WO2002048354A1 - Gene suppresseur de tumeurs identifie sur le chromosome 18 - Google Patents

Gene suppresseur de tumeurs identifie sur le chromosome 18 Download PDF

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Publication number
WO2002048354A1
WO2002048354A1 PCT/AU2001/001623 AU0101623W WO0248354A1 WO 2002048354 A1 WO2002048354 A1 WO 2002048354A1 AU 0101623 W AU0101623 W AU 0101623W WO 0248354 A1 WO0248354 A1 WO 0248354A1
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tsg18
nucleic acid
seq
polypeptide
set forth
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PCT/AU2001/001623
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English (en)
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David Frederick Callen
Scott Anthony Whitmore
Gabriel Kremmidiotis
Marina Kochetkova
Joanna Crawford
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Bionomics Limited
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Priority to AU2002221342A priority Critical patent/AU2002221342A1/en
Publication of WO2002048354A1 publication Critical patent/WO2002048354A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the identification of a novel gene, TSG18, with a role in suppressing cellular proliferation via a tumour suppressor mechanism as well as a role in immune/autoimmune/inflammatory functions and associated disorders.
  • the invention is also concerned with the therapy of pathologies shown to be associated with TSG18, the screening of drugs to treat these pathologies, and the diagnosis and prognosis of pathologies shown to be associated with TSG18.
  • Tumour suppressor genes were first identified in the childhood cancer retinoblastoma. Both inherited and sporadic forms of this cancer exist, with the familial form inherited as a highly penetrant autosomal dominant trait, which was mapped to chromosome 13ql4 by genetic linkage analysis (Sparkes et al . , 1983). The observation that bilateral retinoblastoma was characteristic of the inherited disease and occurred at an early age, whereas unilateral retinoblastoma was characteristic of the sporadic form and occurred at a later age, led to the hypothesis that the tumour arises from two mutational steps (Knudson, 1971) .
  • tumour suppressor genes firmly establish the fact that a general mechanism in human cancer is the inactivation of tumour suppressor genes by LOH. Indeed LOH in tumour DNA is now taken as being strongly indicative of the presence and inactivation of a tumour suppressor gene.
  • breast cancer is the most common malignancy seen in women, affecting approximately 10% of females in the Western world.
  • the route to breast cancer is not as well mapped as that of colon cancer due in part to the histological stages of breast cancer development being less well defined.
  • breast cancer is derived from the epithelial lining of terminal mammary ducts or lobuli.
  • Hormonal influences, such as those exerted by oestrogen are believed to be important because of the marked increase in breast cancer incidence in post- enopausal women, but the initial steps in breast cancer development probably occur before the onset of menopause.
  • colon carcinoma it is believed that a number of genes need to become involved in a stepwise progression during breast tumourigenesis .
  • Cytogenetic studies have implicated loss of the long arm of chromosome 16 as an early event in breast carcinogenesis since it is found in tumours with few or no other cytogenetic abnormalities. Alterations in chromosome 1 and 16 have also been seen in several cases of duetal carcinoma i situ (DCIS), the preinvasive stage of ductal breast carcinoma. In addition, LOH studies on DCIS samples identified loss of 16q markers in 29 to 89% of the cases tested (Chen et al . , 1996; Radford efc al., 1995). Together, these findings suggest the presence of a tumour suppressor gene mapping to the long arm of chromosome 16 that is critically involved in the early development of a large proportion of breast cancers.
  • DCIS duetal carcinoma i situ
  • TSG16 gene was identified from a region of restricted LOH at 16q24.3 (International Patent Application Number PCT/AU00/01329, the contents of which are incorporated herein by reference) .
  • This gene encodes a polypeptide active in suppressing cellular proliferation.
  • TSG16 functions as a tumour suppressor gene as well as having a role in immune/autoimmune/inflammatory disorders.
  • TSG16 the TSG18 gene may also be implicated in tumour suppression as well as other functions related to TSG16, such as those associated with immune/a toimmune/inflammatory responses .
  • the present invention provides an isolated mammalian nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, which encodes a polypeptide active in suppressing cellular functions associated with cancer.
  • cellular functions associated with cancer include but are not restricted to, singly or in combination, cell proliferation, cell cycle, cell survival, invasion and growth receptor responses.
  • the suppression of these cellular functions is frequently referred to as tumour suppression function and the genes which encode proteins having this function as tumour suppressor genes.
  • the invention also encompasses an isolated mammalian nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l and which encodes a polypeptide active in suppressing cellular functions associated with cancer. These include, but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses.
  • variants will have preferably at least about 85%, and most preferably at least about 95% sequence identity to the nucleotide sequence encoding TSG18.
  • a particular aspect of the invention encompasses a variant of SEQ ID NO:l which has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% sequence identity to SEQ ID NO:l. Any one of the polynucleotide variants described above can encode an amino acid sequence, which contains at least one functional or structural characteristic of TSG18.
  • sequence identity is calculated using the BLASTN algorithm with the BLOSSUM62 default matrix.
  • the invention also encompasses an isolated mammalian nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer, including but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.
  • hybridization with 32 P labelled probes will most preferably occur at 42°C in 750 mM NaCl, 75 mM trisodium citrate, 2% SDS, 50% formamide,
  • the invention also provides an isolated mammalian nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. Still further, the invention encompasses an isolated mammalian nucleic acid molecule wherein the amino acid sequence has at least 70%, preferably 85%, and most preferably 95%, sequence identity to the sequence set forth in SEQ ID NO: 2. Preferably, sequence identity is determined using the BLASTP algorithm with the BLOSSUM62 default matrix.
  • the invention also encompasses an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.
  • nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 1
  • the invention in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.
  • the invention is concerned with an isolated nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.
  • an isolated nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO: 2.
  • an isolated nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l.
  • an isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.
  • an isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l from base 215 to base 6,401.
  • the invention provides an isolated gene comprising the nucleotide sequence set forth in SEQ ID NO:l and TSG18 control elements, particularly the cis and trans elements which act in breast tissue.
  • the nucleotide sequences of the present invention can be engineered using methods accepted in the art so as to alter TSG18-encoding sequences for a variety of purposes. These include, but are not limited to, modification of the cloning, processing, and/or expression of the gene product. PCR reassembly of gene fragments and the use of synthetic oligonucleotides allow the engineering of TSG18 nucleotide sequences. For example, oligonucleotide- mediated site-directed mutagenesis can introduce mutations that create new restriction sites, alter glycosylation patterns and produce splice variants etc.
  • the invention includes each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring TSG18, and all such variations are to be considered as being specifically disclosed.
  • the polynucleotides of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified, or may contain non-natural or derivatised nucleotide bases as will be appreciated by those skilled in the art . Such modifications include labels, methylation, intercalators, alkylators and modified linkages. In some instances it may be advantageous to produce nucleotide sequences encoding TSG18 or its derivatives possessing a substantially different codon usage than that of the naturally occurring TSG18.
  • codons may be selected to increase the rate of expression of the peptide in a particular prokaryotic or eukaryotic host corresponding with the frequency that particular codons are utilized by the host.
  • Other reasons to alter the nucleotide sequence encoding TSG18 and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA molecules, which encode TSG18 and its derivatives, or fragments thereof, entirely by synthetic chemistry.
  • Synthetic sequences may be inserted into expression vectors and cell systems that contain the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host . These elements may include regulatory sequences, promoters, 5' and 3 ' untranslated regions and specific initiation signals (such as an ATG initiation codon and Kozak consensus sequence) which allow more efficient translation of sequences encoding TSG18.
  • additional control signals may not be needed.
  • exogenous translational control signals as described above should be provided by the vector.
  • Such signals may be of various origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf et al . , 1994).
  • the present invention allows for the preparation of purified TSG18 polypeptide or protein, from the polynucleotides of the present invention or variants thereof.
  • host cells may be transfected with a DNA molecule as described above.
  • said host cells are transfected with an expression vector comprising a DNA molecule according to the invention.
  • expression vector/host systems may be utilized to contain and express sequences encoding TSG18. These include, but are not limited to, microorganisms such as bacteria transformed with plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); or mouse or other animal or human tissue cell systems.
  • Mammalian cells can also be used to express the TSG18 protein using various expression vectors including plasmid, cosmid and viral systems such as adenoviral, retroviral or vaccinia virus expression systems.
  • the invention is not limited by the host cell employed.
  • the polynucleotide sequences, or variants thereof, of the present invention can be stably expressed in cell lines to allow long term production of recombinant proteins in mammalian systems.
  • Sequences encoding TSG18 can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • the selectable marker confers resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode TSG18 may be designed to contain signal sequences which direct secretion of TSG18 through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, glycosylation, phosphorylation, and acylation.
  • Post-translational cleavage of a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells having specific cellular machinery and characteristic mechanisms for post- translational activities e.g., CHO or HeLa cells
  • ATCC American Type Culture Collection
  • vectors which direct high levels of expression of TSG18 may be used such as those containing the T5 or T7 inducible bacteriophage promoter.
  • the present invention also includes the use of the expression systems described above in generating and isolating fusion proteins which contain important functional domains of the protein. These fusion proteins are used for binding, structural and functional studies as well as for the generation of appropriate antibodies.
  • the appropriate TSG18 cDNA sequence is inserted into a vector which contains a nucleotide sequence encoding another peptide (for example, glutathionine succinyl transferase) .
  • the fusion protein is expressed and recovered from prokaryotic or eukaryotic cells.
  • the fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the TSG18 protein obtained by enzymatic cleavage of the fusion protein.
  • TSG18 may also be produced by direct peptide synthesis using solid-phase techniques. Automated synthesis may be achieved by using the ABI 431A Peptide Synthesizer (Perkin-Elmer) . Various fragments of TSG18 may be synthesized separately and then combined to produce the full length molecule. According to the present invention there is provided
  • an isolated mammalian polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, active in suppressing cellular functions associated with cancer, including but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses .
  • the invention also encompasses an isolated mammalian polypeptide active in suppressing cellular functions associated with cancer, including but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses and having at least 70%, more preferably at least 85%, and most preferably at least 95%, sequence identity with the amino acid sequence set forth in SEQ ID NO: 2.
  • sequence identity is determined using the BLASTP algorithm with the BLOSSUM62 default matrix.
  • the invention provides an isolated polypeptide, comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.
  • the invention provides an isolated polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% identity with the amino acid sequence set forth in SEQ ID NO: 2.
  • polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2.
  • a method of preparing a polypeptide comprising the steps of :
  • TSG18 protein or fragments thereof can then be used in further biochemical analyses to establish secondary and tertiary structure for example by x-ray crystallography of TSG18 protein or by nuclear magnetic resonance (NMR) . Determination of structure allows for the rational design of pharmaceuticals to interact with the protein, alter protein charge configuration or charge interaction with other proteins, or to alter its function in the cell.
  • NMR nuclear magnetic resonance
  • TSG18 Chemical and structural similarity in the context of sequences and motifs, exists between regions of both TSG18 and TSG16 and the ankyrin repeat containing family of proteins including BARD1 and 1KB.
  • TSG16 interacts via its ankyrin repeats with members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family, which are proteins that bind to STAT (signal transducer and activator of transcription) proteins to inhibit the immunological responses mediated by cytokine signalling.
  • PIAS activated signal transducer and activator of transcription
  • STAT signal transducer and activator of transcription
  • TSG18 shows significant homology to TSG16, particularly across its ankyrin domains, TSG18 is likely to also interact with members of the PIAS family. Therefore abnormalities of TSG18 function may be associated not only with cancer but also with immune diseases including autoimmune/inflammatory disorders .
  • the invention has provided the nucleotide and protein sequence of the TSG18 gene and therefore enables therapeutic methods for the treatment of all diseases shown to be associated with abnormalities of TSG18 function, including cancer and immune/autoimmune/inflammatory disorders and also enables methods for the diagnosis or prognosis of all diseases shown to be associated with abnormalities of TSG18 function.
  • cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, and stomach.
  • Other cancers may include those of the adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland.
  • Immune/autoimmune/inflammatory disorders include acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophili
  • TSG18 In the treatment of diseases shown to be associated with decreased TSG18 expression and/or activity, it is desirable to increase the activity and/or expression of TSG18. In the treatment of disorders shown to be associated with increased TSG18 expression and/or activity, it is desirable to decrease the activity and/or expression of TSG18.
  • Enhancing TSG18 gene or protein function Enhancing, stimulating or re-activating TSG18 gene or protein function can be achieved in a variety of ways.
  • administration of an isolated DNA molecule, as described above, to a subject in need of such treatment may be initiated.
  • TSG18 is administered to a subject to treat or prevent a disorder shown to be associated with decreased activity and/or expression of TSG18.
  • a vector capable of expressing TSG18 or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder shown to be associated with decreased activity and/or expression of
  • TSG18 including, but not limited to, those described above.
  • Transducing retroviral vectors are often used for somatic cell gene therapy because of their high efficiency of infection and stable integration and expression.
  • the full length TSG18 gene, or portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest.
  • Other viral vectors can be used and include, as is known in the art, adenoviruses, adeno-associated virus, vaccinia virus, papovaviruses, lentiviruses and retroviruses of avian, murine and human origin.
  • Gene therapy would be carried out according to established methods (Friedman, 1991; Culver, 1996) .
  • a vector containing a copy of the TSG18 gene linked to expression control elements and capable of replicating inside the cells is prepared.
  • the vector may be replication deficient and may require helper cells for replication and use in gene therapy.
  • Gene transfer using non-viral methods of infection can also be used. These methods include direct injection of DNA, uptake of naked DNA in the presence of calcium phosphate, electroporation, protoplast fusion or liposome delivery. Gene transfer can also be achieved by delivery as a part of a human artificial chromosome or receptor- mediated gene transfer. This involves linking the DNA to a targeting molecule that will bind to specific cell- surface receptors to induce endocytosis and transfer of the DNA into mammalian cells .
  • One such technique uses poly-L-lysine to link asialoglycoprotein to DNA.
  • An adenovirus is also added to the complex to disrupt the lysosomes and thus allow the DNA to avoid degradation and move to the nucleus.
  • the invention provides a method for the treatment of a disorder shown to be associated with decreased activity and/or expression of TSG18, comprising administering a polypeptide as described above, or an agonist thereof, to a subject in need of such treatment .
  • the invention provides the use of a polypeptide as described above, or an agonist thereof, in the manufacture of a medicament for the treatment of a disorder shown to be associated with decreased activity and/or expression of TSG18.
  • composition comprising a polypeptide as described above, typically a substantially purified TSG18, and a pharmaceutically acceptable carrier may be administered.
  • compositions in accordance with the present invention are prepared by mixing TSG18 or active fragments or variants thereof having the desired degree of purity, with acceptable carriers, excipients, or stabilizers which are well known.
  • Acceptable carriers, excipients or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including absorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitrol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG) .
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including absorbic acid
  • a suitable agonist may also include a small molecule that can mimic the function of TSG18.
  • Inhibiting the function of a mutated gene or protein can be achieved in a variety of ways .
  • a method of treating a disorder shown to be associated with increased activity and/or expression of TSG18 comprising administering an antagonist of TSG18 to a subject in need of such treatment .
  • an antagonist of TSG18 in the manufacture of a medicament for the treatment of a disorder shovm to be associated with increased activity and/or expression of TSG18.
  • an isolated DNA molecule which is the complement of any one of the DNA molecules described above and which encodes an RNA molecule that hybridises with the mRNA encoded by TSG18, may be administered to a subject in need of such treatment.
  • an isolated DNA molecule which is the complement of a DNA molecule of the invention and which encodes an RNA molecule that hybridises with the mRNA encoded by TSG18, in the manufacture of a medicament for the treatment of a disorder shown to be associated with increased activity and/or expression of TSG18.
  • a vector expressing the complement of the polynucleotide encoding TSG18 may be administered to a subject to treat or prevent a disorder shown to be associated with increased activity and/or expression of TSG18 including, but not limited to, those described above.
  • Antisense strategies may use a variety of approaches including the use of antisense oligonucleotides, ribozymes, DNAzy es, injection of antisense RNA and transfection of antisense RNA expression vectors. Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (For example, see Goldman et al . , 1997).
  • TSG18 comprising administering an antagonist of TSG18 to a subject in need of such treatment.
  • an antagonist of TSG18 in the manufacture of a medicament for the treatment of a disorder shown to be associated with increased activity and/or expression of TSG18.
  • purified protein according to the invention may be used to produce antibodies which specifically bind TSG18. These antibodies may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues that express TSG18. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric and single chain antibodies as would be understood by the person skilled in the art .
  • various hosts including rabbits, rats, goats, mice, humans, and others may be immunized by injection with a protein of the invention or with any fragment or oligopeptide thereof, which has immunogenic properties.
  • Various adjuvants may be used to increase immunological response and include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin.
  • Adjuvants used in humans include BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • the oligopeptides, peptides, or fragments used to induce antibodies to TSG18 have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of amino acids from these proteins may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced. Monoclonal antibodies to TSG18 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Antibodies include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • Antibodies may also 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. (For example, see Orlandi et al., 1989; Winter et al., 1991).
  • Antibody fragments which contain specific binding sites for TSG18 may also be generated.
  • such fragments include, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments .
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (For example, see Huse et al . , 1989).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between a protein and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed.
  • any of the genes, proteins, antagonists, antibodies, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents may be made by those skilled in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, therapeutic efficacy with lower dosages of each agent may be possible, thus reducing the potential for adverse side effects.
  • Drug screening any of the genes, proteins, antagonists, antibodies, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents may be made by those skilled in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, therapeutic efficacy with lower dosages of each agent may be possible, thus reducing the potential for adverse side effects.
  • peptides of the invention are useful for screening of candidate pharmaceutical agents in a variety of techniques for the treatment of TSG18-related disorders.
  • Such techniques include, but are not limited to, utilising eukaryotic or prokaryotic host cells that are stably transformed with recombinant molecules expressing the TSG18 polypeptide or fragment thereof, preferably in competitive binding assays.
  • Binding assays will measure for the formation of complexes between the TSG18 polypeptide, or fragments thereof, and the agent being tested, or will measure the degree to which an agent being tested will interfere with the formation of a complex between the TSG18 polypeptide, or fragment thereof, and a known ligand.
  • Another technique for drug screening provides high- throughput screening for compounds having suitable binding affinity to the TSG18 polypeptide (see PCT published application WO84/03564) .
  • large numbers of small peptide test compounds can be synthesised on a solid substrate and can be assayed through TSG18 polypeptide binding and washing. Bound TSG18 polypeptide is then detected by methods well known in the art .
  • purified polypeptides can be coated directly onto plates to identify interacting test compounds .
  • An additional method for drug screening involves the use of host eukaryotic cell lines which carry mutations in the TSG18 gene. The host cell lines are also defective at the polypeptide level. Other cell lines may be used where the gene expression of TSG18 can be switched off. The host cell lines or cells are grown in the presence of various drug compounds and the rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of defective cells.
  • Mutant TSG18 polypeptide may also be used for screening compounds developed as a result of combinatorial library technology. This provides a way to test a large number of different substances for their ability to modulate activity of a polypeptide.
  • the use of peptide libraries is preferred (see patent WO97/02048) with such libraries and their use known in the art.
  • a substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature.
  • Non- peptide "small molecules" are often preferred for many in vivo pharmaceutical applications.
  • a mimic or mimetic of the substance may be designed for pharmaceutical use.
  • the design of mimetics based on a known pharmaceutically active compound ("lead" compound) is a common approach to the development of novel pharmaceuticals. This is often desirable where the original active compound is difficult or expensive to synthesise or where it provides an unsuitable method of administration.
  • particular parts of the original active compound that are important in determining the target property are identified. These parts or residues constituting the active region of the compound are known as its pharmacophore .
  • the pharmacophore structure is modelled according to its physical properties using data from a range of sources including x-ray diffraction data and NMR.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be added. The selection can be made such that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, does not degrade in vivo and retains the biological activity of the lead compound. Further optimisation or modification can be carried out to select one or more final mimetics useful for in vivo or clinical testing.
  • anti-idiotypic antibodies anti-ids
  • the binding site of the anti-ids would be expected to be an analogue of the original binding site.
  • the anti-id could then be used to isolate peptides from chemically or biologically produced peptide banks.
  • any of the therapeutic methods described above may 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 sequences of the invention may be used for the diagnosis or prognosis of these disorders, or a predisposition to such disorders.
  • disorders include, but are not limited to cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, and stomach.
  • cancers may include those of the adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland.
  • Immune/autoimmune/inflammatory disorders include acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis. autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophili
  • the polynucleotides that may be used for diagnostic or prognostic purposes include oligonucleotide sequences, genomic DNA and complementary RNA and DNA molecules.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which mutations in TSG18 or abnormal expression of TSG18 may be correlated with disease.
  • Genomic DNA used for the diagnosis or prognosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material.
  • the DNA may be isolated and used directly for detection of a specific sequence or may be amplified by the polymerase chain reaction (PCR) prior to analysis.
  • PCR polymerase chain reaction
  • RNA or cDNA may also be used, with or without PCR amplification.
  • RNAse protection To detect a specific nucleic acid sequence, direct nucleotide sequencing, reverse transcriptase PCR (RT-PCR) , hybridization using specific oligonucleotides, restriction enzyme digest and mapping, PCR mapping, RNAse protection, and various other methods may be employed. Oligonucleotides specific to particular sequences can be chemically synthesized and labeled radioactively or non-radioactively and hybridised to individual samples immobilized on membranes or other solid-supports or in solution. The presence, absence or excess expression of TSG18 may then be visualized using methods such as autoradiography, fluorometry, or colorimetry.
  • RT-PCR reverse transcriptase PCR
  • the nucleotide sequences encoding TSG18 may be useful in assays that detect the presence of associated disorders, particularly those mentioned previously.
  • the nucleotide sequences encoding TSG18 may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding TSG18 in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • the nucleotide sequence of the TSG18 gene can be compared between normal tissue and diseased tissue in order to establish whether the patient expresses a mutant gene.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding TSG18, under conditions suitable for hybridization or amplification.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used.
  • Another method to identify a normal or standard profile for expression of TSG18 is through quantitative RT-PCR studies. RNA isolated from body cells of a normal individual, particularly RNA isolated from tumour cells, is reverse transcribed and real-time PCR using oligonucleotides specific for the TSG18 gene is conducted to establish a normal level of expression of the gene.
  • Standard values obtained in both these examples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays or quantitative RT-PCR studies may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding TSG18 or closely related molecules may be used to identify nucleic acid sequences which encode TSG18.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding TSG18, allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the TSG18 encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:1 or from genomic sequences including promoters, enhancers, and introns of the TSG18 gene (SEQ ID Numbers: 3-11) .
  • Means for producing specific hybridization probes for DNAs encoding TSG18 include the cloning of polynucleotide sequences encoding TSG18 or TSG18 derivatives into vectors for the production of mRNA probes . Such vectors are known in the art, and are commercially available.
  • Hybridization probes may be labelled by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, or other methods known in the art .
  • a polypeptide as described above in the diagnosis or prognosis of a disorder shown to be associated with TSG18, or a predisposition to such disorders.
  • diagnosis or prognosis can be achieved by monitoring differences in the electrophoretic mobility of normal and mutant proteins. Such an approach will be particularly useful in identifying mutants in which charge substitutions are present, or in which insertions, deletions or substitutions have resulted in a significant change in the electrophoretic migration of the resultant protein.
  • diagnosis may be based upon differences in the proteolytic cleavage patterns of normal and mutant proteins, differences in molar ratios of the various amino acid residues, or by functional assays demonstrating altered function of the gene products.
  • antibodies that specifically bind TSG18 may be used for the diagnosis or prognosis of disorders characterized by abnormal expression of TSG18, or in assays to monitor patients being treated with TSG18 or agonists, antagonists, or inhibitors of TSG18.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic or prognostic assays for TSG18 include methods that utilize the antibody and a label to detect TSG18 in human body fluids or in extracts of cells or tissues .
  • the antibodies may be used with or without modification, and may be labelled by covalent or non-covalent attachment of a reporter molecule.
  • TSG18 A variety of protocols for measuring TSG18, including ⁇ LISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of TSG18 expression.
  • Normal or standard values for TSG18 expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to TSG18 under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of TSG18 expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Once an individual has been diagnosed with a disorder, effective treatments can be initiated.
  • TSG18 may include administering a selective agonist to the mutant TSG18 so as to restore its function to a normal level or introduction of wild-type TSG18, particularly through gene therapy approaches as described above.
  • a vector capable of expressing the appropriate full length TSG18 gene or a fragment or derivative thereof may be administered.
  • substantially purified TSG18 polypeptide and a pharmaceutically acceptable carrier may be administered as described above or drugs which can replace the function of, or mimic the action of TSG18 may be administered.
  • the affected individual may be treated with a selective antagonist such as an antibody to the relevant protein or an antisense (complement) probe to the corresponding gene as described above, or through the use of drugs which may block the action of TSG18.
  • a selective antagonist such as an antibody to the relevant protein or an antisense (complement) probe to the corresponding gene as described above, or through the use of drugs which may block the action of TSG18.
  • cDNAs, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose or prognose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art. (For example, see Schena et al., 1996; Heller et al., 1997).
  • the present invention also provides for the production of genetically modified (knock-out, knock-in and transgenic), non-human animal models transformed with the DNA molecules of the invention. These animals are useful for the study of the TSG18 gene function, to study the mechanisms of disease as related to the TSG18 gene, for the screening of candidate pharmaceutical compounds, for the creation of explanted mammalian cell cultures which express the protein or mutant protein and for the evaluation of potential therapeutic interventions.
  • the TSG18 gene may have been inactivated by knock-out deletion, and knock-out genetically modified non-human animals are therefore provided.
  • Animal species which are suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates such as monkeys and chimpanzees.
  • genetically modified mice and rats are highly desirable due to their relative ease of maintenance and shorter life spans.
  • transgenic yeast or invertebrates may be suitable and preferred because they allow for rapid screening and provide for much easier handling.
  • non-human primates may be desired due to their similarity with humans.
  • a mutant human gene as genomic or minigene cDNA constructs using wild type or mutant or artificial promoter elements or insertion of artificially modified fragments of the endogenous gene by homologous recombination.
  • the modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase.
  • a mutant version of TSG18 can be inserted into a mouse germ line using standard techniques of oocyte microinjection or transfection or microinjection into embryonic stem cells.
  • homologous recombination using embryonic stem cells may be applied.
  • one or more copies of the mutant or wild type TSG18 gene can be inserted into the pronucleus of a just-fertilized mouse oocyte. This oocyte is then reimplanted into a pseudo-pregnant foster mother. The liveborn mice can then be screened for integrants using analysis of tail DNA for the presence of human TSG18 gene sequences .
  • the transgene can be either a complete genomic sequence injected as a YAC, BAC, PAC or other chromosome DNA fragment, a cDNA with either the natural promoter or a heterologous promoter, or a minigene containing all of the coding region and other elements found to be necessary for optimum expression.
  • a nucleic acid encoding a mutant TSG18 polypeptide which cannot form a complex with a wild- ype protein with which wild-type TSG18 does form a complex.
  • the protein is a member of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins, particularly PIAS1 or PIAS3.
  • a mutant TSG18 polypeptide which cannot form a complex with a wild-type protein with which wild- type TSG18 does form a complex.
  • the protein is a member of the PIAS family, particularly PIAS1 or PIAS3.
  • a complex of wild-type TSG18 and a PIAS protein particularly PIASl or PIAS3.
  • a complex of wild-type TSG18 and SUMO E3 ligase an interaction mediated by the binding of TSG18 to PIASl.
  • Figure 1A-1G Genomic sequence of TSG18. Intron sequences are indicated in lowercase letters whereas exonic sequences are in uppercase. Intervals between larger introns are shortened by stretches of v's. The sequence of intron 1 is not known at this time.
  • Figure 2. Northern blot analysis of the TSG18 gene.
  • RNA size markers are indicated on the left of the blot while the size of the mRNA species corresponding to TSG18 are indicated on the right of the blot.
  • 1 Heart; 2: Brain; 3: Placenta; 4: Lung; 5: Liver; 6: Skeletal Muscle; 7: Kidney; 8: Pancreas .
  • Figure 3 Amino acid sequence alignment between the
  • TSG18 and TSG16 proteins are boxed.
  • the ankyrin domain of TSG18 is highlighted in bold italics.
  • the tblastn algorithm and the BLOSSUM62 default matrix were used.
  • the TSG16 gene identified a chromosome 16 specific BAC (accession number ACO09098) in addition to two BAC clones from unknown chromosomal origin (accession numbers AC023256 and AC021009) . As these 2 BAC clones were 100% homologous to TSG16 sequences, they most likely arise from chromosome 16 also.
  • cDNA clones were identified that had identical 3 ' origins and were derived from a variety of tissues. Further homology searches of the non-redundant database identified additional cDNA clones. These included AB020681, which originated at the same 3' end as the previously identified EST cluster, as well as AK024808 which appeared to be located further 5' to these clones. Although AB020681 and AK024808 did not appear to overlap with each other, both showed homology to the TSG16 amino acid sequence. This suggests that they both are derived from a TSG16 homologous gene located on chromosome 18, namely TSG18. To confirm this, additional database screening was attempted to link the AB020681 and AK024808 cDNA clones.
  • the TSG18 gene is 9,035 base pairs in length (SEQ ID NO:l) and is composed of 11 exons that span approximately 80 Kb of genomic DNA.
  • Table 1 shows the genomic structure of the gene indicating the size of exons and introns. Analysis of exons 1 to 11 indicate an open reading frame of 6,186 nucleotides with a start codon in exon 1 at base 215 and a stop codon in exon 11 at base 6,401. This defines a protein of 2,062 amino acids (SEQ ID NO:2). Partial genomic DNA sequences indicating exon/intron junctions for TSG18 are set forth in Figure 1A-1G and SEQ ID Numbers: 3-11.
  • cDNA clones are present in dbEST which belong to the TSG18 gene.
  • An observation of the tissues these cDNA clones were derived from indicates that the gene is expressed in the aorta, brain, breast, eye, germ cells, heart, kidney, lymphatic tissue, pancreas, placenta, prostate, smooth muscle, stomach, testis, tonsil, uterus, bone marrow, lung, ovary and thymus.
  • the TSG18 nucleotide sequence also detects a number of mouse cDNA clones as well as clones derived from cow, rat, chicken, frog and pig cDNA libraries. The homology is as high as 91%, which suggests that this gene is highly conserved across species.
  • TSG16 and TSG18 indicate a high degree of conservation at both the physical and sequence level between the two proteins across their entire length. Both genes are of similar size (2,663 amino acids and 2,062 amino acids respectively), both genes span large genomic intervals, both genes share an extremely large exon (6,578 nucleotides and 4,721 nucleotides respectively) and both genes have extremely high exon/intron structure conservation. Amino acid sequence alignment between the two genes indicates that both have the same 5' and 3' termini, with the difference in length dictated by the fact that TSG18 has a shorter "large" exon. Sequence identity between TSG16 and TSG18 ranged from 22-67% across their entire lengths ( Figure 3) with highest homology occurring between the ankyrin domains present in each gene (74% identity and 87% similarity) .
  • TSG18 The amino acid sequence of TSG18 was used for in silico analysis to identify regions of homology to previously characterised proteins other than TSG16. Initially the BLASTP program was used to search for homologous sequences in the GenBank non-redundant protein database (http://www.ncbi.nlm.nih.gov/index.html). The search identified a number of proteins that exhibited homology to TSG18 in the region containing an ankyrin repeat motif (ANK), including Ankyrin 1, BARDl and IKB-R.
  • ANK ankyrin repeat motif
  • Analyses of the TSG18 protein using the PfScan program confirmed the presence of an ankyrin repeat domain with a PfScan score of 38.382.
  • Ankyrin 1 contains 23 ANK repeats and exhibits a PfScan score of 249.73
  • BARDl with 3 ANK repeats exhibits a score of 36.2
  • I ⁇ - ⁇ with 4 ANK repeats a score of 41.9.
  • Ankyrin repeats have been identified in over 400 proteins ranging from transcription factors to toxins.
  • the main function of ANK domains is to provide a site for protein-protein interactions.
  • the ANK repeat unit contains 33 amino acids with a conserved consensus of XGXTPLHXAAXXGHXXXV/AXXLLXXGAXXN/DXXXX (where X can be any amino acid) .
  • the number of repeats within a protein can vary widely from 3 in the rat Vlp to 23 in the human Ankyrin protein.
  • TSG18, like TSG16 contains 3 ANK repeat units.
  • X-ray structural analysis of the human p53 binding protein (53BP2), iKB- , and the yeast protein Swi6 ankyrin domains indicate that the ANK domain is an L- shaped structure, which consists of ⁇ -hairpins and ⁇ - helices.
  • the ⁇ -helices create a pit which is surrounded by ⁇ -helical protrusions thus providing a docking site for interacting proteins.
  • the highest BLASTP homology scores for TSG18 to characterized proteins were obtained with the ANK domains present in the proteins BARDl and KB-R.
  • homology extended to those residues, which are often non- conserved within the ANK motif. This suggests that TSG18, BARDl and/or IKB-R may have common protein interaction partners .
  • BNLS nuclear localisation signals
  • TSG18 is a nuclear protein that contains an ANK domain which may mediate interactions with proteins that also interact with the proteins BARDl and/or IKB-R.
  • BARDl has been shown to interact with the tumour suppressor protein BRCAl via its RING finger motif.
  • the ankyrin domain of BARDl is responsible for the interaction with CstF-50, a member of the Cleavage stimulation factor complex.
  • This complex along with RNA polymerase II, has been shown to be involved in polyadenylation of mRNA precursors whereby the CstF complex specifies the site of processing (Takagaki et al., 1989).
  • BARDl and BRCAl also interact with RNA polymerase II and the BARDl/CstF-50 interaction has been shown to inhibit polyadenylation in vitro (Kleiman and Manley, 1999) .
  • NF-KB transcription factors include a collection of proteins conserved from humans to Drosophila (reviewed in Gilmore, 1999) . These transcription factors are notably absent in yeast and C. elegans, probably as a result of the primary function of these factors, which is to control a variety of physiological aspects of immune responses, inflammation, and growth and development.
  • the NF-KB proteins are related through a highly conserved DNA-binding/dimerisation domain called the Rel homology (RH) domain.
  • RH Rel homology
  • NF-KB transcription factors bind to 10 base pair DNA sites (KB sites) as dimers.
  • the activity of NF-KB is tightly regulated by interaction with inhibitory KB proteins.
  • Activation of IKK leads to the phosphorylation of two specific serine residues near the N terminus of l ⁇ B- ⁇ , which targets l ⁇ B- ⁇ for ubiquitination and degradation by the proteasome.
  • the unmasked NF-KB can then enter the nucleus to activate target gene expression.
  • There is a body of evidence linking deregulated NF-KB activity to oncogenesis in mammalian systems (reviewed in Gilmore et al . , 1999).
  • alterations affecting the expression or function of the 1KB family members Bcl-3, iKB- ⁇ and iKB- ⁇ have also been observed in several cancers.
  • tumour cells that display constitutively high levels of nuclear NF-KB activity due to hyperactivation of the NF-KB signaling pathway or to inactivating mutations in the regulatory 1KB subunits (reviewed in Rayet and Gelinas, 1999) .
  • the IKB-R protein was originally cloned by differential expression from a human lung epithelial cell line and has been shown to inhibit the DNA binding ability of an NF-KB complex present in nuclear extracts prepared from interleukin-1 activated HeLa cells (Ray et al., 1995) . It is therefore possible that this member of the 1KB family may play an important role in the regulation of NF- KB function in epithelial cells.
  • TSG18 may be a key protein in either or both of the pathways to which these important proteins belong, particularly in epithelial cells. Based on past studies it is possible that TSG18, like TSG16, may form a link connecting the BARD1/BRCA1 and NF-KB/IKB pathways .
  • Mammalian expression vectors containing TSG18 cDNA can be transfected into breast or other carcinoma cell lines that have lesions in the gene. Phenotypic reversion in cultures (eg cell morphology, growth of transformants in soft-agar, growth rate) and in animals (eg tumourigenicity in nude mice) is examined. These studies can utilise wild-type or mutant forms of TSG18. Deletion and missense mutants of TSG18 can be constructed by in vitro mutagenesis.
  • TSG18 protein to bind known and unknown protein can be examined. Due to the presence of an ANK domain region in TSG18 it is most likely that this gene participates in protein/protein interactions and procedures such as the yeast two-hybrid system are used to discover and identify any functional partners.
  • the principle behind the yeast two-hybrid procedure is that many eukaryotic transcriptional activators, including those in yeast, consist of two discrete modular domains. The first is a DNA-binding domain that binds to a specific promoter sequence and the second is an activation domain that directs the RNA polymerase II complex to transcribe the gene downstream of the DNA binding site. Both domains are required for transcriptional activation as neither domain can activate transcription on its own.
  • the gene of interest or parts thereof (BAIT)
  • BAIT the gene of interest or parts thereof
  • a second gene, or number of genes, such as those from a cDNA library (TARGET) is cloned so that it is expressed as a fusion to an activation domain. Interaction of the protein of interest with its binding partner brings the DNA-binding peptide together with the activation domain and initiates transcription of the reporter genes.
  • the first reporter gene will select for yeast cells that contain interacting proteins (this reporter is usually a nutritional gene required for growth on selective media) .
  • the second reporter is used for confirmation and while being expressed in response to interacting proteins it is usually not required for growth.
  • yeast two- hybrid analysis using the displayGREEN-BASICTM Two-Hybrid System kit identified TSG16 ANK domain interacting proteins. From sequence analysis of these clones, members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins were identified to be interacting with the ANK domain of TSG16, in particular PIASl and PIAS3.
  • the PIASl protein has been found to bind to p53 (Kayho et al . , 2001), a tumour suppressor protein that plays a critical role in carcinogenesis.
  • the amount of p53 and its transcriptional activity are increased in response to genotoxic stress through mechanisms such as phosphorylation, acetylation and sumoylation.
  • Sumoylation involves the binding of an ubiquitin-like protein (SUMO- 1/sentrin/PICl) to target proteins.
  • SUMO-1 conjugation to substrate protein appears to occur as in the ubiquitination reaction, with the E3 enzyme (SUMO ligase) being a key enzyme for recognition of the substrate to be sumoylated.
  • TSG16 A component of this enzyme has recently been shown to be the PIASl protein (Kahyo et al., 2001).
  • TSG16 As TSG16 also bind PIASl, it suggests that the either the TSG16 protein is a component of the SUMO E3 ligase complex and is involved in sumoylation of proteins such as p53, or PIAS proteins may affect the transcriptional activity of a range of tumour suppressor molecules, including p53 and TSG16.
  • the PIAS family of proteins have also been shown to specifically inhibit STAT (signal transducer and activator of transcription) protein signaling (Liu et al . , 1998).
  • STAT proteins are a family of cytoplasmic transcription factors that become activated, by tyrosine phosphorylation, following the binding of cytokines to their cell surface receptors. After phosphorylation, STATs dimerise, translocate to the nucleus and bind specific DNA elements in the promoters of responsive genes to activate transcription. STATl for example, plays an important role in mediating interferon-gamma (IFN- ⁇ ) , interleukin-6 (IL-6) type cytokine and epidermal growth factor (EGF) -dependant biological responses while STAT3 activation has been shown to be linked to oncogenic transformation.
  • IFN- ⁇ interferon-gamma
  • IL-6 interleukin-6
  • EGF epidermal growth factor
  • IFN- ⁇ is a cytokine that plays a fundamental role in several aspects of the immune response (Boehm et al., 1997) .
  • Other properties include stimulation of bactericidal activity of phagocytes, stimulation of antigen presentation through class I and II major histocompatability complex molecules, as well as affects on cell proliferation and apoptosis.
  • IFN- ⁇ is able to mediate activation of an antiviral state and cause cell growth arrest at the Gi phase of the cell cycle.
  • the IFN- ⁇ response has recently been postulated to be part of an endogenous tumour surveillance system (Kaplan et al . , 1998).
  • STATl interacts with the tumour suppressor BRCAl. This leads to differential activation of transcription of a subset of IFN- ⁇ target genes leading to growth inhibition by this cytokine, with one of these genes being the cyclin-dependent kinase inhibitor, p2lWAFl (Ouchi et al . , 2000). It has been further shown that p2lWAFl activation is impaired in breast cancer cells lacking a functional BRCAl protein. Thus it is possible that the disturbance of the p21WAFl induction provides an early growth advantage to nascent tumour cells, which allows them to bypass the initial antitumour actions of IFN- ⁇ .
  • EGF and IL-6 type cytokines also mediate their actions in part through the STAT pathway. While EGF is a mitogen for many cells, growth of some cultured cell lines, containing high numbers of EGF receptors, are inhibited by EGF. This growth inhibition has been shown for A431 cells to be mediated by the activation of STATl (via specific receptor kinase activity) and NF-KB (via 1KB degradation), which drive p21WAFl gene expression (Ohtsubo et al . , 2000).
  • the IL-6 type cytokines signal through the common receptor subunit gpl30 and are involved in the regulation of many processes including gene expression, cell proliferation and differentiation. IL-6 has also been shown to stimulate inflammatory responses during wound healing.
  • PIAS proteins can modulate steroid receptor-dependent transcriptional activation and also have an established role in the negative regulation of STAT signaling.
  • PIASl binds to the STATl dimer; this has been proposed to mask the DNA-binding activity of STATl (Liao et al . , 2000).
  • Recent studies have suggested that the PIAS family of proteins may function to regulate other transcriptional responses (Moilanen et al . , 1999). Therefore the recruitment of PIASl to different transcription factors only after ligand stimulation may allow the targeting of PIASl to a specific transcriptional response induced by the corresponding signal.
  • TSG16 may play a role in all PIAS associated functions.
  • TSG16 and/or TSG18 may: (1) form part of the SUMO E3 ligase complex that sumoylates protein targets such as p53; or (2) be additional tumour suppressor genes (other than p53) that are sumoylated by PIASl containing SUMO E3 ligase complexes; or (3) represent a novel family of proteins involved in modulation of the STAT signaling pathway. Such signals are linked to immunological responses, including those associated with tumour suppression.
  • TSG18 interacting genes and proteins can also be studied such that these partners can also be targets for drug discovery.
  • TSG18 recombinant proteins can be produced in bacterial, yeast, insect and/or mammalian cells and used in crystallographical and NMR studies. Together with molecular modeling of the protein, structure-driven drug design can be facilitated.
  • TSG18 The knowledge of the nucleotide and amino acid sequence of TSG18 allows for the production of antibodies, which selectively bind to TSG18 protein or fragments thereof. Following the identification of mutations in the gene, antibodies can also be made to selectively bind and distinguish mutant from normal protein. Antibodies specific for mutagenised epitopes are especially useful in cell culture assays to screen for malignant cells at different stages of malignant development. These antibodies may also be used to screen malignant cells, which have been treated with pharmaceutical agents to evaluate the therapeutic potential of the agent.
  • short peptides can be designed homologous to the TSG18 amino acid sequence. Such peptides are typically 10 to 15 amino acids in length. These peptides should be designed in regions of least homology to the mouse orthologue to avoid cross species interactions in further down-stream experiments such as monoclonal antibody production. Synthetic peptides can then be conjugated to biotin (Sulfo-NHS-LC Biotin) using standard protocols supplied with commercially available kits such as the PIERCETM kit (PIERCE) .
  • PIERCETM kit PIERCE
  • Biotinylated peptides are subsequently co plexed with avidin in solution and for each peptide complex, 2 rabbits are immunized with 4 doses of antigen (200 ⁇ g per dose) in intervals of three weeks between doses. The initial dose is mixed with Freund's Complete adjuvant while subsequent doses are combined with Freund's Immuno-adjuvant. After completion of the immunization, rabbits are test bled and reactivity of sera assayed by dot blot with serial dilutions of the original peptides. If rabbits show significant reactivity compared with pre-im une sera, they are then sacrificed and the blood collected such that immune sera can separated for further experiments.
  • Monoclonal antibodies can be prepared for TSG18 in the following manner. Immunogen comprising intact TSG18 protein or TSG18 peptides (wild type or mutant) is injected in Freund's adjuvant into mice with each mouse receiving four injections of 10 to 100 ug of immunogen. After the fourth injection blood samples taken from the mice are examined for the presence of antibody to the immunogen. Immune mice are sacrificed, their spleens removed and single cell suspensions are prepared (Harlow and Lane, 1988). The spleen cells serve as a source of lymphocytes, which are then fused with a permanently growing myeloma partner cell (Kohler and Milstein, 1975) .
  • Cells are plated at a density of 2X10 5 cells/well in 96 well plates and individual wells are examined for growth. These wells are then tested for the presence of TSG18 specific antibodies by ELISA or RIA using wild type or mutant TSG18 target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality. Clones with the desired specificity are expanded and grown as ascites in mice followed by purification using affinity chromatography using Protein A Sepharose, ion-exchange chromatography or variations and combinations of these techniques.
  • tumour suppressor gene TSG18
  • TSG18 The tumour suppressor gene, TSG18, is implicated in cancers that arise from a number of tissues due to its ubiquitous expression pattern.
  • this gene is implicated in other disease states due to the presence of specific functional domains within its encoded protein.
  • the novel DNA molecules of the present invention are therefore useful in methods for the early detection of disease susceptible individuals as well as in diagnostic, prognostic and therapeutic procedures associated with these disease states.

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Abstract

L'invention concerne une molécule d'acide nucléique isolée comprenant la séquence nucléotidique exposée dans la séquence SEQ ID NO:1, ou des fragments actifs de cette séquence, codant un polypeptide actif dans la suppression de la prolifération cellulaire. Plus particulièrement, TSC18 a une fonction de gène suppresseur de tumeurs et joue également un rôle dans les troubles immunitaires/auto-immunitaires/inflammatoires. L'invention concerne également des variants de ces molécules d'ADN permettant de retenir leur fonction, des polypeptides codés par ces molécules d'ADN et des anticorps associés, ainsi que l'utilisation de ces molécules dans des applications diagnostiques, pronostiques et thérapeutiques et d'autres applications, telles que l'identification de substances pharmaceutiques d'intérêt potentiel.
PCT/AU2001/001623 2000-12-14 2001-12-14 Gene suppresseur de tumeurs identifie sur le chromosome 18 WO2002048354A1 (fr)

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AU2002221342A AU2002221342A1 (en) 2000-12-14 2001-12-14 Tumour suppressor gene identified on chromosome 18

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AUPR2049 2000-12-14
AUPR2049A AUPR204900A0 (en) 2000-12-14 2000-12-14 A novel gene

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WO2002048354A1 true WO2002048354A1 (fr) 2002-06-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003073105A2 (fr) * 2002-02-28 2003-09-04 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Utilisation du plag1 et du plagl2 dans le diagnostic du cancer et le criblage de médicaments

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001032861A1 (fr) * 1999-10-29 2001-05-10 Women's And Children's Hospital Genes suppresseurs de tumeurs du chromosome 16

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2001032861A1 (fr) * 1999-10-29 2001-05-10 Women's And Children's Hospital Genes suppresseurs de tumeurs du chromosome 16

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DATABASE GENBANK [online] 22 February 2000 (2000-02-22), SUGANO ET AL., accession no. EMBL Database accession no. (AK000060) *
DATABASE GENBANK [online] 29 September 2000 (2000-09-29), ISOGAI ET AL., accession no. EMBL Database accession no. (AK021430) *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003073105A2 (fr) * 2002-02-28 2003-09-04 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Utilisation du plag1 et du plagl2 dans le diagnostic du cancer et le criblage de médicaments
WO2003073105A3 (fr) * 2002-02-28 2004-04-01 Vlaams Interuniv Inst Biotech Utilisation du plag1 et du plagl2 dans le diagnostic du cancer et le criblage de médicaments

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AU2002221342A1 (en) 2002-06-24
AUPR204900A0 (en) 2001-01-11

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