WO1995011923A1 - A novel tumor marker and novel method of isolating same - Google Patents

A novel tumor marker and novel method of isolating same Download PDF

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WO1995011923A1
WO1995011923A1 PCT/US1994/012502 US9412502W WO9511923A1 WO 1995011923 A1 WO1995011923 A1 WO 1995011923A1 US 9412502 W US9412502 W US 9412502W WO 9511923 A1 WO9511923 A1 WO 9511923A1
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tci
leu
protein
gly
thr
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PCT/US1994/012502
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Lan Bo Chen
Shideng Bao
Yuan Liu
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Dana-Farber Cancer Institute, Inc.
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Priority to EP94932133A priority Critical patent/EP0725799A1/en
Priority to US08/448,388 priority patent/US5889159A/en
Publication of WO1995011923A1 publication Critical patent/WO1995011923A1/en

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    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [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
    • C07K16/3046Stomach, Intestines
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    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [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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    • C12Q2600/158Expression markers

Definitions

  • This invention relates to proteins that serve as tumor markers for human carcinoma and to methods of isolating differentially expressed genes.
  • Tumor markers for human tumor cells have been largely limited to activated oncogenes and their products, for example, the myc, ras, fos, and erbB2 genes and their encoded oncoproteins.
  • activated anti-oncogenes such as RB, p53, and DCC, have been identified in normal cells but do not appear to be present in tumor cells.
  • Oncogene and anti-oncogene products have proven difficult to use as consistent predictors of tumor and normal tissue, respectively, due to the relatively low level of expression of the genes encoding these proteins.
  • a tumor marker which is not only differentially expressed in tumor and normal tissue, but also consistently detectable in human tumor tissue and consistently absent in the corresponding normal tissue.
  • a common method used to identify genes differentially or uniquely expressed in tumors, in cells responding to growth factors, and in differentiated cell types such as, among others, T cells, adipocytes, neurons, and hepatocytes is the subtractive hybridization technique (S.W. Lee et al., Proc. Natl. Acad. Sci. USA £50:4699, 1983).
  • a method of differential display of eukaryotic mRNA by means of the polymerase chain reaction (PCR) has recently been developed (P. Liang et al., Science 257:967. 1992). This method
  • SUBSTITUTE SHEET /RULE 26 utilizes oligo dT linked to two additional bases as the primer for reverse transcription driven by reverse transcriptase. cDNA fragments are then amplified by Taq DNA polymerase-based PCR using an oligo dT primer along with one additional primer. The amplified cDNAs are then resolved by DNA sequencing gels. There is a need in the art for a streamlined and simplified process for isolating cDNAs corresponding to differentially expressed mRNAs.
  • TCI SEQ ID NO:4
  • TCI tumor marker
  • the invention thus encompasses the TCI protein (SEQ ID NO:4), which is useful as a tumor marker for tumor diagnosis and therapy, particularly for colorectal, breast, and gastrointestinal tumors, and for metastatic tumors emanating from these tumor types.
  • TCI is also a useful marker in general for tumor cell invasion and metastasis.
  • mRNA encoding TCI is not expressed in most cultured tumor cells, i.e., in vitro , but is expressed once these cells are grown in vivo . Because later stage and deeply invasive tumors contain higher levels of TCI protein than other tumor tissues, TCI appears to be a particularly useful marker for later stage cancers.
  • TCI protein may also serve as a target in tumor targeted therapy to prevent tumor cell metastasis and thus invasion of additional organs.
  • a polypeptide fragment of the TCI protein may be used as an antagonist of TCI biological activity; e.g., where TCI biological activity includes invasion and metastasis, the polypeptide fragment may be administered to a patient afflicted with the tumor in order to inhibit the spread of the tumor to other tissues.
  • a truncated portion of TCI which retains the invasive and metastatic biological activities of the full- length molecule will be useful for screening for antagonists of TCI activity. Potentially useful polypeptides are described herein.
  • the invention also encompasses nucleotide probes based on the TCI nucleotide sequence; e.g., 10, 20, 30, 40, etc. nucleotides in length. Such probes are useful for PCR-based tumor detection and in situ hybridization of tumor tissue sections. In addition, probes whose nucleotide sequences are based on homologies with other genes or proteins having sequences related to TCI, i.e., genes of the TCI family, two of which are described herein, are useful for detecting additional genes belonging to the TCI family of genes.
  • the invention thus also encompasses methods of screening for agents which inhibit expression of the TCI gene (SEQ ID NO:3) in vitro, comprising exposing a metastatic cell line in which TCI mRNA is detectable in cultured cells to an agent suspected of inhibiting production of the TCI mRNA; and determining the level of TCI mRNA in the exposed cell line, wherein a decrease in the level of TCI mRNA after exposure of the cell line to the agent is indicative of inhibition of TCI mRNA production.
  • the screening method may include in vitro screening of a metastatic cell line in which TCI protein is detectable in cultured cells to an agent suspected of inhibiting production of the TCI protein; and determining the level of TCI protein in the cell line, wherein a decrease in the level of TCI protein after exposure of the cell line to the agent is indicative of inhibition of TCI protein production.
  • the invention also encompasses in vivo methods of screening for agents which inhibit expression of the TCI gene, comprising exposing a mammal having tumor cells in which TCI mRNA or protein is detectable to an agent suspected of inhibiting production of TCI mRNA or protein; and determining the level of TCI mRNA or protein in tumor cells of the exposed mammal. A decrease in the level of TCI mRNA or protein after exposure of the mammal to the agent is indicative of inhibition of TCI gene expression.
  • These screening methods are particularly applicable to breast tumor cells, colon tumor cells, or tumor cells of the gastrointestinal tract.
  • the invention also encompasses a pharmaceutical composition for use in treating a late stage cancer, comprising an effective amount of an inhibitor of TCI, and a method of treating late stage cancer, comprising administering to a mammal afflicted with a late stage cancer a therapeutically effective amount of an inhibitor of TCI.
  • Late stage cancers include those which have become deeply invasive in a tissue or which have metastasized to other tissues.
  • TCI is detectable in patient blood, urine, sputum or other body fluid using a monoclonal antibody specific for a TCI epitope.
  • the invention also encompasses antibodies specific for TCI, which can easily be prepared in a kit form.
  • Monoclonal antibodies specific for TCI may be used for tumor imaging to localize tumor position and size.
  • TCl-specific monoclonal antibodies are also useful as screening and diagnostic agents in immunohistochemical staining of tissue sections to distinguish tumor cells from normal cells.
  • anti-TCl antibodies are particularly useful where they recognize cells which produce the TCI protein when such cells are paraffin-embedded and/or formalin-fixed.
  • One example of such an antibody is the monoclonal antibody anti-TCl-1 produced by the hybridoma deposited with the American Type Culture Collection as ATCC Deposit No. HB 11481.
  • the invention also features a novel method, called palindromic PCR, for identifying and isolating a gene, e.g., a gene which is differentially expressed in different types of tissues.
  • the method is based on the use of short DNA primers and corresponding palindromic nucleotide sequences in the nucleotide sequence to be isolated.
  • the invention encompasses a method for producing a double stranded cDNA that includes the steps of contacting an mRNA with a DNA primer under stringent hybridization conditions to form a first hybrid molecule, the primer having a length of from 8 to 12 nucleotides and, preferably, 9 to 11 nucleotides; subjecting the first hybrid molecule to an enzyme having reverse transcriptase activity, to produce a first DNA strand complementary to at least a portion of the mRNA; contacting the first DNA strand with the primer under stringent hybridization conditions to form a second hybrid molecule; and subjecting the second hybrid molecule to an enzyme having DNA polymerase activity, to produce a second DNA strand complementary to the first DNA strand.
  • the method also includes the step of amplifying the first and second DNA strands.
  • a single enzyme provides both the reverse transcriptase activity and the DNA polymerase activity.
  • a suitable such enzyme is rTth DNA polymerase from the thermophilic eubacterium Thermus th ⁇ rmophilu ⁇ .
  • palindromic nucleotide sequences means that a double stranded DNA molecule contains a specific DNA sequence in both its coding strand and its anti-parallel strand, when those strands are read in the same direction, e.g., 5 1 to 3'.
  • the specific sequence of the DNA primer is arbitrary in that it is based upon individual judgment. In some instances, the sequence can be entirely random or partly random for one or more bases. Preferably, the GC content of the primer is between 40% and 60%, most preferably about 50%. In other instances, the arbitrary sequence can be selected to contain a specific ratio of each deoxynucleotide. The arbitrary sequence can also be selected to contain, or not to contain, a recognition site for a specific restriction endonuclease.
  • the DNA primer can contain a sequence that is known to be a "consensus sequence" of an mRNA of known sequence.
  • a "consensus sequence” is a sequence that has been found in a gene family of proteins having a similar function or similar properties.
  • the use of a primer that includes a consensus sequence may result in the cloning of additional members of a desired gene family.
  • Palindromic PCR enables genes that are altered in their frequency of expression, as well as those that are constitutively or differentially expressed, to be identified by simple visual inspection and isolated.
  • the method also allows the cloning and sequencing of selected RNAs, so that the investigator may determine the relative desirability of the gene product prior to screening a comprehensive cDNA library for the full length gene product.
  • Fig. 1 is a polyacrylamide gel of size-separated cDNAs that were reverse transcribed from paired mRNAs from colon carcinoma (T) and adjacent normal colon tissue (N) and subsequently amplified.
  • Fig. 2A is a gel in which the TCI cDNA fragment identified in Fig. 1 was recovered and re-amplified.
  • Fig. 2B is a Northern Blot of three pairs of RNA from colon carcinoma (T) and their adjacent normal colon tissue (N) probed with 32P-labeled TCI cDNA.
  • Fig. 3 shows the nucleotide sequence (described herein as SEQ ID NO:l) and corresponding amino acid sequence (SEQ ID NO:2) of the 636 bp partial TCI clone.
  • Fig. 4 shows the nucleotide sequence (SEQ ID NO:3) and corresponding amino acid sequence (SEQ ID NO:4) of the full- length TCI gene and protein.
  • Fig. 5 is a sequence comparison of the four internal homologous domains of TCI (SEQ ID NO:4), each approximately 135 amino acids.
  • Fig. 6A is a schematic representation of the four repeats of TCI.
  • Fig. 6B is a proposed schematic arrangement of the four repeated domains and the N- and C-terminal domains.
  • Fig. 7 shows the amino acid sequence identity between TCI (SEQ ID NO:4) and Big-h3 (SEQ ID NO:17).
  • Fig. 8 is a Northern Blot of five pairs of RNA from colon carcinoma (T) and adjacent normal colon tissue (N) probed with 32P-labeled Big-h3 cDNA, the bottom panel representing control RNA probed with 32P-labeled B-actin.
  • Fig. 9 shows amino acid sequence homology between TCI (SEQ ID NO:4) and Fasciclin I from Grasshopper (GrF) (SEQ ID NO:18) and Drosophila (DrF) (SEQ ID NO:19).
  • Fig. 10 is a Schematic representation showing that, on average, in every 202 bases of sequence in one strand of cDNA, there is one 9-base sequence exactly palindromic to that in a region of its antiparallel strand.
  • Fig. 11 shows the relationship between the palindromic frequency and the number of bases in a putative DNA primer, as determined by cDNA Matrix analysis.
  • Fig. 12 is a schematic representation of the method of the invention, palindromic PCR, driven by the enzyme rTth DNA polymerase with one DNA primer in one reaction tube; the dotted line indicates mRNA and the solid line indicates cDNA; the short jagged line represents the single DNA primer.
  • Fig. 13A shows the effect of the length of the DNA primer on the cDNA amplification patterns; the length and nucleotide sequence of each primer are: A, 8-mer (5'- TGTCGAGA) ; B' , 9-mer (5'-TGTCCAGAC) ; C , 10-mer (5'- TGTCCAGATG) (SEQ ID NO:5); D', 11-mer (5'-TGTCCAGATGC) (SEQ ID NO:6); E', 12-mer (5'-TGTCCAGATGAC) (SEQ ID NO:7).
  • Fig. 13B shows the effect of the GC content of the DNA primer on the cDNA amplification patterns; the GC content and nucleotide sequence of each primer (10-mer) are: A', 40% (5'-TGTCCAGATA) (SEQ ID NO:8); B', 50% (5'-TGTCCAGATG) (SEQ ID NO:5); C, 60% (5'-TGTCCAGACG) (SEQ ID NO:9): D', 70% (5'- TGTCCAGCCG) (SEQ ID NO:10); E', 80% (5'-TGTCCCGCCG) (SEQ ID NO:ll); F' , 90% (5'-TGCCCGGCCG) (SEQ ID NO:12).
  • A' 40% (5'-TGTCCAGATA) (SEQ ID NO:8); B', 50% (5'-TGTCCAGATG) (SEQ ID NO:5); C, 60% (5'-TGTCCAGACG) (SEQ ID NO:9): D', 70% (5'- TGTCCAGCCG) (S
  • FIG. 13C shows the effect of the sequence specificity of the DNA primer on the cDNA amplification patterns; 10-mer primers with the same GC content but different sequences are: A', 5'-TGATGCACTC (SEQ ID NO:13); B', 5'-TGAGCTACTC (SEQ ID NO:14); C, 5'-TGACTGACTC (SEQ ID NO:15).
  • Fig. 13D shows palindromic PCR performed by rTth DNA polymerase (A) with reverse transcription cycles (RT cycles) and (B) without RT cycles.
  • Fig. 14 shows the identification of differentially expressed genes in human colon carcinoma.
  • Fig. 15 shows reamplification of the TCI cDNA fragment isolated from colon carcinoma; the PCR product was analyzed on a 1.0% agarose gel; a 0.63 Kb cDNA fragment (arrow) was detected.
  • Fig. 16 is an autoradiogram of DNA sequencing gels showing the presence of PP1 primer sequence (5'-CTGATCCATG) (SEQ ID NO:16) at the 5'-end of both strands of the TCI cDNA fragment; cloning sites are indicated by arrows, sequences below arrows are pBS (KS) vector sequences reading from T3 primer and T7 primer.
  • Fig. 17 is a Northern Blot of 24 pairs of colon carcinoma (T) and their adjacent normal tissue (N) probed with 32P-labeled TCI cDNA.
  • Fig. 18 shows the Tumor/Normal RNA Ratio from Northern Blot results of Fig. 17.
  • Fig. 19 is a Northern Blot of RNA from carcinoma cells which result from metastasis from colon carcinoma to liver (LM) and their adjacent normal liver (NL) probed with 32 P- labeled TCI cDNA.
  • Fig. 20 shows a Northern Blot of RNA from breast cancer cell line MCF-7 (1) and colon cancer cell line CX-1 (2) cultured in vitro, and MCF-7 tumor (3) and CX-l tumor (4) grown in vivo in nude mice.
  • Fig. 21 shows staining of formalin-fixed and paraffin- embedded colon tumor tissue sections using the monoclonal antibody anti-TCl-1 and avidin-biotin-peroxidase detection.
  • Fig. 22 shows staining as described in Fig. 21, except that panels A, C, D represent breast invasive ductal carcinoma and panel B, normal breast tissue.
  • Fig. 23 shows staining as in Fig. 13, except that panels A and B represent gastric carcinoma, and panels C and D, deeply invasive colon carcinoma.
  • Fig. 24 is a Western Blot analysis of protein samples from two pairs of colon carcinoma and their adjacent normal colon (A) and two pairs of breast carcinoma and their adjacent normal breast (B) , using a monoclonal antibody against TCI protein as a probe.
  • Fig. 25A shows the ethidium bromide staining pattern of an RNA gel in which the same amount of RNA from JMN (1) and JMN1B (2) cells is loaded per lane.
  • Fig. 25B is a Northern Blot analysis of RNA from malignant mesothelioma cells JMN1B (2) and JMN (1) using TCI cDNA as a probe.
  • Fig. 26 is a Western blot using a monoclonal antibody against TCI to probe JMN1B cells grown in conditioned medium and whole cell lysate.
  • Fig. 27 shows JMN1B cells fixed with paraformaldehyde without subsequent permeabilization in panels A and B, and JMN1B cells fixed with paraformaldehyde and then permeabilized in panels C and D.
  • Figs. 28A-28D show the corrected nucleotide sequence and corresponding amino acid sequence of the full length TCI gene and protein. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • TCI (SEQ ID NO:4) is a novel protein that is found in invasive and metastatic tumor cells.
  • the nucleotide sequence is a novel protein that is found in invasive and metastatic tumor cells. The nucleotide sequence
  • SEQ ID NO:3 (SEQ ID NO:3) encoding TCI was found using a novel technique described herein as palindromic PCR, a technique which enables identification and cloning of a gene that is differentially expressed in tissues. Cloning and sequencing of the gene encoding TCI and characterization of the protein is described below, along with examples of how the protein is detected in invasive and metastatic cancers. Examples describing additional uses of the TCI protein and its fragments, the nucleotide sequence encoding TCI and fragments thereof, and antibodies specific for TCI are also included.
  • the identification, cloning, and differential detection of expression of the TCI gene was performed as follows.
  • a 636 bp cDNA fragment (SEQ ID N0:1) containing TCI sequences was identified and isolated by a rapid method termed palindromic PCR, described herein, from human surgical colon carcinoma tissue. Briefly, paired mRNAs were isolated from colon carcinoma tissue and adjacent normal colon tissue from the same patient, then matched mRNAs were reverse transcribed to cDNA and subsequently amplified by the palindromic PCR method described herein, which utilizes one DNA primer. Both reverse transcription and PCR reactions were driven by a single enzyme, rTth DNA polymerase, in a single tube.
  • 35 S or 33 P-labeled PCR cDNA fragments were resolved on a DNA sequencing gel.
  • paired mRNAs from colon carcinoma (T) and adjacent normal colon tissue (N) were reverse transcribed to cDNA and subsequently amplified by palindromic PCR.
  • 35 S-labeled PCR cDNA fragments were then resolved on a DNA sequencing gel.
  • a differential cDNA band (TCI) appeared to be present only in the tumor sample.
  • TCI cDNA fragment was recovered from the sequencing gel and then reamplified with the same palindromic primer.
  • This 636 bp fragment is identified with a horizontal arrow in Fig. 2A.
  • FIG. 2B is a Northern Blot of three pairs of RNA from colon carcinoma (T) and their adjacent normal colon tissue (N) probed with 32 P-labeled TCI cDNA. TCI mRNA was over-expressed in all three cases of colon carcinoma, whereas only very weak TCI message appeared in the adjacent normal tissue. In the bottom panel of the blot, control RNA was blotted with 32 P-labeled cDNA encoding B- actin.
  • TCI mRNA size is about 3.6Kb.
  • This first TCI cDNA fragment was cloned into a pBluescript plasmid DNA vector strategies. Nucleotide sequence analysis revealed that this fragment contained 636bp with nucleotide sequences corresponding to the primer sequence at both 5'-ends of the double-stranded DNA (Fig. 3 and SEQ ID NO: 1) . The corresponding predicted amino acid sequence is shown in Fig. 3 and provided in SEQ ID NO: 2.
  • a search of the GenBank database with this cDNA fragment revealed that TCI is a novel gene.
  • Nucleotide sequence analysis of the 636bp TCI cDNA fragment obtained by the described differential display method revealed that it contained a partial open reading frame. Therefore, this 636bp cDNA fragment was used as probe to screen a cDNA library. Several overlapping clones were obtained and contained a 2997bp sequence. To obtain the complete open reading frame for TCI, a modified 5'-end RACE technique was used to amplify the TCI coding regions. The nucleotide and deduced amino acid sequence of full-length TCI is shown in Fig. 4 and provided in SEQ ID NOS: 3 and 4.
  • the N-terminal signal sequence is underlined; one predicted N- linked glycosylation site (NDT) is boxed and a polyadenylation signal (AATAAA) is indicated.
  • the cDNA contains 3126bp with a potential polyadenylation sequences (AATAAA) at the 3'-end, beginning at residue 2963.
  • the open reading frame (ORF) encodes a 777-amino acid protein with a calculated molecular weight of 86kD.
  • the TCI protein contains an amino-terminal signal peptide or secretory leader signal (ALPARILALALALAL) , and one predicted site of N-linked glycosylation at amino acid residue 605 (NDT) .
  • ALPARILALALALAL secretory leader signal
  • C-C Ce okine B family motif
  • TCI contained four internal homologous domains of approximately 135 amino acids. A comparison of these repeats is shown in Fig. 5. Each boxed amino acid is identical with at least one other residue at that same position. The interdomain homologies range from 32% (between domains 2 and 4) to 18% (between domains 1 and 3) .
  • Some amino acid sequence such as TLF— AP— TNEAF, NGVIHXID are highly
  • V S are used herein to indicate that alanine or valine, and threonine or serine, respectively, may be found at these positions.
  • the notation X is used herein to indicate that this position may include any amino acid.
  • Each repeat starts with the most divergent sequence. The four repeats occur between residues 139-537 and are uninterrupted by non-homologous domains. A schematic representation of the four repeats of TCI is shown in Fig. 6A. The four homologous repeats suggest a tetrameric structure (Mclachlan 1980; Zinn et al, 1988) with two binding sites, one at each intrachain dimer.
  • the four repeats of TCI may serve as ligand binding sites, with the N-terminal or C-terminal domains serving as the functional domain.
  • One possible arrangement of the four repeated domains and the N- and C-terminal domains is shown schematically in Fig. 6B.
  • the nucleotide and corresponding amino acid sequence of the TCI gene and protein with a corrected leader signal sequence are given in Figs. 28A-28D.
  • Palindromic PCR Described below is a novel technique used to identify the TCI mRNA and prepare TCI cDNA.
  • sequence of bases in a coding and antisense strand of a cDNA molecule are, in a sense, "mirror images" of one another, we have found that with surprising frequency a short sequence of bases, e.g. 9 or 10, in one strand will be found to have an exact copy in its anti-parallel strand.
  • a short sequence of bases e.g. 9 or 10
  • DNA Matrix DNA Strider 1.2
  • DNA Matrix Single strand cDNA (the mRNA strand) and its anti-parallel strand were compared, each from the 5' to 3' end by the DNA Matrix program. For example, as illustrated in Fig.
  • Table 1 presents the statistical data showing the palindromic frequency related to the number of bases in the searched segment.
  • rTth DNA polymerase from the thermophilic eubacterium Thermus thermophilus
  • rTth DNA polymerase possesses a very efficient reverse transcriptase activity in the presence of MnCl 2 and a thermostable DNA polymerase activity in the presence of MgCl 2 .
  • the rTth DNA polymerase has been observed to be greater than 100-fold more efficient in coupled reverse transcription and PCR than the analogous DNA polymerase, Taq (T. W. Myers et al., Biochemistry 3i):7661, 1991) .
  • an appropriate primer would allow anchored annealing to some regions of certain mRNA species that contain sequence complementary to the palindromic primer. This subpopulation of mRNAs is likely to be reverse transcribed by rTth DNA polymerase.
  • a "Palindromic" primer apparently has a greater probability of anchoring to the coding regions of mRNA than oligodT primer.
  • the same primer can anneal to some regions of the first strand cDNA and function as the "Downstream primer” in a PCR reaction.
  • the same primer can also function as the "Upstream primer.”
  • the primer anchors to first strand cDNAs the annealing position to various cDNA molecules should, in principle, be at different distances in different molecules from the first annealing position. Therefore, the amplified cDNA fragments from various mRNAs will be of different sizes.
  • these PCR- generated cDNA fragments are labeled with 35 S-dATP or 33 P- dATP, they can be resolved as a ladder by DNA sequencing gels. A display of cDNAs originating from various mRNAs can then be visualized after autoradiography.
  • the selection of the specific palindromic primer depends on three important factors: the length, the GC content, and the sequence specificity.
  • DNA Matrix analysis has indicated that the ideal length of a primer for an appropriate palindromic frequency is from 9 to 11 bases. Therefore, a set of primers from 8 base to 12 base in length with 50% GC content was chosen for study. Our results showed that 9, 10, and 11 base primers gave an appropriate number of cDNA fragments readily resolvable by DNA sequencing gels (Fig. 13) .
  • a set of 10-mer primers with GC content ranging from 40% to 90% was tested. The results suggested that a GC content from 40% to 80% is acceptable (Fig. 13) .
  • rTth DNA polymerase allows the detection of IL-la mRNA, which has a very low copy number, in 80pg of total cellular RNA (T.W. Myers et al., Biochemistry 30:7661. 1991) .
  • the higher efficiency of rTth DNA polymerase ensures that the palindromic PCR method of the invention provides high sensitivity.
  • rTth polymerase is thermostable, it can also be used to perform several RT cycles (reverse transcription cycles) , which means several copies of first strand cDNA can be obtained from a single copy of mRNA.
  • RT cycles reverse transcription cycles
  • the sensitivity of the method is increased by performing multiple RT cycles using rTth polymerase (Fig. 13) .
  • the method of the invention was tested in a search for differences in mRNA expression between human colon carcinoma and the adjacent normal epithelium from a surgical specimen.
  • Paired mRNA preparations were reverse transcribed with a palindromic primer 5'-CTGATCCATG (designated as PP-l primer) (SEQ ID NO:16) in the presence of MnCl 2 followed by PCR with the same primer in the presence of MgCl 2 using rTth DNA polymerase.
  • the reaction products were then analyzed by DNA sequencing gels. About 70-110 amplified cDNA fragments ranging from 100-700 bases from both preparations were detected (Fig. 14) . Whereas overall cDNA patterns between tumor and normal tissue are similar, significant differences were detected by this method.
  • Mix B which consisting of 12.5 ⁇ l of dd H 2 0, 2.0 ⁇ l of 10X chelating buffer (50% glycerol (v/v) , lOOmM Tris-HCl, pH 8.3, 1M KC1, 0.5% Tween 20), 2.0 ⁇ l of 25 mM MgCl 2 solution, 2.50 mM dNTP and 2.0 ⁇ l of 35 S-dATP (or 33 P-dATP) is dispensed in the amount of 20 ⁇ l into each 5.0 ⁇ l RT reaction mixture.
  • the samples are mixed and spun briefly and then overlaid with 25 ⁇ l of mineral oil.
  • the polymerase chain reaction is then started: 94°C for 40 sec, 40°C for 2 min., 72°C for 35 sec. (for 40 cycles, hold at 72°C for 4 min.), and then 4°C.
  • cDNA analysis 7 ⁇ l of a PCR sample is mixed with 4 ⁇ l of sequencing loading buffer, samples are incubated at 80°C for 3 min., and then placed on ice. 4.5 ⁇ l of the sample is loaded on a 6%-8% agarose DNA sequencing gel.
  • a gel slice containing a desirable cDNA band (such as TCI) was soaked in 200 ⁇ l of ddH 2 0 for 20 min and then separated from 3M paper with a clean forcep or a plastic pipette tip. The gel was removed and pounded with an autoclaved plastic pipette tip. Elution buffer (20 ⁇ l) was added and the mixture was vortexed and left at room temperature for 4 hrs or overnight. After centrifugation, cDNA fragments in 10 ⁇ l eluent were reamplified by rTth DNA polymerase with the same palindromic primer, as described.
  • a desirable cDNA band such as TCI
  • telomeres After one 40-cycle PCR, the reamplified cDNA could be detected by agrose gels stained with ethidium bromide. The amount of cDNA generated was sufficient for cloning and preparing a probe for Northern Blot analysis.
  • Fig. 15 shows the gel obtained when the TCI cDNA band was subjected to elution and reamplification. Total PCR product of the TCI fragment was 2.5 ⁇ g.
  • the reamplified TCI cDNA fragment was treated with T4 DNA polymerase and cloned into pBluescript plasmid DNA vector at Smal site by blunt end ligation.
  • the nucleotide sequence of the TCI fragment (SEQ ID NO:l) showed that a sequence identical to the PP1 primer (SEQ ID NO:16) is indeed present at the 5'-end of both strands of the TCI fragment (Fig. 16) . This result confirms that the 5'-ends of both complementary chains of the TCI cDNA fragment used the same palindromic primer during palindromic PCR as discussed above.
  • the method of the invention differs from other methods in a number of ways.
  • palindromic PCR only a single primer (9-11 bases) is used and is sufficient to prime reverse transcription as well as to support subsequent PCR for a display of nearly 100 cDNA species. Because the pattern of amplified cDNAs depends on the sequence of the single palindromic primer, the species of mRNAs that are subjected to amplification can readily be controlled by a proper sequence of the palindromic primer. If a group or family of genes shares certain sequences, a primer can be chosen from such a sequence, and a specific display of this set of mRNAs can readily be performed. Likewise, computer analysis of the Genebank database may reveal additional sequences useful as a primer shared by a set of related genes. The use of such a primer by the method of the invention would allow the display for the expression of a given set of genes. Palindromic PCR provides an easy, sensitive and economical way to identify and isolate differentially expressed genes related to tumor and other disease.
  • TCI DNA Differential expression of TCI DNA in normal tissue and tumor cells.
  • TCI mRNA in colon carcinoma tissue confirmed the differential expression of TCI mRNA in colon carcinoma tissue, and the absence of TCI mRNA in the corresponding normal tissue. Evaluation of the expression of TCI mRNA in additional cases of colon carcinoma at different stages was also undertaken. Surgical specimens of 24 cases of human primary colon carcinoma and 6 cases of liver metastases were examined by Northern hybridization of total RNA with 32 P- labeled TCI probe.
  • T colon carcinoma
  • N adjacent normal tissue
  • TCI mRNA levels vary in different cases of carcinoma.
  • Panels I and II show A: TCI mRNA and B: Control; Panel III shows TCI mRNA and control (Actin) mRNA.
  • Fig. 18 shows the Tumor/Normal RNA Ratio from Northern Blot results of Fig. 17.
  • the horizontal line indicates the mean Tumor/Normal ratio.
  • TCI mRNA was abundantly expressed in all 24 cases of primary colon carcinoma and 6 cases of liver metastases, whereas only a small amount of TCI mRNA was detected in a few cases of paired adjacent normal tissue.
  • the mRNA level of TCI was much greater in primary colon carcinoma than in paired adjacent normal colonic epithelium in all 24 cases.
  • the Tumor/Normal ratio varied from 5.6 to 92, and the mean Tumor/Normal ratio being 32.
  • the Tumor/Normal ratio when plotted against the Duke's stage of disease, gave evidence for increasing TCI expression with increasing stage of colon carcinoma.
  • Fig. 19 shows a Northern Blot of RNA from metastatic colon carcinoma to liver (LM) and their adjacent normal liver (NL) probed with 32 P-labeled TCI cDNA.
  • TCI mRNA was expressed only in metastatic tumor in 5 of 6 samples. Only one sample of normal liver tissue expressed a very weak TCI message. The Tumor/Normal ratio is greater than 64.
  • TCI mRNA in cultured cancer cells and in vivo tumor cells was analyzed and is described below. TCI was overexpressed in tumor tissue in vivo. The expression of TCI mRNA in cultured cancer cell lines in vitro was examined by Northern Blot analysis.
  • RNAs isolated from twelve colon cancer cell lines (HT29, Clone A, MIP101, CX-1, Morser, CCL227, CCL228, etc.) derived from different stage of human colon carcinoma, two melanoma cell lines (LOX, A2058) , one breast cancer cell line (MCF-7) , two cervical cancer cell lines (Hela, A431) , three bladder cancer cell lines (EJ, T24, MB49) , one pancreas cancer cell line (CRL1420) , two hepatoma cell lines (HepG2, HepG3) and four normal cell lines (FS-2, MRC-5, 498A, CV-1) were screened by Northern Blot analysis. However, the TCI transcript could not be detected in all of these cell lines.
  • FIG. 20 shows a Northern Blot of RNA from breast cancer cell line MCF-7 (1) and colon cancer cell line CX-1 (2) cultured in vitro, and MCF-7 tumor (3) and CX-1 tumor (4) grown in vivo in nude mice. TCI mRNA in colon cancer cell line CX-1 and breast cancer cell line MCF-7 cultured in vitro could not be detected by Northern Blot analysis.
  • TCI mRNA was detectable by Northern Blot analysis, the TCI mRNA levels being dramatically increased in vivo . This result suggests that TCI gene expression was turned on or dramatically increased in the tumor cells in vivo.
  • the differential expression of the TCI gene appears to be related to invasion and metastasis of tumor cells in vivo.
  • the regulation of TCI gene expression in vivo and in vitro could be a very important model to understand tumorigenesis and tumor malignant behavior.
  • TCI protein in in vivo tumor cells, cultured carcinoma cells, and in corresponding normal cells was examined, and is described below.
  • the TCI gene (SEQ ID NO:3) was cloned into a plasmid expression vector, and recombinant TCI protein (SEQ ID NO:4) was expressed in bacteria.
  • Several monoclonal antibodies against the bacterially-produced TCI protein were raised, as will be described below.
  • a variety of formalin-fixed and paraffin- embedded tumor tissue sections were examined by immunohistochemical staining with a mouse monoclonal anti-TCl antibody anti-TCl-1 using an avidin-biotinylated-peroxidase detection technique. Strong positive staining of TCI was found in primary colon carcinoma (Fig.
  • TCI protein level in tumor tissue is much greater than the level of TCI in adjacent normal tissue (Figs. 21B, 23B) .
  • Fig. 24 is a Western Blot analysis of protein samples from two pairs of colon carcinoma and their adjacent normal colon (A) and two pairs of breast carcinoma and their adjacent normal breast (B) , using a monoclonal antibody against TCI protein as a probe.
  • a major 86kd protein (arrow) was detected by anti-TCl antibody in tumor samples (T) but not in normal samples (N) .
  • the Western Blot analysis confirmed that tumor tissue contained significantly more TCI protein than the corresponding adjacent normal tissue.
  • Fig. 25 is a Northern Blot analysis of RNA from malignant mesothelioma cells JMN1B and JMN using TCI cDNA as a probe.
  • Panel 25B demonstrate that TCI mRNA level in JMN1B cells (2) is much greater than that in JMN cells (1) .
  • Panel 25A shows the ethidium bromide staining pattern of an RNA gel in which the same amount of JMN (1) and JMNIB (2) RNA is loaded per lane.
  • TCI mRNA level is much higher in JMNIB than in JMN, the JMNIB/JMN ratio being approximately 14. Higher expression of TCI mRNA in JMNIB could be related to the observed greater tumorigenicity of JMNIB cells. It has been found that JMNIB cells can secrete an "EGF-like" growth factor called transformed mesothelial growth factor (TMGF) that satisfies the EGF requirement of normal human mesothelial cells. The difference in the levels of TCI mRNA in JMNIB and JMN cells provides an ideal cell model to understand the regulation of TCI expression and its relation to tumorigenicity.
  • TMGF transformed mesothelial growth factor
  • TCI protein (SEQ ID NO:4) contained a secretory leader signal at its N-terminus.
  • the secretion of TCI protein was confirmed by Wester Blot analysis of conditioned medium of JMNIB cells. JMNIB cells were cultured in regular medium until 90% confluent, then cultured in serum free medium for two days. This serum free conditioned medium was analyzed by immunoblotting with anti-TCl monoclonal antibody.
  • Fig. 26 is a Western blot analysis using a monoclonal antibody against TCI to probe JMNIB cells grown in conditioned medium and whole cell lysate.
  • Two major bands (about 86kd and 104kd) were recognized by anti-TCl antibody both in JMNIB cell conditioned medium (1) and whole cell lysate (2) .
  • Numbers on the left indicate the position of molecular weight standards in kilodalton.
  • the protein size of the lower molecular weight 86kd band is consistent with that of deduced TCI protein, whereas the higher molecular weight 104kd band is consistent with a TCI glycoprotein.
  • Human malignant mesothelioma cell line JMNIB can express abundant TCI. This cell line was used to study the distribution and localization of TCI protein by immunofluorescent staining with an anti-TCl monoclonal antibody followed by Rhodamine conjugated goat anti-mouse IgG secondary antibody.
  • JMNIB cells were fixed with paraformaldehyde without subsequent permeabilization, the positive staining was seen on the cell surface or outside of the cell (Fig. 27, panels A,B) , which confirms the secretion of TCI protein.
  • JMNIB cells were fixed with paraformaldehyde and then permeabilized, positive staining appeared in the Golgi complex and the endoplasmic reticulum (ER) in the cell (Fig.
  • TCI may be related to tumor malignant behavior such as invasion and metastases. These observations include the following: TCI is significantly overexpressed in tumor tissue; TCI is a secreted protein; later stage tumor expresses higher levels of TCI; deeper invasive tumor contains higher levels of TCI protein; TCI expression turns off in cultured tumor cells in vitro and turns on again after cells grow tumor tissue in vivo. These observations indicate that the function of TCI is not related to tumor cell proliferation, but is more likely involved in tumor malignant behavior in vivo, such as invasion and metastases.
  • TCI is a member of a Family of Proteins.
  • TCI Several stretches of amino acids GSFTXFAPSNEAW, TLXAPTNEAFEKXP, ATNGWHXIDXV, LYXGQXLETXGGKXLRVFVYR, HYPNGXVTVNCAR are highly conserved between TCI and Big-h3.
  • Northern Blot analysis showed that the TCI gene is expressed from a larger transcript than Big-h3, and DNA sequence analysis indicated that TCI contains a longer open reading frame encoding a higher molecular weight protein than the Big-h3 gene. It has been found that Big-h3 also contains four internal repeats. The amino acid sequence homology and structural similarity between TCI and Big-h3 indicate their functional similarity and relationship.
  • Fig. 8 is a Northern Blot of five pairs of RNA from colon carcinoma (T) and adjacent normal colon tissue (N) probed with 32 P-labeled Big-h3 cDNA. The blot shows Big-h3 mRNA level in colon carcinoma to be much higher than that in adjacent normal tissue.
  • the bottom panel represents control RNA probed with 32 P-labeled B-actin.
  • TCI mRNA In contrast to the expression pattern of TCI mRNA, which is shown to be largely restricted to in vivo tumor tissue, Big-h3 mRNA is not only expressed in the tumor tissue, but also expressed in the cultured tumor cell lines and some normal cell lines. Though TCI and Big-h3 shared significant homology, their responses to growth factors are distinctly different.
  • Fasciclin I, II, III are extrinsic membrane glycoproteins involved in the growth cone guidance during nervous system development in the insect embryo.
  • a search of NBRF protein database revealed a significant homologous domain between TCI and Fasciclin I from Grasshopper and Drosophila.
  • One TCI domain of 204 amino acids shared 30% identity with Grasshopper Fasciclin I, and shared 25% identity with Drosophila Fasciclin.
  • Fig. 9 shows amino acid sequence homology between TCI (SEQ ID NO:4) and Fasciclin I from Grasshopper (GrF) (SEQ ID NO:18) and Drosophila (DrF) (SEQ ID NO:19).
  • Fasciclin Boxed amino acids are identical with at least one other amino acid at that same position. It has been found that Fasciclin also contained four internal homologous domains, each consisting of approximately 150 amino acids. The domains of TCI and Fasciclin I share some highly conserved amino acid stretches such as
  • TXF PTNXAF
  • VXHWDXXLXP VXHWDXXLXP
  • Fasciclin is TXF— APTNXA— F. All four internal repeats in TCI
  • the invention also includes methods of screening for agents which inhibit TCI gene expression, whether such inhibition be at the transcriptional or translational level.
  • Screening methods, according to the invention, for agents which inhibit expression of the TCI gene in vitro will include exposing a metastatic cell line in which TCI mRNA is detectable in culture to an agent suspected of inhibiting production of the TCI mRNA; and determining the level of TCI mRNA in the exposed cell line, wherein a decrease in the level of TCI mRNA after exposure of the cell line to the agent is indicative of inhibition of TCI mRNA production.
  • such screening methods may include in vitro screening of a metastatic cell line in which TCI protein is detectable in culture to an agent suspected of inhibiting production of the TCI protein; and determining the level of TCI protein in the cell line, wherein a decrease in the level of TCI protein after exposure of the cell line to the agent is indicative of inhibition of TCI protein production.
  • the invention also encompasses in vivo methods of screening for agents which inhibit expression of the TCI gene, comprising exposing a mammal having tumor cells in which TCI mRNA or protein is detectable to an agent suspected of inhibiting production of TCI mRNA or protein; and determining the level of TCI mRNA or protein in tumor cells of the exposed mammal. A decrease in the level of TCI mRNA or protein after exposure of the mammal to the agent is indicative of inhibition of TCI gene expression.
  • agents can be screened in vitro or in vivo as follows.
  • a metastatic cell line e.g., JMNIB
  • JMNIB metastatic cell line
  • a mammal afflicted with a late stage cancer particularly one of breast cancer, colon cancer, or cancer of the gastrointestinal tract, is exposed to the agent at a dosage and for a time sufficient to inhibit expression of TCI.
  • a late stage cancer is defined by the Duke's stage of the cancer; i.e., late stage cancers correspond to Duke's stages 3-4.
  • the amount or dosage of the agent which is effective to inhibit TCI expression may be determined using serial dilutions of the agent.
  • the level of TCI mRNA or protein may be determined using an aliquot of cells from the cell culture or the in vivo tumor and performing Northern Blot analysis or Western Blot analysis, respectively.
  • the agent will be considered inhibitory if the level of TCI mRNA or protein decreases by more than 50%, and preferably more than 70-80%, relative to the same cell line which has not been exposed to the agent.
  • TCI inhibitory fragments include, but are not limited to, T4F
  • the invention also encompasses the treatment of late stage cancers by administration to a mammal afflicted with a late stage cancer one or more of the above-selected inhibitory agents.
  • Late stage cancers particularly those of the breast, colon, or gastrointestinal tract, are treated according to the invention by administering the inhibitory agent to a mammal afflicted with a late stage cancer in an amount and for a time sufficient to decrease the level of TCI protein or mRNA.
  • the mode of administration may be intravenously, intraperitoneally, by intramuscular or intradermal injection, or orally. Administration may be by single dose, or may be continuous or intermittent.
  • the dosage of inhibitory agent is that dosage which is effective to inhibit TCI production, i.e., within the range of 10 ⁇ g/kg body weight - 100 gm/kg body weight, preferably, within the range of 1 mg/kg body weight - 1 gm/kg body weight, most preferably 10-100 mg/kg body weight.
  • An anti-TCl antibody is produced according to Kohler and
  • Hybridoraas produced by the above process are selected for anti-TCl antibodies using the TCI as an antigen in an ELISA assay.
  • the single type of immunoglobulin produced by a such a hybridoma is specific for a single antigenic determinant, or epitope, on the TCI antigen.
  • TCl-specific antibodies for example, anti-TCl-l produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under the ATCC number HB 11481, are unique in that they recognize the TCI protein, more specifically an epitope of the TCI protein, in formaldehyde- fixed and paraffin-embedded tumor cells which bear TCI.
  • ATCC American Type Culture Collection
  • the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1,14 and 35 USC 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited microorganism, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicants' assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • ADDRESSEE Weingarten, Schurgin, Gagnebin & Hayes
  • B STREET: Ten Post Office Square
  • MOLECULE TYPE protein
  • CAA CAG ATT TTG GGC ACC AAA AAG AAA TAC TTC AGC ACT TGT AAG AAC 246 Gin Gin He Leu Gly Thr Lys Lys Lys Tyr Phe Ser Thr Cys Lys Asn 55 60 65
  • TAC CAC ATC TTA AAT ACT CTC CAG TGT TCT GAG TCT ATT ATG GGA GGA 1014 Tyr His He Leu Asn Thr Leu Gin Cys Ser Glu Ser He Met Gly Gly 310 315 320 GCA GTC TTT GAG ACG CTG GAA GGA AAT ACA ATT GAG ATA GGA TGT GAC 1062
  • AGC AAA ATC TTT CTG AAA GAA GTA AAT GAT ACA CTT CTG GTG AAT GAA 1878 Ser Lys He Phe Leu Lys Glu Val Asn Asp Thr Leu Leu Val Asn Glu
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • SEQUENCE DESCRIPTION SEQ ID NO:18:

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Abstract

The invention encompasses a novel tumor marker which is present on tumor cells and absent on correponding normal cells, nucleic acid encoding the tumor marker, and a novel method of isolating DNA encoding the tumor marker or a gene which is differentially expressed in tissues.

Description

A NOVEL TUMOR MARKER AND NOVEL METHOD OF ISOLATING SAME
FIELD OF THE INVENTION This invention relates to proteins that serve as tumor markers for human carcinoma and to methods of isolating differentially expressed genes.
GOVERNMENT RIGHTS
This invention was made in part with U.S. Government support. Therefore, the U.S. Government has certain rights in the invention.
BACKGROUND OF THE INVENTION Tumor markers for human tumor cells have been largely limited to activated oncogenes and their products, for example, the myc, ras, fos, and erbB2 genes and their encoded oncoproteins. In addition, activated anti-oncogenes, such as RB, p53, and DCC, have been identified in normal cells but do not appear to be present in tumor cells. Oncogene and anti-oncogene products have proven difficult to use as consistent predictors of tumor and normal tissue, respectively, due to the relatively low level of expression of the genes encoding these proteins. Thus, there is a need in the art for a tumor marker which is not only differentially expressed in tumor and normal tissue, but also consistently detectable in human tumor tissue and consistently absent in the corresponding normal tissue.
A common method used to identify genes differentially or uniquely expressed in tumors, in cells responding to growth factors, and in differentiated cell types such as, among others, T cells, adipocytes, neurons, and hepatocytes is the subtractive hybridization technique (S.W. Lee et al., Proc. Natl. Acad. Sci. USA £50:4699, 1983). A method of differential display of eukaryotic mRNA by means of the polymerase chain reaction (PCR) has recently been developed (P. Liang et al., Science 257:967. 1992). This method
SUBSTITUTE SHEET /RULE 26) utilizes oligo dT linked to two additional bases as the primer for reverse transcription driven by reverse transcriptase. cDNA fragments are then amplified by Taq DNA polymerase-based PCR using an oligo dT primer along with one additional primer. The amplified cDNAs are then resolved by DNA sequencing gels. There is a need in the art for a streamlined and simplified process for isolating cDNAs corresponding to differentially expressed mRNAs.
SUMMARY OF THE INVENTION The invention is based on the discovery of a novel protein, TCI (SEQ ID NO:4), which is a tumor marker, particularly for invasive and metastatic tumors, and the gene encoding this protein.
The invention thus encompasses the TCI protein (SEQ ID NO:4), which is useful as a tumor marker for tumor diagnosis and therapy, particularly for colorectal, breast, and gastrointestinal tumors, and for metastatic tumors emanating from these tumor types. TCI is also a useful marker in general for tumor cell invasion and metastasis. mRNA encoding TCI is not expressed in most cultured tumor cells, i.e., in vitro , but is expressed once these cells are grown in vivo . Because later stage and deeply invasive tumors contain higher levels of TCI protein than other tumor tissues, TCI appears to be a particularly useful marker for later stage cancers.
TCI protein may also serve as a target in tumor targeted therapy to prevent tumor cell metastasis and thus invasion of additional organs. For example, a polypeptide fragment of the TCI protein may be used as an antagonist of TCI biological activity; e.g., where TCI biological activity includes invasion and metastasis, the polypeptide fragment may be administered to a patient afflicted with the tumor in order to inhibit the spread of the tumor to other tissues. Alternatively, a truncated portion of TCI which retains the invasive and metastatic biological activities of the full- length molecule will be useful for screening for antagonists of TCI activity. Potentially useful polypeptides are described herein.
The invention also encompasses nucleotide probes based on the TCI nucleotide sequence; e.g., 10, 20, 30, 40, etc. nucleotides in length. Such probes are useful for PCR-based tumor detection and in situ hybridization of tumor tissue sections. In addition, probes whose nucleotide sequences are based on homologies with other genes or proteins having sequences related to TCI, i.e., genes of the TCI family, two of which are described herein, are useful for detecting additional genes belonging to the TCI family of genes.
The invention thus also encompasses methods of screening for agents which inhibit expression of the TCI gene (SEQ ID NO:3) in vitro, comprising exposing a metastatic cell line in which TCI mRNA is detectable in cultured cells to an agent suspected of inhibiting production of the TCI mRNA; and determining the level of TCI mRNA in the exposed cell line, wherein a decrease in the level of TCI mRNA after exposure of the cell line to the agent is indicative of inhibition of TCI mRNA production.
Alternatively, the screening method may include in vitro screening of a metastatic cell line in which TCI protein is detectable in cultured cells to an agent suspected of inhibiting production of the TCI protein; and determining the level of TCI protein in the cell line, wherein a decrease in the level of TCI protein after exposure of the cell line to the agent is indicative of inhibition of TCI protein production.
The invention also encompasses in vivo methods of screening for agents which inhibit expression of the TCI gene, comprising exposing a mammal having tumor cells in which TCI mRNA or protein is detectable to an agent suspected of inhibiting production of TCI mRNA or protein; and determining the level of TCI mRNA or protein in tumor cells of the exposed mammal. A decrease in the level of TCI mRNA or protein after exposure of the mammal to the agent is indicative of inhibition of TCI gene expression. These screening methods are particularly applicable to breast tumor cells, colon tumor cells, or tumor cells of the gastrointestinal tract.
The invention also encompasses a pharmaceutical composition for use in treating a late stage cancer, comprising an effective amount of an inhibitor of TCI, and a method of treating late stage cancer, comprising administering to a mammal afflicted with a late stage cancer a therapeutically effective amount of an inhibitor of TCI. Late stage cancers include those which have become deeply invasive in a tissue or which have metastasized to other tissues.
TCI is detectable in patient blood, urine, sputum or other body fluid using a monoclonal antibody specific for a TCI epitope. Thus, the invention also encompasses antibodies specific for TCI, which can easily be prepared in a kit form. Monoclonal antibodies specific for TCI may be used for tumor imaging to localize tumor position and size. TCl-specific monoclonal antibodies are also useful as screening and diagnostic agents in immunohistochemical staining of tissue sections to distinguish tumor cells from normal cells. Thus, anti-TCl antibodies are particularly useful where they recognize cells which produce the TCI protein when such cells are paraffin-embedded and/or formalin-fixed. One example of such an antibody is the monoclonal antibody anti-TCl-1 produced by the hybridoma deposited with the American Type Culture Collection as ATCC Deposit No. HB 11481.
In another aspect, the invention also features a novel method, called palindromic PCR, for identifying and isolating a gene, e.g., a gene which is differentially expressed in different types of tissues. The method is based on the use of short DNA primers and corresponding palindromic nucleotide sequences in the nucleotide sequence to be isolated.
Thus, the invention encompasses a method for producing a double stranded cDNA that includes the steps of contacting an mRNA with a DNA primer under stringent hybridization conditions to form a first hybrid molecule, the primer having a length of from 8 to 12 nucleotides and, preferably, 9 to 11 nucleotides; subjecting the first hybrid molecule to an enzyme having reverse transcriptase activity, to produce a first DNA strand complementary to at least a portion of the mRNA; contacting the first DNA strand with the primer under stringent hybridization conditions to form a second hybrid molecule; and subjecting the second hybrid molecule to an enzyme having DNA polymerase activity, to produce a second DNA strand complementary to the first DNA strand. Preferably, the method also includes the step of amplifying the first and second DNA strands.
In preferred embodiments, a single enzyme provides both the reverse transcriptase activity and the DNA polymerase activity. One example of a suitable such enzyme is rTth DNA polymerase from the thermophilic eubacterium Thermus thβrmophiluε .
As used herein, the term "palindromic nucleotide sequences" means that a double stranded DNA molecule contains a specific DNA sequence in both its coding strand and its anti-parallel strand, when those strands are read in the same direction, e.g., 51 to 3'.
The specific sequence of the DNA primer is arbitrary in that it is based upon individual judgment. In some instances, the sequence can be entirely random or partly random for one or more bases. Preferably, the GC content of the primer is between 40% and 60%, most preferably about 50%. In other instances, the arbitrary sequence can be selected to contain a specific ratio of each deoxynucleotide. The arbitrary sequence can also be selected to contain, or not to contain, a recognition site for a specific restriction endonuclease.
The DNA primer can contain a sequence that is known to be a "consensus sequence" of an mRNA of known sequence. As defined herein, a "consensus sequence" is a sequence that has been found in a gene family of proteins having a similar function or similar properties. The use of a primer that includes a consensus sequence may result in the cloning of additional members of a desired gene family. Palindromic PCR enables genes that are altered in their frequency of expression, as well as those that are constitutively or differentially expressed, to be identified by simple visual inspection and isolated. The method also allows the cloning and sequencing of selected RNAs, so that the investigator may determine the relative desirability of the gene product prior to screening a comprehensive cDNA library for the full length gene product.
Further objects and advantages of the invention will be apparent in light of the following description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a polyacrylamide gel of size-separated cDNAs that were reverse transcribed from paired mRNAs from colon carcinoma (T) and adjacent normal colon tissue (N) and subsequently amplified.
Fig. 2A is a gel in which the TCI cDNA fragment identified in Fig. 1 was recovered and re-amplified.
Fig. 2B is a Northern Blot of three pairs of RNA from colon carcinoma (T) and their adjacent normal colon tissue (N) probed with 32P-labeled TCI cDNA.
Fig. 3 shows the nucleotide sequence (described herein as SEQ ID NO:l) and corresponding amino acid sequence (SEQ ID NO:2) of the 636 bp partial TCI clone. Fig. 4 shows the nucleotide sequence (SEQ ID NO:3) and corresponding amino acid sequence (SEQ ID NO:4) of the full- length TCI gene and protein.
Fig. 5 is a sequence comparison of the four internal homologous domains of TCI (SEQ ID NO:4), each approximately 135 amino acids.
Fig. 6A is a schematic representation of the four repeats of TCI.
Fig. 6B is a proposed schematic arrangement of the four repeated domains and the N- and C-terminal domains.
Fig. 7 shows the amino acid sequence identity between TCI (SEQ ID NO:4) and Big-h3 (SEQ ID NO:17).
Fig. 8 is a Northern Blot of five pairs of RNA from colon carcinoma (T) and adjacent normal colon tissue (N) probed with 32P-labeled Big-h3 cDNA, the bottom panel representing control RNA probed with 32P-labeled B-actin.
Fig. 9 shows amino acid sequence homology between TCI (SEQ ID NO:4) and Fasciclin I from Grasshopper (GrF) (SEQ ID NO:18) and Drosophila (DrF) (SEQ ID NO:19). Fig. 10 is a Schematic representation showing that, on average, in every 202 bases of sequence in one strand of cDNA, there is one 9-base sequence exactly palindromic to that in a region of its antiparallel strand.
Fig. 11 shows the relationship between the palindromic frequency and the number of bases in a putative DNA primer, as determined by cDNA Matrix analysis.
Fig. 12 is a schematic representation of the method of the invention, palindromic PCR, driven by the enzyme rTth DNA polymerase with one DNA primer in one reaction tube; the dotted line indicates mRNA and the solid line indicates cDNA; the short jagged line represents the single DNA primer.
Fig. 13A shows the effect of the length of the DNA primer on the cDNA amplification patterns; the length and nucleotide sequence of each primer are: A, 8-mer (5'- TGTCGAGA) ; B' , 9-mer (5'-TGTCCAGAC) ; C , 10-mer (5'- TGTCCAGATG) (SEQ ID NO:5); D', 11-mer (5'-TGTCCAGATGC) (SEQ ID NO:6); E', 12-mer (5'-TGTCCAGATGAC) (SEQ ID NO:7).
Fig. 13B shows the effect of the GC content of the DNA primer on the cDNA amplification patterns; the GC content and nucleotide sequence of each primer (10-mer) are: A', 40% (5'-TGTCCAGATA) (SEQ ID NO:8); B', 50% (5'-TGTCCAGATG) (SEQ ID NO:5); C, 60% (5'-TGTCCAGACG) (SEQ ID NO:9): D', 70% (5'- TGTCCAGCCG) (SEQ ID NO:10); E', 80% (5'-TGTCCCGCCG) (SEQ ID NO:ll); F' , 90% (5'-TGCCCGGCCG) (SEQ ID NO:12). Fig. 13C shows the effect of the sequence specificity of the DNA primer on the cDNA amplification patterns; 10-mer primers with the same GC content but different sequences are: A', 5'-TGATGCACTC (SEQ ID NO:13); B', 5'-TGAGCTACTC (SEQ ID NO:14); C, 5'-TGACTGACTC (SEQ ID NO:15). Fig. 13D shows palindromic PCR performed by rTth DNA polymerase (A) with reverse transcription cycles (RT cycles) and (B) without RT cycles.
Fig. 14 shows the identification of differentially expressed genes in human colon carcinoma. Fig. 15 shows reamplification of the TCI cDNA fragment isolated from colon carcinoma; the PCR product was analyzed on a 1.0% agarose gel; a 0.63 Kb cDNA fragment (arrow) was detected.
Fig. 16 is an autoradiogram of DNA sequencing gels showing the presence of PP1 primer sequence (5'-CTGATCCATG) (SEQ ID NO:16) at the 5'-end of both strands of the TCI cDNA fragment; cloning sites are indicated by arrows, sequences below arrows are pBS (KS) vector sequences reading from T3 primer and T7 primer. Fig. 17 is a Northern Blot of 24 pairs of colon carcinoma (T) and their adjacent normal tissue (N) probed with 32P-labeled TCI cDNA.
Fig. 18 shows the Tumor/Normal RNA Ratio from Northern Blot results of Fig. 17. Fig. 19 is a Northern Blot of RNA from carcinoma cells which result from metastasis from colon carcinoma to liver (LM) and their adjacent normal liver (NL) probed with 32P- labeled TCI cDNA.
Fig. 20 shows a Northern Blot of RNA from breast cancer cell line MCF-7 (1) and colon cancer cell line CX-1 (2) cultured in vitro, and MCF-7 tumor (3) and CX-l tumor (4) grown in vivo in nude mice.
Fig. 21 shows staining of formalin-fixed and paraffin- embedded colon tumor tissue sections using the monoclonal antibody anti-TCl-1 and avidin-biotin-peroxidase detection. Fig. 22 shows staining as described in Fig. 21, except that panels A, C, D represent breast invasive ductal carcinoma and panel B, normal breast tissue.
Fig. 23 shows staining as in Fig. 13, except that panels A and B represent gastric carcinoma, and panels C and D, deeply invasive colon carcinoma.
Fig. 24 is a Western Blot analysis of protein samples from two pairs of colon carcinoma and their adjacent normal colon (A) and two pairs of breast carcinoma and their adjacent normal breast (B) , using a monoclonal antibody against TCI protein as a probe.
Fig. 25A shows the ethidium bromide staining pattern of an RNA gel in which the same amount of RNA from JMN (1) and JMN1B (2) cells is loaded per lane.
Fig. 25B is a Northern Blot analysis of RNA from malignant mesothelioma cells JMN1B (2) and JMN (1) using TCI cDNA as a probe.
Fig. 26 is a Western blot using a monoclonal antibody against TCI to probe JMN1B cells grown in conditioned medium and whole cell lysate. Fig. 27 shows JMN1B cells fixed with paraformaldehyde without subsequent permeabilization in panels A and B, and JMN1B cells fixed with paraformaldehyde and then permeabilized in panels C and D.
Figs. 28A-28D show the corrected nucleotide sequence and corresponding amino acid sequence of the full length TCI gene and protein. DESCRIPTION OF THE PREFERRED EMBODIMENTS
TCI (SEQ ID NO:4) is a novel protein that is found in invasive and metastatic tumor cells. The nucleotide sequence
(SEQ ID NO:3) encoding TCI was found using a novel technique described herein as palindromic PCR, a technique which enables identification and cloning of a gene that is differentially expressed in tissues. Cloning and sequencing of the gene encoding TCI and characterization of the protein is described below, along with examples of how the protein is detected in invasive and metastatic cancers. Examples describing additional uses of the TCI protein and its fragments, the nucleotide sequence encoding TCI and fragments thereof, and antibodies specific for TCI are also included.
Identification, Cloning and Detection of Expression of the TCI Gene
The identification, cloning, and differential detection of expression of the TCI gene (SEQ ID NO:3) was performed as follows. A 636 bp cDNA fragment (SEQ ID N0:1) containing TCI sequences was identified and isolated by a rapid method termed palindromic PCR, described herein, from human surgical colon carcinoma tissue. Briefly, paired mRNAs were isolated from colon carcinoma tissue and adjacent normal colon tissue from the same patient, then matched mRNAs were reverse transcribed to cDNA and subsequently amplified by the palindromic PCR method described herein, which utilizes one DNA primer. Both reverse transcription and PCR reactions were driven by a single enzyme, rTth DNA polymerase, in a single tube. 35S or 33P-labeled PCR cDNA fragments were resolved on a DNA sequencing gel. As shown in Fig. 1, paired mRNAs from colon carcinoma (T) and adjacent normal colon tissue (N) were reverse transcribed to cDNA and subsequently amplified by palindromic PCR. 35S-labeled PCR cDNA fragments were then resolved on a DNA sequencing gel. A differential cDNA band (TCI) appeared to be present only in the tumor sample. This TCI cDNA fragment was recovered from the sequencing gel and then reamplified with the same palindromic primer. This 636 bp fragment is identified with a horizontal arrow in Fig. 2A.
TCI gene expression was examined in colon carcinoma cells and in the corresponding adjacent colon tissue, and the results were as follows. Fig. 2B is a Northern Blot of three pairs of RNA from colon carcinoma (T) and their adjacent normal colon tissue (N) probed with 32P-labeled TCI cDNA. TCI mRNA was over-expressed in all three cases of colon carcinoma, whereas only very weak TCI message appeared in the adjacent normal tissue. In the bottom panel of the blot, control RNA was blotted with 32P-labeled cDNA encoding B- actin.
Northern Blot analysis of several pairs of Tumor/Normal total RNA using a 32P-labeled TCI cDNA probe revealed that the TCI mRNA size is about 3.6Kb. This first TCI cDNA fragment was cloned into a pBluescript plasmid DNA vector strategies. Nucleotide sequence analysis revealed that this fragment contained 636bp with nucleotide sequences corresponding to the primer sequence at both 5'-ends of the double-stranded DNA (Fig. 3 and SEQ ID NO: 1) . The corresponding predicted amino acid sequence is shown in Fig. 3 and provided in SEQ ID NO: 2. A search of the GenBank database with this cDNA fragment revealed that TCI is a novel gene. Nucleotide sequence analysis of the 636bp TCI cDNA fragment obtained by the described differential display method revealed that it contained a partial open reading frame. Therefore, this 636bp cDNA fragment was used as probe to screen a cDNA library. Several overlapping clones were obtained and contained a 2997bp sequence. To obtain the complete open reading frame for TCI, a modified 5'-end RACE technique was used to amplify the TCI coding regions. The nucleotide and deduced amino acid sequence of full-length TCI is shown in Fig. 4 and provided in SEQ ID NOS: 3 and 4. The N-terminal signal sequence is underlined; one predicted N- linked glycosylation site (NDT) is boxed and a polyadenylation signal (AATAAA) is indicated. The cDNA contains 3126bp with a potential polyadenylation sequences (AATAAA) at the 3'-end, beginning at residue 2963. The open reading frame (ORF) encodes a 777-amino acid protein with a calculated molecular weight of 86kD. The TCI protein contains an amino-terminal signal peptide or secretory leader signal (ALPARILALALALAL) , and one predicted site of N-linked glycosylation at amino acid residue 605 (NDT) . One Ce okine B family motif (C-C) was found at amino acid residue 85 (C-C) of TCI.
Analysis of the deduced amino acid sequence (SEQ ID NO:4) revealed that TCI contained four internal homologous domains of approximately 135 amino acids. A comparison of these repeats is shown in Fig. 5. Each boxed amino acid is identical with at least one other residue at that same position. The interdomain homologies range from 32% (between domains 2 and 4) to 18% (between domains 1 and 3) . Some amino acid sequence such as TLF— AP— TNEAF, NGVIHXID are highly
conserved between all four repeats. The notati .ons — A and— T
V S are used herein to indicate that alanine or valine, and threonine or serine, respectively, may be found at these positions. In addition, the notation X is used herein to indicate that this position may include any amino acid. Each repeat starts with the most divergent sequence. The four repeats occur between residues 139-537 and are uninterrupted by non-homologous domains. A schematic representation of the four repeats of TCI is shown in Fig. 6A. The four homologous repeats suggest a tetrameric structure (Mclachlan 1980; Zinn et al, 1988) with two binding sites, one at each intrachain dimer. The four repeats of TCI may serve as ligand binding sites, with the N-terminal or C-terminal domains serving as the functional domain. One possible arrangement of the four repeated domains and the N- and C-terminal domains is shown schematically in Fig. 6B. The nucleotide and corresponding amino acid sequence of the TCI gene and protein with a corrected leader signal sequence are given in Figs. 28A-28D.
Palindromic PCR Described below is a novel technique used to identify the TCI mRNA and prepare TCI cDNA. Although the sequence of bases in a coding and antisense strand of a cDNA molecule are, in a sense, "mirror images" of one another, we have found that with surprising frequency a short sequence of bases, e.g. 9 or 10, in one strand will be found to have an exact copy in its anti-parallel strand. We call these sequences "palindromic" sequences. This phenomenon has been used to develop a method of cDNA isolation and amplification.
In order to determine the frequency of occurrence or "palindromic frequency" of these anti-parallel repeats, a computer program called DNA Matrix (DNA Strider 1.2) was used to analyze double stranded cDNAs which were randomly selected from the GenBank database. DNA matrix analysis revealed the palindromic frequency of double strand cDNA to be surprisingly high and led to our development of a relationship between the number of bases in the chosen sequence, the "palindromic bases," and the palindromic frequency. Single strand cDNA (the mRNA strand) and its anti-parallel strand were compared, each from the 5' to 3' end by the DNA Matrix program. For example, as illustrated in Fig. 10, on the average, in every 202 bases of sequence in one strand of cDNA, there is one 9-base sequence that is exactly duplicated to that in another region of its antiparallel strand. The palindromic frequency found in native cDNA is much higher than that which would be calculated from random composition, suggesting that the nucleotide composition of double-stranded cDNA follows certain palindromic rules. As shown in Fig. 11, the palindromic frequency dramatically decreases when the number of bases in the searched segment increases. The key numbers of bases which lead to dramatic change of palindromic frequency are 9, 10 and 11 bases. This, then, is the theoretical basis for designing a primer for use in the DNA isolation and amplification method of the invention, palindromic PCR.
Table 1 presents the statistical data showing the palindromic frequency related to the number of bases in the searched segment.
Table 1
Palindromic Frequency Related to
No. of Bases in Searched Segment as
Revealed by cDNA Matrix
Analysis
No. Bases in Average Length to Find
Searched Segment One X-Base Palindromic Palindromic Frequenc
( X Bases ) Sequence
7 bases 18 bases 0.4
9 bases 202 bases 0.048
11 bases 872 bases 0.015
13 bases >1996 bases <0.007
The principle of the method of palindromic PCR is shown in schematic representation in Fig. 12. The general strategy is to use a single primer and one enzyme combining both reverse transcriptase and DNA polymerase activities, e.g., rTth DNA polymerase (from the thermophilic eubacterium Thermus thermophilus) , to perform both reverse transcription and polymerase chain reaction in one reaction tube. rTth DNA polymerase possesses a very efficient reverse transcriptase activity in the presence of MnCl2 and a thermostable DNA polymerase activity in the presence of MgCl2. The rTth DNA polymerase has been observed to be greater than 100-fold more efficient in coupled reverse transcription and PCR than the analogous DNA polymerase, Taq (T. W. Myers et al., Biochemistry 3i):7661, 1991) . In this reaction, an appropriate primer would allow anchored annealing to some regions of certain mRNA species that contain sequence complementary to the palindromic primer. This subpopulation of mRNAs is likely to be reverse transcribed by rTth DNA polymerase. A "Palindromic" primer apparently has a greater probability of anchoring to the coding regions of mRNA than oligodT primer. Once mRNAs are reverse transcribed to form a first strand cDNA species, the same primer can anneal to some regions of the first strand cDNA and function as the "Downstream primer" in a PCR reaction. The same primer can also function as the "Upstream primer." When the primer anchors to first strand cDNAs, the annealing position to various cDNA molecules should, in principle, be at different distances in different molecules from the first annealing position. Therefore, the amplified cDNA fragments from various mRNAs will be of different sizes. Once these PCR- generated cDNA fragments are labeled with 35S-dATP or 33P- dATP, they can be resolved as a ladder by DNA sequencing gels. A display of cDNAs originating from various mRNAs can then be visualized after autoradiography.
The selection of the specific palindromic primer depends on three important factors: the length, the GC content, and the sequence specificity. DNA Matrix analysis has indicated that the ideal length of a primer for an appropriate palindromic frequency is from 9 to 11 bases. Therefore, a set of primers from 8 base to 12 base in length with 50% GC content was chosen for study. Our results showed that 9, 10, and 11 base primers gave an appropriate number of cDNA fragments readily resolvable by DNA sequencing gels (Fig. 13) . To identify the GC content of the primer most suitable for this method, a set of 10-mer primers with GC content ranging from 40% to 90% was tested. The results suggested that a GC content from 40% to 80% is acceptable (Fig. 13) . However, primers with 40% to 60% GC content appear to yield better results. To examine the effect of the specific sequence of the primer, 10-mer primers of different sequences each having 50% GC content was tested. As predicted, different primers gave rise to different cDNA patterns (Fig. 13) . As little a difference as three bases led to totally different cDNA profiles. cDNA patterns generated by palindromic PCR are highly stable. When the same conditions were used but the experiments repeated at different times, the patterns of the amplified cDNA fragments were highly reproduced, indicating the reliability of this method.
In order to be sure of detecting mRNAs with a low copy number, it was necessary to determine the sensitivity of this method. It has been reported that the amplification driven by rTth DNA polymerase is at least 100-fold greater than that by Taq polymerase. rTth DNA polymerase allows the detection of IL-la mRNA, which has a very low copy number, in 80pg of total cellular RNA (T.W. Myers et al., Biochemistry 30:7661. 1991) . Thus, the higher efficiency of rTth DNA polymerase ensures that the palindromic PCR method of the invention provides high sensitivity. In addition, because rTth polymerase is thermostable, it can also be used to perform several RT cycles (reverse transcription cycles) , which means several copies of first strand cDNA can be obtained from a single copy of mRNA. The sensitivity of the method is increased by performing multiple RT cycles using rTth polymerase (Fig. 13) .
The method of the invention was tested in a search for differences in mRNA expression between human colon carcinoma and the adjacent normal epithelium from a surgical specimen. Paired mRNA preparations were reverse transcribed with a palindromic primer 5'-CTGATCCATG (designated as PP-l primer) (SEQ ID NO:16) in the presence of MnCl2 followed by PCR with the same primer in the presence of MgCl2 using rTth DNA polymerase. The reaction products were then analyzed by DNA sequencing gels. About 70-110 amplified cDNA fragments ranging from 100-700 bases from both preparations were detected (Fig. 14) . Whereas overall cDNA patterns between tumor and normal tissue are similar, significant differences were detected by this method. Most cDNA bands showed the same intensity between tumor and normal preparations, but two cDNA bands designated as TCI and TC2 appeared with increased intensities in tumor tissue (Fig. 14) . A sample reaction protocol is described below. To 1.1 μl of double distilled (dd) H20 is added 0.5 μl of 10X rTth DNA polymerase reverse transcriptase (RT) buffer (lOOmM Tris-Hcl, pH 8.3, 900mM KC1) , 0.5 μl of lOmM MnCl2, 0.4 μl of 2.50mM dNTP, 1.0 μl (0.50 μg) of one palindromic primer (9-11 mer), and 1.0 μl (100 ng) of mRNA to form Mix A in a total vol. of 4.5 μl. Mix A is heated in a 0.5 ml PCR tube at 65°C for 6 min and then at 37°C for 8 min. Next, 0.5 μl (1.25 unit) of rTth DNA polymerase is added, the reaction mixture is mixed well, spun briefly, incubated at 70°C for 12 min and then placed on ice. Mix B which consisting of 12.5 μl of dd H20, 2.0 μl of 10X chelating buffer (50% glycerol (v/v) , lOOmM Tris-HCl, pH 8.3, 1M KC1, 0.5% Tween 20), 2.0 μl of 25 mM MgCl2 solution, 2.50 mM dNTP and 2.0 μl of 35S-dATP (or 33P-dATP) is dispensed in the amount of 20 μl into each 5.0 μl RT reaction mixture. The samples are mixed and spun briefly and then overlaid with 25 μl of mineral oil. The polymerase chain reaction is then started: 94°C for 40 sec, 40°C for 2 min., 72°C for 35 sec. (for 40 cycles, hold at 72°C for 4 min.), and then 4°C.
For cDNA analysis, 7 μl of a PCR sample is mixed with 4 μl of sequencing loading buffer, samples are incubated at 80°C for 3 min., and then placed on ice. 4.5 μl of the sample is loaded on a 6%-8% agarose DNA sequencing gel.
A gel slice containing a desirable cDNA band (such as TCI) was soaked in 200 μl of ddH20 for 20 min and then separated from 3M paper with a clean forcep or a plastic pipette tip. The gel was removed and pounded with an autoclaved plastic pipette tip. Elution buffer (20 μl) was added and the mixture was vortexed and left at room temperature for 4 hrs or overnight. After centrifugation, cDNA fragments in 10 μl eluent were reamplified by rTth DNA polymerase with the same palindromic primer, as described. After one 40-cycle PCR, the reamplified cDNA could be detected by agrose gels stained with ethidium bromide. The amount of cDNA generated was sufficient for cloning and preparing a probe for Northern Blot analysis. Fig. 15 shows the gel obtained when the TCI cDNA band was subjected to elution and reamplification. Total PCR product of the TCI fragment was 2.5 μg.
The reamplified TCI cDNA fragment was treated with T4 DNA polymerase and cloned into pBluescript plasmid DNA vector at Smal site by blunt end ligation. The nucleotide sequence of the TCI fragment (SEQ ID NO:l) showed that a sequence identical to the PP1 primer (SEQ ID NO:16) is indeed present at the 5'-end of both strands of the TCI fragment (Fig. 16) . This result confirms that the 5'-ends of both complementary chains of the TCI cDNA fragment used the same palindromic primer during palindromic PCR as discussed above. It also implies that the same palindromic primer sequence is present at the 5'-ends of both strands for every PCR product in the same reaction. These results establish that a single 9- 11 base palindromic primer can effectively prime reverse transcription and then serve as both a "Downstream primer" and an "Upstream primer" in palindromic PCR amplification.
The method of the invention differs from other methods in a number of ways. In palindromic PCR, only a single primer (9-11 bases) is used and is sufficient to prime reverse transcription as well as to support subsequent PCR for a display of nearly 100 cDNA species. Because the pattern of amplified cDNAs depends on the sequence of the single palindromic primer, the species of mRNAs that are subjected to amplification can readily be controlled by a proper sequence of the palindromic primer. If a group or family of genes shares certain sequences, a primer can be chosen from such a sequence, and a specific display of this set of mRNAs can readily be performed. Likewise, computer analysis of the Genebank database may reveal additional sequences useful as a primer shared by a set of related genes. The use of such a primer by the method of the invention would allow the display for the expression of a given set of genes. Palindromic PCR provides an easy, sensitive and economical way to identify and isolate differentially expressed genes related to tumor and other disease.
Differential expression of TCI DNA in normal tissue and tumor cells.
Northern Blot analysis, as described above, confirmed the differential expression of TCI mRNA in colon carcinoma tissue, and the absence of TCI mRNA in the corresponding normal tissue. Evaluation of the expression of TCI mRNA in additional cases of colon carcinoma at different stages was also undertaken. Surgical specimens of 24 cases of human primary colon carcinoma and 6 cases of liver metastases were examined by Northern hybridization of total RNA with 32P- labeled TCI probe.
A Northern Blot of 24 pairs of colon carcinoma (T) and their adjacent normal tissue (N) probed with 32P-labeled TCI cDNA is shown in Fig. 17. It is evident from the results that the level of TCI mRNA in tumor tissue is much greater than the level in adjacent normal tissue in all 24 cases. The TCI mRNA levels vary in different cases of carcinoma. Panels I and II show A: TCI mRNA and B: Control; Panel III shows TCI mRNA and control (Actin) mRNA.
Fig. 18 shows the Tumor/Normal RNA Ratio from Northern Blot results of Fig. 17. The horizontal line indicates the mean Tumor/Normal ratio. TCI mRNA was abundantly expressed in all 24 cases of primary colon carcinoma and 6 cases of liver metastases, whereas only a small amount of TCI mRNA was detected in a few cases of paired adjacent normal tissue. The mRNA level of TCI was much greater in primary colon carcinoma than in paired adjacent normal colonic epithelium in all 24 cases. The Tumor/Normal ratio varied from 5.6 to 92, and the mean Tumor/Normal ratio being 32. The Tumor/Normal ratio, when plotted against the Duke's stage of disease, gave evidence for increasing TCI expression with increasing stage of colon carcinoma.
In all six cases of paired colon carcinoma metastatic to liver, the TCI mRNA level was much higher in metastatic tumor than in adjacent normal liver tissue. Fig. 19 shows a Northern Blot of RNA from metastatic colon carcinoma to liver (LM) and their adjacent normal liver (NL) probed with 32P-labeled TCI cDNA. TCI mRNA was expressed only in metastatic tumor in 5 of 6 samples. Only one sample of normal liver tissue expressed a very weak TCI message. The Tumor/Normal ratio is greater than 64. These results suggested that differential expression of TCI may be associated with human colorectal cancer progression and biological aggressiveness of the disease.
In vivo and in vitro expression of TCI mRNA
The expression of TCI mRNA in cultured cancer cells and in vivo tumor cells was analyzed and is described below. TCI was overexpressed in tumor tissue in vivo. The expression of TCI mRNA in cultured cancer cell lines in vitro was examined by Northern Blot analysis. RNAs isolated from twelve colon cancer cell lines (HT29, Clone A, MIP101, CX-1, Morser, CCL227, CCL228, etc.) derived from different stage of human colon carcinoma, two melanoma cell lines (LOX, A2058) , one breast cancer cell line (MCF-7) , two cervical cancer cell lines (Hela, A431) , three bladder cancer cell lines (EJ, T24, MB49) , one pancreas cancer cell line (CRL1420) , two hepatoma cell lines (HepG2, HepG3) and four normal cell lines (FS-2, MRC-5, 498A, CV-1) were screened by Northern Blot analysis. However, the TCI transcript could not be detected in all of these cell lines. This result suggested that TCI expression was dramatically decreased or indeed turned off in cultured cancer cells. However, after cultured cancer cells were injected into nude mice to grow tumor in vivo, TCI mRNA expression turned on again and its mRNA level could be detected by Northern Blot analysis. Fig. 20 shows a Northern Blot of RNA from breast cancer cell line MCF-7 (1) and colon cancer cell line CX-1 (2) cultured in vitro, and MCF-7 tumor (3) and CX-1 tumor (4) grown in vivo in nude mice. TCI mRNA in colon cancer cell line CX-1 and breast cancer cell line MCF-7 cultured in vitro could not be detected by Northern Blot analysis. After cultured CX-1 and HT29 cells were injected into nude mice to form tumors in vivo, TCI mRNA was detectable by Northern Blot analysis, the TCI mRNA levels being dramatically increased in vivo . This result suggests that TCI gene expression was turned on or dramatically increased in the tumor cells in vivo. Thus, the differential expression of the TCI gene appears to be related to invasion and metastasis of tumor cells in vivo. The regulation of TCI gene expression in vivo and in vitro could be a very important model to understand tumorigenesis and tumor malignant behavior.
Expression of TCI protein
The expression of TCI protein in in vivo tumor cells, cultured carcinoma cells, and in corresponding normal cells was examined, and is described below. The TCI gene (SEQ ID NO:3) was cloned into a plasmid expression vector, and recombinant TCI protein (SEQ ID NO:4) was expressed in bacteria. Several monoclonal antibodies against the bacterially-produced TCI protein were raised, as will be described below. A variety of formalin-fixed and paraffin- embedded tumor tissue sections were examined by immunohistochemical staining with a mouse monoclonal anti-TCl antibody anti-TCl-1 using an avidin-biotinylated-peroxidase detection technique. Strong positive staining of TCI was found in primary colon carcinoma (Fig. 21, panel A) , colon carcinoma metastatic to liver (Fig. 21, panel C) and lymph node (Fig. 21, panel D) , breast carcinoma (Fig. 22, panels A,C,D) and gastric carcinoma (Fig. 23, panels A,B) . The TCI protein level in tumor tissue is much greater than the level of TCI in adjacent normal tissue (Figs. 21B, 23B) . These results, which represent the staining of sixteen cases of different stages of primary colon carcinoma, eight cases of colon tumor metastatic to liver and lymph node, fourteen cases of breast carcinoma and five cases of gastric carcinoma, suggested the following three conclusions. First, the TCI protein level appeared to be different in different types of carcinoma, with protein levels being highest in breast carcinoma. Second, the advance edge of the deeper invasive tumor appeared stain stronger for TCI, suggesting a greater prevalence of TCI protein at the advance edge of the tissue. Third, the move advanced stages of tumor appeared to contain more TCI protein.
Fig. 24 is a Western Blot analysis of protein samples from two pairs of colon carcinoma and their adjacent normal colon (A) and two pairs of breast carcinoma and their adjacent normal breast (B) , using a monoclonal antibody against TCI protein as a probe. A major 86kd protein (arrow) was detected by anti-TCl antibody in tumor samples (T) but not in normal samples (N) . The Western Blot analysis confirmed that tumor tissue contained significantly more TCI protein than the corresponding adjacent normal tissue.
TCI gene expression
The presence of TCI mRNA and protein in malignant mesothelioma cells was examined, and is described below. More than 42 cell lines have been screened for TCI gene expression by Northern Blot analysis. However, only two cell lines, JMN1B and JMN, express detectable mRNA by Northern Blot analysis. JMN1B and JMN are malignant mesothelioma, JMN1B being a subline of JMN cells with showing enhanced tumorigenicity after passage of JMN cells through a nude mouse. Fig. 25 is a Northern Blot analysis of RNA from malignant mesothelioma cells JMN1B and JMN using TCI cDNA as a probe. The results presented in Panel 25B demonstrate that TCI mRNA level in JMN1B cells (2) is much greater than that in JMN cells (1) . Panel 25A shows the ethidium bromide staining pattern of an RNA gel in which the same amount of JMN (1) and JMNIB (2) RNA is loaded per lane.
The Northern Blot analysis revealed that the TCI mRNA level is much higher in JMNIB than in JMN, the JMNIB/JMN ratio being approximately 14. Higher expression of TCI mRNA in JMNIB could be related to the observed greater tumorigenicity of JMNIB cells. It has been found that JMNIB cells can secrete an "EGF-like" growth factor called transformed mesothelial growth factor (TMGF) that satisfies the EGF requirement of normal human mesothelial cells. The difference in the levels of TCI mRNA in JMNIB and JMN cells provides an ideal cell model to understand the regulation of TCI expression and its relation to tumorigenicity.
Sequence analysis of the deduced amino acid sequence has revealed that the TCI protein (SEQ ID NO:4) contained a secretory leader signal at its N-terminus. The secretion of TCI protein was confirmed by Wester Blot analysis of conditioned medium of JMNIB cells. JMNIB cells were cultured in regular medium until 90% confluent, then cultured in serum free medium for two days. This serum free conditioned medium was analyzed by immunoblotting with anti-TCl monoclonal antibody. Fig. 26 is a Western blot analysis using a monoclonal antibody against TCI to probe JMNIB cells grown in conditioned medium and whole cell lysate. Two major bands (about 86kd and 104kd) were recognized by anti-TCl antibody both in JMNIB cell conditioned medium (1) and whole cell lysate (2) . Numbers on the left indicate the position of molecular weight standards in kilodalton. The protein size of the lower molecular weight 86kd band is consistent with that of deduced TCI protein, whereas the higher molecular weight 104kd band is consistent with a TCI glycoprotein. There is one predicted site of N-linked glycosylation at the amino acid residue 605(NDT) of deduced TCI protein sequence. There are 60 threonine residues and 36 serine residues in the deduced TCI sequence, each of which is a potential site of O-linked glycosylation. Human malignant mesothelioma cell line JMNIB can express abundant TCI. This cell line was used to study the distribution and localization of TCI protein by immunofluorescent staining with an anti-TCl monoclonal antibody followed by Rhodamine conjugated goat anti-mouse IgG secondary antibody. When JMNIB cells were fixed with paraformaldehyde without subsequent permeabilization, the positive staining was seen on the cell surface or outside of the cell (Fig. 27, panels A,B) , which confirms the secretion of TCI protein. When JMNIB cells were fixed with paraformaldehyde and then permeabilized, positive staining appeared in the Golgi complex and the endoplasmic reticulum (ER) in the cell (Fig. 27, panels C,D) , suggesting that TCI protein is synthesized in the ER and Golgi complex. The staining in the Golgi complex is clearly evident, indicating that glycosylation of TCI protein may be located in the Golgi complex. The TCI protein distribution pattern also suggests that TCI is a secreted glycoprotein.
Without being bound to one theory as to the biological function of TCI, observations as to the prevalence and expression of TCI mRNA and protein indicate that TCI may be related to tumor malignant behavior such as invasion and metastases. These observations include the following: TCI is significantly overexpressed in tumor tissue; TCI is a secreted protein; later stage tumor expresses higher levels of TCI; deeper invasive tumor contains higher levels of TCI protein; TCI expression turns off in cultured tumor cells in vitro and turns on again after cells grow tumor tissue in vivo. These observations indicate that the function of TCI is not related to tumor cell proliferation, but is more likely involved in tumor malignant behavior in vivo, such as invasion and metastases.
TCI is a member of a Family of Proteins.
A FASTA search of the GenBank and EMBL database with the TCI open reading frame indicated that the protein is unique. However, TCI whole protein shared 45% sequence identity with a TGF-beta inducible gene, Big-h3, at the amino acid level, suggesting that TCI and Big-h3 may belong to a new gene family. The identity between TCI (SEQ ID NO:4) and Big-h3 (SEQ ID NO:11) at the amino acid level is shown in Fig. 7. In Fig. 7, identical amino acids between TCI and Big-h3 are boxed. Several stretches of amino acids GSFTXFAPSNEAW, TLXAPTNEAFEKXP, ATNGWHXIDXV, LYXGQXLETXGGKXLRVFVYR, HYPNGXVTVNCAR are highly conserved between TCI and Big-h3. Northern Blot analysis showed that the TCI gene is expressed from a larger transcript than Big-h3, and DNA sequence analysis indicated that TCI contains a longer open reading frame encoding a higher molecular weight protein than the Big-h3 gene. It has been found that Big-h3 also contains four internal repeats. The amino acid sequence homology and structural similarity between TCI and Big-h3 indicate their functional similarity and relationship. We found that Big-h3 mRNA is also much more abundant in colon carcinoma tissue than in adjacent normal colon tissue (Fig. 7) . Fig. 8 is a Northern Blot of five pairs of RNA from colon carcinoma (T) and adjacent normal colon tissue (N) probed with 32P-labeled Big-h3 cDNA. The blot shows Big-h3 mRNA level in colon carcinoma to be much higher than that in adjacent normal tissue. The bottom panel represents control RNA probed with 32P-labeled B-actin.
In contrast to the expression pattern of TCI mRNA, which is shown to be largely restricted to in vivo tumor tissue, Big-h3 mRNA is not only expressed in the tumor tissue, but also expressed in the cultured tumor cell lines and some normal cell lines. Though TCI and Big-h3 shared significant homology, their responses to growth factors are distinctly different.
Fasciclin I, II, III are extrinsic membrane glycoproteins involved in the growth cone guidance during nervous system development in the insect embryo. A search of NBRF protein database revealed a significant homologous domain between TCI and Fasciclin I from Grasshopper and Drosophila. One TCI domain of 204 amino acids (amino acid residue 503-706) shared 30% identity with Grasshopper Fasciclin I, and shared 25% identity with Drosophila Fasciclin. Fig. 9 shows amino acid sequence homology between TCI (SEQ ID NO:4) and Fasciclin I from Grasshopper (GrF) (SEQ ID NO:18) and Drosophila (DrF) (SEQ ID NO:19). Boxed amino acids are identical with at least one other amino acid at that same position. It has been found that Fasciclin also contained four internal homologous domains, each consisting of approximately 150 amino acids. The domains of TCI and Fasciclin I share some highly conserved amino acid stretches such as
TXF—PTNXAF, and VXHWDXXLXP. A The most conserved sequence among TCI, Big-h3 and
Fasciclin is TXF— APTNXA— F. All four internal repeats in TCI
V W or Big-h3 or Fasciclin I also share the most conserved sequence TXF— AP— TNXA— F. This sequence appears to be an
important motif of this gene family.
Screening for antagonists to TCI function.
The invention also includes methods of screening for agents which inhibit TCI gene expression, whether such inhibition be at the transcriptional or translational level.
Screening methods, according to the invention, for agents which inhibit expression of the TCI gene in vitro will include exposing a metastatic cell line in which TCI mRNA is detectable in culture to an agent suspected of inhibiting production of the TCI mRNA; and determining the level of TCI mRNA in the exposed cell line, wherein a decrease in the level of TCI mRNA after exposure of the cell line to the agent is indicative of inhibition of TCI mRNA production. Alternatively, such screening methods may include in vitro screening of a metastatic cell line in which TCI protein is detectable in culture to an agent suspected of inhibiting production of the TCI protein; and determining the level of TCI protein in the cell line, wherein a decrease in the level of TCI protein after exposure of the cell line to the agent is indicative of inhibition of TCI protein production.
The invention also encompasses in vivo methods of screening for agents which inhibit expression of the TCI gene, comprising exposing a mammal having tumor cells in which TCI mRNA or protein is detectable to an agent suspected of inhibiting production of TCI mRNA or protein; and determining the level of TCI mRNA or protein in tumor cells of the exposed mammal. A decrease in the level of TCI mRNA or protein after exposure of the mammal to the agent is indicative of inhibition of TCI gene expression.
According to the invention, agents can be screened in vitro or in vivo as follows. For in vitro screening, a metastatic cell line, e.g., JMNIB, may be cultured in vitro and exposed to an agent suspected of inhibiting TCI expression in an amount and for a time sufficient to inhibit such expression. For in vivo screening, a mammal afflicted with a late stage cancer, particularly one of breast cancer, colon cancer, or cancer of the gastrointestinal tract, is exposed to the agent at a dosage and for a time sufficient to inhibit expression of TCI. A late stage cancer is defined by the Duke's stage of the cancer; i.e., late stage cancers correspond to Duke's stages 3-4. The amount or dosage of the agent which is effective to inhibit TCI expression may be determined using serial dilutions of the agent. The level of TCI mRNA or protein may be determined using an aliquot of cells from the cell culture or the in vivo tumor and performing Northern Blot analysis or Western Blot analysis, respectively. The agent will be considered inhibitory if the level of TCI mRNA or protein decreases by more than 50%, and preferably more than 70-80%, relative to the same cell line which has not been exposed to the agent.
Examples of potential inhibitors of TCI mRNA or protein production include but are not limited to antisense RNA, competitive inhibitors of the TCI protein such as fragments of the TCI protein itself, or antibodies to TCI protein. Candidate TCI inhibitory fragments include, but are not limited to, T4F|P N A and NG - HX- .
Use of anatagonists to TCI functions.
The invention also encompasses the treatment of late stage cancers by administration to a mammal afflicted with a late stage cancer one or more of the above-selected inhibitory agents. Late stage cancers, particularly those of the breast, colon, or gastrointestinal tract, are treated according to the invention by administering the inhibitory agent to a mammal afflicted with a late stage cancer in an amount and for a time sufficient to decrease the level of TCI protein or mRNA. The mode of administration may be intravenously, intraperitoneally, by intramuscular or intradermal injection, or orally. Administration may be by single dose, or may be continuous or intermittent. The dosage of inhibitory agent is that dosage which is effective to inhibit TCI production, i.e., within the range of 10 μg/kg body weight - 100 gm/kg body weight, preferably, within the range of 1 mg/kg body weight - 1 gm/kg body weight, most preferably 10-100 mg/kg body weight.
Production of monoclonal antibodies reactive with TCI. An anti-TCl antibody is produced according to Kohler and
Milstein, Nature, 256:495-497 (1975), Eur. J. Immunol. 6:511- 519 (1976) , both of which are hereby incorporated by reference, using the TCI protein or a fragment thereof as the immunizing antigen. Hybridoraas produced by the above process are selected for anti-TCl antibodies using the TCI as an antigen in an ELISA assay. The single type of immunoglobulin produced by a such a hybridoma is specific for a single antigenic determinant, or epitope, on the TCI antigen. Certain TCl-specific antibodies, for example, anti-TCl-l produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under the ATCC number HB 11481, are unique in that they recognize the TCI protein, more specifically an epitope of the TCI protein, in formaldehyde- fixed and paraffin-embedded tumor cells which bear TCI.
Deposits
The following samples were deposited on October 29,
1993, with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852.
Deposit ATCC Accession No.
TCI gene in pBluescript 75599 plasmid DNA vector
Hybridoma TC-1 HB 11481
Applicants' assignee, Dana-Farber Cancer Institute,
Inc. , represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1,14 and 35 USC 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited microorganism, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicants' assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
OTHER EMBODIMENTS
The invention is not limited to those embodiments described herein, but may encompass modifications and variations which do not depart from the spirit of the invention. While the invention has been described in connection with specific embodiments thereof, it will be understood that further modifications are within the scope of the following claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Chen, Lan Bo Bao, Shideng Liu, Yuan (ii) TITLE OF INVENTION: A NOVEL TUMOR MARKER AND NOVEL METHOD OF
ISOLATING SAME (iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Weingarten, Schurgin, Gagnebin & Hayes (B) STREET: Ten Post Office Square
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02109 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/146,488
(B) FILING DATE: 29-OCT-1993
(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Heine, Holliday C.
(B) REGISTRATION NUMBER: 34,346
(C) REFERENCE/DOCKET NUMBER: DFCI-333XX (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 543-2290 (B) TELEFAX: (617) 451-0313
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..636 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: CTG ATC CAT GGG AAC CAG ATT GCA ACA AAT GGT GTT GTC CAT GTC ATT 48 Leu He His Gly Asn Gin He Ala Thr Asn Gly Val Val His Val He 1 5 10 15 GAC CGT GTG CTT ACA CAA ATT GGT ACC TCA ATT CAA GAC TTC ATT GAA 96
Asp Arg Val Leu Thr Gin He Gly Thr Ser He Gin Asp Phe He Glu
20 25 30
GCA GAA GAT GAC CTT TCA TCT TTT AGA GCA GCT GCC ATC ACA TCG GAC 144 Ala Glu Asp Asp Leu Ser Ser Phe Arg Ala Ala Ala He Thr Ser Asp 35 40 45
ATA TTG GAG GCC CTT GGA AGA GAC GGT CAC TTC ACA CTC TTT GCT CCC 192 He Leu Glu Ala Leu Gly Arg Asp Gly His Phe Thr Leu Phe Ala Pro
50 55 60
ACC AAT GAG GCT TTT GAG AAA CTT CCA CGA GGT GTC CTA GAA AGG ATC 240 Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg Gly Val Leu Glu Arg He
65 70 75 80
ATG GGA GAC AAA GTG GCT TCC GAA GCT CTT ATG AAG TAC CAC ATC TTA 288 Met Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys Tyr His He Leu 85 90 95 AAT ACT CTC CAG TGT TCT GAG TCT ATT ATG GGA GGA GCA GTC TTT GAG 336
Asn Thr Leu Gin Cys Ser Glu Ser He Met Gly Gly Ala Val Phe Glu
100 105 110
ACG CTG GAA GGA AAT ACA ATT GAG ATA GGA TGT GAC GGT GAC AGT ATA 384 Thr Leu Glu Gly Asn Thr He Glu He Gly Cys Asp Gly Asp Ser He 115 120 125
ACA GTA AAT GGA ATC AAA ATG GTG AAC AAA AAG GAT ATT GTG ACA AAT 432 Thr Val Asn Gly He Lys Met Val Asn Lys Lys Asp He Val Thr Asn
130 135 140
AAT GGT GTG ATC CAT TTG ATT GAT CAG GTC CTA ATT CCT GAT TCT GCC 480 Asn Gly Val He His Leu He Asp Gin Val Leu He Pro Asp Ser Ala
145 150 155 160
AAA CAA GTT ATT GAG CTG GCT GGA AAA CAG CAA ACC ACC TTC ACG GAT 528 Lys Gin Val He Glu Leu Ala Gly Lys Gin Gin Thr Thr Phe Thr Asp 165 170 175 CTT GTG GCC CAA TTA GGC TTG GCA TCT GCT CTG AGG CCA GAT GGA GAA 576
Leu Val Ala Gin Leu Gly Leu Ala Ser Ala Leu Arg Pro Asp Gly Glu
180 185 190
TAC ACT TTG CTG GCA CCT GTG AAT AAT GCA TTT TCT GAT GAT ACT CTC 624 Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser Asp Asp Thr Leu 195 200 205
AGC ATG GAT CAG 636
Ser Met Asp Gin 210 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Leu He His Gly Asn Gin He Ala Thr Asn Gly Val Val His Val He 1 5 10 15 Asp Arg Val Leu Thr Gin He Gly Thr Ser He Gin Asp Phe He Glu
20 25 30
Ala Glu Asp Asp Leu Ser Ser Phe Arg Ala Ala Ala He Thr Ser Asp
35 40 45
He Leu Glu Ala Leu Gly Arg Asp Gly His Phe Thr Leu Phe Ala Pro 50 55 60
Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg Gly Val Leu Glu Arg He
65 70 75 80
Met Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys Tyr His He Leu
85 90 95 Asn Thr Leu Gin Cys Ser Glu Ser He Met Gly Gly Ala Val Phe Glu
100 105 110
Thr Leu Glu Gly Asn Thr He Glu He Gly Cys Asp Gly Asp Ser He
115 120 125
Thr Val Asn Gly He Lys Met Val Asn Lys Lys Asp He Val Thr Asn 130 135 140
Asn Gly Val He His Leu He Asp Gin Val Leu He Pro Asp Ser Ala
145 150 155 160
Lys Gin Val He Glu Leu Ala Gly Lys Gin Gin Thr Thr Phe Thr Asp
165 170 175 Leu Val Ala Gin Leu Gly Leu Ala Ser Ala Leu Arg Pro Asp Gly Glu
180 185 190
Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser Asp Asp Thr Leu
195 200 205
Ser Met Asp Gin 210
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3126 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 43.-2376 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCCACCATGT AGCCCGCGTC ACCGTTCTGC GCATTCCGCA GC ATG GCT CTG CCT 54
Met Ala Leu Pro 1 GCC CGA ATC CTC GCT CTG GCC CTC GCA CTG GCG CTC GGA CCC GCC GTG 102 Ala Arg He Leu Ala Leu Ala Leu Ala Leu Ala Leu Gly Pro Ala Val
5 10 15 20
ACA CTG GCC AAC CCG GCG AGA ACG CCG TAC GAG CTG GTA CTC CAG AAG 150 Thr Leu Ala Asn Pro Ala Arg Thr Pro Tyr Glu Leu Val Leu Gin Lys 25 30 35 AGC TCG GCA CGA GGG GGT CGG GAC CAA GGC CCA AAT GTC TGT GCC CTT 198
Ser Ser Ala Arg Gly Gly Arg Asp Gin Gly Pro Asn Val Cys Ala Leu
40 45 50
CAA CAG ATT TTG GGC ACC AAA AAG AAA TAC TTC AGC ACT TGT AAG AAC 246 Gin Gin He Leu Gly Thr Lys Lys Lys Tyr Phe Ser Thr Cys Lys Asn 55 60 65
TGG TAT AAA AAG TCC ATC TGT GGA CAG AAA ACG ACT GTG TTA TAT GAA 294 Trp Tyr Lys Lys Ser He Cys Gly Gin Lys Thr Thr Val Leu Tyr Glu
70 75 80
TGT TGC CCT GGT TAT ATG AGA ATG GAA GGA ATG AAA GGC TGC CCA GCA 342 Cys Cys Pro Gly Tyr Met Arg Met Glu Gly Met Lys Gly Cys Pro Ala
85 90 95 100
GTT TTG CCC ATT GAC CAT GTT TAT GGC ACT CTG GGC ATC GTG GGA GCC 390 Val Leu Pro He Asp His Val Tyr Gly Thr Leu Gly He Val Gly Ala 105 110 115 ACC ACA ACG CAG CGC TAT TCT GAC GCC TCA AAA CTG AGG GAG GAG ATC 438
Thr Thr Thr Gin Arg Tyr Ser Asp Ala Ser Lys Leu Arg Glu Glu He
120 125 130
GAG GGA AAG GGA TCC TTC ACT TAC TTT GCA CCG AGT AAT GAG GCT TGG 486 Glu Gly Lys Gly Ser Phe Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp 135 140 145
GAC AAC TTG GAT TCT GAT ATC CGT AGA GGT TTG GAG AGC AAC GTG AAT 534 Asp Asn Leu Asp Ser Asp He Arg Arg Gly Leu Glu Ser Asn Val Asn
150 155 160
GTT GAA TTA CTG AAT GCT TTA CAT AGT CAC ATG ATT AAT AAG AGA ATG 582 Val Glu Leu Leu Asn Ala Leu His Ser His Met He Asn Lys Arg Met
165 170 175 180
TTG ACC AAG GAC TTA AAA AAT GGC ATG ATT ATT CCT TCA ATG TAT AAC 630 Leu Thr Lys Asp Leu Lys Asn Gly Met He He Pro Ser Met Tyr Asn 185 190 195 AAT TTG GGG CTT TTC ATT AAC CAT TAT CCT AAT GGG GTT GTC ACT GTT 678 Asn Leu Gly Leu Phe He Asn His Tyr Pro Asn Gly Val Val Thr Val
200 205 210
AAT TGT GCT CGA ATC ATC CAT GGG AAC CAG ATT GCA ACA AAT GGT GTT 726 Asn Cys Ala Arg He He His Gly Asn Gin He Ala Thr Asn Gly Val
215 220 225
GTC CAT GTC ATT GAC CGT GTG CTT ACA CAA ATT GGT ACC TCA ATT CAA 774 Val His Val He Asp Arg Val Leu Thr Gin He Gly Thr Ser He Gin 230 235 240 GAC TTC ATT GAA GCA GAA GAT GAC CTT TCA TCT TTT AGA GCA GCT GCC 822
Asp Phe He Glu Ala Glu Asp Asp Leu Ser Ser Phe Arg Ala Ala Ala 245 250 255 260
ATC ACA TCG GAC ATA TTG GAG GCC CTT GGA AGA GAC GGT CAC TTC ACA 870 He Thr Ser Asp He Leu Glu Ala Leu Gly Arg Asp Gly His Phe Thr 265 270 275
CTC TTT GCT CCC ACC AAT GAG GCT TTT GAG AAA CTT CCA CGA GGT GTC 918 Leu Phe Ala Pro Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg Gly Val
280 285 290
CTA GAA AGG ATC ATG GGA GAC AAA GTG GCT TCC GAA GCT CTT ATG AAG 966 Leu Glu Arg He Met Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys
295 300 305
TAC CAC ATC TTA AAT ACT CTC CAG TGT TCT GAG TCT ATT ATG GGA GGA 1014 Tyr His He Leu Asn Thr Leu Gin Cys Ser Glu Ser He Met Gly Gly 310 315 320 GCA GTC TTT GAG ACG CTG GAA GGA AAT ACA ATT GAG ATA GGA TGT GAC 1062
Ala Val Phe Glu Thr Leu Glu Gly Asn Thr He Glu He Gly Cys Asp 325 330 335 340
GGT GAC AGT ATA ACA GTA AAT GGA ATC AAA ATG GTG AAC AAA AAG GAT 1110 Gly Asp Ser He Thr Val Asn Gly He Lys Met Val Asn Lys Lys Asp 345 350 355
ATT GTG ACA AAT AAT GGT GTG ATC CAT TTG ATT GAT CAG GTC CTA ATT 1158 He Val Thr Asn Asn Gly Val He His Leu He Asp Gin Val Leu He
360 365 370
CCT GAT TCT GCC AAA CAA GTT ATT GAG CTG GCT GGA AAA CAG CAA ACC 1206 Pro Asp Ser Ala Lys Gin Val He Glu Leu Ala Gly Lys Gin Gin Thr
375 380 385
ACC TTC ACG GAT CTT GTG GCC CAA TTA GGC TTG GCA TCT GCT CTG AGG 1254 Thr Phe Thr Asp Leu Val Ala Gin Leu Gly Leu Ala Ser Ala Leu Arg 390 395 400 CCA GAT GGA GAA TAC ACT TTG CTG GCA CCT GTG AAT AAT GCA TTT TCT 1302
Pro Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser 405 410 415 420 GAT GAT ACT CTC AGC ATG GAT CAG CGC CTC CTT AAA TTA ATT CTG CAG 1350 Asp Asp Thr Leu Ser Met Asp Gin Arg Leu Leu Lys Leu He Leu Gin
425 430 435
AAT CAC ATA TTG AAA GTA AAA GTT GGC CTT AAT GAG CTT TAC AAC GGG 1398 Asn His He Leu Lys Val Lys Val Gly Leu Asn Glu Leu Tyr Asn Gly
440 445 450
CAA ATA CTG GAA ACC ATC GGA GGC AAA CAG CTC AGA GTC TTC GTA TAT 1446 Gin He Leu Glu Thr He Gly Gly Lys Gin Leu Arg Val Phe Val Tyr 455 460 465 CGT ACA GCT GTC TGC ATT GAA AAT TCA TGC ATG GAG AAA GGG AGT AAG 1494
Arg Thr Ala Val Cys He Glu Asn Ser Cys Met Glu Lys Gly Ser Lys
470 475 480
CAA GGG AGA AAC GGT GCG ATT CAC ATA TTC CGC GAG ATC ATC AAG CCA 1542 Gin Gly Arg Asn Gly Ala He His He Phe Arg Glu He He Lys Pro 485 490 495 500
GCA GAG AAA TCC CTC CAT GAA AAG TTA AAA CAA GAT AAG CGC TTT ACG 1590 Ala Glu Lys Ser Leu His Glu Lys Leu Lys Gin Asp Lys Arg Phe Thr
505 510 515
ACC TTC CTC AGC CTA CTT GAA GCT GCA GAC TTG AAA GAG CTC CTG ACA 1638 Thr Phe Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys Glu Leu Leu Thr
520 525 530
CAA CCT GGA GAC TGG ACA TTA TTT GTG CCA ACC AAT GAT GCT TTT AAG 1686 Gin Pro Gly Asp Trp Thr Leu Phe Val Pro Thr Asn Asp Ala Phe Lys 535 540 545 GGA ATG ACT AGT GAA GAA AAA GAA ATT CTG ATA CGG GAC AAA AAT GCT 1734
Gly Met Thr Ser Glu Glu Lys Glu He Leu He Arg Asp Lys Asn Ala
550 555 560
CTT CAA AAC ATC ATT CTT TAT CAC CTG ACA CCA GGA GTT TTC ATT GGA 1782 Leu Gin Asn He He Leu Tyr His Leu Thr Pro Gly Val Phe He Gly 565 570 575 580
AAA GGA TTT GAA CCT GGT GTT ACT AAC ATT TTA AAG ACC ACA CAA GGA 1830 Lys Gly Phe Glu Pro Gly Val Thr Asn He Leu Lys Thr Thr Gin Gly
585 590 595
AGC AAA ATC TTT CTG AAA GAA GTA AAT GAT ACA CTT CTG GTG AAT GAA 1878 Ser Lys He Phe Leu Lys Glu Val Asn Asp Thr Leu Leu Val Asn Glu
600 605 610
TTG AAA TCA AAA GAA TCT GAC ATC ATG ACA ACA AAT GGT GTA ATT CAT 1926 Leu Lys Ser Lys Glu Ser Asp He Met Thr Thr Asn Gly Val He His 615 620 625 GTT GTA GAT AAA CTC CTC TAT CCA GCA GAC ACA CCT GTT GGA AAT GAT 1974
Val Val Asp Lys Leu Leu Tyr Pro Ala Asp Thr Pro Val Gly Asn Asp 630 635 640 CAA CTG CTG GAA ATA CTT AAT AAA TTA ATC AAA TAC ATC CAA ATT AAG 2022 Gin Leu Leu Glu He Leu Asn Lys Leu He Lys Tyr He Gin He Lys 645 650 655 660
TTT GTT CGT GGT AGC ACC TTC AAA GAA ATC CCC GTG ACT GTC TAT AGA 2070 Phe Val Arg Gly Ser Thr Phe Lys Glu He Pro Val Thr Val Tyr Arg
665 670 675
CCC ACA CTA ACA AAA GTC AAA ATT GAA GGT GAA CCT GAA TTC AGA CTG 2118 Pro Thr Leu Thr Lys Val Lys He Glu Gly Glu Pro Glu Phe Arg Leu 680 685 690 ATT AAA GAA GGT GAA ACA ATA ACT GAA GTG ATC CAT GGA GAG CCA ATT 2166
He Lys Glu Gly Glu Thr He Thr Glu Val He His Gly Glu Pro He
695 700 705
ATT AAA AAA TAC ACC AAA ATC ATT GAT GGA GTG CCT GTG GAA ATA ACT 2214 He Lys Lys Tyr Thr Lys He He Asp Gly Val Pro Val Glu He Thr 710 715 720
GAA AAA GAG ACA CGA GAA GAA CGA ATC ATT ACA GGT CCT GAA ATA AAA 2262 Glu Lys Glu Thr Arg Glu Glu Arg He He Thr Gly Pro Glu He Lys 725 730 735 740
TAC ACT AGG ATT TCT ACT GGA GGT GGA GAA ACA GAA GAA ACT CTG AAG 2310 Tyr Thr Arg He Ser Thr Gly Gly Gly Glu Thr Glu Glu Thr Leu Lys
745 750 755
AAA TTG TTA CAA GAA GAC ACA CCC GTG AGG AAG TTG CAA GCC AAC AAA 2358 Lys Leu Leu Gin Glu Asp Thr Pro Val Arg Lys Leu Gin Ala Asn Lys 760 765 770 AAA AGT TCA AGG ATC TAGAAGACGA TTAAGGGAAG GTCGTTCTCA GTGAAAATCC 2413
Lys Ser Ser Arg He
775 AAAAACCAGA AAAAAATGTT TATACAACCC TAAGTCAATA ACCTGACCTT AGAAAATTGT 2473
GAGAGCCAAG TTGACTTCAG GAACTGAAAC ATCAGCACAA AGAAGCAATC ATCAAATAAT 2533 TCTGAACACA AATTTAATAT TTTTTTTTCT GAATGAGAAA CATGAGGGAA ATTGTGGAGT 2593
TAGCCTCCTG TGGTAAAGGA ATTGAAGAAA ATATAACACC TTACACCCTT TTTCATCTTG 2653
ACATTAAAAG TTCTGGCTAA CTTTGGAATC CATTAGAGAA AAATCCTTGT CACCAGATTC 2713
ATTACAATTC AAATCGAAGA GTTGTGAACT GTTATCCCAT TGAAAAGACC GAGCCTTGTA 2773
TGTATGTTAT GGATACATAA AATGCACGCA AGCCATTATC TCTCCATGGG AAGCTAAGTT 2833 ATAAAAATAG GTGCTTGGTG TACAAAACTT TTTATGATCA AAAGGCTTTG CACATTTCTA 2893
TATGAGTGGG TTTACTGGTA AATTATGTTA TTTTTTACAA CTAATTTTGT ACTCTCAGAA 2953
TGTTTGTCAT ATGCTTCTTG CAATGCATAT TTTTTAATCT CAAACGTTTC AATAAAACCA 3013
TTTTTCAGAT ATAAAGAGAA TTACTTCAAA TTGAGTAATT CAGAAAAACT CAAGATTTAA 3073
GTTAAAAAGT GGTTTGGACT TGGGAATAGG ACTTTATACC TCTTTCTCGT GCC 3126
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 777 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ala Leu Pro Ala Arg He Leu Ala Leu Ala Leu Ala Leu Ala Leu
1 5 10 15
Gly Pro Ala Val Thr Leu Ala Asn Pro Ala Arg Thr Pro Tyr Glu Leu
20 25 30
Val Leu Gin Lys Ser Ser Ala Arg Gly Gly Arg Asp Gin Gly Pro Asn 35 40 45
Val Cys Ala Leu Gin Gin He Leu Gly Thr Lys Lys Lys Tyr Phe Ser
50 55 60
Thr Cys Lys Asn Trp Tyr Lys Lys Ser He Cys Gly Gin Lys Thr Thr 65 70 75 80 Val Leu Tyr Glu Cys Cys Pro Gly Tyr Met Arg Met Glu Gly Met Lys
85 90 95
Gly Cys Pro Ala Val Leu Pro He Asp His Val Tyr Gly Thr Leu Gly
100 105 110
He Val Gly Ala Thr Thr Thr Gin Arg Tyr Ser Asp Ala Ser Lys Leu 115 120 125
Arg Glu Glu He Glu Gly Lys Gly Ser Phe Thr Tyr Phe Ala Pro Ser
130 135 140
Asn Glu Ala Trp Asp Asn Leu Asp Ser Asp He Arg Arg Gly Leu Glu 145 150 155 160 Ser Asn Val Asn Val Glu Leu Leu Asn Ala Leu His Ser His Met He
165 170 175
Asn Lys Arg Met Leu Thr Lys Asp Leu Lys Asn Gly Met He He Pro
180 185 190
Ser Met Tyr Asn Asn Leu Gly Leu Phe He Asn His Tyr Pro Asn Gly 195 200 205
Val Val Thr Val Asn Cys Ala Arg He He His Gly Asn Gin He Ala
210 215 220
Thr Asn Gly Val Val His Val He Asp Arg Val Leu Thr Gin He Gly 225 230 235 240 Thr Ser He Gin Asp Phe He Glu Ala Glu Asp Asp Leu Ser Ser Phe
245 250 255
Arg Ala Ala Ala He Thr Ser Asp He Leu Glu Ala Leu Gly Arg Asp
260 265 270
Gly His Phe Thr Leu Phe Ala Pro Thr Asn Glu Ala Phe Glu Lys Leu 275 280 285
Pro Arg Gly Val Leu Glu Arg He Met Gly Asp Lys Val Ala Ser Glu
290 295 300
Ala Leu Met Lys Tyr His He Leu Asn Thr Leu Gin Cys Ser Glu Ser 305 310 315 320 He Met Gly Gly Ala Val Phe Glu Thr Leu Glu Gly Asn Thr He Glu
325 330 335
He Gly Cys Asp Gly Asp Ser He Thr Val Asn Gly He Lys Met Val 340 345 350 Asn Lys Lys Asp He Val Thr Asn Asn Gly Val He His Leu He Asp
355 360 365
Gin Val Leu He Pro Asp Ser Ala Lys Gin Val He Glu Leu Ala Gly
370 375 380
Lys Gin Gin Thr Thr Phe Thr Asp Leu Val Ala Gin Leu Gly Leu Ala 385 390 395 400
Ser Ala Leu Arg Pro Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn
405 410 415
Asn Ala Phe Ser Asp Asp Thr Leu Ser Met Asp Gin Arg Leu Leu Lys 420 425 430 Leu He Leu Gin Asn His He Leu Lys Val Lys Val Gly Leu Asn Glu
435 440 445
Leu Tyr Asn Gly Gin He Leu Glu Thr He Gly Gly Lys Gin Leu Arg
450 455 460
Val Phe Val Tyr Arg Thr Ala Val Cys He Glu Asn Ser Cys Met Glu 465 470 475 480
Lys Gly Ser Lys Gin Gly Arg Asn Gly Ala He His He Phe Arg Glu
485 490 495
He He Lys Pro Ala Glu Lys Ser Leu His Glu Lys Leu Lys Gin Asp 500 505 510 Lys Arg Phe Thr Thr Phe Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys
515 520 525
Glu Leu Leu Thr Gin Pro Gly Asp Trp Thr Leu Phe Val Pro Thr Asn
530 535 540
Asp Ala Phe Lys Gly Met Thr Ser Glu Glu Lys Glu He Leu He Arg 545 550 555 560
Asp Lys Asn Ala Leu Gin Asn He He Leu Tyr His Leu Thr Pro Gly
565 570 575
Val Phe He Gly Lys Gly Phe Glu Pro Gly Val Thr Asn He Leu Lys 580 585 590 Thr Thr Gin Gly Ser Lys He Phe Leu Lys Glu Val Asn Asp Thr Leu
595 600 605
Leu Val Asn Glu Leu Lys Ser Lys Glu Ser Asp He Met Thr Thr Asn
610 615 620
Gly Val He His Val Val Asp Lys Leu Leu Tyr Pro Ala Asp Thr Pro 625 630 635 640
Val Gly Asn Asp Gin Leu Leu Glu He Leu Asn Lys Leu He Lys Tyr
645 650 655
He Gin He Lys Phe Val Arg Gly Ser Thr Phe Lys Glu He Pro Val 660 665 670 Thr Val Tyr Arg Pro Thr Leu Thr Lys Val Lys He Glu Gly Glu Pro
675 680 685
Glu Phe Arg Leu He Lys Glu Gly Glu Thr He Thr Glu Val He His
690 695 700 Gly Glu Pro He He Lys Lys Tyr Thr Lys He He Asp Gly Val Pro
705 710 715 720
Val Glu He Thr Glu Lys Glu Thr Arg Glu Glu Arg He He Thr Gly
725 730 735
Pro Glu He Lys Tyr Thr Arg He Ser Thr Gly Gly Gly Glu Thr Glu 740 745 750
Glu Thr Leu Lys Lys Leu Leu Gin Glu Asp Thr Pro Val Arg Lys Leu
755 760 765
Gin Ala Asn Lys Lys Ser Ser Arg He 770 775
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TGTCCAGATG
10
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TGTCCAGATG C 11
(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGTCCAGATG AC 12
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGTCCAGATA
10
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TGTCCAGACG 10
(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TGTCCAGCCG 10
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TGTCCCGCCG
10
(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TGCCCGGCCG 10
(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TGATGCACTC 10 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TGAGCTACTC 10
(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TGACTGACTC 10
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTGATCCATG
10
(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 683 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Ala Leu Phe Val Arg Leu Leu Ala Leu Ala Leu Ala Leu Ala Leu 1 5 10 15
Gly Pro Ala Ala Thr Leu Ala Gly Pro Ala Lys Ser Pro Tyr Gin Leu 20 25 30
Pro Leu Gin His Ser Arg Leu Arg Gly Arg Gin His Gly Pro Asn Val
35 40 45
Cys Ala Val Thr Lys Val He Gly Thr Asn Arg Lys Tyr Phe Thr Asn 50 55 60 Cys Lys Gin Trp Tyr Gin Arg Lys He Cys Gly Lys Ser Thr Val He
65 70 75 80
Ser Tyr Glu Cys Cys Pro Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly
85 90 95
Cys Pro Ala Ala Leu Pro Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val 100 105 110
Val Gly Ser Thr Thr Thr Gin Leu Tyr Thr Asp Arg Thr Glu Lys Leu
115 120 125
Arg Pro Glu Met Glu Gly Pro Gly Ser Phe Thr He Phe Ala Pro Ser 130 135 140 Asn Glu Ala Trp Ala Ser Leu Pro Ala Glu Val Leu Val Ser Leu Val
145 150 155 160
Ser Asn Val Asn He Glu Leu Leu Asn Ala Leu Arg Tyr His Met Val
165 170 175
Gly Arg Arg Val Leu Thr Asp Glu Leu Lys His Gly Met Thr Leu Thr 180 185 190
Ser Met Tyr Gin Asn Ser Asn He Gin He His His Tyr Pro Asn Gly
195 200 205
He Val Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His Ala 210 215 220 Thr Asn Gly Val Val His Leu He Asp Lys Val He Ser Thr He Thr
225 230 235 240
Asn Asn He Gin Gin He He Glu He Glu Asp Thr Phe Glu Thr Leu
245 250 255
Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr Met Leu Glu Gly Asn 260 265 270
Gly Gin Tyr Thr Leu Leu Ala Pro Thr Asn Glu Ala Phe Glu Lys He
275 280 285
Pro Ser Glu Thr Leu Asn Arg He Leu Gly Asp Pro Glu Ala Leu Arg 290 295 300 Asp Leu Leu Asn Asn His He Leu Lys Ser Ala Met Cys Ala Glu Ala
305 310 315 320
He Val Ala Gly Leu Ser Val Glu Thr Leu Glu Gly Thr Thr Leu Glu
325 330 335 Val Gly Cys Ser Gly Asp Met Leu Thr He Asn Gly Lys Ala He He
340 345 350
Ser Asn Lys Asp He Leu Ala Thr Asn Gly Val He His Tyr He Asp
355 360 365
Glu Leu Leu He Pro Asp Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala 370 375 380
Glu Ser Asp Val Ser Thr Ala He Asp Leu Phe Arg Gin Ala Gly Leu
385 390 395 400
Gly Asn His Leu Ser Gly Ser Glu Arg Leu Thr Leu Leu Ala Pro Leu
405 410 415 Asn Ser Val Phe Lys Asp Gly Thr Pro Pro He Asp Ala His Thr Arg
420 425 430
Asn Leu Leu Arg Asn His He He Lys Asp Gin Leu Ala Ser Lys Tyr
435 440 445
Leu Tyr His Gly Gin Thr Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg 450 455 460
Val Phe Val Tyr Arg Asn Ser Leu Cys He Glu Asn Ser Cys He Ala
465 470 475 480
Ala His Asp Lys Arg Gly Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg
485 490 495 Val Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp
500 505 510
Asn Arg Phe Ser Met Leu Val Ala Ala He Gin Ser Ala Gly Leu Thr
515 520 525
Glu Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn 530 535 540
Glu Ala Phe Arg Ala Leu Pro Pro Arg Glu Ser Arg Arg Leu Leu Gly
545 550 555 560
Asp Ala Lys Glu Leu Ala Asn He Leu Lys Tyr His He Gly Asp Glu
565 570 575 He Leu Val Ser Gly Gly He Gly Ala Leu Val Arg Leu Lys Ser Leu
580 585 590
Gin Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val
595 600 605
Asn Lys Glu Pro Val Ala Glu Pro Asp He Met Ala Thr Asn Gly Val 610 615 620
Val His Val He Thr Asn Val Leu Gin Pro Pro Ala Asn Arg Pro Gin 625 630 635 640
Glu Arg Gly Asp Glu Leu Ala Asp Ser Ala Leu Glu He Phe Lys Gin 645 650 655 Ala Ser Ala Phe Ser Arg Ala Ser Gin Arg Ser Val Arg Leu Ala Val
660 665 670
Pro Tyr Gin Lys Leu Leu Glu Arg Met Lys His 675 680
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Gly Glu Lys Ser Leu Glu Tyr Lys He Arg Asp Asp Pro Asp Leu Ser 1 5 10 15
Gin Phe Tyr Ser Trp Leu Glu His Asn Glu Val Ala Asn Ser Thr Leu 20 25 30 Gin Leu Arg Gin Val Thr Val Phe Ala Pro Thr Asn Leu Ala Gin Phe
35 40 45
Asn Tyr Lys Ala Arg Asp Gly Asp Glu Asn He He Leu Tyr His Met
50 55 60
Thr Asn Leu Ala His Ser Leu Asp Gin Leu Gly His Lys Val Asn Ser 65 70 75 80
Glu Leu Asp Gly Asn Pro Pro Leu Trp He Thr Arg Arg Arg Asp Thr
85 90 95
He Phe Val Asn Asn Ala Arg Val Leu Thr Glu Arg Ser Asn Tyr Glu 100 105 110 Ala Val Asn Arg His Gly Lys Lys Gin Val Leu His Val Val Asp Ser
115 120 125
Val Leu Glu Pro Val Trp Ser Thr Ser Gly Gin Leu Tyr Asn Pro Asp
130 135 140
Ala Phe Gin Phe Leu Asn Gin Ser Glu Asn Leu Asp Leu Gly Leu His 145 150 155 160
Arg Val Arg Ser Phe Arg Gin Arg Val Phe Gin Asn Gin Lys Gin Asn
165 170 175
Asp Phe Lys Leu Glu Gly Lys His Thr Phe Phe He Pro Val Asp Glu 180 185 190 Gly Phe Lys Pro Leu Pro Arg Pro Glu Lys He Asp Gin Lys
195 200 205 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ala Ala Ala Asp Leu Ala Asp Lys Leu Arg Asp Asp Ser Glu Leu Ser 1 5 10 15
Gin Phe Tyr Ser Leu Leu Glu Ser Asn Gin He Ala Asn Ser Thr Leu 20 25 30
Ser Leu Arg Ser Cys Thr He Phe Val Pro Thr Asn Glu Ala Phe Gin
35 40 45
Arg Tyr Lys Ser Lys Thr Ala His Val Leu Tyr His He Thr Thr Glu 50 55 60 Ala Tyr Thr Gin Lys Arg Leu Pro Asn Thr Val Ser Ser Asp Met Ala
65 70 75 80
Gly Asn Pro Pro Leu Tyr He Thr Lys Asn Ser Asn Gly Asp He Phe
85 90 95
Val Gly Asn Ala Arg He He Pro Ser Leu Ser Val Glu Thr Asn Ser 100 105 110
Asp Gly Lys Arg Gin He Met His He He Asp Glu Val Leu Glu Pro
115 120 125
Leu Thr Val Lys Ala Gly His Ser Asp Thr Pro Asn Asn Pro Asn Ala 130 135 140 Leu Lys Phe Leu Lys Asn Ala Glu Glu Phe Asn Val Asp Asn He Gly
145 150 155 160
Val Arg Thr Tyr Arg Ser Gin Val Thr Met Ala Lys Lys Glu Ser Val
165 170 175
Tyr Asp Ala Ala Gly Gin His Thr Phe Leu Val Pro Val Asp Glu Gly 180 185 190
Phe Lys Leu Ser Ala Arg Ser Ser 195 200

Claims

CLAIMS What is claimed is:
1. A monoclonal antibody that binds to an epitope of TCI in formalin- fixed or paraffin-embedded tissues.
2. A monoclonal antibody produced by the hybridoma cell line ATCC No.
HB 11481, or a monoclonal antibody that binds to the same antigenic determinant as a monoclonal antibody produced by the hybridoma cell line ATCC No. HB 11481.
3. A method of screening for agents that inhibit expression of the TCI gene in vitro, comprising exposing a metastatic cell line in which TCI mRNA is detectable in culture to an agent suspected of inhibiting production of said TCI mRNA; and determining the level of TCI mRNA in said exposed cell line, wherein a decrease in the level of TCI mRNA after exposure of said cell line to said agent is indicative of inhibition of said TCI mRNA production.
4. A method of screening for agents that inhibit expression of the TCI protein in vitro, comprising exposing a metastatic cell line in which TCI protein is detectable in culture to an agent suspected of inhibiting production of said TCI protein; and determining the level of TCI protein in said exposed cell line, wherein a decrease in the level of TCI protein after exposure of said cell line to said agent is indicative of inhibition of said TCI protein production.
5. The method of claim 3, said cell line comprising JMNIB.
6. The method of claim 4, said cell line comprising JMNIB.
7. A method of screening for agents that inhibit expression of the TCI gene in vivo, comprising exposing a mammal having tumor cells in which TCI mRNA is detectable to an agent suspected of inhibiting production of said TCI mRNA; and determining the level of TCI mRNA in tumor cells of said exposed mammal, wherein a decrease in the level of TCI mRNA after exposure of said mammal to said agent is indicative of inhibition of said TCI mRNA production.
8. A method of screening for agents that inhibit production of TCI protein in vivo, comprising exposing a mammal having tumor cells in which TCI protein is detectable to an agent suspected of inhibiting production of said TCI protein; and determining the level of TCI protein in tumor cells of said exposed mammal, wherein a decrease in the level of TCI protein after exposure of said mammal to said agent is indicative of inhibition of said TCI protein production.
9. The method of claim 7, wherein said tumor cells are breast tumor cells, colon tumor cells, or tumor cells of the gastrointestinal tract.
10. The method of claim 8, wherein said tumor cells are breast tumor cells, colon tumor cells, or tumor cells of the gastrointestinal tract.
11. A pharmaceutical composition for use in treating a late stage cancer comprising an effective amount of an inhibitor of TCI.
12. The pharmaceutical composition of claim 11 wherein said late stage cancer is one of breast cancer, colon cancer, or gastrointestinal cancer.
13. A pharmaceutical composition for use in preventing tumor cell metastasis comprising an effective amount of an inhibitor of TCI.
14. A method for detecting a tumor in a subject, comprising detecting the presence of tumor marker protein TCI in a sample of body fluid from said subject.
15. A method for detecting a in a subject comprising the steps of: providing a sample of body fluid from said subject; contacting said sample with a monoclonal antibody specific for an epitope of tumor marker protein TCI; and detecting the presence of TCI protein in said sample, wherein the presence of TCI protein in said sample is indicative of the presence of a tumor in said subject.
16. The method of claim 14 or 15, wherein said body fluid is selected from the group consisting of blood, urine and sputum.
17. A method for detecting a tumor in a subject, comprising detecting the presence of tumor marker protein TCI, or of mRNA encoding tumor marker protein TCI, in a sample of a tissue section from said subject.
18. A method for detecting an invasive or metastatic tumor comprising the steps of: providing a sample of a formalin-fixed or paraffin-embedded tissue section from said subject; contacting said sample with a monoclonal antibody specific for an epitope of tumor marker protein TCI in formalin-fixed or paraffin- embedded tissue sections; and determining the level of TCI protein in said sample, wherein the level of TCI protein in said sample is related to the presence of an invasive or metastatic tumor in said subject.
19. The method of claim 17 or 18, wherein said tissue is breast, colon, or gastrointestinal tract tissue.
20. The method of claim 18, wherein said monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line ATCC No. HB 11481.
21. The method of claim 18, wherein said monoclonal antibody is a monoclonal antibody that binds to the same antigenic determinant as a monoclonal antibody produced by the hybridoma cell line ATCC No. HB 11481.
22. A kit for diagnosis of an invasive or metastatic tumor in a subject, comprising the monoclonal antibody of claim 1 or claim 2.
PCT/US1994/012502 1993-10-29 1994-10-31 A novel tumor marker and novel method of isolating same WO1995011923A1 (en)

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EP94932133A EP0725799A1 (en) 1993-10-29 1994-10-31 A novel tumor marker and novel method of isolating same
US08/448,388 US5889159A (en) 1993-10-29 1994-10-31 Monoclonal antibodies to tumor marker TC1 and methods for use of same

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EP (1) EP0725799A1 (en)
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