WO2001055170A1 - Mob-5/hmob-5 as a cancer diagnostic marker - Google Patents
Mob-5/hmob-5 as a cancer diagnostic marker Download PDFInfo
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- WO2001055170A1 WO2001055170A1 PCT/US2001/002680 US0102680W WO0155170A1 WO 2001055170 A1 WO2001055170 A1 WO 2001055170A1 US 0102680 W US0102680 W US 0102680W WO 0155170 A1 WO0155170 A1 WO 0155170A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- This invention relates to the discovery of a novel gene, designated mob-5, an immediate transcriptional target of oncogenic ras.
- the invention further relates to the detection of mob-5 expression and/or the presence ofthe Mob-5 protein as potential markers for the early diagnosis of cancer.
- Ras oncogenes cooperate with several other proto-oncogenes or mutant p53 tumor suppressor genes to transform mammalian cells (Hinds, et al. 1989).
- Ras proteins are among the most important molecular switch molecules that relay mitogenic or differentiation signals from the cell surface to the nucleus where selective genes are activated. Oncogenic mutations lock the Ras proteins into a permanent "on" position, leading to unregulated cell proliferation which is a hallmark of cancer.
- Ras proteins function, whether directly or through other signaling molecules, to control expression of genes that are important for cell growth and differentiation (Kern et al., 1991; Hunter, 1995). Progress has been made toward understanding Ras signaling pathway from growth factor receptor through activation of a cascade of protein kinases (Boguski and McCormick, 1993). In contrast, much less is known about the downstream genes that are transcriptionally activated by Ras.
- This invention relates to the discovery of a novel gene, designated mob-5, as an immediate transcriptional target of oncogenic ras.
- the gene encodes a secreted protein of 183 amino acids, confirmed by immunoassays in oncogenic ras transformed cells and colorectal cancer cells from patient tissues. Normal ras activation cannot induce mob-5 expression.
- PSA prostate specific antigen
- Mob-5 has tumor-specificity in addition to restricted expression in normal tissues. Therefore, the unique characteristics of tumor-specificity, specificity of activation by Ras, and secretion make mob-5 expression and/or Mob-5 presence markers for the early diagnosis of cancer.
- the invention provides an isolated nucleic acid comprising the nucleic acid set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO: 7 and SEQ ID NO: 9.
- the invention also provides purified polypeptides having the sequence set forth in the Sequence Listing as any of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 10.
- This invention in one aspect, relates to a method of detecting the presence of cancer in a patient comprising: a) contacting a sample from the patient with an antibody to Mob-5; and b) detecting the binding ofthe antibody with an antigen in the sample, wherein binding of antigen to the antibody indicates the presence of Mob-5 antigen in the sample and wherein Mob-5 antigen in the sample indicates the presence of cancer in the patient, thereby detecting the presence of cancer in the patient.
- the invention further provides a method of classifying a cancer as a colorectal cancer comprising: a) obtaining a sample from a patient diagnosed with cancer; b) contacting the sample with an antibody to Mob-5; and c) detecting the binding ofthe antibody with an antigen in the sample, wherein the binding ofthe antibody to the antigen indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- the invention also provides a method of classifying a cancer as a colorectal cancer comprising: a) obtaining a sample from a patient diagnosed with cancer; b) contacting the sample with a Mob-5 antigen; and c) detecting the binding ofthe antigen with an antibody in the sample, wherein the binding ofthe antigen to the antibody indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- a method of determining the effectiveness of an anti-cancer therapy comprising: a) obtaining a sample from a patient undergoing anti-cancer therapy, and b) monitoring the sample for expression of a mob-5 gene, whereby inhibition ofthe expression ofthe mob-5 gene indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- the mvention also provides a method of determining the effectiveness of an anti-cancer therapy comprising: a) obtaining a sample from a patient undergoing anti- cancer therapy, and b) monitoring the sample for active mob-5 gene product, whereby inhibition ofthe activity ofthe mob-5 gene product indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- Also provided by this invention is a method of screening agents for anti-cancer activity comprising; a) administering the agent to a cancer cell; and b)monitoring the expression oi a. mob-5 gene in the cell, whereby an inhibition of the expression of the mob-5 gene indicates the agent has anti-cancer activity, thereby screening the agent for anti-cancer activity.
- a method of screening agents for anti- cancer activity comprising; a) administering the agent to a cancer cell; and b) monitoring the activity of a Mob-5 gene product in the cell, whereby an inhibition of the activity ofthe Mob-5 gene product indicates the agent has anti-cancer activity, thereby screening the agent for anti-cancer activity.
- Also provided by this invention is a method of inhibiting a cellular transformation phenotype induced by an oncogene whose product functions upstream of a mob-5 gene product, comprising inhibiting formation of an active mob-5 gene product in a cell containing the upstream oncogene so as to inhibit the expression ofthe cellular transformation phenotype ofthe upstream oncogene.
- a method of inhibiting a cellular transformation phenotype induced by an oncogene whose product functions upstream of a mob-5 gene product comprising inhibiting an active mob-5 gene product in a cell containing the upstream oncogene so as to inhibit the expression ofthe cellular transformation phenotype ofthe upstream oncogene.
- a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting an active Mob-5 gene product in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- a method of determining the effectiveness of an anti-cancer therapy comprising: a) obtaining a sample from a patient undergoing anti-cancer therapy, and b) monitoring the sample for the receptor for the mob-5 gene product, whereby a decreased amount ofthe receptor for the mob-5 gene product indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- Also provided by this invention is a method of determining the effectiveness of an anti-cancer therapy comprising: a) obtaining a sample from a patient undergoing anti-cancer therapy, and b) monitoring the sample for expression of a gene encoding a Mob-5 receptor, whereby inhibition ofthe expression ofthe gene encoding the Mob-5 receptor indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- the invention further provides a method of screening agents for anti-cancer activity comprising; a) administering the agent to a cancer cell; and b) monitoring the expression of a gene encoding a Mob-5 receptor in the cell, whereby an inhibition of the expression ofthe gene encoding a Mob-5 receptor indicates the agent has anti- cancer activity, thereby screening the agent for anti-cancer activity.
- Also provided by this invention is a method of screening agents for anti-cancer activity comprising; a) administering the agent to a cancer cell; and b) monitoring the activity of a Mob-5 receptor in the cell, whereby an inhibition ofthe activity ofthe Mob-5 receptor indicates the agent has anti-cancer activity, thereby screening the agent for anti-cancer activity.
- a method of inhibiting a cellular transformation phenotype induced by an oncogene whose product functions upstream of a. mob-5 gene product comprising inhibiting formation of an active Mob-5 receptor in a cell containing the upstream oncogene so as to inhibit the expression ofthe cellular transformation phenotype ofthe upstream oncogene.
- a method of inhibiting a cellular transformation phenotype induced by an oncogene whose product functions upstream of a mob-5 gene product comprising inhibiting an active Mob-5 receptor in a cell containing the upstream oncogene so as to inhibit the expression ofthe cellular transformation phenotype of the upstream oncogene.
- Also provided is a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting the formation of active Mob-5 receptor in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- the invention further provides a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting an active Mob-5 receptor in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- the invention also provides a method of detecting the presence of cancer in a patient comprising: a) contacting a sample from the patient with an antibody to a Mob- 5 receptor; and b) detecting the binding ofthe antibody with an antigen in the sample, wherein binding of antigen to the antibody indicates the presence of Mob-5 receptor antigen in the sample and wherein Mob-5 receptor antigen in the sample indicates the presence of cancer in the patient, thereby detecting the presence of cancer in the patient.
- a method of classifying a cancer as a colorectal cancer comprising: a) obtaining a sample from a patient diagnosed with cancer; b) contacting the sample with an antibody to a Mob-5 receptor; and c) detecting the binding ofthe antibody with an antigen in the sample, wherein the binding ofthe antibody to the antigen indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- Also provided is a method of classifying a cancer as a colorectal cancer comprising: a) obtaining a sample from a patient diagnosed with cancer; b) contacting the sample with a Mob-5 receptor antigen; and c) detecting the binding of the antigen with an antibody in the sample, wherein the binding ofthe antigen to the antibody indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- Figure 1 illustrates the identification of mob-5 as an oncogenic ras target gene by differential display.
- FIG. 2 illustrates that the activation of mob-5 expression by oncogenic h-Ras is blocked by farnesyl transferase inhibitor (FTI)
- FTI farnesyl transferase inhibitor
- Figure 3 illustrates the cDNA sequence of mob-5 and its predicted protein sequence alignment with Mda-7 and IL-10
- A The full length cDNA of mob-5 and the predicted Mob-5 protein sequence.
- the full length cDNA of mob-5 was isolated from cDNA library of Rat-1 (ras) cells using mob-5 cDNA probe from differential display (flanked by the arbitrary primer and anchored oligo-dT primer underlined at the 3' untranslated region). The putative hydrophobic signal peptide located at the N-terminus of Mob-5 protein is also underlined.
- B The protein sequence alignment of Mob-5 and Mda-7 was analyzed by the BLAST program from Genbank.
- Figure 4 illustrates Mob-5 expression can be activated by both oncogenic h-ras and k- ras and the gene encodes a 23 KD secreted protein which binds in a species-specific manner to the cell surface of oncogenic ras transformed fibroblasts and epithelial cells
- hMob-5 is also a secreted protein.
- the entire hMob-5 coding region with its native signal peptide sequence was fused at the C-terminus with AP and the hMob-5- AP from the cell culture medium was detected by direct Western blot analysis with antibody to either hMob-5 and AP. AP alone was used as a control for the antibody specificity.
- Figure 5 illustrates tumor specific expression of hMob-5.
- Total RNA from five colorectal cancer patients were analyzed by RT-PCR for the 800 bp coding region of hmob-5.
- Figure 6 illustrates the identification of cMob-5, a cancer-specific homolog of Mob-5 that has an internal deletion of 53 amino acids.
- CMob5 specific primers were used to detect cancer specific expression of cmob5 mRNA in colon cancer tissues.
- Figure 7 illustrates the protein sequence of human Mob-5 where the underlined S (Serine) is a single amino acid insertion between codon 14 and 15 of MDA-7, which makes human Mob-5 different from MDA-7.
- the bold amino acids correspond to the 53 amino acids that are deleted from the cancer-specific Mob-5 (cMob-5).
- cMob-5 does not contain the underlined serine amino acid residue.
- nucleic acid includes multiple copies ofthe nucleic acid and can also include more than one particular species of nucleic acid molecule.
- the invention provides isolated nucleic acids comprising the nucleic acids set forth in the Sequence Listing as SEQ ID NO: 1 (corresponding to a cDNA encoding a rat Mob-5 protein), SEQ ID NO:3 (corresponding to a cDNA encoding the cancer specific human Mob-5 protein homolog with an internal deletion of 53 amino acid residues, referred to as cMob-5.)and SEQ ID NO: 5 (corresponding to a cDNA encoding a human Mob-5 protein) SEQ ID NO:7 (corresponding to a nucleic acid encoding a rat Mob-5 -AP fusion protein and SEQ ID NO: 9 (corresponding to a nucleic acid encoding a human Mob-5-AP fusion protein.).
- SEQ ID NO: 1 corresponding to a cDNA encoding a rat Mob-5 protein
- SEQ ID NO:3 corresponding to a cDNA encoding the cancer specific human Mob-5 protein homolog with an internal deletion of 53 amino acid residues
- nucleic acid refers to single-or multiple stranded molecules which may be DNA or RNA, or any combination thereof, including modifications to those nucleic acids.
- the nucleic acid may represent a coding strand or its complement, or any combination thereof.
- Nucleic acids may be identical in sequence to the sequences which are naturally occurring for any ofthe novel genes discussed herein or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure.
- Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methy lphosphonate deoxynucleotides).
- genes and nucleic acids provided for by the present invention may be obtained in any number of ways.
- a DNA molecule encoding a Mob-5 protein can be isolated from the organism in which it is normally found.
- a genomic DNA or cDNA library can be constructed and screened for the presence of the gene or nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the const ction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA).
- the gene or nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps.
- the sequence encoding specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art.
- PCR primers can be designed which span the amino acid position or positions and which can substitute any amino acid for another amino acid.
- a nucleic acid can be amplified and inserted into the wild-type Mob-5 protein coding sequence in order to obtain any of a number of possible combinations of amino acids at any position ofthe Mob-5 protein.
- one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis. General methods are set forth in Smith, M. "In vitro mutagenesis" Ann. Rev.
- Mob-5 protein is to synthesize a recombinant DNA molecule which encodes the Mob-5 protein.
- oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis.
- a nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand.
- One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector.
- Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together.
- Cunningham, et al "Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis," Science, 243:1330-1336 (1989) have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al., Proc. Nat. Acad. Sci.
- the invention also provides for the isolated nucleic acids of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7 and SEQ ID NO: 9 in a vector suitable for expressing the nucleic acid.
- a nucleic acid encoding a particular Mob-5 protein of interest, or a region of that nucleic acid is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild-type and/or modified Mob-5 protein.
- the vector is contemplated to have the necessary functional elements that direct and regulate transcription ofthe inserted gene, or nucleic acid.
- These functional elements include, but are not limited to, a promoter, regions upstream or downstream ofthe promoter, such as enhancers that may regulate the transcriptional activity ofthe promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression ofthe inserted gene or hybrid gene. (See generally, Sambrook et al).
- E. coli Esscherichia coli
- Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
- bacilli such as Bacillus subtilis
- enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
- prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
- any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
- the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation.
- an amino terminal methionine can be provided by insertion of a Met codon 5' and in- frame with the downstream nucleic acid insert.
- the carboxy-terminal extension ofthe nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures.
- yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha- factor leader region (encoded by the MF"-1 gene) is routinely used to direct protein secretion from yeast. (Brake, et al, " ⁇ -Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae.” Proc. Nat.
- the leader region of pre-pro-alpha- factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence.
- the nucleic acid coding sequence can be fused in-frame to the pre-pro-alpha- factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
- the nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals.
- the nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or beta-galactosidase, used to facilitate purification ofthe fusion protein by affinity chromatography.
- a second protein coding sequence such as Sj26 or beta-galactosidase
- the insertion of protease cleavage sites to separate the components ofthe fusion protein is applicable to constructs used for expression in yeast. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems.
- Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
- Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal.
- the vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification.
- the chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence ofthe vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA co ⁇ esponding to the insert coding sequence can be confirmed by Northern blot analysis.
- suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc.
- Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
- Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.
- the vectors containing the nucleic acid segments of interest can be transfe ⁇ ed into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation may be used for other cellular hosts.
- vectors for the expression of genes or nucleic acids in mammalian cells those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexin 1, and eosinophil major basic protein, can be employed.
- the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acids in mammalian cells (such as COS-7).
- Insect cells also permit the expression of mammalian proteins. Recombinant proteins produced in insect cells with baculovirus vectors undergo post-translational modifications similar to that of wild-type proteins.
- baculovirus vectors useful for the expression of active proteins in insect cells are characterized by insertion of the protein coding sequence downstream of the Autographica californica nuclear polyhedrosis virus (AcNPV) promoter for the gene encoding polyhedrin, the major occlusion protein.
- Cultured insect cells such as Spodoptera frugiperda cell lines are transfected with a mixture of viral and plasmid DNAs and the viral progeny are plated.
- Deletion or insertional inactivation ofthe polyhedrin gene results in the production of occlusion negative viruses which form plaques that are distinctively different from those of wild-type occlusion positive viruses. These distinctive plaque morphologies allow visual screening for recombinant viruses in which the AcNPV gene has been replaced with a hybrid gene of choice. High quantity expression and production ofthe Mob-5 protein can also be achieved by transgenic animal technology by which animals can be made to produce Mob-5 in serum, milk, etc in large amounts.
- the invention also provides for the vectors containing the contemplated nucleic acids in a host suitable for expressing the nucleic acids.
- the vectors containing the nucleic acid segments of interest can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation, transduction, and electroporation are commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or hpofection mediated transfection or electroporation may be used for other cellular hosts.
- the genes or nucleic acids ofthe present invention can be operatively linked to one or more ofthe functional elements that direct and regulate transcription ofthe inserted gene as discussed above and the gene or nucleic acid can be expressed.
- a gene or nucleic acid can be operatively linked to a bacterial or phage promoter and used to direct the transcription ofthe gene or nucleic acid in vitro.
- a further example includes using a gene or nucleic acid provided herein in a coupled transcription-translation system where the gene directs transcription and the RNA thereby produced is used as a template for translation to produce a polypeptide.
- the products of these reactions can be used in many applications such as using labeled RNAs as probes and using polypeptides to generate antibodies or in a procedure where the polypeptides are being administered to a cell or a patient.
- Expression ofthe gene or nucleic acid, in combination with a vector can be by either in vivo or in vitro.
- In vivo synthesis comprises transforming prokaryotic or eukaryotic cells that can serve as host cells for the vector.
- expression of the gene or nucleic acid can occur in an in vitro expression system.
- in vitro transcription systems are commercially available which are routinely used to synthesize relatively large amounts of mRNA.
- the nucleic acid encoding the Mob-5 protein would be cloned into an expression vector adjacent to a transcription promoter.
- the Bluescript II cloning and expression vectors contain multiple cloning sites which are flanked by strong prokaryotic transcription promoters.
- Kits are available which contain all the necessary reagents for in vitro synthesis of an RNA from a DNA template such as the Bluescript vectors. (Stratagene Cloning Systems, La Jolla, CA). RNA produced in vitro by a system such as this can then be translated in vitro to produce the desired Mob-5 protein. (Stratagene Cloning Systems, La Jolla, CA).
- the present invention also contemplates nucleic acid probes for detecting the mob-5 gene wherein the DNA probe hybridizes to the nucleotide sequence set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7 and or SEQ ID NO:9.
- nucleic acid probe refers to a nucleic acid fragment that selectively hybridizes under stringent conditions with a nucleic acid comprising a nucleic acid set forth in a sequence listed herein. This hybridization must be specific. The degree of complementarity between the hybridizing nucleic acid and the sequence to which it hybridizes should be at least enough to exclude hybridization with a nucleic acid encoding an unrelated protein.
- Allelic variants can be identified and isolated by nucleic acid hybridization techniques. Probes selective to the nucleic acid set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7 and SEQ ID NO:9 can be synthesized and used to probe the nucleic acid from various cells, tissues, libraries etc. High sequence complementarity and stringent hybridization conditions can be selected such that the probe selectively hybridizes to allelic variants ofthe sequence set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO: 7 and SEQ ID NO: 9.
- the selectively hybridizing nucleic acids ofthe invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99%) complementarity with the segment ofthe sequence to which it hybridizes. This percent complementarity can be based preferably on a nucleotide-by-nucleotide comparison ofthe two strands.
- the nucleic acids can be at least 12, 50, 100, 150, 200, 300, 500, 750, or 1000 nucleotides in length.
- the nucleic acid can be a coding sequence for the mob-5 proteins or fragments thereof that can be used as a probe or primer for detecting the presence of these genes.
- the invention provides compositions including at least two nucleic acids which hybridize with different regions so as to amplify a desired region.
- target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions.
- the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other bacteria.
- the invention provides examples of nucleic acids unique to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9 in the Sequence Listing so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid.
- washing conditions refers to the washing conditions used in a hybridization protocol.
- the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5-20°C below the calculated T m ofthe nucleic acid hybrid under study.
- the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein coding nucleic acid of interest and then washed under conditions of different stringencies.
- the T m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate T m of 54°C.
- the invention provides the polypeptides encoded by the nucleic acids set forth in SEQ ID NO: 1, SEQ ID NO:3 , SEQ ID NO:5 ,SEQ ID NO: 7 and SEQ ID NO: 9 as well as the polypeptides set forth in the Sequence Listing as SEQ ID NO:2, SEQ ID NO:4 , SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 10.
- the invention also provides fragments of unmodified and modified Mob-5 proteins. Fragments of Mob-5 and cMob-5 that do not include a signal peptide sequence are also contemplated.
- the polypeptide fragments ofthe present invention can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof
- an expression system capable of producing the polypeptide fragments thereof
- one skilled in the art can determine the active regions of a Mob-5 protein which can interact with another protein and cause a biological effect associated with the Mob-5 protein.
- amino acids found to not contribute to either the activity, binding specificity, or other biological effect associated with the Mob-5 protein can be deleted and /or substituted without a loss in the respective activity.
- the fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acid residues, provided the activity ofthe peptide is not significantly altered or impaired.
- Mob-5 polypeptides can also be obtained in any of a number of procedures well known in the art.
- One method of producing a polypeptide is to link two peptides or polypeptides together by protein chemistry techniques.
- peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmefhyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
- Fmoc 9-fluorenylmefhyloxycarbonyl
- Boc tert -butyloxycarbonoyl
- a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a hybrid peptide can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
- peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a larger polypeptide.
- the peptide or polypeptide can be independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form a larger protein via similar peptide condensation reactions.
- enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et al. Biochemistry, 30:4151 (1991)).
- native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. "Synthesis of Proteins by Native Chemical Ligation," Science, 266:776-779 (1994)).
- the first step is the chemoselective reaction of an unprotected synthetic peptide- -fhioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
- Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Clark-Lewis et al.
- unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result ofthe chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al. Science, 256:221 (1992)).
- This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton et al. "Techniques in Protein Chemistry IV," Academic Press, New York, pp. 257-267 (1992)).
- the Mob-5 polypeptides of this invention an also be fused to another protein such as alkaline phosphatase for detection methods.
- This fusion polypeptide can be utilized in an ELISA or Western blot to detect the receptor for Mob-5.
- the Mob5- alkaline phosphatase (Mob-5 AP) polypeptides of this invention can be used to measure Mob-5 in an ELISA-based assay.
- plates can be coated with an anti-Mob- 5 antibody. Samples can then be added to the plates that contain a given amount of Mob-5 AP which will bind to the plates. Therefore, in the absence of antigen, Mob- SAP should bind to the plates and produce the maximal amount of alkaline phosphatase activity.
- the antigen may compete with Mob-5 AP for the Mob-5 antibody on the plates. The presence of antigen will competitively inhibit the binding of Mob-5 AP.
- purified antibodies that selectively or specifically bind to the Mob-5 polypeptides provided and contemplated herein, for example, purified antibodies which selectively or specifically bind to a polypeptide encoded by the nucleic acid set forth in any of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO:5 ,SEQ ID NO: 7 and SEQ ID NO: 9, as well as purified antibodies which selectively or specifically bind to the polypeptides set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 10.
- the antibody (either polyclonal or monoclonal) can be raised to any ofthe polypeptides provided and contemplated herein, both naturally occurring and recombinant polypeptides, and immunogenic fragments, thereof.
- the antibody can be used in techniques or procedures such as diagnostics, treatment, or vaccination.
- Anti-idiotypic antibodies and affinity matured antibodies are also considered.
- the purified antibodies of this invention include monoclonal antibodies which can be used for diagnostic or analytical purposes.
- the monoclonal antibody could be utilized in a clinical testing kit to monitor levels of Mob-5 in human tissues or secretions.
- Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, "Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. (See, for example, Kelly et al.
- Humanized and chimeric antibodies are also contemplated in this invention.
- Heterologous antibodies can be made by well known methods (See, for example, US Patents 5545806, 5569825,5625126, 5633425, 5661016, 5770429, 5789650, and 5814318)
- the phrase "specifically binds" with the polypeptide refers to a binding reaction which is determinative ofthe presence ofthe protein in a heterogeneous population of proteins and other biologies.
- the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample.
- Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
- a variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane "Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
- the present invention further provides a method of detecting the presence of cancer in a patient comprising: contacting a sample from the patient with an antibody to Mob-5, detecting the binding ofthe antibody with an antigen in the sample, wherein binding ofthe antigen to the antibody indicates the presence of Mob-5 antigen in the sample and wherein Mob-5 antigen in the sample indicates the presence of cancer in the patient, thereby detecting the presence of cancer in the patient.
- a method of detecting the presence of cancer in a patient comprising:contacting a sample from the patient with Mob-5 antigen and detecting the binding ofthe antigen with an antibody in the sample, wherein binding of antibody to the antigen indicates the presence of Mob-5 antibody in the sample and wherein Mob-5 antibody in the sample indicates the presence of cancer in the patient, thereby detecting the presence of cancer in the patient.
- cancer refers to or describes the physiological condition, preferably in a mammalian subject, that is typically characterized by unregulated cell growth.
- examples of cancer include but are not limited to r ⁇ s-induced cancers, colorectal cancer, carcinoma, lymphoma, sarcoma, blastoma and leukemia. More particular examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breast cancer, prostrate carcinoma, rhabdomyosarcoma, colon carcinoma, and head and neck cancer. While the term “cancer” as used herein is not limited to any one specific form ofthe disease, it is believed that the methods ofthe invention will be particularly effective for cancers which are found to be accompanied by increased levels of Mob-5 expression.
- the "subject" or "patient” of this method can be any animal.
- the animal ofthe present invention is a human.
- non-human animals which can be treated by the methods of this invention can include, but are not limited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits.
- the sample of this invention can be from any organism and can be, but is not limited to, peripheral blood, plasma, urine, saliva, gastric secretion, feces, bone ma ⁇ ow specimens, primary tumors, embedded tissue sections, frozen tissue sections, cell preparations, cytological preparations, exfoliate samples (e.g., sputum), fine needle aspirations, amnion cells, fresh tissue, dry tissue, and cultured cells or tissue. It is further contemplated that the biological sample of this invention can also be whole cells or cell organelles (e.g., nuclei).
- the sample can be unfixed or fixed according to standard protocols widely available in the art and can also be embedded in a suitable medium for preparation ofthe sample. For example, the sample can be embedded in paraffin or other suitable medium (e.g., epoxy or acrylamide) to facilitate preparation of the biological specimen for the detection methods of this invention.
- antibody is used herein in a broad sense and includes intact immunoglobulin molecules and fragments or polymers of those immunoglobulin molecules, so long as they exhibit any ofthe desired properties described herein.
- Antibodies are typically proteins which exhibit binding specificity to a specific antigen.
- Native antibodies are usually heterotetrameric glycoproteins, composed of two light (L) chains and two heavy (H) chains.
- the heavy and light chains are typically identical, but not necessarily so.
- each light chain is linked to a heavy chain by one or more covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
- Each heavy and light chain also typically has regularly spaced intrachain disulfide bridges.
- Each heavy chain typically has at one end a variable domain (V(H)) followed by a number of constant domains.
- Each light chain typically has a variable domain at one end (V(E)) and a constant domain at its other end; the constant domain ofthe light chain is typically aligned with the first constant domain ofthe heavy chain, and the light chain variable domain is typically aligned with the variable domain ofthe heavy chain.
- Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
- the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
- immunoglobulins can typically be assigned to different classes. There are approximately five major classes of immunoglobulins: IgA, IgD, Ig ⁇ , IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
- the heavy chain constant domains that co ⁇ espond to the different classes of immunoglobulins are called ⁇ , ⁇ , e, ⁇ , and ⁇ , respectively.
- variable is used herein to describe certain portions ofthe variable domains which differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
- variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
- CDRs complementarity determining regions
- FR framework
- the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ . -sheet structure.
- the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al, "Sequences of Proteins of Immunological Interest” National Institutes of Health, Bethesda, Md. (1987)).
- the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation ofthe antibody in antibody-dependent cellular toxicity.
- the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
- the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion ofthe heavy and/or light chain may be identical with or homologous to co ⁇ esponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder ofthe chain(s) may be identical with or homologous to co ⁇ esponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (See, U.S.
- the present invention also provides a monoclonal antibody that binds to human Mob-5 and to rat Mob-5. This monoclonal antibody is produced from hybridoma clone L-14.
- antigen when used in the detection context generally means detecting the antigen, specifically Mob-5, cMob-5 (a cancer specific homolog of Mob-5 that has an internal deletion of 53 amino acids), the Mob-5 receptor, or a fragment thereof.
- the antigens of this invention can also be used to detect antibodies to Mob-5, the Mob-5 receptor or fragments thereof.
- the antigen may exist on the cell surface as well as be detectable in a body fluid.
- One example ofthe method of detecting the antigen is performed by contacting a fluid or tissue sample from the patient with an amount of a an antibody, possibly purified, reactive with the antigen, cells containing the antigen, or fragments ofthe antigen, and detecting the reaction ofthe antibody with the antigen.
- the fluid sample of this method can comprise any body fluid which would contain the antigen or a cell containing the antigen, such as blood, plasma, serum, saliva and urine, sputum, mucus and the like.
- An antibody used to detect the antigens of this invention is preferably specifically reactive with the antigen.
- the step of detecting the binding ofthe antibody with the antigen can be further aided, in appropriate instances, by the use of a secondary antibody or other ligand which is reactive, either specifically with a different epitope or nonspecifically with the ligand or reacted antibody.
- the antibody can be labeled with a detectable marker.
- Enzyme immunoassays such as immunofluorescence assays (IF A), enzyme linked immunosorbent assays (ELISA) and immunoblotting can be readily adapted to accomplish the detection ofthe antigen.
- An ELISA method effective for the detection of the antigen can, for example, be as follows: (1) bind the antibody to a substrate; (2) contact the bound antibody with a fluid or tissue sample containing the antigen; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change.
- Other assays for detecting the binding of an antibody to an antigen can be used.
- the present invention further provides a kit for detecting the binding of an antibody to the Mob-5 or the Mob-5 receptor, or a fragment thereof.
- the kit can detect the presence of an antigen specifically reactive with the antibody or an immunoreactive fragment thereof.
- the kit can include an antibody bound to a substrate, a secondary antibody reactive with the antigen and a reagent for detecting a reaction ofthe secondary antibody with the antigen.
- Such a kit can be an ELISA kit and can comprise the substrate, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as described above.
- the diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein.
- kits of the present invention can be selected from those available in the art in accord with the specific diagnostic method practiced in the kit. Such kits can be used to detect the binding ofthe antibody with Mob-5 and Mob-5 receptor, or a fragment thereof, in tissue and fluid samples from a patient.
- the prognostic evaluation can determine what type of anti-cancer therapy to employ at different stages of cancer depending on the amounts of Mob-5 antigen detected in the patient's sample.
- the invention also provides for a method of classifying a cancer as a colorectal cancer comprising, obtaining a sample from a patient diagnosed with cancer, contacting the sample with an antibody to Mob-5, and detecting the binding of the antibody with an antigen in the sample, wherein the binding ofthe antibody to the antigen indicated the cancer is colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- classifying is meant to determine that the expression of Mob-5 co ⁇ esponds to a specific type of cancer, such as colorectal cancer and not to another type of cancer, thus classifying the cancer.
- a specific type of cancer such as colorectal cancer
- the detection of Mob-5 in the sample can be combined with the detection of another specific tumor marker to confirm that the cancer is the specific type of cancer.
- diagnostic is meant that one skilled in the art has determined either by blood test, immunoassay, ultrasound, urinalysis, magnetic resonance imaging, physical examination, biopsy or any other diagnostic method that the patient has cancer.
- the invention further provides a method of classifying a cancer as a colorectal cancer comprising, obtaining a sample from a patient diagnosed with cancer, contacting the sample with a Mob-5 antigen, and detecting the binding ofthe antigen with an antibody in the sample, wherein the binding ofthe antigen to the antibody indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- the detection of binding of Mob-5 antigen to Mob-5 antibody in the sample can be accomplished by utilizing immunological detection methods previously discussed or the capture immunoassay as described in the Examples.
- Also provided by the present invention is a method of determining the effectiveness of an anti-cancer therapy comprising, obtaining a sample from a patient undergoing anti-cancer therapy, and monitoring the sample for expression of a mob-5 gene, whereby inhibition ofthe expression ofthe mob-5 gene indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- Also provided by the present invention is a method of determining the effectiveness of an anti-cancer therapy comprising, obtaining a sample from a patient undergoing anti-cancer therapy, and monitoring the sample for expression of a Mob-5 protein, such as Mob-5 or cMob-5 whereby inhibition ofthe expression ofthe Mob-5 or cMob-5 protein indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- a Mob-5 protein such as Mob-5 or cMob-5
- Expression refers to the transcription and translation of a gene to yield the encoded protein, in particular a Mob-5 protein or a Mob-5 receptor protein .
- a Mob-5 protein or a Mob-5 receptor protein One of ordinary skill in the art will know how to monitor the level of expression of nucleic acids as well as proteins throughout the anti-cancer therapy in order to determine the effectiveness of an anti-cancer treatment. If the levels ofthe mob-5 gene or the activity ofthe Mob-5 protein decrease when compared to the levels measured prior to the anti- cancer therapy, the anti-cancer therapy may be effective.
- One skilled in the art may determine how effective the anti-cancer therapy is by co ⁇ elating the decreases in mob- 5 gene or Mob-5 protein expression with a particular stage of cancer. Similarly, decreases in mob-5 receptor gene or Mob-5 receptor expression can be co ⁇ elated with a particular stage of cancer.
- the nucleic acids of this invention can be detected with a probe capable of hybridizing to the nucleic acid of a cell or a sample.
- This probe can be a nucleic acid comprising the nucleotide sequence of a coding strand or its complementary strand or the nucleotide sequence of a sense strand or antisense strand, or a fragment thereof.
- the nucleic acid can comprise the nucleic acid ofthe mob-5 gene, or a sequence associated with a gene that is associated with the mob-5 gene, such as the mob-5 receptor gene.
- the probe of this invention can be either DNA or RNA and can bind either DNA or RNA, or both, in the biological sample.
- the probe can be the coding or complementary strand of a complete gene or gene fragment.
- the nucleotide sequence of the probe can be any sequence having sufficient complementarity to a nucleic acid sequence in the biological sample to allow for hybridization ofthe probe to the target nucleic acid in the biological sample under a desired hybridization condition.
- the probe will hybridize only to the nucleic acid target of interest in the sample and will not bind non-specifically to other nucleic acids in the sample or other regions ofthe target nucleic acid in the sample.
- the hybridization conditions can be varied according to the degree of stringency desired in the hybridization. For example, if the hybridization conditions are for high stringency, the probe will bind only to the nucleic acid sequences in the sample with which it has a very high degree of complementarity.
- Low stringency hybridization conditions will allow for hybridization ofthe probe to nucleic acid sequences in the sample which have some complementarity but which are not as highly complementary to the probe sequence as would be required for hybridization to occur at high stringency. Since sequence divergence can exist between individuals for cancer or tumor-related genes, one skilled in the art can take these population differences into account when optimizing hybridization conditions.
- the hybridization conditions will therefore vary depending on the biological sample, probe type and target. An artisan will know how to optimize hybridization conditions for a particular application ofthe present method.
- the nucleic acid probes of this invention can be modified nucleic acids.
- modified nucleotides are well known in the art and include, but are not limited to, thio-modified deoxynucleotide triphosphates and borano-modified deoxynucleotide triphosphates (Eckstein and Gish, Trends in Biochem. Sci., 14:97-100 (1989) and Porter Nucleic Acids Research, 25:1611-1617 (1997)).
- the nucleic acid probe can be commercially obtained or can be synthesized according to standard nucleotide synthesizing protocols well known in the art. Alternatively, the probe can be produced by isolation and purification of a nucleic acid sequence from biological materials according to methods standard in the art of molecular biology (Sambrook et al. 1989. Molecular Cloning: A Laboratoiy Manual, 2d Ed., Cold Spring Harbor Laboratory Pres, Cold Spring Harbor, NY). The nucleic acid probe can be amplified according to well known procedure for amplification of nucleic acid (e.g., polymerase chain reaction). Furthermore, the probe of this invention can be linked to any ofthe detectable moieties of this invention by protocols standard in the art.
- the detectable moieties to which the nucleic acid probe of this invention can be linked to include, but are not limited to, a hapten, biotin, digoxigenin, fluorescein isothiocyanate (FITC), dinitrophenyl, amino methyl coumarin acetic acid, acetylaminofluorene and mercury-sulfhydryl-ligand complexes, as well as any other molecule or compound which can be linked to a probe and detected either directly or indirectly according to the methods described herein.
- FITC fluorescein isothiocyanate
- a probe such as a nucleic acid probe or an antibody
- a detectable moiety that can be directly detected, such as a flurorochrome or a dye, such as a chromogenic dye, and the use of secondary reagents to detect the probe is not strictly required.
- the present invention also includes methods for oligonucleotide hybridization wherein the hybridized oligonucleotide is used as a primer for an enzyme catalyzed elongation reaction such as in situ PCR and primed in situ labeling reactions, whereby haptenized nucleotides are incorporated in situ.
- methods for in situ hybridization employing synthetic peptide nucleic acid (PNA) oligonucleotide probes (Nielsen et al., 1991. "Sequence- selective recognition of DNA by strand displacement with a thymine-substituted polyamide.” Science 254:1497-1500; Egholm et al., 1993.
- PNA synthetic peptide nucleic acid
- PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules. Nature 365:566-568).
- the levels of protein in this invention can be detected by ELISA, FIA, immunob lotting or any other immunodetection method. These methods can be combined with histochemical or microscopic analysis to determine the levels of proteins in samples.
- the patients of this invention undergoing anti-cancer therapy can include patients undergoing surgery, chemotherapy, radiotherapy, immunotherapy or any combination thereof.
- chemotherapeutic agents include cisplatin, 5- fluorouracil and S-l.
- Immunotherapeutics methods include administration of interleukin-2 and interferon- ⁇ .
- a method of determining the effectiveness of an anti-cancer therapy comprising, obtaining a sample from a patient undergoing anti-cancer therapy, and monitoring the sample for active mob-5 gene product, whereby inhibition of the activity ofthe mob-5 gene product indicates the anti- cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- active mob-5 gene product is meant a product ofthe mob-5 gene which can exert a biological function associated with the mob-5 gene.
- An active mob-5 gene product may act as a transcriptional activator to elicit downstream effects.
- the mob-5 gene product may also function from outside ofthe cell either as a ligand which could bind to its cell surface receptor and participate in signal transduction, or it may function through the interaction with other secreted proteins and extracellular matrix molecules outside of cells. Any effect associated with mob-5 associated cancers is also contemplated by this invention.
- inhibitor is familiar to one skilled in the art and is used herein to describe any reduction in the activity ofthe mob-5 gene product.
- the degree of inhibition does not have to be complete, such as completely inhibiting the activity of the mob-5 gene product, and therefore comprises any inhibition ofthe activity ofthe mob-5 gene product relative to the activity ofthe mob-5 gene product in a similar environment in the absence ofthe inhibiting compound.
- the invention also provides a method of screening agents for anti-cancer activity comprising, administering the agent to a cancer cell, monitoring the expression of a mob-5 gene in the cell, whereby an inhibition ofthe expression of the mob-5 gene indicates the agent has anti-cancer activity, thereby screening the agent for anti-cancer activity.
- the "cells” of this invention include any cell type, cancerous or noncancerous, that may express or be affected by the expression of a mob-5 gene or the activity of a Mob-5 protein. Examples include, but are not limited to, colorectal cancer cells, pancreatic cancer cells, gastrointestinal cancer cells and prostate cancer cells.
- Also provided is a method of screening agents for anti-cancer activity comprising, administering the agent to a cancer cell, monitoring the activity of a mob-5 gene product in the cell, whereby an inhibition ofthe activity ofthe mob-5 gene product indicates the agent has anti-cancer activity, thereby screening the agent for anti- cancer activity.
- the invention also provides a method of inhibiting a cellular transformation phenotype induced by an oncogene whose product functions upstream of a mob-5 gene product, comprising inhibiting formation of an active mob-5 gene product in a cell containing the upstream oncogene so as to inhibit the expression of the cellular transformation phenotype ofthe upstream oncogene.
- cellular transformation phenotype means that cells have undergone at least some transformation characterized by one or more morphologic or biochemical changes such as loss of contact inhibition, increased rate of glycolysis, alterations ofthe cell surface and other changes that would be known to one skilled in the art.
- oncogene refers to genes that produce products involved in altering cellular metabolism and often stimulate unregulated growth associated with malignant transformation of cells.
- oncogenes include but are not limited to, ras, src, myc, and fas.
- the cellular transformation phenotype of this invention can be induced by a ras oncogene product.
- inhibiting in the context of gene expression, is familiar to one skilled in the art and is used herein to describe any compound or composition which inhibits or decreases the expression of an active mob-5 gene product.
- the degree of inhibition does not have to be complete, such as completely inhibiting the expression of a mob-5 gene product, and therefore comprises any inhibition ofthe expression ofthe mob-5 gene product relative to the expression ofthe mob-5 gene product in a similar environment in the absence ofthe inhibiting compound.
- the cellular transformation phenotype of this invention can be inhibited by a nucleic acid antisense to the mob-5 gene.
- Antisense technology is well known in the art and describes a mechanism whereby a nucleic acid comprising a nucleotide sequence which is in a complementary, "antisense” orientation with respect to a coding or “sense” sequence of an endogenous gene, is introduced into a cell, whereby a duplex may form between the antisense sequence and its complementary sense sequence. The formation of this duplex may result in inactivation ofthe endogenous gene.
- the antisense nucleic acid can inhibit gene expression by forming an RNA/RNA duplex between the antisense RNA and the RNA transcribed from a target gene.
- the precise mechanism by which this duplex formation decreases the production ofthe protein encoded by the endogenous gene most likely involves binding of complementary regions of the normal sense mRNA and the antisense RNA strand with duplex formation in a manner that blocks RNA processing and translation.
- Alternative mechanisms include the formation of a triplex between the antisense RNA and duplex DNA or the formation of a DNA-RNA duplex with subsequent degradation of DNA-RNA hybrids by RNAse H.
- an antisense effect can result from certain DNA-based oligonucleotides via triple-helix formation between the oligomer and double-stranded DNA which results in the repression of gene transcription.
- Antisense nucleic acid can be produced for any relevant endogenous gene for which the coding sequence has been or can be determined according to well known methods.
- a nucleic acid encoding an antisense RNA can be selected based on the protein desired to be inhibited or decreased in cells, by providing an RNA that will selectively bind to the cellular mRNA encoding such protein. Binding ofthe antisense molecule to the target mRNA may incapacitate the mRNAs, thus preventing its translation into a functional protein. The antisense RNA/mRNA complexes can then become a target for RNAse-H and are eventually degraded by the host cell RNAse-H. Control regions, such as enhancers and promoters, can be selected for antisense RNA targeting according to the cell or tissue in which it is to be expressed, as is known in the art.
- Preferable antisense-encoding constructs can encode full-length complements to target sequences; however, smaller length sequences down to oligonucleotide size can be utilized.
- the antisense-encoding constructs can encode full-length complements to the mob-5 gene, smaller length sequences or oligonucleotide sequences.
- the invention also provides a method of inhibiting a cellular transformation phenotype induced by an oncogene whose product functions upstream of a mob-5 gene product, comprising inhibiting an active mob-5 gene product in a cell containing the upstream oncogene so as to inhibit the expression ofthe cellular transformation phenotype ofthe upstream oncogene.
- an active mob-5 gene product can be inhibited by an antibody to the mob-5 gene product.
- the mob-5 gene product of this invention can be inhibited by ligand binding to the protein. Ligand binding includes reversible and nonreversible binding.
- the present invention also provides a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting the formation of active Mob-5 protein in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- the formation of an active Mob-5 protein can be inhibited by a nucleic acid antisense to mob-5.
- a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting an active Mob-5 protein in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- An active Mob-5 protein in a cell can be inhibited by an antibody to Mob- 5.
- the present invention also provides a method of determining the effectiveness of an anti-cancer therapy comprising, obtaining a sample from a patient undergoing anti-cancer therapy, and monitoring the sample for the receptor for the mob-5 gene product, whereby a decreased amount ofthe receptor for the mob-5 gene product indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- the invention also provides a method of determining the effectiveness of an anti-cancer therapy comprising, obtaining a sample from a patient undergoing anti- cancer therapy, and monitoring the sample for expression of a gene encoding a Mob-5 receptor, whereby inhibition ofthe expression ofthe gene encoding the Mob-5 receptor indicates the anti-cancer therapy is effective, thereby determining the effectiveness of an anti-cancer therapy.
- a method of screening agents for anti-cancer activity comprising, administering the agent to a cancer cell, monitoring the expression of a gene encoding a Mob-5 receptor in the cell, whereby an inhibition of the expression ofthe gene encoding a Mob-5 receptor indicates the agent has anti- cancer activity, thereby screening the agent for anti-cancer activity.
- a method of screening agents for anti-cancer activity comprising, administering the agent to a cancer cell, monitoring the activity of a Mob-5 receptor in the cell, whereby an inhibition ofthe expression ofthe Mob-5 receptor indicates the agent has anti-cancer activity, thereby screening the agent for anti-cancer activity.
- the cellular transformation phenotype can be induced by a ras oncogene product.
- the cellular transformation phenotype can be inhibited by a nucleic acid antisense to a Mob-5 receptor gene.
- the cellular transformation phenotype can be induced by a ras oncogene product.
- An active Mob-5 receptor in a cell can be inhibited by an antibody to the Mob-5 receptor.
- the present invention also provides a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting the formation of active Mob-5 receptor in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- a nucleic acid antisense to the Mob-5 receptor gene can be utilized to inhibit the formation of an active Mob-5 receptor.
- Also provided by the present invention is a method for inhibiting a mob-5 induced cellular transformation phenotype comprising inhibiting an active Mob-5 receptor in a cell so as to inhibit the expression ofthe mob-5 induced cellular transformation phenotype.
- An active Mob-5 receptor can be inhibiting by an antibody to the Mob-5 receptor.
- the present invention also provides a method of inhibiting a mob-5 induced cellular transformation phenotype, wherein the active Mob-5 receptor is inhibited by administering to the cell an altered Mob-5 which binds to the Mob-5 receptor, whereby binding ofthe altered Mob-5 to the receptor inhibits the binding of Mob-5 to the receptor, thereby inhibiting the expression ofthe mob-5 induced cellular transformation phenotype.
- the invention also provides a method of detecting the presence of cancer in a patient comprising, contacting a sample from the patient with an antibody to a Mob-5 receptor, detecting the binding ofthe antibody with an antigen in serum, wherein binding of antigen to the antibody indicates the presence of Mob-5 receptor antigen in the sample and wherein Mob-5 receptor antigen in the sample indicates the presence of cancer in the patient, thereby detecting the presence of cancer in the patient.
- the invention also provides a method of detecting the presence of cancer in a patient comprising, contacting a sample from the patient with a Mob-5 receptor antigen; detecting the binding ofthe antigen with an antibody in the sample, wherein binding of antigen to the antibody indicates the presence of Mob-5 receptor antibody in the sample and wherein Mob-5 receptor antibody in the sample indicates the presence of cancer in the patient, thereby detecting the presence of cancer in the patient.
- the present invention also provides a method of detecting the Mob-5 receptor comprising, contacting a cell with a Mob5-AP fusion protein, detecting the binding of the Mob5-AP fusion protein to the cell, wherein binding ofthe Mob5-AP to the cell indicates the cell is a Mob-5 receptor expressing cell, thus detecting the Mob-5 receptor.
- Mob5 AP fusion proteins can be made as described in the Examples and utilized in receptor binding assays described in the Examples and by Flanagan and Leder (1990) to determine if a cell expresses a cell surface receptor for a Mob-5 protein.
- a method of classifying a cancer as a colorectal cancer comprising, obtaining a sample from a patient diagnosed with cancer, contacting the sample with an antibody to a Mob-5 receptor, and detecting the binding ofthe antibody with an antigen in the sample, wherein the binding ofthe antibody to the antigen indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- the present invention also provides a method of classifying a cancer as a colorectal cancer comprising, obtaining a sample from a patient diagnosed with cancer, contacting the sample with a Mob-5 receptor antigen, and detecting the binding ofthe antigen with an antibody in the sample, wherein the binding ofthe antigen to the antibody indicates the cancer is a colorectal cancer, thereby classifying the cancer as a colorectal cancer.
- Ratl, Ratl(ras), Rat-l:iRas Liang et al., 1994; Zhang et al., 1996)
- RIE, RIE(h-ras) and RIE(k-ras) were routinely grown in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies Inc. Grand Island, N.Y.) with 10% bovine calf serum (HyClone, Logan, UT) and 1% penicillin- streptomycin (Life Technologies Inc. Grand Island, N.Y.) at 37 °C with 10% CO 2 .
- DMEM Dulbecco's Modified Eagle Medium
- bovine calf serum HyClone, Logan, UT
- penicillin- streptomycin Life Technologies Inc. Grand Island, N.Y.
- 293T cells and their derivatives were maintained under the same conditions as above except 10% fetal bovine serum (HyClone, Logan, UT) was used in place of 10% bovine calf serum. Serum starvation and restimulation ofthe Rat-1 cells in the absence and presence of cyclohexamide (Sigma, St. Louis, MO) was carried out essentially as described previously (Zhang, et al., 1997).
- RNA purification, automated differential display screening and Northern blot analvsis Total RNA from cells were purified using the RNApure reagent following the manufacture instruction (GenHunter Corp., Arlington, TN). DNase I treatment of RNA prior to differential display was carried out using the MessageClean kit (GenHunter Corp., Nashville, TN). Differential display PCR reactions were setup in 96-well PCR plates (Perkin-Elmer) by the Beckman Biomek 2000 automated liquid dispensing workstation using one-base anchored oligo-dT primers and rationally designed arbitrary 13mers (Liang et al., 1994b) from the RNAimage kits (GenHunter Corp., Arlington, TN).
- the cDNAs were amplified in the presence of 33 P [ ⁇ -dATP] (New England Nuclear, Boston, MA) using Taq DNA polymerase (Qiagene) and separated on 6% denaturing polyacrylamide gels (National Diagnostic, Atlanta, GA).
- mob-5 One ofthe 14 ras inducible genes, designated mob-5 was novel and the gene was identified in both screenings using either inducible ras oncogene or inhibitor which blocks ras signaling ( Figure 1 A and IB).
- the 394 bp mob-5 cDNA was reamplified from the differential display gels, cloned and used as a probe to successfully verify the h-ras induction ofthe gene ( Figure IC).
- Mob-1 a known ras target gene previously identified (Liang et al., 1994a; Zhang et al, 1997) was used as a control for ras induction.
- mob-5 mRNA was downregulated as early as 24 hours after drug treatment and this downregulation was coupled temporally to reversion to more normal cellular morphology. mob-5 cannot be induced by serum in non-transformed Rat-1 cells, nor can its expression be detected in major tissues from normal adult rats
- FTI farnesyltransferase inhibitors
- Rat-1 (ras) cells were treated with 5 ⁇ M of FTI and the mob-5 mRNA expression and cellular morphology were monitored following the drug addition.
- mob-5 mRNA expression was essentially abolished in 24 hours ( Figure 2A), when the cells begin to lose the transformed cellular morphology.
- FTI was able to completely block the induction of mob-5 expression by IPTG in Rat-l :iRas cells ( Figure 2B).
- mob-5 encodes a secreted protein
- the full length mob-5 cDNA was isolated from a cDNA library of h-ras transformed rat embryo fibroblasts (Liang et al. , 1994a).
- the 1.2 kb mob-5 cDNA encodes an open reading frame of 183 amino acids with an in- frame stop codon 27 bases upstream of the putative translation initiation codon ( Figure 3A).
- the 394 bp mob-5 cDNA sequence identified by differential display lies at the 3' end of the mob-5 full length cDNA.
- the predicted Mob-5 protein exhibits 68 % sequence identity to Mda-7, a gene isolated as a differentiation associated gene from human melanoma cells treated with interferon- ⁇ and Mezerein (Jiang et al. , 1995; Figure 3B).
- the only other gene that showed significant degree of homology (23 % identity) to Mob-5 over most of the coding regions is interleukin 10 (IL-10) which is an potent anti-inflammatory cytokine ( Figure 3C; Moore et al. , 1990)
- IL-10 interleukin 10
- the Mob-5 protein sequence contains a typical signal peptide sequence at its N-terminus, suggesting that Mob-5 is a secreted protein, although previous study failed to demonstrate that Mda-7 was secreted.
- Mob-5 may function from outside ofthe cell either as a ligand which could bind to its cell surface receptor and participate in signal transduction or it may function through the interaction v ith other secreted proteins and extracellular matrix molecules outside of cells.
- One approach is to determine if Mob-5 interacts with other proteins in vivo.
- K-ras and H-ras transformed RIE cells can be metabolically labeled with radioactive 35S-methionine and 35S-cysteine. The culture media ofthe cells will be immunoprecipitated with Mob-5 antibody.
- the precipitated protein(s) are then separated by SDS-PAGE and visualized by autoradiography to evaluate what other protein species are co-precipitated with Mob-5.
- preimmune serum is used in parallel.
- the protein(s) that specifically interact with Mob-5 may be further studied by affinity purification and microsequencing ofthe purified polypeptides.
- the obtained sequence information can then be either analyzed by database search for homology to known proteins or used to design degenerate oligonucleotide primers to amplify part ofthe co ⁇ esponding cDNA of interest.
- Mob-5 acts as an autocrine growth factor downstream of activated Ras.
- Ras transformed RIE cells can be used in a receptor binding assay.
- APtag technology Felanagan and leder, 1990
- Mob-5-alkaline phosphatase (AP) fusion protein was employed to create a Mob-5-alkaline phosphatase (AP) fusion protein, which can be used as an affinity agent for the receptor analysis.
- This strategy has been successfully used to identify and clone a number of important cell surface receptors, including those of leptin (Tartaglia et al, 1995) and semaphorin III (He and Tessier- Lavigne, 1997).
- Mob-5-AP was secreted at high levels in 293T cells stably transfected with the recombinant plasmid.
- the secreted 90 KD Mob-5-AP can be readily detected from the culture media by either the alkaline phosphatase assay or direct Western blot analysis using anti-Mob-5 antibody ( Figure 4A).
- the construct ofthe Mob-5-AP fusion protein was also verified by Western blot using antibody to the 67 KD human placental secreted AP ( Figure 4A, right panel).
- Mob-5 receptor study Rat-1 cells before and after transformation by oncogenic h-ras were incubated with the cell culture media containing an equal amount of either the Mob-5-AP fusion protein or secreted AP itself. Unlike the AP alone control, Mob-5-AP was found to be preferentially bound to the cell surface ofthe h-ras transformed Rat-1 cells, but not of the parental control ( Figure 4B). Thus, Mob-5/Mob-5 receptor may represent a novel putative autocrine loop coordinately activated by ras oncogenes.
- hMob-5 Over expression of hMob-5 in colon cancer and species specificity ofthe Mob-5R With a high degree of homology to Mob-5, it is likely that the hMob-5 is the human homolog of Mob-5. hMob-5 was found to be over expressed in nearly all human colon cancer specimens analyzed. Using RT-PCR, the entire coding region of hMob-5 was amplified from human colon cancer tissues and hMob-5-AP was constructed and expressed in 293T cells.
- hMob-5-AP directed by hMob-5's native signal peptide was found to be secreted at a high level into the culture medium and could readily detected by both AP activity assay (range from 0.7 to 1.7 unit/ml) and direct Western blot using polyclonal antibody against hMob- 5( Figure 4D). Further confirmation ofthe 90 KD HMob-5-AP fusion protein was obtained by Western blot using antibody to AP itself ( Figure 4D).
- the putative Mob-5 receptor was also found to be differentially expressed between the normal and h-ras transformed RIE cells using Mob-5-AP fusion protein as a ligand (Figure 4E, left panel).
- the receptor binding assay was conducted using hMob-5-AP in place of Mob-SAP.
- hMob-5-AP did not exhibit any preferential cell surface binding to the RIE cells before and after transformation by h-ras oncogene ( Figure 4E, right panel).
- mob-5 expression is activated by both oncogenic h-ras and k-ras in intestinal epithelial cells Cancer arises from the epithelial cells rather than fibroblasts and K-ras mutations have been tightly linked with cancers of gastric intestinal origins (Kiaris and Spandidos, 1995). Supporting the importance of mob-5 in ras mediated oncogenesis, mob-5 expression was shown to be activated by both h-ras and k-ras oncogenes in rat intestinal epithelial cells (RIE). As in fibroblasts, Mob-5-AP binds specifically only to the ras transformed RIE cells but not to the non-transformed parental control.
- RIE rat intestinal epithelial cells
- the lambda ZAP II Vector/Gigapack cloning kit was used to construct the cDNA library following the instruction provided by the manufacturer. 500,000 plaques were screened for full length mob-5 cDNA using ⁇ 32 P-labeled cDNA probe from differential display. Positive plaques were excised as phagemids and sequenced by the molecular biology core facility of Dana-Farber Cancer Institute, Boston.
- the entire hmob-5 coding region was amplified from RNA isolated from human colon cancer tissues by RT-PCR based on the published sequence (Jiang et al., 1995).
- the cDNA synthesis was carried out in a total volume of 20 ⁇ l containing up 1 mg of DNA-free total RNA in the presence of 4 ⁇ of 5xRT buffer (GenHunter), with 20 ⁇ M dNTP, 1 ⁇ M of oligo-dT20 primer (GenHunter) and 200 units of MMLV reverse transcriptase (GenHunter) at 37 °C for 1 hour.
- cMob-5 cDNA was cloned by RT-PCR from colorectal cancer tissues. PCR primers used were as indicated in Figure 6 (upper panel), L-Primer (5'- TGCAAAGCCTGTGGACTTTAGCCAG-3')(SEQ ID NO: 17)and R-Primer 1 (5'- CCGCCTGTGTGCACTGTCTCTGATG-3 ' ) (SEQ ID NO : 18) .
- L-Primer 5'- TGCAAAGCCTGTGGACTTTAGCCAG-3'
- R-Primer 1 5'- CCGCCTGTGTGCACTGTCTCTGATG-3 '
- a smaller cDNA of 325 bp was discovered only from the colorectal cancer tissues but not from their adjacent normal controls.
- DNA sequence analysis revealed an internal in-frame deletion of 159 base (53 amino acids) from the hMob-5 coding region.
- the full length cMob-5 cDNA was then obtained by RT-PCR from a colorectal cancer tissue using primer Lmda (5'- CCAGGAGGAACACGAGACTGAGAGATG-3'XSEQ ID NO: 19) and primer Rmda (5'-GGAGGTCCAGGTCTAGACATTC-3') (SEQ ID NO: 20).
- AAGCTTTCCGGATTGGCAAT-3' (SEQ ID NO: 14).
- the PCR product was first subcloned into PCR-TRAP vector (GenHunter, Ten, TN) and the E ⁇ HI- H dlll insert was then excised, purified and ligated into corresponding sites of the 6X- ⁇ is tag expression vector pQE32 (Qiagen, Chatsworth, CA).
- hMob-5-6XHis tagged protein the coding region of hMob-, excluding the putative signal peptide sequence (codon 1-44), was amplified by PCR using primers Lhis-hMob5 (5'-GGATCCABBTATCAGGGG-3')(SEQ ID NO: 15) and Rhis-hMob5 (5'-AAGCTTGTAGAATTTCTGCATC-3') (SEQ ID NO: 16).
- Lhis-hMob5 5'-GGATCCABBTATCAGGGG-3'
- Rhis-hMob5 5'-AAGCTTGTAGAATTTCTGCATC-3'
- the recombinant plasmids were transformed into Escherichia coli TGI cells to produce N-terminal 6X-His tagged Mob-5 and hMob-5.
- the purified proteins were dialyzed in PBS to remove the denaturant and then injected into New Zealand White rabbits to produce polyclonal antibodies (HRP Inc., Denver, PA).
- mob-5 mammalian expression construct A 680-bp BspHI-Bgl II restriction fragment containing the entire coding region of mob-5 was generated by PCR using cloned mob-5 cDNA as a template. The targeting construct was subsequently inserted between the Nco I and Bam I sites of retroviral vector pMFG-S (Danos and Mulligan, 1988; Dranoff, G. et al. 1993) and sequenced to ensure the sequence accuracy.
- Mob-5-AP and h.Mob-5-AP fusion and receptor binding assay Both the entire Mob- 5 and hMob-5 coding regions including their signal peptides were amplified by PCR and subsequently inserted into the Bgl II site of APtag2 cloning vector (Flanagan and Leder, 1990; GenHunter Corp. , Nashville TN) to allow the C-terminal fusion of the AP.
- the recombinant plasmids and APtag4 which express the secreted AP alone (Flanagan and Leder, 1990) were stably transfected into 293T cells. The expression of the fusion proteins, AP activity assay and receptor binding were essentially as described before (Flanagan and Leder, 1990 ).
- the cells were seed at 5 X 10 5 per well in 6- well plates in duplicates. After cells reach confluence, receptor binding assay was carried out with 1 ml of either the AP fusion protein- or AP alone- containing media ( all were 0.7 unit/ml) produced by the 293T cells.
- Retroviral Infection Recombinant plasmids were transfected into target cells by the standard calcium phosphate precipitation method.
- pMFG-S-Mob-5 was cotransfected with pCMV-Neo vector at a rate of 10: 1 (lO ⁇ g of pCMV-S-rMob-5: I ⁇ g of pCMV-Neo) into the CRIP packaging cells to produce G418 (lmg/ml) resistant stable cell lines.
- Retroviral infection was carried out essentially as described previously (Zhang et al., 1998).
- GBA Blue A Sepharose beads at high affinity. This allows the use of GBA in a method for detecting Mob-5 in an ELISA. GBA is utilized to capture the antigen (i.e. Mob-5) and a biotinylated monoclonal antibody to Mob-5 is used to detect the antigen. The use of GBA allows for higher capacity and lower costs when compared to the traditional ELISA method.
- the method is performed by diluting a biotinylated Mab- hMob-5 (for example, the monoclonal antibody produced from hybridoma clone L-14) 1 :500 in PBST (Phosphate Buffered Saline, Tween 0.05%); diluting Streptavidin-HRP (Sigma) 1 :500 in PBST; mixing the diluted Mab-hMob-5 with the diluted Streptavidin- HRP in equal volume (for example, 500 ml each); letting the mixture sit for 5-10 minutes; washing the GBA beads with PBST twice; spinning and removing the upper liquid phase with an aspirator; resuspending the washed GBA beads in PBST as a 50% suspension; mixing 100-500 ml of a sample with 100 ml ofthe diluted Mab-hMob-5 and Streptavidin-HRP mixture in a tube; adding 50 ml of resuspended GBA beads to the tube; allowing
- a hMob-5-9XHis medium can be utilized as a positive control.
- the GBA beads may be added to the sample prior to the addition of the biotinylated Mab/ streptavidin- HRP mixture, alkaline phosphatase labeled streptavidin can be used, wash times may vary, spin times may vary and development times may vary.
- wash times may vary
- spin times may vary
- development times may vary.
- hMob-5 Detection of auto-antibody to hMob-5 in colorectal cancer patients
- the antibody capture immunoassay was used to detect the presence of antibody to Mob-5 in the plasma from colorectal cancer patients.
- purified recombinant hMob-5 was blotted onto PVC membrane. After blocking the non-specific binding site with BSA, diluted plasma samples from normal controls and colorectal cancer patients obtained from Brigham- Women's Hospital and the Vanderbilt Clinic were incubated individually with the antigen. The presence ofthe auto-antibody to hMob-5 was detected after binding to the anti-human IgG labeled with HRP. About half of the colorectal cancer patients appeared to contain antibody to hMob-5, while 1 out of 8 normal controls showed a positive result.
- Neuropilin is a receptor for the axonal chemorepellent semaphorin III. Cell 90, 739-751.
- Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11, 2477-2486.
- Viral interleukin 10 is critical for the induction of B cell growth transformation by Epstein-Barr virus. J. Exp. Med. 178, 439-447.
- Mob-1 a ras target gene, is over expressed in colorectal cancer. Oncogene 14, 1607-1610.
Abstract
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AU2001231192A AU2001231192A1 (en) | 2000-01-26 | 2001-01-26 | Mob-5/hmob-5 as a cancer diagnostic marker |
EP01903364A EP1254155A4 (en) | 2000-01-26 | 2001-01-26 | Mob-5/hmob-5 as a cancer diagnostic marker |
US10/182,094 US20040019193A1 (en) | 2001-01-26 | 2001-01-26 | Mob-5/hmob-5 as a cancer diagnostic marker |
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WO2003038129A2 (en) * | 2001-10-30 | 2003-05-08 | Ortho-Clinical Diagnostics, Inc. | Methods for assessing and treating leukemia |
WO2003082255A2 (en) * | 2002-04-03 | 2003-10-09 | Novartis Ag | Use of mob-5 in pain |
WO2005060998A2 (en) * | 2003-12-03 | 2005-07-07 | Schering Corporation | Methods of modulating cytokine activity; related reagents |
EP1767656A2 (en) * | 2001-10-30 | 2007-03-28 | Ortho-Clinical Diagnostics, Inc. | Methods for assessing and treating leukemia |
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JP4145953B2 (en) * | 1993-10-27 | 2008-09-03 | ザ・トラスティーズ・オブ・コランビア・ユニバーシティー・イン・ザ・シティー・オブ・ニューヨーク | MDA-7 gene |
US6262333B1 (en) * | 1998-06-10 | 2001-07-17 | Bayer Corporation | Human genes and gene expression products |
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Non-Patent Citations (4)
Title |
---|
DATABASE GENBANK [online] 1 October 2000 (2000-10-01), ZHANG ET AL: "Identification of a novel ligand-receptor pair constitutively activated by ras oncogenes", XP002940600, Database accession no. Q9JI24 * |
DATABASE GENBANK [online] 8 August 2000 (2000-08-08), ZHANG ET AL.: "Identification of a novel ligand-receptor pair constitutively activated by ras oncogenes", XP002940599, Database accession no. AF269251 * |
J. BIOL. CHEM., vol. 275, no. 32, 2000, pages 24436 - 24443 * |
See also references of EP1254155A4 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2003038129A2 (en) * | 2001-10-30 | 2003-05-08 | Ortho-Clinical Diagnostics, Inc. | Methods for assessing and treating leukemia |
WO2003038129A3 (en) * | 2001-10-30 | 2004-09-10 | Ortho Clinical Diagnostics Inc | Methods for assessing and treating leukemia |
EP1767656A2 (en) * | 2001-10-30 | 2007-03-28 | Ortho-Clinical Diagnostics, Inc. | Methods for assessing and treating leukemia |
EP1767656A3 (en) * | 2001-10-30 | 2007-07-25 | Ortho-Clinical Diagnostics, Inc. | Methods for assessing and treating leukemia |
WO2003082255A2 (en) * | 2002-04-03 | 2003-10-09 | Novartis Ag | Use of mob-5 in pain |
WO2003082255A3 (en) * | 2002-04-03 | 2004-02-05 | Novartis Ag | Use of mob-5 in pain |
WO2005060998A2 (en) * | 2003-12-03 | 2005-07-07 | Schering Corporation | Methods of modulating cytokine activity; related reagents |
WO2005060998A3 (en) * | 2003-12-03 | 2006-03-16 | Schering Corp | Methods of modulating cytokine activity; related reagents |
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EP1254155A1 (en) | 2002-11-06 |
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AU2001231192A1 (en) | 2001-08-07 |
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