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WO2004074441A2 - Amplification ou surexpression de la fusion du type septine mll (msf), septine9 et procedes connexes - Google Patents

Amplification ou surexpression de la fusion du type septine mll (msf), septine9 et procedes connexes Download PDF

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Publication number
WO2004074441A2
WO2004074441A2 PCT/US2004/004468 US2004004468W WO2004074441A2 WO 2004074441 A2 WO2004074441 A2 WO 2004074441A2 US 2004004468 W US2004004468 W US 2004004468W WO 2004074441 A2 WO2004074441 A2 WO 2004074441A2
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gene
protein
cancer
ortholog
foregoing
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PCT/US2004/004468
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WO2004074441A3 (fr
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Cristina Montagna
Kent Hunter
Thomas Ried
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Government Of The United States Of America Represented By The Secretary Department Of Health And Human Services
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Publication of WO2004074441A2 publication Critical patent/WO2004074441A2/fr
Publication of WO2004074441A3 publication Critical patent/WO2004074441A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention pertains to methods of detecting cancer, a method of inhibiting a protein, oligonucleotides for use therein, a method of inducing apoptosis, methods of testing a candidate drug for efficacy as an anti-cancer drug, and methods for evaluating the progression of cancer.
  • Conditional inactivation of the breast cancer susceptibility gene Brcal requires amplification of terminal mouse chromosome 11, which, in some cases, includes the HER2/Neu oncogene in addition to losses on mouse chromosome 4 (Xu et al., Nat. Genet. 22: 37-43 (1999); Weaver et al., Genes Chrom. Cancer 25: 251-260 (1999); and Liyanage et al, Nat. Genet. 14: 312-315 (1996)).
  • the present invention provides methods of detecting cancer in a mammal.
  • One method comprises determining whether or not the mammal has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.
  • amplification of the MSF gene, the Septin9 gene, or the ortholog of either of the foregoing is indicative of cancer.
  • Another method comprises determining whether or not the mammal has an overexpression of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.
  • overexpression of the protein or of the nucleic acid molecule is indicative of cancer.
  • the present invention also provides a method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell.
  • the method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein.
  • oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9. These oligonucleotides are suitable for use in the foregoing methods, as described herein.
  • the present invention also provides a method of inducing apoptosis in a cell, which expresses a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. The method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein.
  • Methods of testing a candidate drug for efficacy as an anti-cancer drug are also provided by the present invention.
  • One method comprises comparing (i) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell, which has an amplification in a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, before administration of the candidate drug to the cell to (ii) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal after administration of the candidate drug to the cell.
  • a decrease in the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.
  • Another method comprises comparing (i) the concentration of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a cell before administration of the candidate drug to the cell to (ii) the concentration of the protein or the nucleic acid molecule after administration of the candidate drug to the cell.
  • a decrease in the concentration of the protein or the nucleic acid molecule upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.
  • the present invention further provides methods for evaluating the progression of cancer in a mammal.
  • One method comprises monitoring the copy number of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal, which has cancer, for a period of time.
  • an increase in the copy number over the period of time indicates a progression of cancer in the mammal
  • a decrease in the copy number over the period of time indicates a regression of cancer in the mammal.
  • Another method comprises monitoring the concentration of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a mammal, which has cancer, for a period of time.
  • an increase in the concentration of the protein or of the nucleic acid molecule over the period of time indicates a progression of cancer in the mammal
  • a decrease in the concentration of the protein or of the nucleic acid molecule over the period of time indicates a regression of cancer in the mammal.
  • Figure 1 represents a listing of the nucleotide and amino acid sequences discussed herein.
  • DETAILED DESCRIPTION OF THE INVENTION [0013] The present invention provides methods of detecting cancer in a mammal.
  • One method comprises determining whether or not the mammal has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, h this method, amplification of the MSF gene, the Septin9 gene, or the ortholog of either of the foregoing is indicative of cancer.
  • Another method comprises determining whether or not the mammal has an overexpression of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.
  • overexpression of the protein or the nucleic acid molecule is indicative of cancer.
  • the Septin9 (Sept9) gene also known in the art as the SL3-3 integration site-1 (Sintl) gene, was first described in Sorensen et al, J Virol. 74(5): 2161-2168 (2000), as a gene in mouse that encodes a protein (Septin9; Sept9; Sintl) of 334 amino acids.
  • the coding sequence of the Septin9 gene and the amino acid sequence of the encoded protein are publicly available at the National Center for Biotechnology Information (NCBI) website as GenBank Accession No. NM_017380 and NP_059076, respectively, and are disclosed herein as SEQ ID NO: 3 and SEQ ID NO: 5, respectively.
  • Septin9 is a member of the Septin protein family, which comprises several proteins known to function in a variety of cellular functions, including cytokinesis, exocytosis, and other processes involving the cytoskeleton (See Field et al., Trends Cell Biol. 9(10): 387-394 (1999); and Cooper et al., J. Cell. Biol. 134(6): 1345-1348 (1996)).
  • Septin9 is evolutionarily conserved, having orthologous proteins in a variety of species, including Aspergillus nidulans, C intestinalis, catfish, cattle, Crytococcus, Drosophila melanogaster, frog, Gallus gallus, Homo sapiens, M.
  • Grisea Medaka, Neurospora crassa, pig, rat, Schizosaccharomyces pombe, and zebrafish.
  • orthologous means deriving from a common ancestor gene but present in different species.
  • orthologs refers to genes, nucleic acid molecules encoded thereby, i.e., mRNA, or proteins encoded thereby that are derived from a common ancestor gene but are present in different species.
  • the MSF gene also known as AF17q25, is the human ortholog of mouse Septin9. MSF was first described in Osaka et al, Proc. Natl. Acad. Sci. USA 96: 6428-6433 (1999), as a gene that encodes a protein of 568 amino acids.
  • the coding sequence of the MSF gene and the amino acid sequence of the protein encoded thereby are publicly available at the NCBI website as GenBank Accession No. XM_113892 and XP_113892, respectively, and are disclosed herein as SEQ ID NO: 4 and SEQ LD NO: 6, respectively.
  • amplification refers to an increase in the copy number of chromosomal sequences, i.e., genes.
  • RNA samples are known in the art. Suitable methods include, for instance, Polymerase Chain Reaction (PCR), microarray analysis, in situ hybridization, and Southern blotting, some of which are described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2 nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989.
  • PCR Polymerase Chain Reaction
  • an oligonucleotide probe designed to hybridize selectively to the gene of which an amplification is being determined i.e., the MSF gene, the Se ⁇ tin9 gene, or the ortholog of either of the foregoing
  • a sample containing genomic DNA obtained from the mammal.
  • the oligonucleotide probe and the genomic DNA of the sample are incubated under conditions that permit selective hybridization.
  • the hybridization is done under high stringency conditions.
  • high stringency conditions it is meant that the probe specifically hybridizes to target sequences of the genomic DNA in an amount that is detectably stronger than non-specific hybridization.
  • High stringency conditions would be conditions, which would distinguish a polynucleotide with an exact complementary sequence of the target sequences of the genomic DNA from those sequences containing only a few small regions (e.g., 3-10 bases) with exact complementary sequence of the targets of the genomic DNA.
  • small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases and high stringency hybridization makes them easily distinguishable.
  • Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe and the target sequences of the genomic DNA and are particularly suitable for detecting amplifications of genomic sequences. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • the way in which amplification of the complex is achieved can be through template-dependent amplification of the genomic DNA sequence that is adjacent to the nucleotide sequence to which the oligonucleotide probe hybridizes.
  • Various template-dependent processes for amplifying such DNA sequence are known in the art, a number of which are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.
  • One of the best-known processes is PCR.
  • the complex - is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis.
  • Preferred enzymes include, for example, DNA polymerases, such as T4 DNA polymerase and TaQMan DNA polymerase (Applied Biosystems, Foster City, CA). Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product, or amplicons, is produced.
  • Other methods for amplification of the genomic DNA sequence include the ligase chain reaction (LCR), which is disclosed in U.S. Patent No. 4,883,750; isothermal amplification, in which restriction endonucleases and ligases are used to achieve the amplification of molecules that contain nucleotide 5'-[ ⁇ -thio]-triphosphates in one strand (Walker et al., Proc.
  • strand displacement amplification SDA
  • RCR repair chain reaction
  • CPR cyclic probe reaction
  • the reaction Upon hybridization, the reaction is treated with RNase H, and the products of the probe are identified as distinctive products, which are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • a number of other amplification processes are contemplated; however, the invention is not limited as to which method is used.
  • the amplicons are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al. (1989), supra. Alternatively, chromatographic techniques can be employed to effect separation.
  • chromatography There are many kinds of chromatography that can be used in the context of the present inventive methods, e.g., adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas cliromatography (Freifelder, Physical Biochemistry Applications to Biochemistry and Molecular Biology, 2 n Ed., Wm. Freeman and Co., New York, N.Y. (1982)).
  • Amplicons must be visualized in order to confirm that hybridization of the oligonucleotide probe with the genomic DNA occurred.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplicons are integrally labeled with radio-, colorimetrically-, or fluorometrically-labeled nucleotides
  • the amplicons then can be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • the oligonucleotide probe that hybridizes can, alternatively, be radio-, colorimetrically-, or fluorometrically-labeled.
  • oligonucleotide probe is brought into contact with the amplicons.
  • This other probe can be conjugated to a chromophore or can be radiolabeled.
  • the other probe is conjugated to a binding partner, such as an antibody or biotin, where the other member of the binding pair carries a detectable moiety (i.e., a label).
  • a binding partner such as an antibody or biotin
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • the oligonucleotide probes described above are limited inasmuch as any oligonucleotide having any nucleotide sequence can be used as long as the oligonucleotide is hybridizable to the MSF gene, the Septin9 gene, or the ortholog of either of the foregoing of the genomic DNA.
  • nucleic acid molecule can be any nucleic acid molecule, e.g., RNA (e.g., mRNA) and cDNA, as long as it is encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • Methods of determining whether or not a mammal has an overexpression of a protein or a nucleic acid molecule are known in the art. Suitable methods include, for instance, Western blotting, in the case that an overexpression of a protein is being determined, and Northern blotting, Reverse transcription-PCR (RT-PCR), and Real-Time PCR, in the case that an overexpression of a RNA or cDNA is being determined. Such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2 nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.
  • real-time PCR is used when determining whether or not a mammal has an overexpression of a nucleic acid molecule encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, hi real-time PCR, which is described in Bustin, J. Mol. Endocrinology 25: 169-193 (2000), PCRs are carried out in 96-well plates in the presence of a fluorogenic oligonucleotide probe that hybridizes to the amplicons. The fluorescent probes are double-labeled with a reporter fluorochrome and a quencher fluorochrome.
  • the Taq polymerase which possesses 5' nuclease activity, cleaves the probe such that the quencher fluorochrome is displaced from the reporter fluorochrome, thereby allowing the latter to emit fluorescence.
  • the resulting increase in emission which is directly proportional to the level of amplicons, is monitored by a spectrophotometer.
  • the cycle of amplification at which a particular level of fluorescence is detected by the spectrophotometer is called the threshold cycle, C T . It is this value that is used to compare levels of amplicons.
  • it is desirable that the real-time PCR is carried out as in Example 2.
  • the immunobinding assays involve obtaining a sample containing the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, a peptide fragment thereof, or an antibody that specifically binds to the protein or peptide fragment thereof, and contacting the sample with an antibody that specifically binds to the protein, peptide or antibody under conditions effective to allow the formation of immunocomplexes.
  • Any suitable antibody can be used in conjunction with the present invention such that the antibody is specific for the protein or peptide fragment thereof encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing or antibody thereto.
  • Such antibodies can be made in accordance with those methods of making antibodies known in the art.
  • fragments of the antibody can be used as long as the fragment specifically binds to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • Such fragments are known in the art to include, for instance, F(ab) 2 ' fragments, single chain antibody variable region fragment (ScFv) chains, and the like.
  • the immunobinding assays for use in the present invention include methods of detecting or quantitating the immune complexes formed upon incubating the sample with the antibody.
  • the antibody used to form the immune complexes can, itself, be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the presence of or the amount of the primary immune complexes to be determined.
  • the first added component that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the first antibody.
  • the second binding ligand is, itself, often an antibody, which can be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two-step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the first antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed.
  • a number of other assays are contemplated; however, the invention is not limited as to which method is used.
  • the cancer detected by the present inventive methods can be any cancer, including, but not limited to, lung cancer, brain cancer, ovarian cancer, uterine cancer, testicular cancer, lymphoma, leukemia, stomach cancer, pancreatic cancer, skin cancer, breast cancer, adenocarcinoma, glio a, bone cancer, and the like.
  • the present inventive methods of detecting cancer are particularly useful for detecting breast cancer or adenocarcinoma.
  • the mammal can be any mammal, including, but not limited to, mammals of the order Rodentia, such as mice, the order Logomorpha, such as rabbits, the order Carnivora, including Felines (cats) and Canines (dogs), the order Artiodactyla, including Bovines (cows) and Swines (pigs), the order Perssodactyla, including Equines (horses), the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • mammals of the order Rodentia such as mice
  • the order Logomorpha such as rabbits
  • the order Carnivora including Felines (cats) and Canines (dogs)
  • the order Artiodactyla including Bovines (cows) and Swines (pigs)
  • the order Perssodactyla including Equines (horses)
  • the order Primates, Ceboids, or Simoids monkeys
  • the present invention also provides a method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell.
  • the method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein.
  • the present invention is a method of inducing apoptosis in a cell, which expresses a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.
  • the method comprises administering to the cell an inhibitor of the protein in an amount that is sufficient to inhibit the protein.
  • the phrase "inhibitor of the protein” refers to any chemical compound, natural or synthetic, that inhibits the function of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • the term “inhibit,” and words stemming therefrom do not necessarily imply 100% or complete inhibition. Rather, there are varying degrees of inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Jh this regard, inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can induce any level of inhibition.
  • the inhibitors of the protein can inhibit at least 10% of the function or activity of the protein in the absence of any inhibitors of the protein. It is more preferred that the inhibitors of the protein achieve at least 50% inhibition. Most preferably, the inhibitor of the protein inhibits 90% or more of the activity of the protein in the absence of any inliibitors of the protein.
  • the function of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing is to hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP).
  • GTP guanosine triphosphate
  • GDP guanosine diphosphate
  • the proteins encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing have GTPase activity.
  • any inhibitor of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can be employed.
  • the inhibitor of the protein can be, for instance, an inhibitor that inhibits the enzymatic activity of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • the inhibitor can be one that is known to inhibit guanosine triphosphatase (GTPase) activity, i.e., a GTPase inhibitor.
  • GTPase guanosine triphosphatase
  • Such inhibitors are known in the art and include non-hydrolyzable GTP analogs.
  • the inhibitor of the protein can be an isolated or purified oligonucleotide that can hybridize to a nucleic acid molecule encoding the protein, such that administration of the oligonucleotide will result in the inhibition of the expression of the protein.
  • the oligonucleotide can be of any length, comprising any number of nucleotides, as long as it can hybridize to the nucleic acid molecule encoding the protein.
  • the oligonucleotide that can hybridize is at least 18 nucleotides in length.
  • the oligonucleotide can be of any nucleotide sequence as long as it can hybridize to the nucleic acid molecule in a manner sufficient to inhibit the expression of the protein. While it is likely that many other oligonucleotides having different sequences are suitable for use in the present inventive methods, the oligonucleotide preferably comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the present invention also provides the isolated or purified oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 8, or SEQ ID NO: 9.
  • isolated as used herein is defined as having been removed from its natural environment.
  • purified as used herein is defined as having removed some or all other constituents.
  • oligonucleotide as used herein is defined as a polymer of DNA or RNA, (i.e., a polynucleotide), which can be single-stranded or double-stranded, synthesized or obtained from natural sources, and which can contain natural, non-natural or altered nucleotides and can contain natural, non-natural or altered internucleotide linkages.
  • a polynucleotide i.e., a polynucleotide
  • the isolated oligonucleotides of the present invention may be suitable in some instances for the isolated oligonucleotides of the present invention to comprise one or more insertions, deletions, and or substitutions. It is, furthermore, preferred that the isolated oligonucleotides of the present invention are synthesized, single-stranded polymers of DNA.
  • oligonucleotides A variety of techniques used to synthesize the present inventive oligonucleotides are known in the art. See, for example, Sambrook et al, 1989, supra; and Lemaitre et al, Proc. Natl. Acad. Sci. USA 84: 648-652 (1987). The oligonucleotides can alternatively by synthesized commercially by companies, such as Eurogentec, Belgium.
  • the inhibitor of the protein can, alternatively, be an antibody, or fragment thereof, that binds specifically to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • Antibodies suitable for use in the present inventive methods of inhibiting a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can be synthesized by methods of making antibodies that are known in the art.
  • fragment can be any fragment that binds specifically to the protein.
  • the fragment can include, for instance, an F(ab 2 )' fragment.
  • the antibodies, and fragments thereof will bind to the protein and prevent its activity by preventing a substrate or another protein from binding to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, wherein the binding of the substrate or other protein is necessary for the function of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • Inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing that are useful in the present inventive methods can be in the form of a salt, which is preferably a pharmaceutically acceptable salt.
  • suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example j3-toluenesulphonic acid.
  • Inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing that can be used in the present inventive methods can be formed as a composition, such as a pharmaceutical composition.
  • Pharmaceutical compositions containing the inhibitor of the protein can comprise more than one active ingredient, such as more than one type of inhibitor of the protein, e.g. a composition comprising a GTPase inhibitor and an isolated or purified oligonucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the pharmaceutical composition can alternatively comprise an inhibitor of the protein in combination with other pharmaceutically active agents or drugs.
  • an isolated or purified oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9 can be co- administered with a chemotherapeutic agent.
  • an antibody that binds specifically to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can be co-administered with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical agents or drugs that are destructive of malignant cells and tissues or are toxic to the causative agent of the cancer being treated, such as a virus, bacterium, or other microorganism.
  • chemotherapeutic agents include, for example, paclitaxel, cisplatin, vincristine, vinblastine, camptothecin, bleomycin, emetine, thioguanine, 5-azacytidine, hydroxyurea, cytosine arabinoside, and the like.
  • the carrier can be any suitable carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. It will be appreciated by one of ordinary skill in the art that, in addition to the following described pharmaceutical composition, the compounds and inhibitors of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
  • compositions described herein for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier will be determined in part by the particular inhibitor of the protein, as well as by the particular method used to administer the inhibitor of the protein. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present inventive methods.
  • the following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting.
  • these routes of administering the compound comprising the inhibitor of the protein are known, and, although more than one route can be used to administer a particular inhibitor of the protein, a particular route can provide a more immediate and more effective response than another route.
  • injectable formulations are among those formulations that are preferred in accordance with the present invention.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).
  • Topical formulations are well-known to those of skill in the art. Such formulations are particularly suitable in the context of the present invention for application to the skin.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the inhibitor dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • the inhibitor of the protein can be made into aerosol formulations to be administered via inhalation.
  • aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the inhibitor of the protein can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, ' hydroxypropylmethylcellulose, or carboxymethyl
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • the parenteral formulations will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • HLB hydrophile-lipophile balance
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the inhibitor of the protein, or compositions comprising such an inhibitor of the protein can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • the inhibitor of the protein of the present inventive methods can be modified in any number of ways, such that the therapeutic efficacy of the inhibitor is increased through the modification.
  • the inhibitor of the protein could be conjugated either directly or indirectly through a linker to a targeting moiety.
  • the practice of conjugating inhibitors to targeting moieties is known in the art. See, for instance, Wadwa et al, J Drug Targeting 3: 111 (1995), and U.S. Patent No. 5,087,616.
  • targeting moiety refers to any molecule or agent that specifically recognizes and binds to a cell-surface receptor, such that the targeting moiety directs the delivery of the inhibitor to a population of cells on which surface the receptor is expressed.
  • Targeting moieties include, but are not limited to, antibodies, or fragments thereof, peptides, hormones, growth factors, cytokines, and any other naturally- or non-naturally-existing ligands, which bind to cell surface receptors.
  • linker refers to any agent or molecule that bridges the inhibitor of the protein to the targeting moiety.
  • the inhibitor of the protein can be modified into a depot form, such that the manner in which the inhibitor of the protein is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No.
  • Depot forms of inhibitors can be, for example, an implantable composition comprising the inhibitor of the protein and a porous material, such as a polymer, wherein the inhibitor is encapsulated by or diffused throughout the porous material.
  • the depot is then implanted into the desired location within the body and the inhibitor of the protein is released from the implant at a predetermined rate by diffusing through the porous material.
  • the present inventive methods can comprise the administration of the inhibitor of the protein, in the presence or absence of an agent that enhances its efficacy.
  • the amount or dose of the compound or inhibitor administered should be sufficient to effect a therapeutic response in the animal over a reasonable time frame.
  • the dose of the inhibitor of the protein should be sufficient to inhibit the protein encoded by the MSF gene, the Septin9 gene or an ortholog of either of the foregoing in a cell within about 1-2 hours, if not 3-4 hours, from the time of administration.
  • the dose will be determined by the efficacy of the particular inhibitor and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • Many assays for determining an administered dose are known in the art.
  • an assay which comprises comparing the extent to which the protein is inhibited in a cell upon administration of a given dose of an inhibitor of the protein to a mammal among a set of mammals of which is each given a different dose of the inhibitor, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which the protein is inhibited upon administration of a certain dose can be assayed by measuring GTPase activity as described in Tu et al, J Biol. Chem. 276: 20160-20166 (2001).
  • the dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inhibitor. Ultimately, the attending physician will decide the dosage of the inhibitor of the protein with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inhibitor to be administered, route of administration, and the severity of the condition being treated.
  • the protein is in a cell
  • the cell can be any cell, which expresses or contains a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • the cell has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.
  • the cell also or alternatively has an overexpression of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.
  • the cell of the present inventive methods also can be a cell of any tissue type, normal or diseased, such as breast tissue, carcinoma tissue, and the like.
  • the cell can be a cultured cell (in vitro) or a cell of a cultured tissue or organ (in situ).
  • the cell can be in a living system (in vivo) or can be the living system, such as when the cell is a unicellular organism.
  • the cell is in a multicellular host. More preferably, the host is a mammal.
  • mammals include those that are discussed herein. Most preferably, the mammal is a human.
  • the cell is in a host that has cancer.
  • the cancer is treated upon inhibiting the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.
  • the term "treat,” and words stemming therefrom, as used herein, can be any level of treatment having a potential benefit or therapeutic effect.
  • the inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can treat cancer to any degree.
  • Methods of testing a candidate drug for efficacy as an anti-cancer drug are also provided by the present invention.
  • One method comprises comparing (i) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell, which has an amplification in a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, before administration of the candidate drug to the cell to (ii) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal after administration of the candidate drug to the cell.
  • a decrease in the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.
  • Another method comprises comparing (i) the concentration of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a cell before administration of the candidate drug to the cell to (ii) the concentration of the protein or the nucleic acid molecule after administration of the candidate drug to the cell.
  • a decrease in the concentration of the protein or the nucleic acid molecule upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.
  • the present invention further provides methods for evaluating the progression of cancer in a mammal
  • One method comprises monitoring the copy number of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal, which has cancer, for a period of time.
  • an increase in the copy number over the period of time indicates a progression of cancer in the mammal
  • a decrease in the copy number over the period of time indicates a regression of cancer in the mammal.
  • Another method comprises monitoring the concentration of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a mammal, which has cancer, for a period of time.
  • an increase in the concentration of the protein or the nucleic acid molecule over the period of time indicates a progression of cancer in the mammal
  • a decrease in the concentration of the protein or the nucleic acid molecule over the period of time indicates a regression of cancer in the mammal.
  • the level of amplification of a MSF gene, a Septin 9 gene or an ortholog of either of the foregoing, as well as the concentration of a protein or a nucleic acid molecule encoded thereby can be determined through any of the methods of detecting an amplification of a gene or an overexpression of a protein or a nucleic acid molecule described herein.
  • the cancer and the cell of the methods of testing a candidate drug and of the methods for evaluating the progression of cancer can be any cancer and any cell, respectively, as described herein.
  • HER2/NEU herceptin-2.Neu
  • Ccndl Cyclier Dl
  • CGH comparative genomic hybridization
  • SKY spectral karyotyping
  • MMTV mouse mammary tumor virus
  • PyVmt Polyoma virus middle T
  • FISH fluorescene in situ hybridization
  • MSF MLL Septin-like Fusion
  • NCBI National Center for Biotechnology Information
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • RPR repair chain reaction
  • CPR cyclic probe reaction
  • RT-PCR reverse transcription - PCR
  • ELISA enzyme - linked immunosorbent assay
  • ScFv single chain antibody variable region fragment
  • GTP guanosine triphosphates
  • GDP guanosine diphosphate
  • ATCC American Type Culture Collection
  • Dmin double minute
  • BAC bacterial artificial chrometics
  • Tumors and cell lines Mammary epithelial cell lines were derived from 15 different MMTV- PyVmt primary tumors and 11 cell lines were derived from their lung metastases.
  • C-myc 83, brt-1, brt-5, brt-10 and pbrt-1 were derived form the MMTV-c-Myc transgenic model and the Brcal conditional knock-out as previously reported (Weaver et al, Chromosomes Cancer 25: 251-260 (1999); and Weaver et al, Oncogene 21: 5097-5107 (2002)).
  • FF2, 4, 5, 7, 8, 9, 10, 12 and 21 cell lines were derived from the Notch4 transgenic mice described earlier (Gallahan et al, Cancer Res.
  • Metaphase chromosomes were prepared from the cell lines at passages 15-20 after colcemid arrest (1 hour, final concentration 0.1 mg/ml) and standard hypotonic treatment and fixation in methanol/acetic acid.
  • the human breast cancer cell lines BT 549, UACC 812, Zr 75-30, UACC 893, T47D, Pc3 and MDA 157 (American Type Culture Collection, (ATCC) Manassas, VA), SUM159 (University of Michigan) and MPE 600 (Vysis, Downers Grove, IL) were cultured as described on the internet at the website for the ATCC.
  • This example demonstrates a method of detecting an amplification of a Septin 9 gene and of a MSF gene.
  • mice BAC clones for FISH analysis were selected using the website for the NCBI: Mouse Genome Resources and the website for the European Bioinformatics Institute for mouse: RP23-147O23, 284L12, 101J23, 341C5, 202G21, 369E6, 382B19, 99M13, 84C12, 2814, 354K2, 333B13, 48A17, and 480Bll (Research Genetics, Carlsbad, CA).
  • the clones were labeled by nick-translation and hybridized to tumor metaphase chromosomes according to standard procedures.
  • mice Nine tumors showed jumping translocations (recipient chromosomes were 1, 4, 5, 6, 7, and X) and 4 tumors showed duplications of distal chromosome 11.
  • Two tumors (158mt, 2571mt) revealed numerous double minute chromosomes (dmin), a cytogenetic feature reflecting the amplification of oncogenes in solid tumors (8-20 dmin per cell in 50% of the cells).
  • Mouse chromosome 11 contains a plethora of breast cancer-associated genes, including the tumor suppressor genes Trp53 and Brcal and the HER2/Neu oncogene, a member of the Egf receptor family. To determine whether these genes were deleted or amplified, FISH analysis with gene-specific BAC clones was performed.
  • BAC clones specific for the cytogenetically identified region of copy number gain on chromosome 11E2 were established. Eleven BAC clones spanning a region of 17 Mbp were selected and hybridized to tumor metaphase chromosomes derived from five different tumors representing the diversity of cytogenetic abnormalities observed. In some tumors (157mt, 404mt), the BAC clones (RP23-284L12 and 84C12), containing sequence homology for Sept9 and Rac3 were present in additional copy numbers, h other tumors (158mt, 2571mt), only the Sept9- specific clone showed copy number increases.
  • the minimally amplified region comprises exclusively BAC clone RP23- 284L12, which is the clone that contains the Sept9 gene. Sequence analysis revealed that no other known genes are present in this genomic clone.
  • cDNA was reverse-transcribed from 2 mg of total RNA by using a first-strand cDNA synthesis kit (Invitrogen, Grand Island, NY) and analyzed with the ABI PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA).
  • Primers and fluorescein/6-carboxy-tetramethyl-rhodamine (FAM/TAMRA) real-time PCR probes (Synthegen, Houston, TX) for the cDNA-specific real-time quantitative PCR assay were designed using Primer express 1.5 software for human GRB2, MSF, and RAC3 genes, and for the mouse Bax, Grb2, HER2/Neu, Rac3, Sept9 and Thbsl genes (see Table 1).
  • the custom designed primers were used along with commercially available primers for mouse glyceraldehyde-3 -phosphate dehydrogenase mRNA and 18S rRNA housekeeping controls (Applied Biosystems, Foster City, CA) to determine relative mRNA expression levels.
  • cDNA derived from normal mouse brain and normal mammary glands were used for generating a standard curve and normalizing data.
  • the normal mammary gland standard was arbitrarily designated as 1.0.
  • cDNA extracted from samples 143mt, 404mt, 257 lmt and pbrt-5 were hybridized to 96-gene cDNA array filters from the mouse cancer pathway finder and cell-cycle series (SuperArray, Bethesda, MD) following the supplier's protocol.
  • this chromosome arm contains several amplicons, with either known (HER2/Neu on chromosome band 17ql3) or suspected oncogenes (Monni et al, Proc. Natl. Acad. Sci. U.S.A. 98: 5711-5716 (2001)).
  • HER2/Neu on chromosome band 17ql3 a known chromosome band 17ql3
  • suspected oncogenes Monni et al, Proc. Natl. Acad. Sci. U.S.A. 98: 5711-5716 (2001)
  • increased copy numbers distal to chromosome band 17q23 also have been frequently observed (Orsetti et al, Oncogene 18: 6262-6270 (1999)).
  • the MSF gene maps to band 17q25.
  • the quantitative PCR clearly established that MSF/Sept9 is highly expressed in six of nine cell lines, and that the overexpression of this gene can occur in the absence of genomic copy number increase.
  • MSF/Sept9 overexpression was accompanied by an increase in RAC3 expression. GRB2 mRNA levels were not increased. This finding establishes that abnormal expression of the MSF/Sept9 gene is not only involved in murine tumorigenesis, but in human carcinomas as well.
  • This example demonstrates a method of inhibiting a protein encoded by a Septin9 gene and a protein encoded by a MSF gene.
  • siRNA small interference RNA primers
  • si-GFP antisense 5'- GAACUUCAGGGUCAGCUUGCCG-dTT-3' SEQ ID NO: 7
  • Sept9 siRNA sense 5'- GUCCACUUUAAUCAAUACC dTT-3' SEQ ID NO: 8
  • antisense 5'- GGUAUUGAUUAAAGUGGAC dTT-3' SEQ ID NO:9
  • Annealing for duplex siRNA formation was performed in annealing buffer (100 mM potassium acetate, 30 mM HEPES- KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C, followed by 1 hr at 37°C.
  • Transfections of reporter plasmid and siRNA were performed with Oligofectamine (Invitrogen, Grand Island, NY) in 6-well plates. Briefly, 3 ml of oligofectamine diluted in Opti-MEM were applied to the siRNA duplex mixture (60 pmole in 3 ml annealing buffer) and incubated for 25 min at room temperature. The siRNA duplex oligofectamine mixture was added to cultured cells (40-50% confluent). The cells were seeded the previous day in tissue culture media with 10% FBS but without antibiotics. Transfection was carried out for 48 hr.
  • Septins play a role in multiple cellular functions ranging from vesicle transport to cytokinesis.
  • pathway finder array filters that allow one to query the expression levels of 96 genes simultaneously were utilized.
  • One array contained genes involved in cell-cycle regulation, and the second array contained genes that affect different cancer pathways.
  • Four cell lines were tested, three of which were derived from the group of tumors induced by PyVmt overexpression.
  • One tumor, pbrt-5 was derived from conditional Brcal knockouts. The latter and two of the PyVmt models showed 2.8-12.3- fold increased expression levels of Sept9.
  • the two groups of tumors could be distinguished by the expression levels of Thspl and Bax, which increased only in the absence of Sept9 overexpression, i.e., the one PyVmt cell line that did not overexpress Sept9 showed increased levels of Thspl and Bax.
  • the downregulation of Thspl and Bax was confirmed in a larger series of tumors using quantitative reverse transcriptase PCR. It was concluded that the downregulation of Thspl and Bax is linked to the overexpression of Sept9. Upregulation of both of Thspl and Bax induces apoptosis (de Fraipont et al, Trends Mol Med, 7: 401-407 (2001); and Mitchell et al, Cancer Res 60: 6318-6325 (2000)). [0094] This example suggests that inhibition of Septin9 leads to the upregulation of Thspl and Bax, which, in turn, leads to the induction of apoptosis.

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Abstract

L'invention concerne des procédés de détection du cancer chez un mammifère, consistant à déterminer si oui ou non le mammifère présente (i) une amplification du gène MSF, du gène Septine9 ou d'un orthologue d'un des deux gènes précités, ou (ii) une surexpression d'une protéine ou d'une molécule d'acide nucléique, la protéine ou la molécule d'acide nucléique étant codée par un gène MSF, un gène Septine9 ou un orthologue des deux gènes précités. L'amplification du gène ou la surexpression de la protéine ou de la molécule d'acide nucléique indique la présente d'un cancer. L'invention concerne également un procédé d'inhibition d'une protéine codée par un gène MSF, un gène Septine9 ou un orthologue d'un des deux gènes précités, un procédé d'induction de l'apoptose dans une cellule, des procédés de mise à l'épreuve d'un médicament candidat en vue de son efficacité comme médicament anticancéreux, et des procédés d'évaluation de l'évolution du cancer chez un mammifère.
PCT/US2004/004468 2003-02-19 2004-02-18 Amplification ou surexpression de la fusion du type septine mll (msf), septine9 et procedes connexes WO2004074441A2 (fr)

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JP2013090629A (ja) * 2005-04-15 2013-05-16 Epigenomics Ag 細胞増殖性疾患分析のための方法および核酸
CN107663538A (zh) * 2005-04-15 2018-02-06 Epi基因组股份公司 分析细胞增殖性病症的方法和核酸
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CN104046686A (zh) * 2005-04-15 2014-09-17 Epi基因组股份公司 分析细胞增殖性病症的方法和核酸
CN104046686B (zh) * 2005-04-15 2017-07-28 Epi基因组股份公司 分析细胞增殖性病症的方法和核酸
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