WO2001090318A2 - Regulation de l'enzyme similaire a la polyamine oxydase humaine - Google Patents

Regulation de l'enzyme similaire a la polyamine oxydase humaine Download PDF

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Publication number
WO2001090318A2
WO2001090318A2 PCT/EP2001/005969 EP0105969W WO0190318A2 WO 2001090318 A2 WO2001090318 A2 WO 2001090318A2 EP 0105969 W EP0105969 W EP 0105969W WO 0190318 A2 WO0190318 A2 WO 0190318A2
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enzyme
polypeptide
polyamine oxidase
seq
amino acid
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PCT/EP2001/005969
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WO2001090318A3 (fr
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Shyam Ramakrishnan
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Bayer Aktiengesellschaft
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Priority to AU72423/01A priority Critical patent/AU7242301A/en
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Publication of WO2001090318A3 publication Critical patent/WO2001090318A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/03—Oxidoreductases acting on the CH-NH group of donors (1.5) with oxygen as acceptor (1.5.3)
    • C12Y105/03013—N1-Acetylpolyamine oxidase (1.5.3.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/03—Oxidoreductases acting on the CH-NH group of donors (1.5) with oxygen as acceptor (1.5.3)
    • C12Y105/03015—N8-Acetylspermidine oxidase (propane-1,3-diamine-forming) (1.5.3.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the regulation of human polyamine oxidase-like enzyme activity for therapeutic effects.
  • Polyamines are essential for cell growth and differentiation. Intracellular levels are polyamines are regulated by enzymes such as ornithine decarboxylase and S- adenosylmethionine decarboxylase, spermidine/spermine N-acetyltransferase, and polyamine oxidase (Ferioli et al., 1999). Polyamine oxidase is FAD-dependent and transforms N 1 -acetyl derivatives of spermidine and spermine into putrescine and spermidine, respectively, also producing acetamidopropanal and hydrogen peroxide
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 4;
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6;
  • amino acid sequence shown in SEQ ID NO: 6 amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polyamine oxidase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 4;
  • amino acid sequence shown in SEQ ID NO:4 amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6;
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8;
  • Binding between the test compound and the polyamine oxidase-like enzyme polypeptide is detected.
  • a test compound which binds to the polyamine oxidase-like enzyme polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a polyamine oxidase-like enzyme polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 5;
  • nucleotide sequence shown in SEQ ID NO:5 nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 7;
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the polyamine oxidase-like enzyme through interacting with the polyamine oxidase-like enzyme mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a polyamine oxidase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 4;
  • amino acid sequence shown in SEQ ID NO: 6 amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and
  • a polyamine oxidase-like enzyme activity of the polypeptide is detected.
  • a test compound which increases polyamine oxidase-like enzyme activity of the polypeptide relative to polyamine oxidase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases polyamine oxidase-like enzyme activity of the polypeptide relative to polyamine oxidase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • a test compound is contacted with a polyamine oxidase-like enzyme product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 5;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 7; and the nucleotide sequence shown in SEQ ID NO:7.
  • Binding of the test compound to the polyamine oxidase-like enzyme product is detected.
  • a test compound which binds to the polyamine oxidase-like enzyme product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a polyamine oxidase-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 5;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 7; and >
  • Polyamine oxidase-like enzyme activity in the cell is thereby decreased.
  • the invention thus provides reagents and methods for regulating human polyamine oxidase-like enzyme which can be used inter alia, to treat cancers such as leukemias, as well as edema and volume of infarct following both transient focal cerebral ischemia and traumatic brain injury.
  • Fig. 1 shows the DNA-sequence encoding a polyamine oxidase-like enzyme polypeptide.
  • Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.1.
  • Fig. 3 shows the complement of a DNA-sequence encoding a polyamine oxidase- like enzyme polypeptide.
  • Fig. 4 shows the amino acid sequence deduced from the DNA-sequence of Fig. 3.
  • Fig. 5 shows the DNA-sequence encoding a polyamine oxidase-like enzyme polypeptide.
  • Fig. 6 shows the amino acid sequence deduced from the DNA-sequence of Fig. 5.
  • Fig. 7 shows the DNA-sequence encoding a polyamine oxidase-like enzyme polypeptide.
  • Fig. 8 shows the amino acid sequence deduced from the DNA-sequence of Fig. 7.
  • Fig. 9 shows the amino acid sequence of a protein identified by GenBank Accession No. Z35602.
  • Fig. 10 shows the BLASTX alignment of polyamine oxidase-like enzyme polypeptide of Fig. 2 with the protein identified by GenBank Accession No. Z35602 ofFig. 9.
  • Fig.11 shows the BLASTX alignment of polyamine oxidase-like enzyme polypeptide of Fig. 2 with the protein identified by GenBank Accession No. Z35602 of Fig. 9.
  • the invention relates to an isolated polynucleotide encoding a polyamine oxidase- like enzyme polypeptide and being selected from the group consisting of:
  • a polynucleotide encoding a polyamine oxidase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 4; the amino acid sequence shown in SEQ ID NO:4; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8;' and the amino acid sequence shown in SEQ ID NO: 8.
  • a polynucleotide comprising the sequence of SEQ ID NOS: 1, 5 or 7;
  • a novel polyamine oxidase-like enzyme is a discovery of the present invention.
  • Human polyamine oxidase-like enzyme is 36% identical over 156 amino acids to the protein encoded by the C. elegans cosmid clone identified with GenBank Accession No. Z35602 (SEQ ID NO:9) (FIG. 1).
  • Human polyamine oxidase-like enzyme also is 43% identical over 220 amino acids to that protein (FIG. 2).
  • Human polyamine oxidase-like enzyme comprises a functional domain which overlaps the fad-binding domain of the polyamine oxidase fragment (pdb
  • the functional domain is shown as bold and underlined in FIG. 1.
  • Polyamine oxidase-like enzyme polypeptides according to the invention comprise at least 10, 15, 19, 20, 30, 40, 50, 75, 100, 125, 132, 150, 175, 200, 225, 250, 275, 300, 325, or 350 contiguous amino acids of SEQ ID NOS:2, 4, 6, or 8 or a biologically active variant of one of those amino acid sequences, as defined below.
  • An polyamine oxidase-like enzyme polypeptide of the invention therefore can be a portion of an polyamine oxidase-like enzyme molecule, a full-length polyamine oxidase-like enzyme molecule, or a fusion protein comprising all or a portion of an polyamine oxidase-like enzyme molecule.
  • Polyamine oxidase-like enzyme variants which are biologically active, i.e., retain an polyamine oxidase-like enzyme activity, also are polyamine oxidase-like enzyme polypeptides.
  • naturally or non-naturally occurring polyamine oxidase- like enzyme variants have amino acid sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to an amino acid sequence shown in SEQ ID NO:2, 4, 6, or 8. Percent identity between a putative polyamine oxidase-like enzyme variant and an amino acid sequence of SEQ ID NO:2, 4, 6, or 8 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence.
  • Fusion proteins are useful for generating antibodies against polyamine oxidase-like enzyme amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of an polyamine oxidase-like enzyme polypeptide, including its active site and f ⁇ bronectin domains. Methods such as protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • An polyamine oxidase-like enzyme fusion protein comprises two protein segments fused together by means of a peptide bond.
  • Contiguous amino acids for use in a fusion protein can be selected from the amino acid sequences shown in SEQ ID NOS:2, 4, 6, or 8 or from a biologically active variants of those sequences, such as those described above.
  • the first protein segment can comprise at least 10, 15, 19, 20, 30, 40, 50, 75, 100, 125, 132, 150, 175, 200, 225, 250, 275, 300, 325, or 350 contiguous amino acids of SEQ ID NO:2, 4, 6, or 8 or a biologically active variant thereof.
  • a fusion protein comprises the active site of the polyamine oxidase-like enzyme or the functional domain shown in FIG. 1.
  • the first protein segment also can comprise full-length polyamine oxidase-like enzyme.
  • the second protein segment can be a full-length protein or a protein fragment or polypeptide.
  • Proteins commonly used in fusion protein construction include ⁇ - galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transf erase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags.
  • fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the polyamine oxidase-like enzyme polypeptide-encoding sequence and the heterologous protein sequence, so that the polyamine oxidase-like enzyme polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two protein segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises polyamine oxidase-like enzyme coding sequences disclosed herein in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega
  • Species homologs of human polyamine oxidase-like enzyme can be obtained using polyamine oxidase-like enzyme polynucleotides (described below) to make suitable probes or primers to screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of polyamine oxidase-like enzyme, and expressing the cDNAs as is known in the art.
  • Polynucleotides described below
  • An polyamine oxidase-like enzyme polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for an polyamine oxidase-like enzyme polypeptide.
  • Nucleotide sequences encoding the human polyamine oxidase-like enzyme polypeptides shown in SEQ ID NOS:2, 6, and 8 are shown in SEQ ID NOS:l, 5, and 7, respectively.
  • the complement of a nucleotide sequence encoding SEQ ID NO:4 is shown in SEQ ID NO: 3.
  • nucleotide sequences encoding human polyamine oxidase-like enzyme polypeptides, as well as homologous nucleotide sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to the polyamine oxidase-like enzyme coding sequences nucleotide sequences shown in SEQ ID NOS:l, 3, 5, and 7 also are polyamine oxidase-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA
  • species homologs and variants of polyamine oxidase-like enzyme polynucleotides which encode biologically active polyamine oxidase-like enzyme polypeptides also are polyamine oxidase-like enzyme polynucleotides.
  • Variants and homologs of the polyamine oxidase-like enzyme polynucleotides disclosed above also are polyamine oxidase-like enzyme polynucleotides.
  • homologous polyamine oxidase-like enzyme polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known polyamine oxidase-like enzyme polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions-2X SSC (0.3 M NaCl, 0.03
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
  • Species homologs of the polyamine oxidase-like enzyme polynucleotides disclosed herein can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of polyamine oxidase-like enzyme polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double- stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973).
  • Variants of human polyamine oxidase-like enzyme polynucleotides or polyamine oxidase-like enzyme polynucleotides of other species can therefore be identified, for example, by hybridizing a putative homologous polyamine oxidase-like enzyme polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l, 5, or 7 or the complement of SEQ ID NO: 3 to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polyamine oxidase-like enzyme polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to polyamine oxidase-like enzyme polynucleotides or their complements following stringent hybridization and/or wash conditions are also polyamine oxidase-like enzyme polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T m of the hybrid under study.
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
  • a naturally occurring polyamine oxidase-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or synthesized using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated polyamine oxidase-like enzyme polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise polyamine oxidase- like enzyme nucleotide sequences.
  • Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • Polyamine oxidase-like enzyme cDNA molecules can be made with standard molecular biology techniques, using polyamine oxidase-like enzyme mR A as a template. Polyamine oxidase-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polyamine oxidase-like enzyme polynucleotides, using either human genomic DNA or cDNA as a template.
  • polyamine oxidase-like enzyme polynucleotides can be synthesized using synthetic chemistry techniques to synthesize polyamine oxidase-like enzyme polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode an polyamine oxidase-like enzyme polypeptide having, for example, the amino acid sequence shown in SEQ ID NO:2, 4, 6, or 8 or a biologically active variant thereof.
  • the partial sequences of SEQ ID NOS:l, 3, 5, or 7 or their complements can be used to identify the corresponding full length gene from which they were derived.
  • the partial sequences can be nick-translated or end-labeled with 32 P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al, eds., Elsevier Press, N.Y., 1986).
  • a lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript
  • filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters.
  • the filters are hybridized with the labeled probe using hybridization conditions described by Davis et al, 1986.
  • the partial sequences, cloned into lambda or pBluescript can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification.
  • the resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque.
  • the colonies or plaques are selected and expanded, and the DNA is isolated from the colonies for further analysis and sequencing.
  • Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector.
  • Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.
  • the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al, Methods 3, 33-40, 1991).
  • a series of deletion clones are generated, each of which is sequenced.
  • the resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
  • PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human polyamine oxidase-like enzyme to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988).
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68 - 72 °C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations are used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • Polyamine oxidase-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of polyamine oxidase-like enzyme polynucleotides, or by direct chemical synthesis.
  • Polyamine oxidase-like enzyme polypeptides can be purified from cells, including cells which have been transfected with polyamine oxidase-like enzyme expression constructs.
  • Human embryo, joint, sarcoma, immune privileged tissue, and tumor tissue are especially useful sources of polyamine oxidase-like enzyme polypeptides.
  • a purified polyamine oxidase-like enzyme polypeptide is separated from other compounds which normally associate with the polyamine oxidase-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include; 'but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified polyamine oxidase-like enzyme polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS- polyacrylamide gel electrophoresis. Enzymatic activity of the purified preparations can be assayed, for example, as described in the specific examples, below.
  • an polyamine oxidase-like enzyme polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding polyamine oxidase-like enzyme polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N. Y, 1989.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding an polyamine oxidase-like enzyme polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
  • control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding an polyamine oxidase-like enzyme polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the polyamine oxidase-like enzyme polypeptide. For example, when a large quantity of an polyamine oxidase-like enzyme polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E.
  • coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polyamine oxidase- like enzyme polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • BLUESCRIPT Stratagene
  • pIN vectors Van Heeke & Schuster, J Biol. Chem. 264,
  • pGEX vectors can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or Factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • polyamine oxidase-like enzyme polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671- 1680, 1984; Brogue et al, Science 224, 838-843, 1984; Winter et al, Results Probl Cell Differ. 17, 85-105, 1991).
  • These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs or
  • An insect system also can be used to express an polyamine oxidase-like enzyme polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to * express foreign genes in Spodoptera fi-ugiperda cells or in Trichoplusia larvae.
  • Sequences encoding polyamine oxidase-like enzyme polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of polyamine oxidase-like enzyme polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which polyamine oxidase-like enzyme polypeptides can be expressed (Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • a number of viral-based expression systems can be utilized in mammalian host cells.
  • sequences encoding polyamine oxidase-like enzyme polypeptides can be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing an polyamine oxidase- like enzyme polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding polyamine oxidase-like enzyme polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding an polyamine oxidase-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162,
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process an expressed polyamine oxidase-like enzyme polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express polyamine oxidase-like enzyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced polyamine oxidase-like enzyme sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992 supra). Additional selectable genes have been described, for example trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol 55, 121 - 131 , 1995).
  • marker gene expression suggests that the polyamine oxidase-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed.
  • a sequence encoding an polyamine oxidase- like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an polyamine oxidase-like enzyme polypeptide can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding an polyamine oxidase-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the polyamine oxidase-like enzyme polynucleotide.
  • host cells which contain an polyamine oxidase-like enzyme polynucleotide and which express an polyamine oxidase-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding an polyamine oxidase-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding an polyamine oxidase-like enzyme polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding an polyamine oxidase-like enzyme polypeptide to detect transformants which contain an polyamine oxidase-like enzyme polynucleotide.
  • a variety of protocols for detecting and measuring the expression of an polyamine oxidase-like enzyme polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on* an polyamine oxidase-like enzyme polypeptide can be used, or a competitive binding assay can be employed.
  • sequences encoding an polyamine oxidase-like enzyme polypeptide can be cloned into a vector for the production of an mRNA probe.
  • vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase, such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
  • Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding an polyamine oxidase- like enzyme polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode polyamine oxidase-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of polyamine oxidase-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polyamine oxidase-like enzyme polypeptide can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing an polyamine oxidase-like enzyme polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography as described in Porath et al., Prot. Exp. Purif.
  • enterokinase cleavage site provides a means for purifying the polyamine oxidase-like enzyme polypeptide from the fusion protein.
  • Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993).
  • Sequences encoding an polyamine oxidase-like enzyme polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see
  • polyamine oxidase-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence.
  • polyamine oxidase-like enzyme polypeptides can be produced by direct peptide synthesis using solid-phase techniques (Merrif ⁇ eld, J Am.
  • Protein synthesis can be performed using manual techniques or by automation.
  • Peptide Synthesizer Perkin Elmer.
  • Various fragments of polyamine oxidase-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • the composition of a synthetic polyamine oxidase-like enzyme polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the polyamine oxidase-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter polyamine oxidase-like enzyme polypeptide- encoding sequences for a variety of reasons, including modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of an polyamine oxidase-like enzyme polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • An antibody which specifically binds to an epitope of an polyamine oxidase-like enzyme polypeptide can be used therapeutically, as well as in immunochemical assays, including but not limited to Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays including but not limited to Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
  • an antibody which specifically binds to an polyamine oxidase-like enzyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to polyamine oxidase-like enzyme polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate an polyamine oxidase-like enzyme polypeptide from solution.
  • Polyamine oxidase-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • a mammal such as a mouse, rat, rabbit, guinea pig, monkey, or human
  • an polyamine oxidase-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to an polyamine oxidase-like enzyme polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to an polyamine oxidase-like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to polyamine oxidase-like enzyme polypeptides.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15,
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant D ⁇ A methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology. Verhaar et al, 1995, Int. J. Cancer 61, 497-501; ⁇ icholls et al, 1993, J. Immunol. Meth. 165, 81- 91.
  • Antibodies which specifically bind to polyamine oxidase-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837,
  • chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecif ⁇ c, such as the "diabodies" described in WO
  • Antibodies of the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which an polyamine oxidase-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of polyamine oxidase-like enzyme gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.
  • Modifications of polyamine oxidase-like enzyme gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the polyamine oxidase-like enzyme gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an polyamine oxidase-like enzyme polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent polyamine oxidase-like enzyme nucleotides, can provide targeting specificity for polyamine oxidase-like enzyme mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1,- 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polyamine oxidase- like enzyme polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to an polyamine oxidase-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al,
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of an polyamine oxidase-like enzyme polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polyamine oxidase-like enzyme polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).
  • ribozyme cleavage sites within an polyamine oxidase-like enzyme RNA target are initially identified by scanning the RNA molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the polyamine oxidase-like enzyme target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. The suitability of candidate targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related; thus, upon hybridizing to the polyamine oxidase-like enzyme target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polyamine oxidase-like enzyme expression. Alternatively, if it is desired that the cells stably retain the DNA construct, it can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • the DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of polyamine oxidase-like enzyme mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • the invention provides methods for identifying modulators, i.e., candidate or test compounds which bind to polyamine oxidase-like enzyme polypeptides or polynucleotides and/or have a stimulatory or inhibitory effect on, for example, expression or activity of the polyamine oxidase-like enzyme polypeptide or polynucleotide, so as to regulate degradation of the extracellular matrix.
  • Decreased extracellular matrix degradation is useful for preventing or suppressing malignant cells from metastasizing.
  • Increased extracellular matrix degradation may be desired, for example, in developmental disorders characterized by inappropriately low levels of extracellular matrix degradation or in regeneration.
  • the invention provides assays for screening test compounds which bind to or modulate the activity of an polyamine oxidase-like enzyme polypeptide or an polyamine oxidase-like enzyme polynucleotide.
  • a test compound preferably binds to an polyamine oxidase-like enzyme polypeptide or polynucleotide. More preferably, a test compound decreases an polyamine oxidase-like enzyme activity of an polyamine oxidase-like enzyme polypeptide or expression of an polyamine oxidase-like enzyme polynucleotide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to polyamine oxidase-like enzyme polypeptides or polynucleotides or to affect polyamine oxidase-like enzyme activity or polyamine oxidase-like enzyme gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.
  • the test compound is preferably a small molecule which binds to and occupies the active site or the fad-like domain of the polyamine oxidase-like enzyme polypeptide, thereby making the active site or fibronectin domain inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the polyamine oxidase-like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to the polyamine oxidase-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to an polyamine oxidase-like enzyme polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a target polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • BIA Bimolecular Interaction Analysis
  • an polyamine oxidase-like enzyme polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs. For example, in one construct a polynucleotide encoding an polyamine oxidase-like enzyme polypeptide is fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence that encodes an unidentified protein (“prey" or "sample”) is fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • a DNA sequence that encodes an unidentified protein "prey" or "sample”
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the polyamine oxidase-like enzyme polypeptide.
  • a reporter gene e.g., LacZ
  • either the polyamine oxidase-like enzyme polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads
  • test compounds including, but not limited to, latex, polystyrene, or glass beads. Any method known in the art can be used to attach the polyamine oxidase-like enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to an polyamine oxidase-like enzyme polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • an polyamine oxidase-like enzyme polypeptide is a fusion protein comprising a domain that allows the polyamine oxidase-like enzyme polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical,
  • an polyamine oxidase-like enzyme polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polyamine oxidase-like enzyme polypeptides or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to an polyamine oxidase-like enzyme polypeptide polynucleotides, or a test compound, but which do not interfere with a desired binding site, such as the active site or a fibronectin domain of the polyamine oxidase- like enzyme polypeptide can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the polyamine oxidase-like enzyme polypeptide
  • Screening for test compounds which bind to an polyamine oxidase-like enzyme polypeptide or polynucleotide also can be carried out in an intact cell.
  • Any cell which comprises an polyamine oxidase-like enzyme polynucleotide or polypeptide can be used in a cell-based assay system.
  • An polyamine oxidase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line including neoplastic cell lines such as the colon cancer cell lines HCT116, DLD1, HT29, Caco2, SW837, SW480, and RKO, breast cancer cell lines 21-PT, 21- MT, MDA-468, SK-BR3, and BT-474, the A549 lung cancer cell line, and the H392 glioblastoma cell line, can be used.
  • An intact cell is contacted with a test compound. Binding of the test compound to an polyamine oxidase-like enzyme polypeptide or polynucleotide is determined as described above, after lysing the cell to release the polyamine oxidase-like enzyme polypeptide-test compound complex.
  • Test compounds can be tested for the ability to increase or decrease an polyamine oxidase-like enzyme activity of an polyamine oxidase-like enzyme polypeptide.
  • Polyamine oxidase-like enzyme activity can be measured, for example, using the method described in Example 2.
  • Polyamine oxidase-like enzyme activity can be measured after contacting either a purified polyamine oxidase-like enzyme polypeptide, a cell extract, or an intact cell with a test compound.
  • a test compound which decreases polyamine oxidase-like enzyme activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing extracellular matrix degradation.
  • a test compound which increases polyamine oxidase-like enzyme activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing extracellular matrix degradation.
  • test compounds which increase or decrease polyamine oxidase-like enzyme gene expression are identified.
  • An polyamine oxidase-like enzyme polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the polyamine oxidase-like enzyme polynucleotide is determined.
  • the level of expression of polyamine oxidase-like enzyme mRNA or polypeptide in the presence of the test compound is compared to the level of expression of polyamine oxidase-like enzyme mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • test compound when expression of polyamine oxidase-like enzyme mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of polyamine oxidase-like enzyme mRNA or polypeptide is less expression.
  • test compound when expression of the mRNA or protein is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of polyamine oxidase-like enzyme mRNA or polypeptide expression.
  • the level of polyamine oxidase-like enzyme mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or protein. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of an polyamine oxidase-like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into an polyamine oxidase-like enzyme polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses an polyamine oxidase-like enzyme polynucleotide can be used in a cell-based assay system.
  • the polyamine oxidase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • compositions can be used.
  • DLD1, HT29, Caco2, SW837, SW480, and RKO breast cancer cell lines 21 -PT, 21- MT, MDA-468, SK-BR3, and BT-474, the A549 lung cancer cell line, and the H392 glioblastoma cell line, can be used.
  • compositions of the invention can comprise an polyamine oxidase-like enzyme polypeptide, polyamine oxidase-like enzyme polynucleotide, antibodies which specifically bind to an polyamine oxidase-like enzyme polypeptide, or mimetics, agonists, antagonists, or inhibitors of an polyamine oxidase-like enzyme polypeptide.
  • the compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the- viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • the human polyamine oxidase-like enzyme and polynucleotides encoding it can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding human polyamine oxidase-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl.
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of a GPCR also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • Polyamines are thought to be factors which contribute to the pathogenesis of neuronal damage following traumatic brain injury (Dogan et al, 1999a). Inhibition of polyamine oxidation reduces putrescine levels, edema, and volume of infarct following both transient focal cerebral ischemia and traumatic brain injury (Dogan et al, 1999b; Rao et al., 2000). Thus, inhibition of human polyamine oxidase-like enzyme can be useful for treating such conditions.
  • Cancer Compounds interfering with the metabolism of polyamines are potential anticancer agents. Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death- inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins.
  • proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities.
  • Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.
  • the invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a polypeptide- binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • a reagent which affects polyamine oxidase-like enzyme activity can be administered to a human cell, either in vitro or in vivo, to reduce polyamine oxidase-like enzyme activity.
  • the reagent preferably binds to an expression product of a human polyamine oxidase-like enzyme gene. If the expression product is a polypeptide, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells which have been removed from the body.
  • the cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung or liver.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • from about 0.1 ⁇ g to about 10 ⁇ g of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. -Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. USA. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases extracellular matrix degradation relative to that which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in
  • LD 50 the dose lethal to 50% of the population
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.
  • Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of an polyamine oxidase-like enzyme polynucleotide or activity of an polyamine oxidase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of an polyamine oxidase-like enzyme polynucleotide or the activity of an polyamine oxidase-like enzyme polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to polyamine oxidase-like enzyme-specific mRNA, quantitative RT-PCR, immunologic detection of an polyamine oxidase-like enzyme polypeptide, or measurement of polyamine oxidase-like enzyme activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-polyamine oxidase-like enzyme polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells cellular extracts are obtained by conventional methods and the polyamine oxidase- like enzyme activity is measured as described in Ferioli et al. (1999). Briefly, the method of Suzuki et al. (Experientia 40, 838-39, 1984) is used to measure H 2 O 2 production due to N'-monoacetylspermine oxidation. Homovanillic acid is converted into a fluorescent compound in the presence of horseradish peroxidase.
  • SEQ ID NO.: 2 has a polyamine oxidase-like enzyme activity.
  • Purified polyamine oxidase-like enzyme polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Polyamine oxidase-like enzyme polypeptides comprise an amino acid sequence shown in SEQ ID NOS:2, 4, 6, or 8.
  • the test compounds comprise a fluorescent tag.
  • the samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells.
  • Binding of a test compound to an polyamine oxidase-like enzyme polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound was not incubated is identified as a compound which binds to an polyamine oxidase-like enzyme polypeptide.
  • Cellular extracts from cells comprising human polyamine oxidase-like enzyme are contacted with test compounds from a small molecule library and assayed for polyamine oxidase-like enzyme activity. Control extracts, in the absence of a test compound, also are assayed.
  • Human polyamine oxidase-like enzyme activity can be measured, for example, as described in Ferioli et al (1999). Briefly, the method of Suzuki et al. (Expe entia 40, 838-39, 1984) is used to measure H 2 O 2 production due to N 1 -monoacetylspermine oxidation. Homovanillic acid is converted into a fluorescent compound in the presence of horseradish peroxidase. Incubations are performed in the presence of pargyline and aminoguanidine, which inhibits monoamine oxidases and diamine oxidases, respectively. Fluorescence is measured at 323 nm (excitation) and 426 nm (emission).
  • a test compound which decreases polyamine oxidase-like enzyme activity of the extract relative to the control extract by at least 20% is ' identified as an polyamine oxidase-like enzyme inhibitor.
  • test compound is administered to a culture of the breast tumor cell line MDA-468 and incubated at 37 °C for 10 to 45 minutes.
  • a culture of the same type of cells incubated for the same time without the test compound provides a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18,
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled polyamine oxidase-like enzyme-specific probe at 65 ° C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1.
  • a test compound which decreases the polyamine oxidase-like enzyme -specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of polyamine oxidase-like enzyme gene expression.
  • oligonucleotides comprising at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1 is performed on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al, Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electrophoreses and ion exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined using the Limulus Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).
  • An aqueous composition containing the antisense oligonucleotides at a concentration of 0.1-100 ⁇ M is administered to a patient with a traumatic brain injury. Brain edema of the patient is decreased due to decreased polyamine oxidase- like enzyme activity.
  • Ferioli et al. Polyamine oxidase activity in lymphoid tissues of glucocorticoid-treated rats. Biochem. Pharmacol. 58, 1907-14, 1999.
  • the polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects.

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Abstract

L'invention concerne des réactifs régulant l'activité de l'enzyme similaire à la polyamine oxydase humaine et des réactifs se liant à des produits géniques d'enzyme similaire à la polyamine oxydase humaine. On peut utiliser lesdits réactifs, en outre, pour traiter des cancers, tels que la leucémie, ainsi que des oedèmes et le volume de l'infarctus suite à une ischémie cérébrale focale transitoire et une lésion cérébrale traumatique.
PCT/EP2001/005969 2000-05-26 2001-05-25 Regulation de l'enzyme similaire a la polyamine oxydase humaine WO2001090318A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104678002A (zh) * 2013-11-29 2015-06-03 沈阳药科大学 用于评估肺癌疾病的生物标记物
CN109321536A (zh) * 2018-11-13 2019-02-12 中国烟草总公司郑州烟草研究院 一种提高烟叶提取物中氧化酶酶活的提取方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990000195A1 (fr) * 1988-06-30 1990-01-11 Xandra Breakefield Adn codant le type a d'oxydase monoaminee humaine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990000195A1 (fr) * 1988-06-30 1990-01-11 Xandra Breakefield Adn codant le type a d'oxydase monoaminee humaine

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE EM_EST [Online] EMBL; ID AW449768, AC AW449768, 20 February 2000 (2000-02-20) NCI-CGAP: "UI-H-BI3-aks-f-04-0-UI.s1 NCI_CGAP_Sub5 Homo sapiens cDNA clone IMAGE:2735382 3', mRNA sequence." XP002194299 *
DATABASE EM_EST [Online] EMBL; ID HSZZ37702, AC AA332652, 18 April 1997 (1997-04-18) ADAMS M D ET AL.: "EST36627 Embryo, 8 week I Homo sapiens cDNA 5' end similar to similar to monoamine oxidase B" XP002194298 *
DATABASE SWALL [Online] EMBL; YNR2_CAEEL, 15 December 1998 (1998-12-15) GARDNER A: "Hypothetical 60.3 kDa protein R13G10.2 in chromosome III" XP002194300 cited in the application *
HOUEN G: "Mammalian Cu-containing amine oxidases (CAOs): New methods of analysis, structural relationships, and possible functions." APMIS, vol. 107, no. Suppl. 96, 1999, pages 1-46, XP008001880 ISSN: 0903-4641 *
MARTON L J ET AL.: "Polyamines as targets for therapeutic intervention." ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, vol. 35, 1995, pages 55-91, XP008001879 ISBN: 0-8243-0435-7 *
NOVOTNY W F ET AL: "Diamine oxidase is the amiloride-binding protein and is inhibited by amiloride analogues." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 13, 1994, pages 9921-9925, XP002194296 ISSN: 0021-9258 *
SEILER N: "Polyamine oxidase, properties and functions" PROGRESS IN BRAIN RESEARCH, vol. 106, 1995, pages 333-344, XP001064598 *
WANG Y ET AL.: "Cloning and characterization of a human polyamine oxidase that is inducible by polyamine analogue exposure." CANCER RESEARCH, vol. 61, no. 14, 15 July 2001 (2001-07-15), pages 5370-5373, XP002194297 ISSN: 0008-5472 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104678002A (zh) * 2013-11-29 2015-06-03 沈阳药科大学 用于评估肺癌疾病的生物标记物
CN109321536A (zh) * 2018-11-13 2019-02-12 中国烟草总公司郑州烟草研究院 一种提高烟叶提取物中氧化酶酶活的提取方法

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