US20060052584A1 - Human-derived bradeion proteins, DNA coding for the proteins, and uses thereof - Google Patents

Human-derived bradeion proteins, DNA coding for the proteins, and uses thereof Download PDF

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US20060052584A1
US20060052584A1 US11/224,297 US22429705A US2006052584A1 US 20060052584 A1 US20060052584 A1 US 20060052584A1 US 22429705 A US22429705 A US 22429705A US 2006052584 A1 US2006052584 A1 US 2006052584A1
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bradeion
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Manami Tanaka
Tomoo Tanaka
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National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Cranial nerve cells are main elements for controlling survival of higher order animals. Once the neurons are produced, they do not divide at all and only gradually exfoliate or go through necrosis. Exfoliation of the neurons occurs in the normal state but is particularly accelerated by genetic diseases, brain ischemia, or status epilepticus, or under conditions of poor nutrition and low oxygen. Some disorders of cranial nerves associated with aging (e.g., dementia) result from deficiency of an absolute amount of functional neurons caused by accumulation of exfoliated neurons. Thus, the monitoring and control of the exfoliation, as well as regeneration of the functions of neurons, are the most demanding subject to be solved among the aging problems.
  • Cranial nerve cells do not divide at all after the induction phase of differentiation in the process of development, and maintain their functions or is accompanied by gradual deterioration of their functions until the end of the life-time of the individual. They are presumed to have specific division-interrupting and function-maintaining mechanisms although these mechanisms have not yet been clarified.
  • In the central nervous system there exist numbers of unknown proteins and signaling substances, particularly stimulating substances and receptors thereof involved in brain-specific signal transduction, but their details are yet unclear.
  • one object of the present invention is to provide a bradeion protein involved in long-term survival of cranial nerve cells, and DNA coding for the bradeion protein.
  • Another object of the present invention is to provide a vector containing the above-mentioned DNA, and a host cell transformed or transfected with the vector.
  • Still another object of the present invention is to use the DNA or an antibody to the protein for detecting cancers.
  • the present invention provides a human-derived bradeion protein, which has the following properties:
  • analogue refers to a protein that has properties substantially equivalent to those of the human-derived bradeion proteins (for example, at least the properties of (i), (ii), (iii), (vi) and (vii)), or a protein having an amino acid sequence with deletions, substitutions or additions of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO:2 or 4, or a protein having. an amino acid sequence that is substantially the same as that shown in SEQ. ID NO:2 or 4.
  • the analogue of the invention has at least 90%, preferably at least 95%, more preferably at least 97% homology with the amino acid sequence of SEQ ID NO:2 or 4.
  • the present invention provides DNAs comprising nucleotide sequences coding for the above-defined bradeion proteins or analogues thereof, and fragments of the DNAs.
  • cDNA coding for bradeion proteins of the invention may be obtained as follows.
  • Bradeion ⁇ and Bradeion ⁇ proteins were found to have a structure characteristic of an interleukin receptor even in a structure of its transmembranous portion, when subjected to the hydropathy analysis according to Kyte-Doolittle method (J. Mol. Biol., 157 (1): 105-132, 1982). Thus, it has the structure of a transmembrane-type receptor that is presumably involved in the intracellular signaling mechanism.
  • Bradeion ⁇ and Bradeion ⁇ proteins are also similar to the relationship of the tumor suppression genes p53/p73 in that there are two types of expression modes, i.e., ⁇ - and ⁇ -types, and in that either of them is prevalent in various organisms.
  • the cloned or amplified DNA of interest is collected and introduced into an available suitable expression vector.
  • the obtained vector is used to transform a suitable host cell, which is thereafter cultured in a proper medium for expression of the DNA, to isolate and purify the protein of interest.
  • the amino acid sequence of the analogue may include a modification chosen from deletion, substitution or addition of at least one amino acid as long as the analogue has properties substantially equivalent to those of the human Bradeion ⁇ or Bradeion ⁇ .
  • the mutation may be introduced into the amino acid sequence shown in SEQ ID NO:2 or 4 through genetic engineering, for example, by well-known techniques such as site-directed mutagenesis/PCR method (S. N. Ho et al., Gene, 77, 51, 1989); and methods described in Kaoru Saigo and Yumiko Sano (trans.), Current Protocols compact version, Experimental Protocols in Molecular Biology I, June 1997, Maruzen, Tokyo, Japan).
  • substitutions include substitutions between hydrophobic amino acids (Ala, Val, Leu, Ile, etc.), Ser and Thr, Asp and Glu, Asn and Gln, Lys and Arg, and Phe and Tyr.
  • addition include an addition of Met to the N-terminus of a mature protein as seen in products expressed in a bacterial host, and an addition of His-tag, or Met-Lys or Met-Arg sequence to the N-terminus for facilitating induction of a mature protein. It is also possible to truncate some amino acid residues at the carboxyl or amino terminus of the bradeion protein to an extent where bioactivity is not impaired.
  • Proteins without sugar chains are produced when a prokaryotic cell such as bacterium is used as the host cell for expressing DNAs coding for the bradeion proteins or analogues thereof through genetic engineering.
  • products with sugar chains may be produced when the DNAs are expressed in an eurokaryotic cell such as fungus, yeast, insect, plant, or mammalian cell.
  • sugar chains may be formed by introducing an N-binding sugar chain site (generally, Asn-Xaa-Thr/Ser where Xaa is any amino acid other than Pro) into the sequence.
  • the obtained analogue should have bioactivity substantially equivalent to the human bradeions.
  • the present invention further provides an expression vector containing the above-described DNA, and a host cell transformed or transfected with the vector.
  • the vector of the invention may be in the form of, for example, plasmid, phage, or virus.
  • Other types of vectors may also be used as long as they are replicable in a host cell.
  • bacterium plasmids e.g., pBR322, pKC30, pCFM536, etc.
  • phage DNAs e.g., ⁇ phage, etc.
  • yeast plasmids e.g., pG-1, etc.
  • viral DNAs for mammal host cells e.g., baculovirus, vaccinia virus, adenovirus, SV40 and its derivatives, etc.
  • the vector usually contains a replication origin, a selective marker, a promoter, and, if necessary, may contain an enhancer, a transcription termination sequence (terminator), a ribosome-binding site, a polyadenylation signal, etc.
  • the promoter is a regulator sequence for directing a synthesis of mRNA coding for the DNA of the invention.
  • Representative examples of the promoter include adenovirus or SV40 promoter, E.coli lac or trp promoter, phage ⁇ P L promoter, ADH, PHO5, GPD, PGK or AOX1 promoter (for yeast), and a promoter derived from nuclear polyhedrosis virus (for Bombyx mori cell).
  • the selective marker is a gene for providing a phenotype to the host in order to select transformed host cells.
  • exemplary selective markers include kanamycin-resistant gene, ampicillin-resistant gene, tetracycline-resistant gene, and the like (when the vector is used for E.coli ); Leu2, Trp1, Ura3 genes, and the like (when the vector is used for yeasts); and neomycin-resistant gene, thymidine kynase gene, dihydrofolate reductase gene, and the like (when the vector is used for mammalian cells).
  • the human-derived bradeion or an analogue thereof of the invention can be obtained by culturing the host cells which have been transformed or transfected as described above under the control of the promoter, and by collecting the produced protein of interest.
  • the host cell is amplified or grown to a proper cell density.
  • the promoter is induced by shifting the temperature or by chemical induction (with IPTG, etc.).
  • the cell is further cultured for a predetermined period.
  • the protein of interest is secreted extracellularly, it can directly be obtained from the medium.
  • the cell can be disrupted by physical means (e.g., sonication or mechanical disruption) or by chemical means (e.g., lyzozyme or cytolytic agent).
  • Such antibodies may be produced, for example, by methods described in Suguru Matsubasi et al., Biochemical Experimental Methods 15, Introduction to Immunological Experiments, 1982, Japan Scientific Societies Press, Tokyo, Japan; Tatsuo Iwasaki et al., Monoclonal antibody -Hybridoma and ELISA, 1987, Kodansha Scientific, Tokyo, Japan.
  • Humanized monoclonal antibody may also be prepared by using known techniques as described in Teng et al., Proc. Nal. Acad. Sci. USA, 1983, 80:7308-7312, and Kozbor et al., Immunology Today, 1983, 4 (3): 72-79.
  • 5′-terminal primers (described below) were synthesized to systematically screen relevant genes.
  • Gene Trapper Positive Selection system (Life Technologies, Inc.) was used to screen the above-described gene library with the synthesized oligonucleotides and magnet beads.
  • the sequences of the oligonucleotides used were:

Abstract

A human-derived bradeion protein, which has the following properties: (i) it is a transmembranous protein; (ii) it has a structure characteristic of growth hormone and cytokine receptors even in a structure of its transmembranous portion when its structure is determined by a hydrophobicity analysis according to Kyte-Doolittle method; (iii) it is expressed at a high level in a human adult brain, and in less amount in the heart, while it is not expressed in other adult organs or fetus; (iv) it induces programmed cell death (apoptosis) when over-expressed in a cultured human nerve cell lines; (v) it induces termination of cell division and aging when over-expressed in a cultured human normal cell; (vi) it is located in cytoplasm, and forms an intracellular aggregate when overexpressed; and (vii) besides human adult neurons, it is specifically expressed in a human colorectal cancer cell line or in a skin cancer cell line, or an analogue thereof.

Description

    FIELD OF THE INVENTION
  • The present invention relates to a protein involved in long-term survival of cranial nerve-cell, to DNA encoding the protein, and to uses thereof. More particularly, the present invention relates to human-derived bradeion protein or derivatives thereof, to DNA encoding the protein or the derivatives thereof, to a vector containing the DNA, to a host cell transformed or transfected with the vector, to an antibody immunologically reactive with the protein or the derivatives thereof, and to uses of the DNA or the antibody for detecting a cancer.
    • BACKGROUND OF THE INVENTION
  • Cranial nerve cells (neurons) are main elements for controlling survival of higher order animals. Once the neurons are produced, they do not divide at all and only gradually exfoliate or go through necrosis. Exfoliation of the neurons occurs in the normal state but is particularly accelerated by genetic diseases, brain ischemia, or status epilepticus, or under conditions of poor nutrition and low oxygen. Some disorders of cranial nerves associated with aging (e.g., dementia) result from deficiency of an absolute amount of functional neurons caused by accumulation of exfoliated neurons. Thus, the monitoring and control of the exfoliation, as well as regeneration of the functions of neurons, are the most demanding subject to be solved among the aging problems.
  • Cranial nerve cells do not divide at all after the induction phase of differentiation in the process of development, and maintain their functions or is accompanied by gradual deterioration of their functions until the end of the life-time of the individual. They are presumed to have specific division-interrupting and function-maintaining mechanisms although these mechanisms have not yet been clarified. In the central nervous system, there exist numbers of unknown proteins and signaling substances, particularly stimulating substances and receptors thereof involved in brain-specific signal transduction, but their details are yet unclear.
  • Numbers of researches have been conducted worldwide on such an important element that controls the survival of the cranial nerve cells. However, none of the elements was clarified in the substance or molecule level, and, prior to everything, it was necessary to develop techniques for analysis. Recently, the group of Dr. Masashi Yanagisawa and his colleagues of the University of Texas, Medical Research Center (authorized by the Howard Hughes Foundation) has succeeded in developing a technique for randomly screening neuropeptides and receptors thereof by using cultured cells, and they have found a substance (named orexin) that directly binds to and stimulates the aperitive center in the hypothalamus, and identified functions of the substance's receptor (Cell, 92, 573-585, 1998). However, such a systematic screening of substances is rare, and currently, stimulating factors involved in brain-specific signal transduction and receptors thereof are not yet fully clarified.
  • Under such circumstances, the present inventors have now constructed an improved expression gene (cDNA) library, developed a systematic screening technique, and succeeded in extraction and selection of genes specific for cranial nerve cells, thereby accomplishing the present invention.
  • Thus, one object of the present invention is to provide a bradeion protein involved in long-term survival of cranial nerve cells, and DNA coding for the bradeion protein.
  • Another object of the present invention is to provide a vector containing the above-mentioned DNA, and a host cell transformed or transfected with the vector.
  • Still another object of the present invention is to use the DNA or an antibody to the protein for detecting cancers.
    • SUMMARY OF THE INVENTION
  • The present invention provides a human-derived bradeion protein, which has the following properties:
      • (i) it is a transmembranous protein;
      • (ii) it has a structure characteristic of growth hormone and cytokine receptors (even in a structure of its transmembranous portion) when its structure is determined by a hydrophobicity analysis according to Kyte-Doolittle method;
      • (iii) it is expressed at a high level in the human adult brain, in less amount in the heart, while it is not expressed in other adult organs or fetus;
      • (iv) it induces programmed cell death (apoptosis) when over-expressed in cultured human cell lines;
      • (v) it induces termination of cell division and aging when over-expressed in cultured human normal cells;
      • (vi) it is located in cytoplasm, and forms an intracellular aggregate when overexpressed; and
      • (vii) besides human adult neurons, it is specifically expressed in a human colorectal cancer cell line or in a skin cancer cell line,
        or an analogue thereof.
  • The proteins of the invention include Bradeion α and Bradeion β proteins having the amino acid sequences shown in SEQ ID NOS:2 and 4, respectively. These proteins are not the consequence of alternative splicing, but coded in the adjacent area (chromosome 17q23) of human genome. In addition to the above-described properties, Bradeion α induces programmed cell death when DNA coding for Bradeion α or an analogue thereof is introduced into a cultured cancer cell.
  • The term “analogue” as used herein refers to a protein that has properties substantially equivalent to those of the human-derived bradeion proteins (for example, at least the properties of (i), (ii), (iii), (vi) and (vii)), or a protein having an amino acid sequence with deletions, substitutions or additions of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO:2 or 4, or a protein having. an amino acid sequence that is substantially the same as that shown in SEQ. ID NO:2 or 4. Preferably, the analogue of the invention has at least 90%, preferably at least 95%, more preferably at least 97% homology with the amino acid sequence of SEQ ID NO:2 or 4. The analogue of the invention also includes human bradeion proteins modified or mutated in the amino acid level, and bradeion proteins from non-human mammals having properties substantially equivalent to those from humans. Only Bradeion β (similar to the human-derived Bradeion β) was found, for example, in a mouse brain and its homology with the human Bradeion β was 94%. The analogues of the invention may be obtained through DNA recombinant techniques by artificial modifications (e.g., site-directed mutagenesis), as long as the original bioactivity of the human bradeion is not impaired. The analogues of the invention may or may not contain a sugar chain, or they may be chemically modified with an aqueous polymer such as polyethylene glycol.
  • Moreover, the present invention provides DNAs comprising nucleotide sequences coding for the above-defined bradeion proteins or analogues thereof, and fragments of the DNAs.
  • Specific examples of such DNAs or fragments thereof include: DNA comprising the sequence of the nucleotides 129-1943 of SEQ ID NO:1 (i.e., DNA encoding human Bradeion α); DNA fragments having at least 15, preferably at least 20, more preferably at least 30 consecutive nucleotides derived from the nucleotides 129-1943 of SEQ ID NO:1; DNA comprising the full-length nucleotide sequence shown in SEQ ID NO:1; DNA comprising the sequence of the nucleotides 129-1562 of SEQ ID NO:3 (i.e., DNA encoding human Bradeion β); DNA fragments having at least 15, preferably at least 20, more preferably at least 30 consecutive nucleotides derived from the nucleotides 129-1562 of SEQ ID NO:3; and DNA comprising the full-length nucleotide sequence shown in SEQ ID NO:3. In addition, DNA that can hybridize with one of the above-mentioned DNAs under stringent conditions is also encompassed in the present invention. The stringent conditions as mentioned herein refer to such conditions that allow hybridization only when there is at least 90% homology, preferably at least 95% homology, more preferably at least 97% homology with the nucleotide sequence shown in SEQ ID NO:1 (positions 129-1943) or SEQ ID NO:3 (positions 129-1562). Generally, such conditions allow hybridization at a temperature that is lower by about 5° C.-30° C., preferably by about 10° C.-25° C. than the melting temperature (Tm) of the complete hybrid. Stringent conditions that may be used are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), esp. “Conditions for Hybridization of Oligonucleotide Probes”. The DNA and fragments thereof according to the present invention can be used not only for the expression of the bradeion proteins but also as a probe for hybridization or as a primer for PCR.
  • The present invention further provides vectors containing DNAs coding for bradeion proteins or analogues thereof, or fragments of the DNAs. The vector usually contains a promoter that is capable of operably expressing the DNAs. In addition to the promoter, the vector may contain at least one other element such as an origin of replication, an enhancer, a ribosome-binding site, a transcription termination factor (terminator), a selective marker, an RNA splicing site, or a polyadenylation signal.
  • The present invention further provides a host cell that has been transformed or transfected with such a vector. The host cell may be a prokaryotic or eukaryotic cell, preferably an eukaryotic cell, more preferably a mammalian cell such as a human cell line.
  • The present invention also provides an antibody that is immunologically reactive with the above-defined bradeions or analogues thereof. The antibody is preferably one that can specifically immuno-react with the bradeion proteins or analogues thereof, and is a polyclonal or monoclonal antibody.
  • The invention further provides a method for detecting a cancer, comprising detecting the cancer by using the above-defined DNAs or fragments thereof or the above-defined antibodies as tumor markers. Herein, the cancer includes, but is not limited to, a human colorectal cancer and a human skin cancer. The detection can be conducted by hybridization or immunoassay.
  • This specification includes all or-part of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-325380, which is a priority document of the present application.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A and 1B are photographs showing the results of a hydropathy analysis by the Kyte-Doolittle method, giving distributions of hydrophobic and hydrophilic portions in Bradeion α together with distributions in IL2, IL3, IL4 and growth hormone receptors for comparison;
  • FIGS. 2A-C are photographs taken with a confocal laser microscope showing images of labeled cells observed by gene-transfer and over-expression of Bradeion α and Bradeion β genes in cultured NT2 neuron (human undifferentiated nerve cell) and HeLa cell. FIG. 2A shows photographs for NT2 neuron; FIG. 2B shows photographs for NT2 neuron and HeLa; and FIG. 2C shows electron microscopic images of the cells shown in FIG. 2B (18 and 24 hours after the introduction). FIG. 2A shows locations of bradeion genes identified with EGFP (Enhanced Green Fluorescent Protein; CLONTECH Lab., Inc.) (left), locations of bradeion genes in mitochondria (center), and overlaid images of the left and center images (right).
  • FIG. 3A is a photograph showing expression of equal amounts of Bradeion α and Bradeion β genes in human cancer cell lines, giving the results of Northern blot analysis using radiolabeled bradeion genes. Lane 1 shows expression in polymyelocytic leukemia, HL60; Lane 2 shows expression in HeLaS3; Lane 3 shows expression in chronicmyologenous leukemia, K-562; Lane 4 shows expression in lymphoblastic leukemia, MOLT-4; Lane 5 shows expression in Burkitt's lymphoma, Raji; Lane 6 shows expression in colorectal adenocarcinoma, SW480 21, 22; Lane 7 shows expression in lung carcinoma, A549; and Lane 8 shows expression in melanoma, G361. The result of Northern blot analysis using a β-actin gene is shown underneath as a positive control.
  • FIG. 3B shows cancer-specific expression of bradeion genes in colorectal adenocarcinoma cell (T1 to T10); skin cancer cells (T11 to T13); and normal cells (N1 to N2). All specimens are from humans. In the figure, “*” refers to the case where both Bradeion α and Bradeion β genes are detected without gene mutation; “ND” refers to the case where detection was impossible due to denaturation of RNA; “Ad (well)” refers to a well differentiated adenocarcinoma; “Ad (mod)” refers to a moderately differentiated adenocarcinoma; “Muc” refers to a mucinouscarcinoma; and “MM” refers to malignant melanoma. Dukes' stage is based on the Dukes' classification. Codon 12 of human K-ras gene (whose wild type sequence is GGT) is indicated if it has been mutated. This mutation is heterozygous.
  • FIG. 3C is photographs of the results of in situ hybridization of specimens from human cancer tissue, showing stained tissues for T13 and T8 (FIG. 3B) (Antisense: positive control, Sense: negative control).
    • DETAILED DESCRIPTION OF THE INVENTION
  • cDNA coding for bradeion proteins of the invention may be obtained as follows.
  • First, brain tissue is homogenized in a phenol or phenol-chloroform solution containing guanidine isothiocyanate, and subjected to high-speed centrifugation to be separated into an aqueous layer and an organic layer. Total RNA contained in the aqueous layer is precipitated and collected by adding isopropanol, or is collected through sucrose or cesium chloride density-gradient centrifugation. The obtained total RNA is subjected to oligo(dT)-cellulose chromatography to purify mRNA (i.e., poly(A) RNA) therefrom.
  • Then, cDNA is synthesized from the mRNA in the presence of a reverse transcriptase. The cDNA is provided with suitable restriction sites and inserted into a phage or plasmid vector having the identical restriction sites. The thus-obtained vector is used to transform or transfect E.coli to produce a cDNA library.
  • Since the cloned cDNA library includes various DNA fragments with information other than that of the DNA of interest, it is necessary to select the DNA of interest. For this purpose, plaque hybridization or colony hybridization may usually be employed. According to such methods, plaques (in the case of a phage vector) or colonies (in the case of a plasmid vector) formed on agar are transferred to a nitrocellulose membrane or a nylon membrane. After being treated with an alkaline solution, they are bound to a radioactive (32P) or fluorescence labeling DNA probe that is capable of hybridizing with the DNA of interest, and exposed onto an X-ray film, thereby detecting and collecting a plaque or colony containing the DNA of interest. Alternatively, the obtained set of clones may be exposed to an inducer such as isopropyl 1-thio-β-D-galactoside (hereinafter, referred to as “IPTG”) to forcibly express proteins. The proteins are then transferred to a nylon membrane or a cellulose membrane, and a specific antibody for the protein of interest is used to immunologically select the corresponding clones.
  • The cDNA of interest collected from the plaques or colonies that positively reacts with the probe is sequenced by Maxiam-Gilbert method or Sanger-Coulson method.
  • For cloning and sequencing, for example, methods described in Sambrook et al., Molecular Cloning (supra), Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Company Assoc. and John Wiley Interscience, NY, 1992, etc. may be used.
  • Specifically, as will be described later in Examples, cDNA library from human adult brain is constructed and thereafter cDNA coding for the bradeion proteins of the invention are collected from the positive clones. As the result of the sequencing analysis, two types of bradeion genes, i.e., Bradeion α and Bradeion β genes, were found which were presumably produced due to alternative splicing. The nucleotide sequences of these genes are shown in SEQ ID NOS: 1 and 3, respectively, where the coding regions were at the positions 129-1943 and 129-1562, respecitively. The Bradeion α and Bradeion β proteins have amino acid sequences shown in SEQ ID NOS:2 and 4, respectively, as identified from their nucleotide sequences. The Bradeion α DNA has 1815 nucleotides and codes for a protein having 605 amino acids. The Bradeion β DNA has 1434 nucleotides and codes for a protein having 478 amino acids. Met at each position 1 of the amino acid sequences of SEQ ID NOS:2 and 4 may be present or absent. When these nucleotide and amino acid sequences were compared to all sequences deposited with the GenBank, it was found that the Bradeion α and Bradeion β genes and proteins were novel.
  • Bradeion α and Bradeion β proteins were found to have a structure characteristic of an interleukin receptor even in a structure of its transmembranous portion, when subjected to the hydropathy analysis according to Kyte-Doolittle method (J. Mol. Biol., 157 (1): 105-132, 1982). Thus, it has the structure of a transmembrane-type receptor that is presumably involved in the intracellular signaling mechanism. Bradeion α and Bradeion β proteins are also similar to the relationship of the tumor suppression genes p53/p73 in that there are two types of expression modes, i.e., α- and β-types, and in that either of them is prevalent in various organisms. Formation of an intracellular aggregate is very similar to that seen in human nerve retroplasia caused by a triplet repeat gene expressed substance (Igarashi et al., Nature Genetics, 111-117, 1998; Martindale et al., Nature Genetics, 150-154, 1998). Accordingly, it is assumed that the Bradeion α and Bradeion β proteins are greatly associated with specific termination of the division of human nerve cells and/or with maintenance of the function of the nerve cells after development/differentiation in the normal gene expression state.
  • The human-derived bradeion proteins or analogues thereof of the invention may be obtained, for example, by using gene recombinant techniques as follows.
  • Taking account of degeneracy of the genetic codes, a hybridization probe having at least 15, preferably about 20 to about 50 consecutive nucleotides is constructed based on the nucleotide sequence shown in SEQ ID NO:1 or 3 or the amino acid sequence shown in SEQ ID NO:2 or 4. By using this hybridization probe, DNAs coding for the bradeion proteins are screened from a genomic DNA library or cDNA library derived from human or non-human mammal brain tissue. The library may be produced by using commercially available vector such as λ ZAPII or pBluescript® cloning vector (Stratagene Cloning Systems). The plaque or colony containing the DNA of interest is selected through plaque hybridization or colony hybridization.
  • Alternatively, a DNA sequence generally having 15 to 100 consecutive nucleotides complementary to the nucleotides 129-1943 of SEQ ID NO:1 or the nucleotides 129-1562 of SEQ ID NO: 3, is produced as a primer. This primer can be used to conduct polymerase chain reaction (PCR) in the genomic DNA library or cDNA library derived from human or non-human mammalian brain tissue, thereby specifically amplifying the DNA of interest. PCR can be conducted through at least 20 cycles, preferably at least 30 cycles of: denaturation at 94° C. for 1 min.; annealing at 57° C. for 2 min.; and elongation at 70° C. for 3 min. For the PCR, see the techniques described in Protein Nucleic acid and Enzyme, “Frontier of PCR method—Basic to Applied Techniques” vol. 4(5), April, 1996 Supplement, Kyoritsu Shuppan, Tokyo, Japan.
  • The cloned or amplified DNA of interest is collected and introduced into an available suitable expression vector. The obtained vector is used to transform a suitable host cell, which is thereafter cultured in a proper medium for expression of the DNA, to isolate and purify the protein of interest.
  • Examples of the analogue of the present invention include: a protein having a substantially equivalent properties to the human-derived bradeion (especially, at least the above-mentioned properties of (i), (ii), (iii), (vi) and (vii)); or a protein having an amino acid sequence with deletions, substitutions or additions of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO:2 or 4; or a protein having an amino acid sequence that is substantially the same as that shown in SEQ ID NO:2 or 4. Preferably, the analogue has at least 90%, preferably at least 95%, more preferably at least 97% homology with the amino acid sequence shown in SEQ ID NO:2 or 4. This means that the amino acid sequence of the analogue may include a modification chosen from deletion, substitution or addition of at least one amino acid as long as the analogue has properties substantially equivalent to those of the human Bradeion α or Bradeion β. The mutation may be introduced into the amino acid sequence shown in SEQ ID NO:2 or 4 through genetic engineering, for example, by well-known techniques such as site-directed mutagenesis/PCR method (S. N. Ho et al., Gene, 77, 51, 1989); and methods described in Kaoru Saigo and Yumiko Sano (trans.), Current Protocols compact version, Experimental Protocols in Molecular Biology I, June 1997, Maruzen, Tokyo, Japan). Examples of the substitution include substitutions between hydrophobic amino acids (Ala, Val, Leu, Ile, etc.), Ser and Thr, Asp and Glu, Asn and Gln, Lys and Arg, and Phe and Tyr. Examples of the addition include an addition of Met to the N-terminus of a mature protein as seen in products expressed in a bacterial host, and an addition of His-tag, or Met-Lys or Met-Arg sequence to the N-terminus for facilitating induction of a mature protein. It is also possible to truncate some amino acid residues at the carboxyl or amino terminus of the bradeion protein to an extent where bioactivity is not impaired.
  • Proteins without sugar chains are produced when a prokaryotic cell such as bacterium is used as the host cell for expressing DNAs coding for the bradeion proteins or analogues thereof through genetic engineering. On the other hand, products with sugar chains may be produced when the DNAs are expressed in an eurokaryotic cell such as fungus, yeast, insect, plant, or mammalian cell. For example, sugar chains may be formed by introducing an N-binding sugar chain site (generally, Asn-Xaa-Thr/Ser where Xaa is any amino acid other than Pro) into the sequence. In any event, the obtained analogue should have bioactivity substantially equivalent to the human bradeions.
  • Amino groups at the N-terminus or ε-amino groups of lysine of the human bradeion proteins or analogues thereof of the invention may be chemically bound to an aqueous polymer such as polyethylene glycol (PEG). Pegylation is generally known to reduce or suppress antigenicity or immunogenisity upon in vivo administration of a pegylated product.
  • The present invention further provides an expression vector containing the above-described DNA, and a host cell transformed or transfected with the vector.
  • The vector of the invention may be in the form of, for example, plasmid, phage, or virus. Other types of vectors may also be used as long as they are replicable in a host cell. For example, bacterium plasmids (e.g., pBR322, pKC30, pCFM536, etc.), phage DNAs (e.g., λ phage, etc.), yeast plasmids (e.g., pG-1, etc.), or viral DNAs for mammal host cells (e.g., baculovirus, vaccinia virus, adenovirus, SV40 and its derivatives, etc.) may be used.
  • The vector usually contains a replication origin, a selective marker, a promoter, and, if necessary, may contain an enhancer, a transcription termination sequence (terminator), a ribosome-binding site, a polyadenylation signal, etc.
  • Where the vector is used for E.coli, the replication origin is derived from ColE1, R factor, or F factor. Where the vector is used for yeast, the replication origin is derived from, for example, 2 μm DNA or ARS1. Where the vector is used for a mammalian cell, the replication origin is derived from, for example, SV40, adenovirus, or bovine papilloma virus.
  • The promoter is a regulator sequence for directing a synthesis of mRNA coding for the DNA of the invention. Representative examples of the promoter include adenovirus or SV40 promoter, E.coli lac or trp promoter, phage λPL promoter, ADH, PHO5, GPD, PGK or AOX1 promoter (for yeast), and a promoter derived from nuclear polyhedrosis virus (for Bombyx mori cell).
  • The selective marker is a gene for providing a phenotype to the host in order to select transformed host cells. Exemplary selective markers include kanamycin-resistant gene, ampicillin-resistant gene, tetracycline-resistant gene, and the like (when the vector is used for E.coli); Leu2, Trp1, Ura3 genes, and the like (when the vector is used for yeasts); and neomycin-resistant gene, thymidine kynase gene, dihydrofolate reductase gene, and the like (when the vector is used for mammalian cells).
  • Commercially available vectors may be used such as pQE70, pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), and pET-11a (Novagen) as bacterium vectors; and pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG and pSVL SV40 (Pharmacia) as eukaryote vectors.
  • The DNA of the invention may be introduced into the vector by any means. The vector preferably contains a polylinker with various restriction sites, or a unique restriction site. The DNA of the invention is inserted into a particular restriction site(s) of the vector where it has been cleaved with a particular restriction endonuclease(s).
  • The expression vector containing the DNA of the invention with a regulatory sequence is used to transform or transfect a suitable host cell, thereby expressing and producing the human bradeion protein of the invention or an analogue thereof in the host cell.
  • The host cell is, for example, a bacterial cell (e.g., E.coli, streptomyces, or Bacillus subtilis), an eukaryotic cell (e.g., Aspergillus strain), an yeast cell (e.g., Saccharomyces cerevisiae, or methanol-assimilating yeast), an insect cell (e.g., Drosophila S2 or Spodoptera Sf9), and a mammalian cell including cultured human cell (e.g., CHO, COS, BHK, 3T3, or C127).
  • Transformation or tranfection may be conducted by a known method such as calcium chloride/rubidium chloride method, calcium phosphate method, DEAE-dextran-mediated transfection, or electroporation.
  • The human-derived bradeion or an analogue thereof of the invention can be obtained by culturing the host cells which have been transformed or transfected as described above under the control of the promoter, and by collecting the produced protein of interest. The host cell is amplified or grown to a proper cell density. Then, the promoter is induced by shifting the temperature or by chemical induction (with IPTG, etc.). The cell is further cultured for a predetermined period. Where the protein of interest is secreted extracellularly, it can directly be obtained from the medium. Where the protein of interest is present intracellularly, the cell can be disrupted by physical means (e.g., sonication or mechanical disruption) or by chemical means (e.g., lyzozyme or cytolytic agent). Then the protein of interest is purified. The protein may partially or completely be purified from the culture medium containing the recombinant cells or an extracted solution thereof, by using routine techniques such as ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, HPLC, electrophoresis, and chromatofocusing, alone or in combination.
  • The DNA of the invention was gene-transferred into several human cell lines or cancer cell lines to examine the functions of the bradeion proteins. As a result, in addition to the above-described findings, the following facts were found out:
      • (1) when the DNA of the invention is gene-transferred into NT2 neuron (Stratagene), which is a cultured human undifferentiated nerve cell, using Superfect reagent (Qiagen) and over-expressed, the cell death is induced within 18-24 hours.
      • (2) when the DNA is similarly over-expressed in cultured human normal cells, termination of the aging and cell division is induced.
      • (3) when the DNA is gene-transferred into a cultured cancer cell, Bradeion α and Bradeion β genes induce the programmed cell death.
      • (4) in the course of the induced cell death, the bradeion protein is present in cytoplasm, and forms an intracellular aggregate.
  • As a result of studies using cultured human cancer cell lines, the expression of bradeion was detected to be specific for colorectal cancer and malignant melanoma (skin cancer), suggesting that it has strong preferability for tissues and cell types in which bradeion genes are expressed.
  • Based on the above-described findings, the bradeion proteins of the invention seem to allow survival of cranial nerve cells of the central nervous system in non-dividing state via neuro-stimulating transmission. It seems to be important that over-expression of the bradeion genes controls cell fate: apoptosis or carcinogenesis, and that normally the expression ratio of the α-type to the β-type is maintained (at a ratio of 10:1 in a normal cranial nerve cell). It was also suggested that depending on changes in the expression ratio (e.g., 1:1), it may induce development of cancer. Accordingly, the bradeion proteins are presumed to play an important role in controlling cell fate (termination, apoptosis, and/or carcinogenesis), and also to determine long-term survival of cranial nerve cells in non-dividing state in human adult central nervous system. Thus, the bradeion proteins are useful for monitoring exfoliated nerve cells associated with aging, studying necrosis of nerve cells that occurs during brain ischemia and status epilepticus, and understanding the survival mechanism of neurons and pathology of brain. The proteins are also useful for producing novel medicines for treating genetic diseases, and may be applied to genetic diagnosis and gene therapy of cancers.
  • Thus, the proteins of the invention or fragments thereof, antibodies to them, or the DNAs of the invention or fragments thereof may be used for detecting or diagnosing cancers (e.g., human colorectal carcinoma and human skin cancer), for determining the cell fate, and as a targeting molecule in colorectal cancer and malignant melanoma.
  • For the above purposes, a polyclonal or monoclonal antibody that is specific to the proteins of the invention is useful. The antibody, preferably the monoclonal antibody, may be used for diagnosis, vaccination and drug delivery systems.
  • Such antibodies may be produced, for example, by methods described in Suguru Matsubasi et al., Biochemical Experimental Methods 15, Introduction to Immunological Experiments, 1982, Japan Scientific Societies Press, Tokyo, Japan; Tatsuo Iwasaki et al., Monoclonal antibody -Hybridoma and ELISA, 1987, Kodansha Scientific, Tokyo, Japan.
  • The polyclonal antibody can be obtained as follows. First, an antigen solution containing the protein of the invention or a fragment thereof as an antigen is mixed with complete Freund's adjuvant to form an emulsion. The emulsion is then subcutaneously injected into a mammal such as rabbit, mouse, goat, bovine or equine. After about 2 weeks, an antigen solution emulsified in incomplete Freund's adjuvant is similarly injected into the animal, which is then boosted if necessary, and blood is drawn from the animal to collect anti-serum as the polyclonal antibody. The anti-serum is further subjected to ammonium sulfate precipitation or ion exchange chromatography using DEAE cellulose to obtain an IgG fraction. The IgG is subjected to an affinity chromatography on CNBr-activated Sephadex or Sepharose bound to the protein of the invention or a fragment thereof to mono-specifically purify the antibody of interest that is bound through immunologic reaction.
  • The monoclonal antibody can be obtained as follows. First, an antigen-adjuvant emulsion is prepared as described above, which is then intraperitoneally injected into a mouse (e.g., BALB/c) for immunization. The spleen was removed from the mouse to collect spleen cells, which are then fused with a myeloma cell (e.g., X63 or NS-1) in the presence of polyethylene glycol (e.g., PEG 400). Then, an antibody-producing hybridoma is selected in an HAT medium to obtain the monoclonal antibody of interest by a cloning method or alternatively from ascites obtained from the mouse after the intraperitoneal injection. Humanized monoclonal antibody may also be prepared by using known techniques as described in Teng et al., Proc. Nal. Acad. Sci. USA, 1983, 80:7308-7312, and Kozbor et al., Immunology Today, 1983, 4 (3): 72-79.
  • The above-described antibody may be used for diagnosing, for example, cancers in a standard immunoassay such as an enzymatic immunoassay, a radioimmunoassay or a fluorescent antibody method.
  • Where the DNA of the invention or a fragment thereof is used as a probe or a primer, at least 15, preferably at least 20, more preferably at least 30 consecutive nucleotides derived from the nucleotide sequence of SEQ ID NO:1 (the nucleotides 129-1943) or SEQ ID NO:3 (the nucleotides 129-1562) can be prepared using an automatic DNA synthesizer. The obtained nucleotide fragment is labeled with an isotope, a fluorescent substance or the like, thereby preparing a probe or a PCR primer for diagnosis. The conditions employed for hybridization are, for example, described in Sambrook et al., Molecular Cloning (supra), and F. M. Ausubel et al., Short Protocols In Molecular Biology, Third Edition, John Wiley & Sons.
    • EXAMPLES
  • Hereinafter, the present invention will be described by way of examples. The present invention, however, is not intended to be limited to these examples.
    • Example 1 Cloning and Sequencing of cDNA Coding for Human Bradeion
  • First, a cDNA library from a human adult brain was constructed using the plasmid vector pCMV SPORT1 (Life Technologies, Inc., USA) which is capable of linking with a CMV promoter for expressing in an eukaryotic cell. The adult brain was obtained from a 36-year-old white Caucasian American female, and mRNA (poly(A) RNA) was extracted therefrom with TRIzol® reagent (Life Technologies, Inc.) and purified with MESSAGEMAKER® reagent (Life Technologies, Inc.). Then double stranded cDNA synthesis and library construction were initiated by SuperScript plasmid system.
  • The prepared mRNA (poly(A) RNA) was linked with NotI primer adapter at its 3′-terminus. Then, a double stranded cDNA was synthesized according to a standard method using Superscript II reverse transcriptase and T4 DNA polymerase. The 5′-terminus of the cDNA was linked with SalI adapter and 3′-terminus was treated with NotI restriction enzyme so that the cDNA fragment had restriction sites SalI and NotI at each end. The cDNA was separated in sizes by gel filtration chromatography to select and fractionate cDNA having a size of 1 kb or more. The obtained set of cDNA fragments was inserted, by a standard method, into the plasmid vector pCMV SPORT1 that also had been cleaved with SalI and NotI, thereby producing circular plasmids. These plasmids were introduced into E.coli DH12S cells (Life Technologies, Inc.) by an electroporation method and amplified to construct a library.
  • The resulting E.coli strains were grown on ampicillin-containing LB agar medium to form colonies. Biodyne A nylon membrane (Pall Corp., US) treated with 10 mM IPTG was placed in close contact with the colonies and left at 37° C. for 2 hours. The nylon membrane was reacted with the antibody CE5 that specifically recognizes cranial nerve cells [Nature, 296, 34-38, 1982]. Positive clones were selected using picoBlue® Immunoscreening Kit (Stratagene, US).
  • Plasmid DNA was collected from the obtained positive clones to be used as a 32P-labeled probe, which was hybridized with nylon membranes with mRNAs specific for different human organs (MTN blot, CLONTECH Lab., Inc.) to test whether the probe was specific for the brain. The nucleotide sequences of the cDNAs were determined by sequencing analysis and compared with homologous sequences deposited with the GenBank. Only the one that was completely novel was deposited with the GenBank as a sequence of interest. The determined nucleotide sequence of the Bradeion α cDNA is shown in SEQ ID NO: 1 (GenBank Accession No. AB002110). The coding region was at the positions 129-1943. The amino acid sequence of the Bradeion α determined based on this nucleotide sequence is shown in SEQ ID NO:2. DNA containing Bradeion α cDNA was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba-shi, Ibaraki-ken 305-8566, Japan) on Jul. 14, 1998 as FERM P-16897.
  • Based on the above-described sequence of Bradeion α cDNA, 5′-terminal primers (described below) were synthesized to systematically screen relevant genes. For this purpose, Gene Trapper Positive Selection system (Life Technologies, Inc.) was used to screen the above-described gene library with the synthesized oligonucleotides and magnet beads. The sequences of the oligonucleotides used were:
  • 5′-ctgagcaagttcgtgaaggatttc-3′ (SEQ ID NO:5); and
  • 5′-cagtcctctgacaaccagcagta-3′ (SEQ ID NO:6)
  • As a result, a gene was detected which was named Bradeion β and whose nucleotide sequence is shown in SEQ ID NO:3 (GenBank Accession No. AB008753). The coding region was at the positions 129-1562. The amino acid sequence of Bradeion β determined based on this nucleotide sequence is shown in SEQ ID NO:4.
    • Example 2 Characterization of Bradeion α and Bradeion β Proteins
  • (1) Hydropathy Analysis
  • The amino acid sequences of Bradeion α and β proteins determined in Example 1 were subjected to hydropathy analysis by Kyte-Doolittle method (Kyte, J. and Doolittle, R. F. J., J. Mol. Biol., 1982, 157 (1): 105-132). This analysis is one method for predicting a high-order structure of a protein based on its amino acid sequence, whereby the distributions of hydrophobic and hydrophilic portions of the protein can be determined. This analysis therefore allows to study the presence of a three-dimentional structure or a transmembranous domain. FIGS. 1A and 1B show the results of the analysis for the bradeion proteins as well as the results for human-derived IL (interleukin) 2, IL3 and IL4 receptors and growth hormone receptor for comparison. Referring to the figures, the proteins having the sequences of growth hormone and cytokine receptors may be divided roughly into three sections, i.e., an assembly of hydrophobic groups (calculated as positive (“+”) values) as a transmembranous portion (third column from the left, shown in red), an extracellular portion (second column from the left, shown in blue) preceding the transmembranous portion, and a cytoplasmic portion (fourth column from the left, shown in green) following the transmembranous portion. This structure is common among all of the receptors including the bradeion proteins of the invention. However, bradeion proteins did not have a hydrophobic signal peptide (first column from the left, shown in yellow).
  • As a result, the bradeion proteins of the invention were found to be membrane proteins with a structure characteristic of an interleukin receptor even in the structure of a transmembranous portion in the amino acid sequences.
  • (2) Localization of Bradeion Proteins
  • Hybridization with the nylon membranes with mRNAs specific for different human organs (MTN blot, CLONTECH Lab., Inc.) indicated a high level expression only in the human adult brain, and a low level of expression in the heart (≦10% of the expression level in the brain). No expression was seen in other organs or in human fetus. Both of the α- and β-types were expressed in the adult brain. The difference in types was due to the gene duplication in the adjacent area (17q23) of human chromosome. A homologous gene sequence, but only one of the two types of human bradeions (i.e., Bradeion β) existed in the mouse brain (94% homology).
  • (3) Experiment of Overexpression of Bradeion α and Bradeion β Genes in Cultured Human Cell Lines
  • Bradeion α and Bradeion β DNAs were individually gene-transferred into NT2 neuron (Stratagene, US), which is a cultured human undifferentiated nerve cell line, with Superfect reagent (QIAGEN, US) and over-expressed. The results are shown in FIGS. 2A, 2B and 2C.
  • FIG. 2A shows images of the labeled cell observed 24 hours after overexpression of Bradeion α gene (upper panels) and Bradeion β gene (lower panels). The left images show the locations of Bradeion α and Bradeion β. The center images show their locations in mitochondria, and the right images show the overlaid images of the left and center images. All of the images were observed with a confocal laser microscope. As a result, it was found that the location of Bradeion α was consistent with the location in mitochondria (Note: yellow color indicates an overlap of red and green.) while the location of Bradeion β was not consistent with the location in mitochondria.
  • Programmed cell death (apoptosis) was induced within 18-24 hours after the transfection of the Bradeion α gene. In the course of the apoptosis, Bradeion α formed an intracellular aggregate. FIG. 2B shows cell images observed at predetermined points of time after the gene transfer. The left images show the cultured human cell NT2 neuron (Stratagene) and the right images show the human cancer cell line, HeLa. Both cell lines formed intracellular aggregates, resulting in cell death. To confirm this fact, the cells of FIG. 2B were observed with an electron microscope. As shown in FIG. 2C, the presence of apoptosis corpuscles specific for programmed cell death (apoptosis) was confirmed.
  • (4) Correlation of Bradeion α and Bradeion β Genes to Cancers
  • Although the Bradeion α and Bradeion β genes are only expressed in the normal adult brain and the heart (about 10% of the expression level of the brain), their expression was also found in cultured human cancer cell lines. The results are shown in FIGS. 3A, 3B and 3C.
  • FIG. 3A shows the results of Northern blot regarding expression of Bradeion α and Bradeion β genes in different cultured human cancer cells. Specific expressions (signals) were found only in Lane 8 (skin cancer cell line G361) and Lane 6 (colorectal adenocarcinoma SW480).
  • Specimens from human patients (i.e., specimens from pathologic tissues) were used for detection of the cancer-specific expression. As shown in FIG. 3B, the specific expression was observed in 10 specimens having colorectal adenocarcinoma (T1 to T10; indicated as Ad), and in 3 specimens having skin cancer (T11 to T13; indicated as Muc and MM). FIG. 3C shows images of stained cancer cells for confirming the cancer-specific expression.
  • The above results show that the Bradeion α and Bradeion β proteins and the genes encoding them can be used as tumor-specific markers of colorectal cancer.
    • ADVANTAGEOUS EFFECT OF THE INVENTION
  • The bradeion proteins of the invention and the DNA encoding them seem to allow survival of cranial nerve cells of the central nervous system in non-dividing state via neuro-stimulating transmission. Over-expression of the bradeion genes induces apoptosis. Normally, the in vivo expression ratio of Bradeion α and Bradeion β proteins is maintained (at a ratio of 10:1 in a normal cranial nerve cell). It is also suggested that depending on changes in the expression ratio (e.g., 1:1), the development of a cancer may be induced. Accordingly, the bradeion proteins and the DNA encoding them were presumed to act as a cell-lifetime-prolonging, cancer-development-suppressing factor that determines long-term survival of cranial nerve cells in non-dividing state after the development/differentiation of the cells. Thus, they are useful for monitoring exfoliated nerve cells associated with the aging, studying the necrosis of nerve cells that occurs during brain ischemia and status epilepticus, and understanding the survival mechanism of the central nerve cells and pathology of brain. They are also useful for producing novel medicines for treating genetic diseases, and may be applied to genetic diagnosis and gene therapy of cancers.
  • All publications (including patent application) cited herein are incorporated herein by reference in their entirety.
  • The following are information on SEQ ID NOS:1 to 4 described herein:
    gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60 SEQ ID NO:1
    tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120
    cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg 180
    aacctgggat caaccgtttc ctggaggaca ccacggatga tggagaactg agcaagttcg 240
    tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacc tgggcatcca 300
    ggccccaagt cccggagcca aggccccagg ccccggacct ctatgatgat gacctggagt 360
    tcagaccccc ctcgcggccc cagtcctctg acaaccagca gtacttctgt gccccagccc 420
    ctctcagccc atctgccagg ccccgcagcc catgggggga gcttgatccc tatgattcct 480
    ctgaggtaga gcctccagcc ctgcctttgc ctttcagtgg gctgctgcag gaagaccggg 540
    ggcagggagc aggaatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtttgtgt 600
    gtgtgtgtat ctgggaccca tttcagtcct gtgtcagccc tagctccaaa atatctgccc 660
    ccaagggcac tggaaatttg cagtttcagc aagggcagga ggcccagctg gtggcctcag 720
    atgggaactc acagaagtct ggcactgctt ttttaaggct ggggcaaagg cctgaaaggg 780
    agagaagatt ggcgctgggt gccggggccc ctttggctcc tcaccgtgat gcattctgcc 840
    ttcctgtcta ctacgatgac aaggagtatg tgggctttgc aaccctcccc aaccaagtcc 900
    accgaaagtc cgtgaagaaa ggctttgact ttaccctcat ggtggcagga gagtctggcc 960
    tgggcaaatc cacacttgtc aatagcctct tcctcactga tctgtaccgg gaccggaaac 1020
    ttcttggtgc tgaagaaagg atcatgcaaa ctgtggagat cactaagcat gcagtggaca 1080
    tagaaaaaaa aggtgtgagg ctgcggctca ccattgtgga cacaccaagt tttggggatg 1140
    cagtcaacaa cacagagtgt atgtctgact ggaagcctgt ggcagaatac attgatcagc 1200
    agtttgagca gtatttccga gacgagagtg gcctgaaccg aaagaacatc caagacaaca 1260
    gggtgcactg ctgcctgtac ttcatctcac ccttcggcca tgggctccgg ccattggatg 1320
    ttgaattcat gaaggccctg catcagcggg tcaacatcgt gcctatcctg gctaaggcag 1380
    acacactgac acctcccgaa gtggaccaca agaaacgcaa aatccgggag gagattgagc 1440
    attttggaat caagatctat caattcccag actgtgactc tgatgaggat gaggacttca 1500
    aattgcagga ccaagcccta aaggaaagca tcccatttgc agtaattggc agcaacactg 1560
    tagtagaggc cagagggcgg cgagttcggg gtcgactcta cccctggggc atcgtggaag 1620
    tggaaaaccc agggcactgc gactttgtga agctgaggac aatgctggta cgtacccaca 1680
    tgcaggacct gaaggatgtg acacgggaga cacattatga gaactaccgg gcacagtgca 1740
    tccagagcat gacccgcctg gtggtgaatg aacggaatcg caagtatgac cagaagccag 1800
    gacaaagctg gcagggggag atcccaagcc tagccttggg tgagaccaag ccctactttt 1860
    gttcttctat aggccctggg ctcaatctaa gcgggtgctg gggtcctcct cgccttatca 1920
    acccttttct ccctttagca aactgactcg ggaaagtggt accgacttcc ccatccctgc 1980
    tgtcccacca gggacagatc cagaaactga gaagcttatc ccagagaaag attaggagct 2040
    gcggcggata cacgagatac tacaccaaat accaaaacag ataaaggaga actatttact 2100
    ggctttcagc cctggatatt taaatctcct cctcttcttc ctgtccatgc cggcccctcc 2160
    cagcaccagc tctgctcagg ccccttcagc tactgccact tcgccttaca tccctgctga 2220
    ctgcccagag actcagagga aataaagttt aataaatctg taggtggctt ctgg 2274
    Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg SEQ ID NO:2
    1 5 10 15
    Thr Glu Pro Gly Ile Asn Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly
    20 25 30
    Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His
    35 40 45
    Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro
    50 55 60
    Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro
    65 70 75 80
    Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro
    85 90 95
    Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Glu Leu
    100 105 110
    Asp Pro Tyr Asp Ser Ser Glu Val Glu Pro Pro Ala Leu Pro Leu Pro
    115 120 125
    Phe Ser Gly Leu Leu Gln Glu Asp Arg Gly Gln Gly Ala Gly Met Cys
    130 135 140
    Val Cys Val Cys Val Cys Val Cys Val Cys Val Phe Val Cys Val Cys
    145 150 155 160
    Ile Trp Asp Pro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys Ile Ser
    165 170 175
    Ala Pro Lys Gly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln Glu Ala
    180 185 190
    Gln Leu Val Ala Ser Asp Gly Asn Ser Gln Lys Ser Gly Thr Ala Phe
    195 200 205
    Leu Arg Leu Gly Gln Arg Pro Glu Arg Glu Arg Arg Leu Ala Leu Gly
    210 215 220
    Ala Gly Ala Pro Leu Ala Pro His Arg Asp Ala Phe Cys Leu Pro Val
    225 230 235 240
    Tyr Tyr Asp Asp Lys Glu Tyr Val Gly Phe Ala Thr Leu Pro Asn Gln
    245 250 255
    Val His Arg Lys Ser Val Lys Lys Gly Phe Asp Phe Thr Leu Met Val
    260 265 270
    Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu Val Asn Ser Leu Phe
    275 280 285
    Leu Thr Asn Leu Tyr Arg Asp Arg Lys Leu Leu Gly Ala Glu Glu Arg
    290 295 300
    Ile Met Gln Thr Val Glu Ile Thr Lys His Ala Val Asp Ile Glu Lys
    305 310 315 320
    Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp Thr Pro Ser Phe Gly
    325 330 335
    Asp Ala Val Asn Asn Thr Glu Cys Met Ser Asp Trp Lys Pro Val Ala
    340 345 350
    Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu Ser Gly
    355 360 365
    Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys Leu Tyr
    370 375 380
    Phe Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val Glu Phe
    385 390 395 400
    Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu Ala Lys
    405 410 415
    Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg Lys Ile
    420 425 430
    Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe Pro Asp
    435 440 445
    Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln Ala Leu
    450 455 460
    Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val Val Glu
    465 470 475 480
    Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly Ile Val
    485 490 495
    Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu Arg Thr Met
    500 505 510
    Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg Glu Thr
    515 520 525
    His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr Arg Leu
    530 535 540
    Val Val Asn Glu Arg Asn Arg Lys Tyr Asp Gln Lys Pro Gly Gln Ser
    545 550 555 560
    Trp Gln Gly Glu Ile Pro Ser Leu Ala Leu Gly Glu Thr Lys Pro Tyr
    565 570 575
    Phe Cys Ser Ser Ile Gly Pro Gly Leu Asn Leu Ser Gly Cys Trp Gly
    580 585 590
    Pro Pro Arg Leu Ile Asn Pro Phe Leu Pro Leu Ala Asn
    595 600 605
    gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60 SEQ ID NO:3
    tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120
    cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg 180
    aagctgggat caagcgtttc ctggaggaca ccacggatga tggagaactg agcaagttcg 240
    tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacc tgggcatcca 300
    ggccccaagt cccggagcca aggccccagg ccccggacct ctatgatgat gacctggagt 360
    tcagaccccc ctcgcggccc cagtcctctg acaaccagca gtacttctgt gccccagccc 420
    ctctcagccc atctgccagg ccccgcagcc catggggcaa gcttgatccc tatgattcct 480
    ctgaggatga caaggagtat gtgggctttg caaccctccc caaccaagtc caccgaaagt 540
    ccgtgaagaa aggctttgac tttaccctca tggtggcagg agagtctggc ctgggcaaat 600
    ccacacttgt caatagcctc ttcctcactg atctgtaccg ggaccggaaa cttcttggtg 660
    ctgaagagag gatcatgcaa actgtggaga tcactaagca tgcagtggac atagaagaga 720
    agggtgtgag gctgcggctc accattgtgg acacaccagg ttttggggat gcagtcaaca 780
    acacagagtg ctggaagcct gtggcagaat acattgatca gcagtttgag cagtatttcc 840
    gagacgagag tggcctgaac cgaaagaaca tccaagacaa cagggtgcac tgctgcctgt 900
    acttcatctc acccttcggc catgggctcc ggccattgga tgttgaattc atgaaggccc 960
    tgcatcagcg ggtcaacatc gtgcctatcc tggctaaggc agacacactg acacctcccg 1020
    aagtggacca caagaaacgc aaaatccggg aggagattga gcattttgga atcaagatct 1080
    atcaattccc agactgtgac tctgatgagg atgaggactt caaattgcag gaccaagccc 1140
    taaaggaaag catcccattt gcagtaattg gcagcaacac tgtagtagag gccagagggc 1200
    ggcgagttcg gggtcgactc tacccctggg gcatcgtgga agtggaaaac ccagggcact 1260
    gcgactttgt gaagctgagg acaatgctgg tacgtaccca catgcaggac ctgaaggatg 1320
    tgacacggga gacacattat gagaactacc gggcacagtg catccagagc atgacccgcc 1380
    tggtggtgaa ggaacggaat cgcaacaaac tgactcggga aagtggtacc gacttcccca 1440
    tccctgctgt cccaccaggg acagatccag aaactgagaa gcttatccga gagaaagatg 1500
    aggagctgcg gcggatgcag gagatgctac acaaaataca aaaacagatg aaggagaact 1560
    attaactggc tttcagccct ggatatttaa atctcctcct cttcttcctg tccatgccgg 1620
    cccctcccag caccagctct gctcaggccc cttcagctac tgccacttcg cctaacatcc 1680
    ctgctgactg cccagagact cagaggaaat aaagtttaat aaatctgtag gtggc 1735
    Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg SEQ ID NO:4
    1 5 10 15
    Thr Glu Ala Gly Ile Lys Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly
    20 25 30
    Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His
    35 40 45
    Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro
    50 55 60
    Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro
    65 70 75 80
    Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro
    85 90 95
    Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Lys Leu
    100 105 110
    Asp Pro Tyr Asp Ser Ser Glu Asp Asp Lys Glu Tyr Val Gly Phe Ala
    115 120 125
    Thr Leu Pro Asn Gln Val His Arg Lys Ser Val Lys Lys Gly Phe Asp
    130 135 140
    Phe Thr Leu Met Val Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu
    145 150 155 160
    Val Asn Ser Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu
    165 170 175
    Gly Ala Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His Ala
    180 185 190
    Val Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp
    195 200 205
    Thr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Val Lys Pro
    210 215 220
    Val Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu
    225 230 235 240
    Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys
    245 250 255
    Leu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val
    260 265 270
    Glu Phe Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu
    275 280 285
    Ala Lys Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg
    290 295 300
    Lys Ile Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe
    305 310 315 320
    Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln
    325 330 335
    Ala Leu Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val
    340 345 350
    Val Glu Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly
    355 360 365
    Ile Val Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu Arg
    370 375 380
    Thr Met Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg
    385 390 395 400
    Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr
    405 410 415
    Arg Leu Val Val Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg Glu Ser
    420 425 430
    Gly Thr Asp Phe Pro Ile Pro Ala Val Pro Pro Gly Thr Asp Pro Glu
    435 440 445
    Thr Glu Lys Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg Met Asp
    450 455 460
    Glu Met Leu His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr
    465 470 475

Claims (17)

1. A human-derived bradeion protein, which has the following properties:
(i) it is a transmembranous protein;
(ii) it has a structure characteristic of growth hormone and cytokine receptors even in a structure of its transmembranous portion when its structure is determined by a hydrophobicity analysis according to Kyte-Doolittle method;
(iii) it is expressed at a high level in a human adult brain, and in less amount in the heart, while it is not expressed in other adult organs or fetus;
(iv) it induces programmed cell death (apoptosis) when over-expressed in a cultured human nerve cell lines;
(v) it induces termination of cell division and aging when over-expressed in a cultured human normal cell;
(vi) it is located in cytoplasm, and forms an intracellular aggregate when overexpressed; and
(vii) besides human adult neurons, it is specifically expressed in a human colorectal cancer cell line or in a skin cancer cell line,
or an analogue thereof.
2. The human-derived bradeion protein or an analogue thereof according to claim 1, wherein the programmed cell death (apoptosis) is induced when DNA coding for the bradeion protein or the analogue thereof is gene-transferred into a cultured cancer cell.
3. The human-derived bradeion protein or an analogue thereof according to claim 1, comprising an amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence with deletions, substitutions or additions of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 2.
4. The human-derived bradeion protein or an analogue thereof according to claim 1, comprising an amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence with deletions, substitutions or additions of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 4.
5. DNA comprising a nucleotide sequence coding for the bradeion protein or the analogue thereof according to claim 1, or a fragment thereof.
6. The DNA or a fragment thereof according to claim 5, comprising the nucleotide sequence 129-1943 of SEQ ID NO: 1, or a nucleotide sequence of at least 15 consecutive nucleotides derived from the nucleotide sequence 129-1943 of SEQ ID NO: 1.
7. The DNA or a fragment thereof according to claim 5, comprising the nucleotide sequence 129-1562 of SEQ ID NO: 3, or a nucleotide sequence of at least 15 consecutive nucleotides derived from the nucleotide sequence 129-1562 of SEQ ID NO: 3.
8. The DNA according to claim 5, which hybridizes with DNA comprising the nucleotide sequence 129-1943 of SEQ ID NO: 1 under stringent conditions.
9. The DNA according to claim 5, which hybridizes with DNA comprising the nucleotide sequence 129-1562 of SEQ ID NO: 3 under stringent conditions.
10. A vector comprising the isolated DNA according to claim 17.
11. A host cell transformed or transfected with the vector of claim 10.
12. A host cell according to claim 11, which is a prokaryotic or eukaryotic cell.
13. An antibody immunologically reactive with the bradeion protein or the analogue thereof according to claim 1.
14. A method for detecting a cancer, comprising detecting the cancer by using the DNA or the fragment thereof according to claim 5 or the antibody according to claim 13 as a tumor-specific marker.
15. The method according to claim 14, wherein the cancer is human colorectal cancer or human malignant melanoma.
16. The method according to claim 14 or 15, wherein the detection is conducted by hybridization or immunoassay.
17. An isolated DNA that encodes a protein characterized by at least 90% homology with a human-derived bradeion a protein that (A) comprises an amino acid sequence of SEQ ID NO:2 and (B) has the following properties:
(i) it is a transmembranous protein;
(ii) it has a transmembrane portion, an extracellular portion, and a cytoplasmic portion in its molecule as determined by a hydrophobicity analysis according to Kyte-Doolittle method;
(iii) it is expressed in the human adult normal brain and heart; the expression level thereof in the heart being about 10% or lower of that in the brain, while it is not expressed in other adult organs or fetus;
(iv) it induces programmed cell death when over-expressed in a cultured human brain-derived undifferentiated nerve cell line;
(v) it induces termination of cell division and aging when overexpressed in a cultured human brain-derived differentiated nerve cell;
(vi) it is located in cytoplasm in the course of the induced cell death, and forms an intracellular aggregate when overexpressed; and
(vii) it is expressed in a human colorectal cancer cell line or in a human malignant melanoma cell line, but not in leukemia, lymphoma and lung carcinoma.
US11/224,297 1998-11-16 2005-09-13 Human-derived bradeion proteins, DNA coding for the proteins, and uses thereof Abandoned US20060052584A1 (en)

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US10/190,555 US7087406B2 (en) 1998-11-16 2002-07-09 Human-derived bradeion proteins, DNA coding for the proteins, and uses thereof
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US7067650B1 (en) * 2000-11-22 2006-06-27 National Institute Of Advanced Industrial Science And Technology Ribozymes targeting bradeion transcripts and use thereof
EP1374672B8 (en) 2001-02-28 2009-11-04 National Institute of Advanced Industrial Science and Technology Drosophila strain carrying bradeion gene(s) transferred thereinto
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AU2002222647A1 (en) * 2001-12-14 2003-06-30 National Institute Of Advanced Industrial Science And Technology Antibody specific for detection of bradeion
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US6423504B1 (en) 2002-07-23

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