# Patent

## Title
Human Ron-related gene variant associated with cancers

## Abstract

The invention relates to the nucleic acid and polypeptide sequences of three novel human Ron-related gene variants (Ron-V1, Ron-V2, and Ron-V3). The invention also provides a process for producing the polypeptides of the variants, as well as uses for the nucleic acid, polypeptide and antibodies to same in diagnosing human breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma.

## Background
FIELD OF THE INVENTION
The invention relates to the nucleic acid and polypeptide sequences of a novel human Ron-related gene variant, preparation process thereof, and uses of the same in diagnosing cancers, in particular, breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma.
BACKGROUND OF THE INVENTION
Cancer is one of the major causes of death in the world. According to the report from the WHO Fact Sheet-Cancer (2003), cancer accounts for 7.1 million (12.6% of the global total) deaths annually and new cancer cases are expected to be increased from 10 million people in 2000 to 15 million people by 2020. In recent years, much progress has been made toward understanding the molecular and cellular biology of cancers. Many important contributions have been made by the identification of several key genetic factors associated with cancers. However, the treatments of cancers still mainly depend on surgery, chemotherapy and radiotherapy because the molecular mechanisms underlying the pathogenesis of cancers remain largely unclear.
It is interesting to note that receptor tyrosine kinases are a family of proteins reported to be involved in many fundamental cellular processes such as cell cycle, signalling transduction, proliferation, differentiation, adhesion, migration, and apoptosis. An important role of the receptor tyrosine kinases in the development of cancers has been reported previously. Therefore, future strategies for the prevention and treatment of cancers may be focused on the elucidation of the receptor tyrosine kinases which are involved in the pathogenesis of cancers. Ron, a member of the receptor tyrosine kinase family located on human chromosome 3p21 (a region frequently deleted in small cell lung carcinoma), was isolated and identified through the screening of cDNA libraries. Previous studies have indicated that Ron is involved in many genetic mechanisms of the tumor cells (Wang et al., (2003) Carcinogenesis 24:1291-300; Angeloni et al., (2004) J Biol Chem. 279:3726-32; Peace et al., (2005) Cancer Res. 65:1285-93; Camp et al., (2005) Ann Surg Oncol. 12:273-81), and that Ron is a potential target for understanding the pathogenesis of cancers. In addition, the association between gene variants and diseases has been reported previously. Therefore, the discovery of gene variants of Ron may be important for the diagnostic markers of cancers.
SUMMARY OF THE INVENTION
The present invention provides one Ron-related gene variant present in human breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma. The nucleotide sequence of the gene variant and polypeptide sequence encoded thereby can be used for the diagnosis of any diseases associated with this gene variant or cancers, in particular, breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma.
The invention further provides an expression vector and host cell for expressing the variant.
The invention further provides a method for producing the variant.
The invention further provides an antibody that specifically binds to the variant. For example, by selecting a sequence unique to one of the variants, e.g., a peptide fragment that spans one the deletion junctions, an antibody may be generated that binds to one of the variants and not to RON. Preferably, such peptide antigens are at least 17 amino acid long, but in some cases smaller peptides such as 10 mers, 12 mers, or 15 mers may suffice. Longer peptides, such as 20 mers, 50 mers, hundred-mers (and those therebetween) and even up to full length proteins may be used.
The invention also provides methods for detecting the presence of the variant in a mammal.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleic acid sequence (SEQ ID NO:1), amino acid sequence (SEQ ID NO:2) and nucleic acid with corresponding amino acid sequence (SEQ ID NO: 1 & 2) of Ron.
FIG. 2 shows the nucleic acid sequence (SEQ ID NO:3), amino acid sequence (SEQ ID NO:4) and nucleic acid with corresponding amino acid sequence (SEQ ID NO: 3 & 4) of Ron-V1.
FIG. 3 shows the nucleic acid sequence (SEQ ID NO:5), amino acid sequence (SEQ ID NO:6) and nucleic acid with corresponding amino acid sequence (SEQ ID NO: 5 & 6) of Ron-V2.
FIG. 4 shows the nucleic acid sequence (SEQ ID NO:7), amino acid sequence (SEQ ID NO:8) and nucleic acid with corresponding amino acid sequence (SEQ ID NO: 7 & 8) of Ron-V3.
FIG. 5 shows the nucleotide sequence alignment between the human Ron gene (SEQ ID NO: 1) and its related gene variants (Ron-V1, Ron-V2, and Ron-V3, SEQ ID NO: ID 3, 5, 7).
FIG. 6 shows the amino acid sequence alignment between the human Ron protein (SEQ ID NO: 2) and its related gene variants (Ron-V1, Ron-V2, and Ron-V3) (SEQ ID NO: 4, 6, 8).
FIG. 7 shows the expression pattern of Ron gene variants in human cell lines.



DETAILED DESCRIPTION OF THE INVENTION
All technical and scientific terms used herein, unless otherwise defined, have the same meanings as commonly understood by persons skilled in the art.
The term “antibody” used herein denotes intact molecules (a polypeptide or group of polypeptides) as well as fragments thereof, such as Fab, R(ab′)2, and Fv fragments, which are capable of binding epitopes. Antibodies are produced by specialized B cells after stimulation by an antigen. Structurally, antibody consists of four subunits including two heavy chains and two light chains. The internal surface shape and charge distribution of the antibody binding domain is complementary to the features of an antigen. Thus, antibody can specifically act against the antigen in an immune response.
The term “base pair (bp)” used herein denotes nucleotides composed of a purine on one strand of DNA which can be hydrogen bonded to a pyrimidine on the other strand. Thymine (or uracil) and adenine residues are linked by two hydrogen bonds. Cytosine and guanine residues are linked by three hydrogen bonds.
The term “Basic Local Alignment Search Tool (“BLAST”) (BLAST; Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402) used herein denotes programs for evaluation of homologies between a query sequence (amino or nucleic acid) and a test sequence. Specific BLAST programs are described as follows:
(1) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;
(2) BLASTP compares an amino acid query sequence against a protein sequence database;
(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence against a protein sequence database;
(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames; and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
The term “cDNA” used herein denotes nucleic acids synthesized from a mRNA template using reverse transcriptase.
The term “cDNA library” used herein denotes a library composed of complementary DNAs which are reverse-transcribed from mRNAs.
The term “complement” used herein denotes a polynucleotide sequence capable of forming base pairing with another polynucleotide sequence. For example, the sequence 5′-ATGGACTTACT-3′ binds to the complementary sequence 5′-AGTAAGTCCAT-3′.
The term “deletion” used herein denotes a removal of a portion of one or more amino acid residues/nucleotides from a gene.
The term “expressed sequence tags (ESTs)” used herein denotes short (200 to 500 base pairs) nucleotide sequence that derives from either 5′ or 3′ end of a cDNA.
The term “expression vector” used herein denotes nucleic acid constructs which contain a cloning site for introducing the DNA into vector, one or more selectable markers for selecting vectors containing the DNA, an origin of replication for replicating the vector whenever the host cell divides, a terminator sequence, a polyadenylation signal, and a suitable control sequence which can effectively express the DNA in a suitable host. The suitable control sequence may include promoter, enhancer and other regulatory sequences necessary for directing polymerases to transcribe the DNA.
The term “host cell” used herein denotes a cell which is used to receive, maintain, and allow the reproduction of an expression vector comprising DNA. Host cells are transformed or transfected with suitable vectors constructed using recombinant DNA methods. The recombinant DNA introduced with the vector is replicated whenever the cell divides.
The term “insertion” or “addition” used herein denotes the addition of a portion of one or more amino acid residues/nucleotides to a gene.
The term “in silico” used herein denotes a process of using computational methods (e.g., BLAST) to analyze DNA sequences.
The term “polymerase chain reaction (PCR)” used herein denotes a method which increases the copy number of a nucleic acid sequence using a DNA polymerase and a set of primers (about 20 bp oligonucleotides complementary to each strand of DNA) under suitable conditions (successive rounds of primer annealing, strand elongation, and dissociation).
The term “polynucleotide” or “nucleic acid sequence” used herein denotes a sequence of nucleotide (guanine, cytosine, thymine or adenine) in a specific order that can be a natural or synthesized fragment of DNA or RNA. It may be single-stranded or double-stranded.
The term “protein” or “polypeptide” used herein denotes a sequence of amino acids in a specific order that can be encoded by a gene or by a recombinant DNA. It can also be chemically synthesized.
The term “RNA interference (RNAi)” used herein denotes an introduction of homologous double stranded RNA (dsRNA) into a cell to specifically inhibit the expression of a gene.
The term “reverse transcriptase-polymerase chain reaction (RT-PCR)” used herein denotes a process which transcribes mRNA to complementary DNA strand using reverse transcriptase followed by polymerase chain reaction to amplify the specific fragment of DNA sequences.
The term “transformation” used herein denotes a process describing the uptake, incorporation, and expression of exogenous DNA by prokaryotic host cells.
The term “transfection” used herein denotes a process describing the uptake, incorporation, and expression of exogenous DNA by eukaryotic host cells.
The term “variant” used herein denotes a fragment of sequence (nucleotide or amino acid) inserted or deleted by one or more nucleotides/amino acids.
According to the present invention, the polypeptides of one novel human Ron-related gene variant and the nucleic acid sequences encoding the same are provided.
According to the present invention, human Ron cDNA sequence was used to query the human EST databases using BLAST program to search for Ron-related gene variants. Three human cDNA partial sequences (i.e., EST) showing similar to Ron was identified from ESTs deposited in a cDNA database constructed using a SW620 (colon adenocarcinoma) cell line. The cDNA clones, named Ron-V1 (Ron variant 1), Ron-V2 (Ron variant 2), and Ron-V3 (Ron variant 3), were then isolated from the SW620 cDNA library and sequenced. FIGS. 2-4 show the nucleic acid sequences (SEQ ID NOs: 3, 5, & 7) of Ron-V1, Ron-V2, and Ron-V3 and its corresponding amino acid sequences (SEQ ID NOs: 4, 6, & 8) encoded thereby.
The full-length of the Ron-V1 cDNA is a 4000 bp clone containing a 3783 bp open reading frame (ORF) extending from 29 bp to 3811 bp, which corresponds to an encoded protein of 1261 amino acid residues with a predicted molecular mass of 136.1 kDa. The initiation ATG sequence of Ron-V1 is located at nucleotide 29-31 bp. The full-length of the Ron-V2 cDNA is a 3880 bp clone containing a 3663 bp open reading frame (ORF) extending from 29 bp to 3691 bp, which corresponds to an encoded protein of 1221 amino acid residues with a predicted molecular mass of 132 kDa. The initiation ATG sequence of Ron-V2 is located at nucleotide 29-31 bp. The full-length of the Ron-V3 cDNA is a 3853 bp clone containing a 3636 bp open reading frame (ORF) extending from 29 bp to 3664 bp, which corresponds to an encoded protein of 1212 amino acid residues with a predicted molecular mass of 131.1 kDa. The initiation ATG sequence of Ron-V3 is located at nucleotide 29-31 bp. To determine the variations in sequence of Ron-V1, Ron-V2 and Ron-V3 cDNA clones, an alignment of Ron nucleotide/amino acid sequence with Ron-V1, Ron-V2 and Ron-V3 was performed (FIGS. 5 and 6). The results indicate that (1) Ron-V1: three major genetic deletions were found in the aligned nucleotide sequences showing that Ron-V1 has a 255 bp, a 137 bp and a 149 bp deletions in the sequence of Ron from nucleotides 2838-3092, nucleotides 3839-3975 and nucleotides 4118-4266, respectively; (2) Ron-V2: four major genetic deletions were found in the aligned nucleotide sequences showing that Ron-V2 has a 120 bp, a 255 bp, a 137 bp and a 149 bp deletions in the sequence of Ron from nucleotides 2678-2797, nucleotides 2838-3092, nucleotides 3839-3975 and nucleotides 4118-4266, respectively; (3) Ron-V3: four major genetic deletions were found in the aligned nucleotide sequences showing that Ron-V3 has a 147 bp, a 255 bp, a 137 bp and a 149 bp deletions in the sequence of Ron from nucleotides 2678-2824, nucleotides 2838-3092, nucleotides 3839-3975 and nucleotides 4118-4266, respectively. Scanning Ron, Ron-V1, Ron-V2 and Ron-V3 amino acid sequences against protein profile databases including PROSITE (Motif Scan) indicated that Ron protein contains one Sema domain (31-522aa), one Protein_Kinase_ATP (1088-1114aa), one Protein_Kinase_TYR (1204-1216aa), and one Protein_Kinase_DOM (1082-1345aa). Ron-V1 protein contains one Sema domain (31-522aa), one Protein_Kinase_ATP (1003-1029aa), one Protein_Kinase_TYR (1119-1131aa), and one Protein_Kinase_DOM (997-1261aa). Ron-V2 protein contains one Sema domain (31-522aa), one Protein_Kinase_ATP (963-989aa), one Protein_Kinase_TYR (1079-1091aa), and one Protein_Kinase_DOM (957-1221aa). Ron-V3 protein contains one Sema domain (31-522aa), one Protein_Kinase ATP (954-980aa), one Protein_Kinase TYR (1070-1082aa), and one Protein_Kinase_DOM (948-1212aa).
In the present invention, a search of ESTs deposited in dbEST at NCBI was performed to determine the presence of Ron gene variants in silico. The result of in silico analysis showed that one EST (GenBank accession number BG823879) was found to confirm the absence of 255 bp region on Ron-V1, Ron-V2, and Ron-V3 nucleotide sequences; one EST (GenBank accession number AW009348) was found to confirm the absence of 137 bp region on Ron-V1, Ron-V2, and Ron-V3 nucleotide sequences. Therefore, any Ron nucleotide fragments containing the immediately upstream and downstream sequences of the deleted 255 bp region (2837-2838 bp of Ron-V1; 2717-2718 bp of Ron-V2; 2691-2692 bp of Ron-V3) and/or 137 bp region (3583-3584 bp of Ron-V1; 3463-3464 bp of Ron-V2; 3436-3437 bp of Ron-V3) and/or 149 bp region (3725-3726 bp of Ron-V1; 3605-3606 bp of Ron-V2; 3578-3579 bp of Ron-V3) are the “gene targets” which may be used as probes to determine the presence of Ron-V1, Ron-V2, and Ron-V3 under high stringency conditions. An alternative approach is that any set of primers for amplifying the fragment containing the immediately upstream and downstream sequences of the deleted 255 bp region (2837-2838 bp of Ron-V1; 2717-2718 bp of Ron-V2; 2691-2692 bp of Ron-V3) and/or 137 bp region (3583-3584 bp of Ron-V1; 3463-3464 bp of Ron-V2; 3436-3437 bp of Ron-V3) and/or 149 bp region (3725-3726 bp of Ron-V1; 3605-3606 bp of Ron-V2; 3578-3579 bp of Ron-V3) may be used for determining the presence of the variants (Ron-V1, Ron-V2, and Ron-V3). On the other hand, any Ron nucleotide fragments containing the immediately upstream and downstream sequences of the deleted 120 bp region (2678-2679 bp of Ron-V2) and 147 bp region (2678-2679 bp of Ron-V3) are the “gene targets” which may be used as probes for determining the presence of Ron-V2 and Ron-V3, respectively, under high stringency conditions. An alternative approach is that any set of primers for amplifying the fragment containing the immediately upstream and downstream sequences of the deleted 120 bp region (2678-2679 bp of Ron-V2) and 147 bp region (2678-2679 bp of Ron-V3) may be used for determining the presence of Ron-V2 and Ron-V3, respectively.
According to the present invention, the polypeptides of the human Ron-V1, Ron-V2, and Ron-V3 and its fragments thereof may be produced through genetic engineering techniques. In this case, they are produced by appropriate host cells which have been transformed by DNAs that encode for the polypeptides or fragments thereof. The nucleotide sequence encoding the polypeptide of the human Ron-V1, Ron-V2, and Ron-V3 or their fragments thereof are inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence in a suitable host. The nucleic acid sequence is inserted into the vector in a manner that will be expressed under appropriate conditions (e.g., in proper orientation and correct reading frame and with appropriate expression sequences, including an RNA polymerase binding sequence and a ribosomal binding sequence).
Any method that is known to those skilled in the art may be used to construct expression vectors containing sequences encoding the polypeptides of the human Ron-V1, Ron-V2, and Ron-V3 and appropriate transcriptional/translational control elements. These methods may include in vitro recombinant DNA and synthetic techniques, and in vivo genetic recombinants.
A variety of expression vector/host systems may be utilized to express the polypeptide-coding sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vector; yeast transformed with yeast expression vector; insect cell systems infected with virus (e.g., baculovirus); plant cell system transformed with viral expression vector (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV); or animal cell system infected with virus (e.g., vaccina virus, adenovirus, etc.). Preferably, the host cell is a bacterium, and most preferably, the bacterium is E. coli. 
Alternatively, the Polypeptides of the human Ron-V1, Ron-V2, and Ron-V3 or fragments thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Perkin-Elmer).
According to the present invention, the fragments of the polypeptides and nucleic acid sequences of the human Ron-V1, Ron-V2, and Ron-V3 are used as immunogens, primers or probes. Preferable, the purified fragments of the human Ron-V1, Ron-V2, and Ron-V3 are used. The fragments may be produced by enzyme digestion, chemical cleavage of isolated or purified polypeptide or nucleic acid sequences, or chemical synthesis. Thereafter, the fragments may be isolated or purified. Such isolated or purified fragments of the polypeptides and nucleic acid sequences can be used as immunogens, primers or probes.
The present invention further provides the antibodies which specifically bind one or more out-surface epitopes of the polypeptides of the human Ron-V1, Ron-V2, and Ron-V3.
According to the present invention, immunization of mammals with immunogens described herein, preferably humans, rabbits, rats, mice, sheep, goats, cows, or horses, is performed following procedures well known to those skilled in the art, for the purpose of obtaining antisera containing polyclonal antibodies or hybridoma lines secreting monoclonal antibodies.
Monoclonal antibodies can be prepared by standard techniques. Such techniques are disclosed, for example, in U.S. Pat. Nos. 4,271,145 and 4,196,265. An animal is immunized with the immunogen. Hybridomas are prepared by fusing spleen cells from the immunized animal with myeloma cells. The fusion products are screened for those producing antibodies that specifically bind to the immunogen. The positive hybridoma clones are isolated, and the monoclonal antibodies are recovered from those clones.
Immunization regimens for production of both polyclonal and monoclonal antibodies are well-known in the art. The immunogen may be injected by any of a number of routes, including subcutaneous, intravenous, intraperitoneal, intradermal, intramuscular, mucosal, or a combination thereof. The immunogen may be injected in soluble form, aggregate form, attached to a physical carrier, or mixed with an adjuvant, using methods and materials well-known in the art. The antisera and antibodies may be purified using well-known column chromatography methods.
According to the present invention, antibody fragments which contain specific binding sites for the polypeptides or fragments thereof may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments.
The subject invention also provides methods for diagnosing the diseases associated with Ron-V1, Ron-V2, or Ron-V3, particularly breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma, by utilizing the nucleic acid sequences, the polypeptide of the human Ron-V1, Ron-V2, or Ron-V3, or fragments thereof, and the antibodies against the polypeptides.
Since Ron-V1, Ron-V2, or Ron-V3 clones were isolated from a SW620 cDNA library, it is advisable that Ron-V1, Ron-V2, or Ron-V3 serve as a marker for the diagnosis of human colon adenocarcinoma. Thus, the expression levels/patterns of Ron-V1, Ron-V2, or Ron-V3 may be a useful indicator for screening patients suspected of having cancers or more specifically, breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma. This suggests that relative expression levels/patterns (mRNA or protein) may confer an increased susceptibility to cancers, and more preferably, to breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma. Fragments of Ron-V1, Ron-V2, or Ron-V3 transcripts (mRNAs) may be detected by RT-PCR approach. Polypeptides of Ron-V1, Ron-V2, or Ron-V3 may be determined by the binding of antibodies to these polypeptides. These approaches may be performed in accordance with conventional methods well known to persons skilled in the art. On the other hand, a method (RNAi; RNA interference) that is known to those skilled in the art may be used to interfere and degrade the targeted RNA using chemically synthesized double stranded short nucleic acid (about 23 nucleotides dsRNA) molecules (Montgomery et al. (1998) Proc Natl Acad Sci USA. 95:15502-7). According to the present invention, the double stranded short nucleic acid molecules containing the sequences of the “gene targets”: nucleotides 2837-2838, nucleotides 3583-3584, or nucleotides 3725-3726 of Ron-V1; nucleotides 2717-2718, nucleotides 3463-3464, or nucleotides 3605-3606 of Ron-V2; nucleotides 2691-2692, nucleotides 3436-3437, or nucleotides 3578-3579 of Ron-V3, which may be used to specifically interfere and degrade Ron-V1, Ron-V2, and Ron-V3 mRNAs. On the other hand, the double stranded short nucleic acid molecules containing the sequence of the “gene targets”: nucleotides 2678-2679 of Ron-V2 or Ron-V3, may be used to specifically interfere and degrade Ron-V2 and Ron-V3 mRNA, respectively. After RNAi approach was conducted, the decreased transcripts or polypeptides may be used to interpret the presence of Ron-V1, Ron-V2, and Ron-V3 mRNAs.
According to the present invention, the expression of these gene variant mRNAs in sample may be determined by, but not limited to, RT-PCR. Using TRIZOLTM reagents (LIFE TECHNOLOGY®), total RNA may be isolated from patient samples. Tissue samples (e.g., biopsy samples) are powdered under liquid nitrogen before samples (e.g., biopsy samples) are powdered under liquid nitrogen before homogenization. RNA purity and integrity are assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. A set of primers can be designed to amplify the expected size of specific PCR fragments (gene targets) of Ron-V1, Ron-V2, and Ron-V3. For example, one of the primers is a fragment of the sequence (A fragment; forward primer) containing the gene targets: nucleotides 2837-2838, nucleotides 3583-3584, or nucleotides 3725-3726 of Ron-V1; nucleotides 2717-2718, nucleotides 3463-3464, or nucleotides 3605-3606 of Ron-V2; nucleotides 2691-2692, nucleotides 3436-3437, or nucleotides 3578-3579 of Ron-V3, and the other is a fragment (B fragment; reverse primer) designed from a reverse complementary strand of Ron-V1, Ron-V2, and Ron-V3 sequences at any location downstream of “A fragment”. If “B fragment” is designed from a reverse complementary strand of the sequence containing the gene targets: nucleotides 2837-2838, nucleotides 3583-3584, or nucleotides 3725-3726 of Ron-V1; nucleotides 2717-2718, nucleotides 3463-3464, or nucleotides 3605-3606 of Ron-V2; nucleotides 2691-2692, nucleotides 3436-3437, or nucleotides 3578-3579 of Ron-V3, the “A fragment” is a fragment of the sequence at any location upstream of “B fragment”. Alternatively, one primer (A fragment; forward primer) is at any location upstream of the sequence containing “gene targets” and the other is designed from a reverse complementary strand at any location downstream of the sequence containing “gene targets”. PCR fragments are analyzed on a 1% agarose gel using five microliters (10%) of the amplified products. The intensity of the signals may be determined by using the Molecular Analyst program (version 1.4.1; Bio-Rad). Thus, the relative expression level for each amplified PCR product may be calculated based on the intensity of signals.
The RT-PCR experiment may be performed according to the manufacturer instructions (BOEHRINGER MANNHEIM®). A 50 μl reaction mixture containing 2 μl total RNA (0.1 μg/μl), 1 μl each primer (20 pM), 1 μl each dNTP (10 mM), 2.5 μl DTT solution (100 mM), 10 μl 5×RT-PCR buffer, 1 μl enzyme mixture, and 28.5 μl sterile distilled water may be subjected to the conditions such as reverse transcription at 60° C. for 30 minutes followed by 35 cycles of denaturation at 94° C. for 2 minutes, annealing at 60° C. for 2 minutes, and extension at 68° C. for 2 minutes. The RT-PCR analysis may be repeated twice to ensure reproducibility, for a total of three independent experiments.
The expression of gene variants can also be analyzed using Northern Blot hybridization approach. Specific fragments containing the sequences of the “gene targets” of the Ron-V1, Ron-V2, and Ron-V3 may be amplified by polymerase chain reaction (PCR) using primer set designed for RT-PCR. The amplified PCR fragment may be labeled and served as a probe to hybridize the membranes containing total RNAs extracted from the samples under the conditions of 55° C. in a suitable hybridization solution for 3 hours. Blots may be washed twice in 2×SSC, 0.1% SDS at room temperature for 15 minutes each, followed by two washes in 0.1×SSC and 0.1% SDS at 65° C. for 20 minutes each. After these washes, blot may be rinsed briefly in suitable washing buffer and incubated in blocking solution for 30 minutes, and then incubated in suitable antibody solution for 30 minutes. Blots may be washed in washing buffer for 30 minutes and equilibrated in suitable detection buffer before detecting the signals. Alternatively, the presence of the “gene targets” of the gene variants (cDNAs or PCR) can be detected using microarray approach. The cDNAs or PCR products corresponding to the nucleotide sequences of the present invention may be immobilized on a suitable substrate such as a glass slide. Hybridization can be preformed using the labeled mRNAs extracted from samples. After hybridization, nonhybridized mRNAs are removed. The relative abundance of each labeled transcript, hybridizing to a cDNA/PCR product immobilized on the microarray, can be determined by analyzing the scanned images.
According to the present invention, the presence of the polypeptide of Ron-V1, Ron-V2, and Ron-V3 in samples may be determined by, but not limited to, the immunoassay which uses the antibody that specifically binds to the polypeptide. The polypeptides of the gene variants may be expressed in prokaryotic cells by using suitable prokaryotic expression vectors. The cDNA fragments of Ron-V1, Ron-V2, and Ron-V3 genes encoding the amino acid sequences may be PCR amplified using primer set designed with restriction enzyme digestion sites incorporated in the 5′ and 3′ ends. The PCR products can then be enzyme digested, purified, and inserted into the corresponding sites of prokaryotic expression vector in-frame to generate recombinant plasmids. Sequence fidelity of this recombinant DNA can be verified by sequencing. The prokaryotic recombinant plasmids may be transformed into host cells (e.g., E. coli BL21 (DE3)). Recombinant protein synthesis may be stimulated by the addition of 0.4 mM isopropylthiogalactoside (IPTG) for 3 h. The bacterially-expressed proteins may be purified.
The polypeptide of the gene variant may be expressed in animal cells by using eukaryotic expression vectors. Cells may be maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; GIBCO® BRL) at 37° C. in a humidified 5% CO2 atmosphere. Before transfection, the nucleotide sequence of each of the gene variant may be amplified with PCR primers containing restriction enzyme digestion sites and ligated into the corresponding sites of eukaryotic expression vector in-frame. Sequence fidelity of this recombinant DNA can be verified by sequencing. The cells may be plated in 12-well plates one day before transfection at a density of 5×104 cells per well. Transfections may be carried out using Lipofectaminutese Plus transfection reagent according to the manufacturer's instructions (GIBCO® BRL). Three hours following transfection, medium containing the complexes may be replaced with fresh medium. Forty-eight hours after incubation, the cells may be scraped into lysis buffer (0.1 M Tris HCl, pH 8.0, 0.1% Triton X-100) for purification of expressed proteins. After these proteins are purified, monoclonal antibodies against these purified proteins (Ron-V1, Ron-V2, and Ron-V3) may be generated using hybridoma technique according to the conventional methods (de StGroth and Scheidegger, (1980) J Immunol Methods 35:1-21; Cote et al. (1983) Proc Natl Acad Sci USA 80: 2026-30; and Kozbor et al. (1985) J Immunol Methods 81:31-42).
According to the present invention, the presence of the polypeptides of Ron-V1, Ron-V2, and Ron-V3 in samples of the breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma may be determined by a Western blot analysis. Proteins extracted from samples may be separated by SDS-PAGE and transferred to suitable membranes such as polyvinylidene difluoride (PVDF) in transfer buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 20% methanol) with a Trans-Blot apparatus for 1 h at 100 V (e.g., Bio-Rad). The proteins can be immunoblotted with specific antibodies. For example, membrane blotted with extracted proteins may be blocked with suitable buffers such as 3% solution of BSA or 3% solution of nonfat milk powder in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and incubated with monoclonal antibody directed against the polypeptides of gene variants. Unbound antibody is removed by washing with TBST for 5×1 minutes. Bound antibody may be detected using commercial ECL Western blotting detecting reagents.
The following examples are provided for illustration, but not for limiting the invention.
EXAMPLES
Analysis of Human Colon Adenocarcinoma EST Database
Expressed sequence tags (ESTs) generated from the large-scale PCR-based sequencing of the 5′-end of human colon adenocarcinoma cDNA clones were compiled and served as EST databases. Sequence comparisons against the nonredundant nucleotide and protein databases were performed using BLASTN and BLASTX programs, at the National Center for Biotechnology Information (NCBITM) with a significance cutoff of p<10-10. ESTs representing putative Ron-V1, Ron-V2, and Ron-V3 genes were identified during the course of EST generation.
Isolation of cDNA Clones
Three cDNA clones exhibiting EST sequences similar to the Ron gene were isolated from the human colon adenocarcinoma cDNA library and named Ron-V1, Ron-V2, and Ron-V3. The inserts of these clones were subsequently excised in vivo from the λZAP Express vector using the EXASSISTTM/XLOLR helper phage system (STRATAGENE®). Phagemid particles were excised by coinfecting XL1-BLUE MRF' cells with EXASSISTTM helper phage. The excised pBluescript phagemids were used to infect E. coli XLOLR cells, which lack the amber suppressor necessary for EXASSISTTM phage replication. Infected XLOLR cells were selected using kanamycin resistance. Resultant colonies contained the double stranded phagemid vector with the cloned cDNA insert. A single colony was grown overnight in LB-kanamycin, and DNA was purified using a QIAGEN® plasmid purification kit.
Full Length Nucleotide Sequencing and Database Comparisons
Phagemid DNA was sequenced using the Taq dye-deoxy terminator cycle sequencing kit for the APPLIED BIOSYSTEMS 377TM sequencing system (PERKINELMER® Life Sciences). Using the primer-walking approach, full-length sequence was determined. Nucleotide and protein searches were performed using BLAST against the non-redundant database of NCBITM.
In Silico Sequence Similarity Analysis
The coding sequence for each cDNA clones was searched against the dbEST sequence database using the BLAST algorithm at the NCBITM website. ESTs derived from dbEST sequence database were used as a source of information for transcript sequence similarity analysis. The accession numbers of the ESTs matching to the deleted sequence of Ron-V1, Ron-V2, and Ron-V3 were identified.
RT-PCR Expression Pattern Analysis
The expression patterns of Ron-V1, Ron-V2, and Ron-V3 were conducted in human cell lines using RT-PCR. The human cell lines were WI38 (Lung, fetus); A549 (Lung adenocarcinoma); H661 (Large cell carcinoma, lung); H520 (Squamous cell carcinoma, lung); H209 (Small cell carcinoma, lung); JHH-4 (Hepatoma); SUP-T1 (T-cell lymophoblastic lymphoma); Daudi (Burkitt's lymophoma); Ramos (Burkitt's lymophoma); RAJI (Burkitt's lymophoma); ZR75-1 (Breast carcinoma); MDA-MB-231 (Breast adenocarcinoma); Hs 578T (Breast carcinoma); G5T/VGH (Glioblastoma multiforme); TSGH9201 (Gastric carcinoma); Ca Ski (Cervix epidermoid carcinoma); Hela S3 (Cervical epitheloid carcinoma); COLO 320 HSR (Colon adenocarcinoma); SW620 (Colon adenocarcinoma); TSGH8301 (Urinary bladder carcinoma); ES-2 (Ovarian carcinoma); DU145 (Brain prostate carcinoma); MIA paca-2 (Pancreatic carcinoma); CE48T/VGH (Esophagus epidermoid carcinoma); CE81T/VGH (Esophagus carcinoma well differentiated aquamous); HL-CZ (Promonocytic leukemia); Hs 181.tes (Normal testis). The purity and integrity of total RNA extracted from each of the cell lines were assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. The forward and reverse primers for Ron gene variants were: 5′-GACTGAGTGTCTGCTAGCACGG-3′ and 5′-ATGGCAGGGAGTGCATCTACGC-3′. The expected size of Ron-V1, Ron-V2, and Ron-V3 PCR fragments were 660 bp, 540 bp and 513 bp.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; accession No. M33197) was used for internal control. The forward and reverse primers for GAPDH were: 5′-TGGGTGTGAACCATGAGAAG-3′ and 5′-GTGTCGCTGTTGAAGTCAGA-3′. The expected size of GAPDH PCR fragment was 472 bp. Briefly, a 50 μl reaction mixture containing 2 μl total RNA (01 μg/μl), 1 μl each primer (20 pM), 1 μl each dNTP (10 mM), 2.5 μl DTT solution (100 mM), 10 μl 5×RT-PCR buffer, 1 μl enzyme mixture and 28.5 μl sterile redistilled water were subjected to reverse transcription at 60° C. for 30 min followed by 35 cycles of denaturation at 94° C. for 2 min, annealing at 60° C. for 2 min, and extension at 68° C. for 2 min. Five microliters (10%) of the amplified products mixed with 1 μl of loading buffer were separated on a 1% horizontal agarose gel stained with ethidium bromide in 0.5×TAE buffer. The gel was electrophoresed at 100 V for 45 min.
FIG. 7 showed that Ron-V1 (660 bp), Ron-V2 (540 bp), and Ron-V3 (513 bp) mRNAs were RT-PCR amplified using the primers described above. Shown on the left are 100 bp DNA ladder markers. The PCR fragments of Ron-V1 (660 bp), Ron-V2 (540 bp), and Ron-V3 (513 bp) were confirmed by sequencing. Ron-V1 mRNA was expressed in the cell lines of cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus carcinoma, and esophagus epidermoid carcinoma; Ron-V2 mRNA was expressed in the cell lines of breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, colon adenocarcinoma, and urinary bladder carcinoma; Ron-V3 mRNA was expressed in the cell lines of breast carcinoma, colon adenocarcinoma, urinary bladder carcinoma, esophagus carcinoma, and esophagus epidermoid carcinoma. The differential expression pattern among Ron-V1, Ron-V2, and Ron-V3 implied that they may be functionally different and may be a suitable marker for diagnosing human breast carcinoma, breast adenocarcinoma, cervix epidermoid carcinoma, cervix epitheloid carcinoma, colon adenocarcinoma, urinary bladder carcinoma, prostate carcinoma, esophagus epidermoid carcinoma and esophagus carcinoma.
REFERENCES
All references are listed herein for the convenience of the reader. Each is incorporated by reference in its entirety.

    1. Angeloni et al., The soluble sema domain of the RON receptor inhibits macrophage-stimulating protein-induced receptor activation. J. Biol. Chem. 279:3726-32, (2004).
    2. Camp et al., RON, a tyrosine kinase receptor involved in tumor progression and metastasis. Ann Surg Oncol. 12:273-81, (2005).
    3. Peace et al., Ron receptor signaling augments mammary tumor formation and metastasis in a murine model of breast cancer. Cancer Res. 65:1285-93, (2005).
    4. Wang et al., Oncogenic and invasive potentials of human macrophage-stimulating protein receptor, the RON receptor tyrosine kinase. Carcinogenesis 24:1291-300, (2003).
    5. Willett et al., Differential screening of a human chromosome 3 library identifies hepatocyte growth factor-like/macrophage-stimulating protein and its receptor in injured lung. Possible implications for neuroendocrine cell survival. J Clin Invest. 99:2979-91, (1997).

## Claims

1. An isolated nucleic acid encoding the amino acid sequence of SEQ ID NO: 4.

2. The isolated nucleic acid of claim 1, comprising the nucleotide sequence of SEQ ID NO: 3.

3. An isolated nucleic acid consisting of 30 consecutive nucleotides of SEQ ID NO: 3 and that overlaps a dinucleotide selected from the group consisting of nucleotides 2837-2838 of SEQ ID NO: 3, nucleotides 3583-3584 of SEQ ID NO: 3, and nucleotides 3725-3726 of SEQ ID NO: 3.

4. An expression vector comprising the nucleic acid of claims 1 or 2.

5. An isolated host cell comprising said expression vector of claim 4.

6. A method for producing a RON variant polypeptide, comprising the steps of a) culturing said host cell of claim 5 under a condition suitable for the expression of a RON variant polypeptide; and b) recovering said RON variant polypeptide from the host cell culture.