at least 50% identical to the
 amino acid sequence of SEQ ID NO:7. More preferably the amino acid
 sequence of the polypeptide is 75%, 85%, 95%, 98%, and most preferably
 100% identical to the amino acid sequence of SEQ ID NO:7. A cell
 containing a CAA polypeptide-encoding nucleic acid molecule is also within
 the invention, as is a method of making a CAA polypeptide. Such a method
 may involve the following steps: (a) providing cell containing a CAA
 polypeptide-encoding nucleic acid molecule, and (b) culturing it under
 conditions permitting expression of the nucleic acid molecule.
 The invention also features CAA polypeptide binding species, such as an
 antibody or antibody fragment which specifically binds to a CAA
 polypeptide, e.g., a CAA-1-specific antibody. Antibodies specific for a
 CAA polypeptide are useful to for tissue typing and for therapeutic
 applications, e.g., to inhibit the activity of CAA-1.
 Compounds capable of inhibiting expression of a CAA polypeptide may be
 therapeutically useful to treat conditions, e.g., rheumatoid arthritis,
 associated with undesired or pathologic joint inflammation. Accordingly,
 the invention includes a method of screening a candidate compound to
 identify a compound capable of inhibiting expression of a CAA polypeptide,
 e.g., CAA-1, by (a) providing a cell expressing a CAA polypeptide, (b)
 contacting the cell with the candidate compound, and (c) determining the
 amount of expression of the CAA polypeptide by the cell. A decrease in the
 amount of CAA polypeptide expression in the presence of the candidate
 compound compared to that in the absence of the candidate compound
 indicates that the compound inhibits expression of the CAA polypeptide. A
 method of identifying a compound which inhibits the activity of CAA-1 can
 be carried out as follows: (a) providing a cell expressing a CAA
 polypeptide, (b) contacting the cell with the candidate compound, and (c)
 determining the amount of HLA II expression by the cell. A decrease in the
 amount of HLA II expression in the presence of the candidate compound
 compared to that in the absence of the candidate compound indicates that
 the compound inhibits HLA II expression in the cell.
 Methods of treating undesired inflammation such as that associated with
 rheumatoid arthritis and other inflammatory arthropathies are also within
 the invention. Such a method may be carried out by administering to a
 mammal in need of such therapy, e.g. a patient suffering from rheumatoid
 arthritis or other inflammatory arthropathies, an effective amount of a
 CAA-1 polypeptide. For example, the peptide is administered locally at the
 site of a rheumatoid lesion to reduce local inflammation and swelling.
 The invention also includes a non-human transgenic mammal that expresses
 human CAA-1 and non-human transgenic mammal with a null mutation in its
 endogenous CAA-1 gene.

Other features and advantages of the invention will be apparent from the
 following description of the preferred embodiments thereof, and from the
 claims.
 DETAILED DESCRIPTION
 To date, no consistent genetic abnormality has been associated with
 chondrosarcoma. The tumors are heterogeneous and often have a number of
 abnormalities in gene expression. The following examples provide evidence
 of a novel gene, csa-1, that is expressed in a human chondrosarcoma cell
 line and in cartilaginous neoplasms but not in normal cartilage. The level
 of expression of a CSA-1 polypeptide correlates with the histological
 grade of the neoplasm, i.e., an increase in expression indicates a higher
 grade tumor. Detection of a CSA-1 gene product or csa-1 transcript is a
 means by which to distinguish a neoplastic cell from a normal cartilage
 cell.
 CSA polypeptides and CAA polypeptides may be used therapeutically. For
 example, a CAA polypeptide such as CAA-1 may function as a tumor
 suppressor. CAA-1 can also be administered to patients to reduce undesired
 inflammation such as joint inflammation in rheumatoid arthritis.
 CSA-1 plays a role in potentiating chondrogenesis associated with
 chondrosarcoma. Transfecting the nonexpressing or normal cell lines with
 vectors which promote high levels of expression of a CSA polypeptide,
 e.g., CSA-1, followed by transformation of a low grade cell line to a high
 grade cell line indicates that CSA expression potentiates neoplastic
 growth. Inhibitors of CSA-1 expression can slow or inhibit neoplastic
 growth. Transformation and grading of transformed cells is evaluated by
 examining changes in morphology, proliferation, adhesion, and
 invasiveness.
 Two novel genes have been cloned. CSA-1 is expressed in a tumor cell line
 and also in some high grade chondrosarcoma, but not normal cartilage, or
 low, or intermediate grade tumors. A second gene, CAA-1, is expressed in
 normal cartilage and an intermediate grade tumor cell line, and as
 alternative sized messages in a high grade cell line.
 EXAMPLE 1
 Cloning of csa Genes
 Chondrosarcoma cell lines derived from human grade I, II and III
 chondrosarcomas were alternately cultured in monolayer cultures and in
 agarose suspension cultures using standard methods. Total cellular RNA was
 isolated using standard techniques. Three different human chondrosarcoma
 cell lines (AQ, stage II tumor; FS, stage II tumor; and MW, stage I tumor)
 and normal articular cartilage obtained from amputation specimens was
 analyzed.
 Using the differential mRNA display technique, a technique that
 systematically amplifies mRNAs by means of RT-PCR with different sets of
 5' arbitrary primers and 3' oligo-dT anchoring primers, the mRNA patterns
 of different cells and cell types were compared. The PCR products were
 resolved on a denaturing polyacrylamide sequencing gel to display mRNA
 patterns that distinguish one cell type from another. The bands that were
 separated by gel electrophoresis represent the 3'-termini of the cDNAs.
 Therefore, a band that is present in one cell type, e.g., a chondrosarcoma
 cell line or chrondrosarcoma biopsy tissue, but not in the normal
 cartilage tissue or in noncartilage tissue, suggests that the gene is
 differentially expressed in chondrosarcomas.
 cDNA was generated using reverse transcriptase and an oligo-dT primer
 (TTTTTTTTTTTTMN (SEQ ID NO:4), where M can be C, G, or A; N can be C, G,
 A, or T). A PCR reaction was then carried out in triplicate with the same
 oligo-dT primer and a second random ten base pair primer (RNAmap,
 GenHunter Corp., Brookline, Mass.). This combination of primers amplified
 approximately 100 cDNAs from 100-500 base pairs long. The cDNAs were
 separated on a sequencing gel. A band that is present in one or more of
 the cancer cell lines and absent in a normal cell may represent an
 oncogene which is being expressed in the cancer cell but not the normal
 cell. Conversely, a band that is absent in one or more of the cancer cell
 lines and present in a normal cell may represent a tumor suppressor gene
 which is not being expressed because of a mutation or regulatory defect.
 The cDNA of a differentially expressed mRNA was eluted from the sequencing
 gel and reamplified in a PCR reaction with the same primers as was used in
 the differential display reaction and cloned into a pCR.TM. II vector
 (Invitrogen, San Diego, Calif.). Specific mRNAs that were present solely
 in chondrosarcoma cells were identified and the corresponding cDNAs
 cloned. cDNAs were sequenced using the M13 forward and reverse sequencing
 primers, which flank the cloning site of the pCR.TM. vector. Some
 sequences were identical to known genes, e.g., cyclin D2, a cell cycle
 regulatory protein, and PTX3, a member of the pentaxin gene family. Novel
 cDNAs, i.e., those without sequence similarity to known genes, were used
 for Northern blotting to confirm that the corresponding gene is
 differentially expressed in chondrosarcoma cells. Twenty such cDNA probes
 were used to screen for differential expression of mRNA and sequenced
 (TABLE 1). A novel gene, csa-1, was found to be differentially expressed
 in chondrosarcoma cells compared to normal cartilage cells and other cell
 types such as breast, lung, and colon cells.
 TABLE 1
 Clone Gel Type MW(I) FS(II) AQ(III) NI Cart
 FS1 DD - + - -
 N3-0 - + + -
 N3-1 - + + +
 FS2 DD - + - -
 E1 DD - - - +
 N3-2 - + + +
 N3-3 - + + -
 FS3 DD + + + -
 AQ1 DD - - + -
 E2 DD - - - +
 AQ3 DD - - + -
 E6 DD - - - +
 AQ2 DD - - + -
 E7 DD - - - +
 FS10 DD + + + -
 FS11 DD + + + -
 AQ6 DD - - + +
 FS8 DD + + - +
 MW1 DD + - - -
 MW2 DD + - - -
 MW3 DD + - - -
 FS9 DD - + + +
 FS10 DD - + - -
 AQ5 DD - + + +
 DD: Differential Display Gel
 N: Northern Blot
 Cloning of csa-1
 The gene encoding CSA-1 was identified using differential display PCR as
 described above. FS8 is a probe corresponding to one of the differentially
 expressed sequences identified. As shown in TABLE 1, the differential
 display gel from which probe FS8 was isolated indicated that this gene was
 not expressed in the AQ cell line. In a Northern blot assay, the probe
 hybridized to a message approximately 0.85 kb in size in the FS cell line
 as well as in a high grade chondrosarcoma. No message was detected in
 normal cartilage, bovine growth plate, or grade 1 or 2 chondrosarcoma.
 The FS8 probe (specific for CSA-1) which corresponded to the 3' end of the
 csa-1 gene was found to be 250 bases long. 5' Rapid Amplification of cDNA
 (5' RACE) was used to clone the full length gene. A gene specific primer
 was synthesized which is complementary to the probe was made and used as a
 primer to synthesize cDNA using RNA from the FS cell line as a template.
 The RNA was digested away with Rnase H, and an anchor primer was added to
 the 3' end with TdT and dCTP. PCR was performed using the 3' anchor primer
 and a second, nested gene specific primer, thereby yielding double
 stranded DNA which is an extension of the gene fragment from which the
 differential display probe was derived. The 5'RACE generated fragment was
 cloned and sequenced.
 Expression of CSA-1 in chondrosarcoma cells was localized to the nucleus of
 the cells by immunostaining using a rabbit polyclonal antibody specific
 for a CSA-1 polypeptide.
 The sequence of the full length csa-1 cDNA (TABLE 2) was found to have an
 open reading frame (ORF) (TABLE 3 and shown in bold in TABLE 2) encoding a
 fifty-two amino acid gene product, CSA-1 (TABLE 4).
 TABLE 2
 CSA-1 cDNA
 ACTTCCCTGGGTTCACAGCAGGGGTGGAACTGGATTCTTCCTGGATGGGGATCCAGATGG (SEQ ID
 NO:3)
 AGGTGGAGCTGCACCCCTTGTAGAGAATGGCTGCGGGTCCCAGGCCAGGAGCTCCCTGCA
 GGGCGGGGGCTCCCACGATCGTATTGACCTCTGGAAGAAGACAGACACTTTCCCACGGGA
 GCTCCTCTCCAGCCAGAGCTACACTTGGCAAACCTTTGGTCCTAAATGATTATTCACTGA
 ATTGAAGAAATACGGTTTACATATCTTCCAAGTATATATGTAGGGTTGATTTGGGAAGCA
 GAACACAGCAGCCCAAATTTGCTTGTAATGTCTGCGACTACAGCCTGCTGGCCTGCCTTC
 ACTGTCTTGGGGGAAGCTCGGGGAGACCAGGTGGACTGGAGTAGACTGTGCAGAGACACT
 GGTCTGGTGAAGATGTCCAGGAAACCACGAGCCTCCAGCCCATTTTCCAACAACCACCCA
 TCAACACCAAAGAGGTTCCCAAGACAACCCAGAAGGGAAAAGGGACCCGTCAAGGAAGTT
 CCAGGAACAAAAGGCTCTCCCTAAAAGACCACCGCTTCAAAAAAACCTGAGGAATGGAGT
 GGGCCAACACTATCCAGCCACTCTGACCAGCCGAACGAGGAACTCAATCAAAATGCGCCA
 TAGCAGGACCACAAGGGCAAGGAGACCACCGCCTTCTCCAGTGCTTCCTTGGGCAGCCAG
 TAATTCCCAGGCAAGGCCAGAGACTTCAAGTCTATCTGAAAAGTCTCCAGAAGTCTAACC
 CCAGATAAATAGCCAACAGGGTGTAGAGTACGTTTTACACCCAAAGGGTAATGCCCCATG
 GTGATGGAAATAAAATGAACATGTTGTAAAATGAAAAAAAAAAA
 TABLE 3
 CSA-1 coding sequence
 ATGGCTGCGGGTCCCAGGCCAGGAGCTCCCTGCAGGGCGGGGGCTCCCACGATCGTATTG (SEQ ID
 NO:1)
 ACCTCTGGAAGAAGACAGACACTTTCCCACGGGAGCTCCTCTCCAGCCAGAGCTACACTT
 GGCAAACCTTTGGTCCTAAATGATTATTCACTGAATTGAAGAAA
 TABLE 4
 CSA-1 AMINO ACID SEQUENCE
 MAAGPRPGAPCRAGAPTIVLTSGRRQTLSHGSSSATLGKPLVLNDYSLN (SEQ ID NO:2)
 Cloning of caa-1
 The gene encoding CAA-1 was also identified using differential display PCR
 as described above. E1 is a probe corresponding to one of the
 differentially expressed sequences identified. As shown in TABLE 1, E1 was
 expressed in normal cartilage, but not in any of the human chondrosarcoma
 cell lines tested by differential display PCR. A northern blot with probe
 E1 showed expression of a 2.2 kb message in normal articular cartilage and
 the FS cell line. In contrast, 2 alternative sized transcripts (7.5 kb and
 1.2 kb) were detected in the high grade cell line AQ. These data indicate
 that the caa-1 gene may be alternatively spliced or rearranged in the AQ
 cell line. The pattern of expression indicates that CAA-1 functions as a
 tumor suppressor gene.
 Expression of CAA-1 expression was analyzed using Northern blotting and
 RT-PCR in human chondrosarcoma cell lines osteoblasts, normal cartilage,
 muscle, primary chondrosarcoma and a panel of tumors and normal tissues.
 Northern blotting was performed with E1 and with a 1.5 kb 5' RACE
 generated portion of the gene as probes. PCR was performed using primers
 which amplify 306 bp of the caa gene.
 Differential display of mRNA showed expression of a gene in normal
 cartilage and in the FS and AQ cell lines (but not in the MW cell line).
 The differential display probe (E1) was sequenced, found to be novel, and
 used for Northern blot analysis, which revealed an estimated message size
 of 2.2 kb in normal cartilage and FS, but not MW, osteoblast, muscle or
 bovine growth plate.
 5' RACE was performed using oligonucleotide primers complementary to the 5'
 end of clone E1 and yielded a 1.5 kb fragment. In order to obtain the
 remaining 5' portion of the gene, 5' RACE was repeated with new 5' primers
 and an additional 0.5 kb fragment was cloned, and the overlapping gene
 fragments were sequenced.
 Northern blotting was performed with the 1.5 kb fragment and a 2.0 kb
 message was detected in four different samples of FS RNA and in a grade II
 chondrosarcoma (CS) and FS RNA. The full length gene is 1955 nucleotides
 in length, which correlates with the message size seen with Northern
 blotting.
 TABLE 5
 CAA-1 cDNA
 CACGCAAAGCAGTGTGGGTTGATTCTGAGGTGCACTGTGGGAAAGAGCTTGTCGCTGCGG (SEQ ID
 NO:5)
 TGTTGCTGTTGGAGACTCGATTGTTGGTGACAGCGAAAGAACGATAACAAAATGCCGGAG
 CGAGATAGTGAGCCGTTCTCCAACCCTTTGGCCCCCGATGGCCACGATGTGGATGATCCT
 CACTCCTTCCACCAATCAAAACTCACCAATGAAGACTTCAGGAAANTNNTCATGACCCCC
 AGGGNTGCACNTACNTNTGCACCACNTTNTAANTNNNNTCACCATGAGATGCCAAGGGAG
 TACAATGAGGATGAAGACCCAGCTGCACGAAGGAGGAAAAAGAAAAGTTATTATGCCAAG
 CTACGCCAACAAGAAATTGAGAGAGAGAGAGAGCTAGCAGAGAAGTACCGGGATCGTGCC
 AAGGAACGGAGAGATGGAGTGAACAAAGATTATGAAGAAACCGAGCTTATCAGCACCACA
 GCTAACTATAGGGCTGTTGGCCCCACTGCTGAGGCGGACAAATCAGCTRCAGNNRAGAGA
 AGACANWNDAHCNAGGAGTCCAAATTCTTGGGTGGTGACATGGAACACACCCATTTGGTG
 AAAGGCTTGGATTTTGNTNTGCTTCHNAANGTNCGAGCTGAGATTGNCMSCMNANARAAA
 NARGAARANGNNCTGATGGNAAANCCCCMGAAAGAAACCAAGAAAGATGAGGATCCTGAA
 AATAAAATTGAATTTAAAACACGTCTGGGCCGCAATGTTTACCGAATGCTTTTTAAGAGC
 AAAGCATATGAGCGGAATGAGTTGTTCCTGCCGGGCCGCATGGCCTATGTGGTAGACCTG
 GATGATGAGTATGCTGACACAGATATCCCCACCACTCTTATCCCGCAGCAAGGCTGATTG
 CCCCACCATGGAGGCCCAGACCACACTGACCACAAATGACATTGTCATTAGCAAGCTGAC
 CCAGATCCTTTCATACCTGAGGCAGGGAACCCGTAACAAGAAGCTTAAGAAGAAGGATAA
 AGGGAAGCCGGAAGAGAAGAAACCTCCTGAGGCTGACATGAATATTTTTGAAGACATTGG
 GGATTACGTACCCTCCACAACCAAGACACCTCGGGACAAGGAGCGGGAGAGATATCGGGA
 ACGGGAGCGTGATCGGGAAAGAGACAGAGACCGTGACCGAGAGCGAGAGCGAGAACGAGA
 TCGGGAACGAGAGCGAGAGCGGGACCGAGAGAGAGAAGAGGAAAAGAAGAGACACAGCTA
 CTTTGAGAAGCCAAAAGTAGATGATGAGCCCATGGACGTTGACAAAGGACCTGGGTCTAC
 CAAGGAGTTGATCAAGTCCATCAATGAAAAGTTTGCTGGGTCTGCTGGCTGGGAAGGCAC
 AGAATCGCTGAAGAAGCCAGAAGACAAAAAGCAGCTGGGAGATTTCTTTGGCATGTCCAA
 CAGTTATGCAGAGTGCTACCCAGCCACGATGGATGACATGGCTGTGGATAGTGATGAGGA
 GGTGGATTATAGCAAAATGGACCAGGGTAACAAGAAGGGGCCCTTAGGCCGTTGGGACTT
 TGATACCCAGGAAGAATACAGCGAGTATATGAACAACAAAGAAGCTTTGCCCAAGGCTGC
 ATTCCAGTATGGTATCAAAATGTCTGAAGGGCGGAAAACCAGGCGCTTCAAGGAAACCAA
 TGACAAAGCAGAGCTTGATCGCCAGTGGAAGAAGATTAGTGCAATCATTGANGAAGAGGA
 AGAAGATGGAAGCTGATGGGGTTGAAGTCAAAAGACCAAAATACTAATCACTAGTTACAA
 CCAGAGATGCTCCACAAGGATATGCTCCCCACTGTTTTCTTTCTACAATTTCCAAAGGTT
 GCAAGATGTTTTTTTGTGATGAATATAAAATTTTATTGTGTAATTACTTGGTTCCATTAA
 AATTGGTTAACTTGCTAAAAAAAAAA
 TABLE 6
 CAA-1 Coding Sequence
 ATGATGAGTATGCTGACACAGATATCCCCACCACTCTTATCCCGCAGCAAGGCTGATTGC (SEQ ID
 NO:6)
 CCCACCATGGAGGCCCAGACCACACTGACCACAAATGACATTGTCATTAGCAAGCTGACC
 CAGATCCTTTCATACCTGAGGCAGGGAACCCGTAACAAGAAGCTTAAGAAGAAGGATAAA
 GGGAAGCCGGAAGAGAAGAAACCTCCTGAGGCTGACATGAATATTTTTGAAGACATTGGG
 GATTACGTACCCTCCACAACCAAGACACCTCGGGACAAGGAGCGGGAGAGATATCGGGAA
 CGGGAGCGTGATCGGGAAAGAGACAGAGACCGTGACCGAGAGCGAGAGCGAGAACGAGAT
 CGGGAACGAGAGCGAGAGCGGGACCGAGAGAGAGAAGAGGAAAAGAAGAGACACAGCTAC
 TTTGAGAAGCCAAAAGTAGATGATGAGCCCATGGACGTTGACAAAGGACCTGGGTCTACC
 AAGGAGTTGATCAAGTCCATCAATGAAAAGTTTGCTGGGTCTGCTGGCTGGGAAGGCACA
 GAATCGCTGAAGAAGCCAGAAGACAAAAAGCAGCTGGGAGATTTCTTTGGCATGTCCAAC
 AGTTATGCAGAGTGCTACCCAGCCACGATGGATGACATGGCTGTGGATAGTGATGAGGAG
 GTGGATTATAGCAAAATGGACCAGGGTAACAAGAAGGGGCCCTTAGGCCGTTGGGACTTT
 GATACCCAGGAAGAATACAGCGAGTATATGAACAACAAAGAAGCTTTGCCCAAGGCTGCA
 TTCCAGTATGGTATCAAAATGTCTGAAGGGCGGAAAACCAGGCGCTTCAAGGAAACCAAT
 GACAAAGCAGAGCTTGATCGCCAGTGGAAGAAGATTAGTGCAATCATTGANGAAGAGGAA
 GAAGATGGAAGCTGA
 TABLE 7
 CAA-1 Amino Acid Sequence
 Met Met Ser Met Leu Thr Gln Ile Ser Pro Pro Leu Leu Ser Arg (SEQ ID
 NO:7)
 Ser Lys Ala Asp Cys Pro Thr Met Glu Ala Gln Thr Thr Leu Thr
 Thr Asn Asp Ile Val Ile Ser Lys Leu Thr Gln Ile Leu Ser Tyr
 Leu Arg Gln Gly Thr Arg Asn Lys Lys Leu Lys Lys Lys Asp Lys
 Gly Lys Pro Glu Glu Lys Lys Pro Pro Glu Ala Asp Met Asn Ile
 Phe Glu Asp Ile Gly Asp Tyr Val Pro Ser Thr Thr Lys Thr Pro
 Arg Asp Lys Glu Arg Glu Arg Tyr Arg Glu Arg Glu Arg Asp Arg
 Glu Arg Asp Arg Asp Arg Asp Arg Glu Arg Glu Arg Glu Arg Asp
 Arg Glu Arg Glu Arg Glu Arg Asp Arg Glu Arg Glu Glu Glu Lys
 Lys Arg His Ser Tyr Phe Glu Lys Pro Lys Val Asp Asp Glu Pro
 Met Asp Val Asp Lys Gly Pro Gly Ser Thr Lys Glu Leu Ile Lys
 Ser Ile Asn Glu Lys Phe Ala Gly Ser Ala Gly Trp Glu Gly Thr
 Glu Ser Leu Lys Lys Pro Glu Asp Lys Lys Gln Leu Gly Asp Phe
 Phe Gly Met Ser Asn Ser Tyr Ala Glu Cys Tyr Pro Ala Thr Met
 Asp Asp Met Ala Val Asp Ser Asp Glu Glu Val Asp Tyr Ser Lys
 Met Asp Gln Gly Asn Lys Lys Gly Pro Leu Gly Arg Trp Asp Phe
 Asp Thr Gln Glu Glu Tyr Ser Glu Tyr Met Asn Asn Lys Glu Ala
 Leu Pro Lys Ala Ala Phe Gln Tyr Gly Ile Lys Met Ser Glu Gly
 Arg Lys Thr Arg Arg Phe Lys Glu Thr Asn Asp Lys Ala Glu Leu
 Asp Arg Gln Trp Lys Lys Ile Ser Ala Ile Ile Xaa Glu Glu Glu
 Glu Asp Gly Ser
 The longest predicted open reading frame (ORF) is 942 bp. This ORF begins
 with the second in frame start codon, and is preceded by a shorter, 126 bp
 ORF. The predicted protein for the long ORF is 314 amino acids with a
 molecular weight of 37 kDa. The estimated message was 2.1 kb, but only 756
 bp of sequence was reported, which was the largest clone isolated form
 their cDNA library.
 Expression of CAA-1 has been detected with PCR in 4/5 normal cartilage
 specimens, 1/2 grade 0, 3/3 grade I, 3/4 grade II, and 5/5 grade III
 chondrosarcoma. Expression as not detected in colon, breast, renal cell,
 and gastric carcinoma and corresponding normal tissues; osteogenic and
 soft tissue sarcoma; and giant cell tumor.
 CAA-1 functions to regulate an immune response. Regulation of an immune
 response, e.g., inflammation, is critical for normal synovial joint
 physiology and tumor surveillance. HLA class II antigens are necessary for
 antigen presentation to T cells, and interferon gamma has been shown to
 upregulate HLA class II expression in many different normal and tumor
 cells, including chondrocytes and synovial lining cells. In addition,
 chondrocytes have been shown to function as antigen presenting cells.
 CAA-1 functions as a cytokine which inhibits the interferon gamma induced
 upregulation of HLA class II antigens. Thus, chondrocytes express a gene
 which modulates its own ability, as well as cells in surrounding synovium,
 to function as antigen presenting cells. Treating a synovial joint with a
 CAA-1 polypeptide decreases the expression of HLA II antigens. Thus, a
 CAA-1 polypeptide can be administered locally to reduce pathological such
 as that associated with rheumatoid arthritis and other inflammatory
 arthropathies.
 CAA-1 is also expressed in neoplastic cartilage. CAA-1 inhibits interferon
 gamma induced upregulation of HLA Class II. Expression of CAA-1 by tumor
 cells may be a mechanism of escape from immunorecognition, i.e. increased
 CAA-1 expression diminishes the ability of the host to control tumor
 growth through immunologic mechanisms. Treatment of chondrosarcoma by
 inhibiting the expression of CAA-1 or function of the CAA-1 gene product
 enhances the ability of the host immune system to control tumor growth
 through immunologic mechanisms.
 EXAMPLE 2
 Production and Purification of Recombinant CSA and CAA Polypeptides
 To produce recombinant polypeptides, DNA encoding a CSA or CAA polypeptide
 in an appropriate expression vector is transfected into a cell. Standard
 methods for transfecting cells with isolated nucleic acid are well known
 to those skilled in the art of molecular biology. For example, prokaryotic
 or eukaryotic cells in culture can be transfected with the DNA of the
 invention operatively linked to expression control sequences appropriate
 for high-level expression in the cell. Such cells are useful for producing
 large amounts of the CSA-1 or CAA-1, which can be purified and used, e.g.,
 as a therapeutic or for raising anti-CSA-1 or anti-CAA-1 antibodies.
 For example, the recombinant gene product may be expressed as a fusion
 protein and purified using a commercially available expression and
 purification system, e.g., the pFLAG expression system (IBI). Recombinant
 polypeptides are injected into a rabbit or rodent to produce antibodies as
 described below.
 EXAMPLE 3
 Production of Antibodies Specific for CSA or CAA Polypeptides
 Antibodies specific for CSA polypeptides can be obtained by techniques well
 known in the art. Such antibodies can be polyclonal or monoclonal.
 Polyclonal antibodies can be obtained, for example, by the methods
 described in Ghose et al., Methods in Enzymology, Vol. 93, 326-327, 1983.
 For example, a CSA-1 polypeptide (containing 23-24 amino acids, e.g., a
 polypeptide containing RRQTLSHGSSSAC (SEQ ID NO:8) was used as an
 immunogen to stimulate the production of CSA-1-reactive polyclonal
 antibodies in the antisera of a rabbit. Similar methods can be used to
 raise antisera in animals such as goats, sheep, and rodents.
 Monoclonal antibodies useful in the present invention can be obtained by
 the well known process described by Milstein and Kohler in Nature,
 256:495-97, 1975, or as modified by Gerhard, Monoclonal Antibodies, Plenum
 Press, 1980, pages 370-371. Hybridomas are screened to identify those
 producing antibodies that are highly specific for a CSA polypeptide.
 Preferably, the antibody will have an affinity of at least about 10.sup.8
 liters/mole and more preferably, an affinity of at least about 10.sup.9
 liters/mole. The use of such monoclonal antibodies provides a means of
 obtaining greater sensitivity in the assays of the present invention
 compared with the use of polyclonal antibodies.
 EXAMPLE 4
 Transgenic Animals
 CSA polypeptides can also be expressed in transgenic animals. These animals
 represent a model system for the study of disorders that are caused by or
 exacerbated by overexpression or underexpression of a CSA polypeptide, and
 for the development of therapeutic agents that modulate the expression or
 activity of a CSA polypeptide.
 A CSA-1 knockout animal is useful to study CSA-1 function. Immunostaining
 of mouse embryos with anti-CSA-1 antibody showed staining of the
 musculoskeletal precursor. A csa-1 transgene with a null mutation results
 in an animal which does not express the CSA-1 gene, a condition which may
 lead to developmental abnormalities since some genes expressed by tumors
 are also expressed during normal embryological development.
 Alternatively, a transgenic animal overexpressing the CSA-1 is useful to
 study the development chondrosarcoma. Such an animal would be a useful
 tool for evaluating treatment of this tumor.
 Transgenic animals can be farm animals (pigs, goats, sheep, cows, horses,
 rabbits, and the like) rodents (such as rats, guinea pigs, and mice),
 non-human primates (for example, baboons, monkeys, and chimpanzees), and
 domestic animals (for example, dogs and cats). Transgenic mice are
 especially preferred.
 Any technique known in the art can be used to introduce a csa-1 transgene
 into animals to produce the founder lines of transgenic animals. Such
 techniques include, but are not limited to, pronuclear microinjection
 (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ
 lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148, 1985);
 gene targeting into embryonic stem cells (Thompson et al., Cell 56:313,
 1989); and electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803, 1983).
 When it is desired that the csa-1 transgene be integrated into the
 chromosomal site of the endogenous csa-1 gene, gene targeting is
 preferred. Briefly, when such a technique is to be used, vectors
 containing some nucleotide sequences homologous to an endogenous csa-1
 gene are designed for the purpose of integrating, via homologous
 recombination with chromosomal sequences, into and disrupting the function
 of the nucleotide sequence of the endogenous gene. The transgene also can
 be selectively introduced into a particular cell type, thus inactivating
 the endogenous csa-1 gene in only that cell type (Gu et al., Science
 265:103, 1984). The regulatory sequences required for such a cell-type
 specific inactivation will depend upon the particular cell type of
 interest, and will be apparent to those of skill in the art. These
 techniques are useful for preparing "knock outs" having no functional csa
 or caa gene.
 Once transgenic animals have been generated, the expression of the
 recombinant transgene can be assayed utilizing standard techniques.
 Initial screening may be accomplished by Southern blot analysis or PCR
 techniques to determine whether integration of the transgene has taken
 place. The level of mRNA expression of the transgene in the tissues of the
 transgenic animals may also be assessed using techniques which include,
 but are not limited to, Northern blot analysis of tissue samples obtained
 from the animal, in situ hybridization analysis, and RT-PCR. Samples of
 csa or caa gene-expressing tissue can also be evaluated
 immunocytochemically using antibodies specific for the transgene product.
 For a review of techniques that can be used to generate and assess
 transgenic animals, skilled artisans can consult Gordon (Intl. Rev. Cytol.
 115:171-229, 1989), and may obtain additional guidance from, for example:
 Hogan et al. "Manipulating the Mouse Embryo" (Cold Spring Harbor Press,
 Cold Spring Harbor, N.Y., 1986; Krimpenfort et al., Bio/Technology 9:86,
 1991; Palmiter et al., Cell 41:343, 1985; Kraemer et al., "Genetic
 Manipulation of the Early Mammalian Embryo," Cold Spring Harbor Press,
 Cold Spring Harbor, N.Y., 1985; Hammer et al., Nature 315:680, 1985;
 Purcel et al., Science, 244:1281, 1986; Wagner et al., U.S. Pat. No.
 5,175,385; and Krimpenfort et al., U.S. Pat. No. 5,175,384.
 EXAMPLE 5
 Diagnosis of Chondrosarcoma
 The invention includes a method of detecting cartilaginous neoplasms in a
 sample of patient-derived tissue. Detection of csa-1 expression (by
 measuring gene transcripts or gene products) in a patient sample compared
 to a control sample or CSA-1 polypeptide would predict chondrogenesis
 indicative of, e.g., chondrosarcoma. The diagnostic method of the
 invention is carried out by measuring csa gene expression in a tissue,
 e.g, a biopsy, or in a bodily fluid, e.g., blood or plasma. Detection of
 expression and determination of the level of gene expression is measured
 using methods known in the art, e.g., in situ hybridization, Northern blot
 analysis, or Western blot analysis using CSA-1-specific monoclonal or
 polyclonal antibodies. An increase in the level of csa-1 expression per
 cell in the test sample of tissue compared to the level per cell in
 control tissue indicates the presence of a chondrosarcoma in the test
 sample. For example, tissue obtained at an biopsy could be tested for
 CSA-1 expression, e.g., the level of CSA-1 transcript or polypeptide. An
 increased level of CSA-1 transcript or polypeptide (compared to normal
 tissue) indicates a high probability of chondrosarcoma. For example, PCR
 was used to detect expression csa-1 in 15 patient-derived chondrosarcoma
 biopsy samples. In contrast, no csa-1 expression was detected in 3
 patient-derived normal control samples.
 The methods described above can also be used to determine the grade of a
 tumor. Northern blotting and quantitative PCR techniques are used to
 determine the level of expression of csa gene expression. For example,
 elevated CSA-1 expression correlates with a higher grade of tumor.
 The diagnostic procedures described above are useful to identify patients
 in need of therapeutic intervention to reduce or prevent chondrosarcoma.
 EXAMPLE 6
 Methods of Therapy
 Patients with chondrosarcoma can be treated by administering CSA-1
 antisense nucleic acids or ribozymes. Other malignant conditions, which
 are characterized by a increase in CSA-1 expression may be treated in a
 similar manner.
 Antisense therapy is used to inhibit expression of proteins, e.g., CSA-1,
 involved in chondrogenesis, e.g., that associated with chondrosarcoma. For
 example, an antisense strand of csa-1 (either RNA or DNA) is directly
 introduced into the cells in a form that is capable of binding to the mRNA
 transcripts. Alternatively, a vector-containing sequence which, which once
 within the target cells is transcribed into the appropriate antisense
 mRNA, may be administered. Antisense nucleic acids which hybridize to mRNA
 can decrease or inhibit production of the polypeptide product encoded by a
 gene by associating with the normally single-stranded mRNA transcript,
 thereby interfering with translation and thus, expression of the protein.
 Ribozyme therapy can also be used to inhibit gene expression. Ribozymes
 bind to specific mRNA and then cut it at a predetermined cleavage point,
 thereby destroying the transcript. These RNA molecules may be used to
 inhibit expression of a csa gene involved in chondrogenesis associated
 with chondrosarcoma according to methods known in the art (Sullivan et
 al., 1994, J. Invest. Derm. 103:85S-89S; Czubayko et al., 1994, J. Biol.
 Chem. 269:21358-21363; Mahieu et al, 1994, Blood 84:3758-65; Kobayashi et
 al. 1994, Cancer Res. 54:1271-1275).
 Another therapeutic approach to inhibiting the expression of proteins or
 polypeptides is the production of intracellularly expressed antibodies
 which, when expressed in a cell, bind to and prevent the transport and
 surface expression of target proteins. Intracellular antibodies may be
 expressed in a cell using known techniques (Chen et al., 1994, Hum. Gene
 Ther. 5:595-601).
 Gene therapy may be carried out by administering to a patient a nucleic
 acid encoding a therapeutic polypeptide, e.g., a tumor suppressor gene
 such as caa-1, by standard vectors and/or gene delivery systems. Suitable
 gene delivery systems may include liposomes, receptor-mediated delivery
 systems, naked DNA, and viral vectors such as herpes viruses,
 retroviruses, adenoviruses and adeno-associated viruses, among others.
 As is discussed above, undesired or pathological inflammation such as that
 associated with rheumatoid arthritis and other inflammatory arthropathies
 can be treated by inhibiting CAA-1 expression. Antisense therapy and
 ribozyme therapy can be used to inhibit CAA-1 expression, and
 intracellular immunization using DNA encoding an anti-CAA-1 antibody can
 be used to inhibit function of the gene product.
 A therapeutic composition may include one or more compounds, e.g., nucleic
 acids or immunosuppressive agents, and a pharmaceutically acceptable
 carrier. The therapeutic composition may also include a gene delivery
 system as described above. Pharmaceutically acceptable carriers are
 biologically compatible vehicles which are suitable for administration to
 an animal: e.g., physiological saline. A therapeutically effective amount
 of a compound is an amount which is capable of producing a medically
 desirable result in a treated animal, e.g., inhibition of expression of a
 target gene, e.g., a cell surface or secreted protein, or inhibition of
 cell activity, e.g., proliferation, migration, antigen presentation,
 antibody production, or cytokine production.
 Parenteral administration, such as intravenous, subcutaneous,
 intramuscular, and intraperitoneal delivery routes, may be used to deliver
 the compound, with intravenous administration being the preferred route.
 Dosages for any one patient depends upon many factors, including the
 patient's size, body surface area, age, the particular compound to be
 administered, sex, time and route of administration, general health, and
 other drugs being administered concurrently. Dosages of the compound to be
 administered will vary (doses of immunosuppressive agents are expected to
 be in the range of doses used for administration of other
 immunosuppressive agents known in the art). A preferred dosage for
 intravenous administration of nucleic acids is from approximately 10.sup.6
 to 10.sup.22 copies of the nucleic acid molecule. Alternatively, the
 compound may be administered via a timed-release implant placed in close
 proximity to diseased tissue or a surgical site after removal of
 neoplastic tissue.
 CSA polypeptides or CAA polypeptides, e.g., a CAA-1 polypeptide, may be
 administered to the patient intravenously in a pharmaceutically acceptable
 carrier such as physiological saline. Standard methods for intracellular
 delivery of peptides can be used, e.g. packaged in liposomes. Such methods
 are well known to those of ordinary skill in the art. It is expected that
 an intravenous dosage of approximately 1 to 100 .mu.moles of the
 polypeptide of the invention would be administered per kg of body weight
 per day. The compositions of the invention are useful for parenteral
 administration, such as intravenous, subcutaneous, intramuscular, and
 intraperitoneal. Alternatively, a CAA polypeptide, e.g., CAA-1, can be
 administered as an implant for slow release at the site of an inflammatory
 lesion.
 DNA (csa-1 encoding DNA, tumor cell-specific promoters, and vectors) of the
 invention may be introduced into target cells of the patient by standard
 vectors and/or gene delivery systems. Suitable gene delivery systems may
 include liposomes, receptor-mediated delivery systems, naked DNA, and
 viral vectors such as herpes viruses, retroviruses, and adenoviruses,
 among others. For example, the DNA of the invention under the control of a
 strong constitutive promoter may be administered locally using an
 adenovirus delivery system.
 The DNA of the invention may be administered in a pharmaceutically
 acceptable carrier. The therapeutic composition may also include a gene
 delivery system as described above. Pharmaceutically acceptable carriers
 are biologically compatible vehicles which are suitable for administration
 to an animal e.g., physiological saline. A therapeutically effective
 amount is an amount of the nucleic acid of the invention which is capable
 of producing a medically desirable result in a treated animal.
 As is well known in the medical arts, dosage for any given patient depends
 upon many factors, including the patient's size, body surface area, age,
 the particular compound to be administered, sex, time and route of
 administration, general health, and other drugs being administered
 concurrently. Dosages for the compounds of the invention will vary, but a
 preferred dosage for intravenous administration is from approximately
 10.sup.6 to 10.sup.22 copies of the nucleic acid molecule. Determination
 of optimal dosage is well within the abilities of a pharmacologist of
 ordinary skill. Drugs which inhibit the CSA-1 promoter may also be
 administered as described above to decrease the level of expression CSA-1
 in tissues.
 EXAMPLE 7
 Identification of Compounds that Decrease csa or caa Gene Expression
 A method of screening candidate compounds to identify compounds capable of
 inhibiting csa gene, e.g. csa-1, expression includes the following steps:
 providing a chondrosarcoma cell; contacting the cell with a candidate
 compound; and determining the amount of csa-1 expression in the cell,
 e.g., by immunostaining to detect a CSA-1 polypeptide or in situ
 hybridization, PCR, or Northern blotting to detect csa-1 transcripts. A
 decrease in the amount of csa-1 expression in cells exposed to the
 candidate compound compared to the amount of expression in cells in the
 absence of compound indicates that the compound inhibits expression of
 csa-1 in chondrosarcoma cells.
 Compounds that inhibit csa-1 expression can also be identified by
 contacting the csa-1 promoter linked to a reporter gene with a candidate
 compound and measuring the level of expression of the reporter gene in the
 presence and absence of the compound. An decreased level of expression in
 the presence of the compound compared to that in its presence indicates
 that the compound inhibits expression of csa-1.
 The screening methods described above can also be used to identify
 compounds which inhibit expression of a caa gene such as caa-1.
 Other embodiments are within the following claims.